were observed (Van Rie et al., 1989). Hence, there is a complexity in toxin-membrane interactions in the insect midgut environment. Binding of toxin to the ...
EFFECT OF RANDOM MUTAGENESIS OF NATIVE cry1Ac AND cry2 ON THE TOXICITY AGAINST Plutella xylostella
ATUL KUMAR UPADHYAY
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005
DECEMBER, 2009
ii
EFFECT OF RANDOM MUTAGENESIS OF NATIVE cry1Ac AND cry2 ON THE TOXICITY AGAINST Plutella xylostella
Thesis submitted to the University of Agricultural Sciences, Dharwad in partial fulfilment of the requirements for the Degree of
Master of Science (Agriculture) in
PLANT BIOTECHNOLOGY
By ATUL KUMAR UPADHYAY
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD - 580 005
DECEMBER, 2009
iii
DEPARTMENT OF BIOTECHNOLOGY COLLEGE OF AGRICULTURE, DHARWAD UNIVERSITY OF AGRICULTURAL SCIENCES, DHARWAD
CERTIFICATE This is to certify that the thesis entitled “EFFECT OF RANDOM MUTAGENESIS OF NATIVE cry1Ac AND cry2 ON THE TOXICITY AGAINST Plutella xylostella” submitted by Mr. ATUL KUMAR UPADHYAY, for the degree of MASTER OF SCIENCE (AGRICULTURE) in PLANT BIOTECHNOLOGY to the University of
Agricultural Sciences, Dharwad, is a record of research work carried out by him during the period of his study in this university, under my guidance and supervision, and the thesis has not previously formed the basis for the award of any degree, diploma, associateship, fellowship or other similar titles.
(P. U. KRISHNARAJ)
DHARWAD DECEMBER, 2009
MAJOR ADVISOR
Approved by : Chairman :
Members :
____________________________ (P. U. KRISHNARAJ)
1. __________________________ (A. R. ALAGAWADI)
2. __________________________ (A. S. VASTRAD)
3. __________________________ (RAMESH BHAT)
iv
Affectionately Dedicated to My Beloved
Parents Shri A. N. Upadhyay Smt. Meera Upadhyay, Brother Rahul Kumar Upadhyay & Sisters Priyanka and Vandana Upadhyay
v
ACKNOWLEDGEMENT No scientific endeavour is a result of an individual effort. The task of acknowledging the cooperation that was offered to me through out my study by my teachers, friends and family member gives me a great sense of pleasure and I feel scanty of word to magnitude their cooperation. I express my deep gratitude to Dr. P. U. KRISHNARAJ, Professor and Head, Department of Biotechnology, University of Agricultural Sciences, Dharwad and chairman of my Advisory Committee, who has been the prime driving force throughout my study. I wish my profound sense of reverence to him for having kindled imagination, nutured scientific temper and offered an academic niche during the course of investigation. It has been very good fortune to have an excellent Advisory Committee of Dr. A. R. ALAGWADI, Dean (Agri,)College of Bijapur, UAS, Dharwad, Dr. A. S. VASTRAD, Associate Professor, Department of entomology and Dr. RAMESH BHAT, Associate Professor, Department of Biotechnology, UAS, Dharwad who have unflinching cooperation and timely suggestions throughout my course of investigation. In a personal note, I wish to express my sincere gratitude and indebtedness to my parents Shri A. N. Upadhyay and Smt. Meera Upadhyay, sisters and brother for their care and support, I find
vi
acknowledge inadequate to quantity the sacrifices, love, affection and constant confidence instilled in me by them and my gratitudes are beyond words. I am strongly beholden to my friends Safi, Malik Ahamed Pasha, Ashwini, Amit, Chidu, Supriya, Ramesh, Sakthi, Rajkumar, Mangala, Mohan, Yadav, Prashant, Verma, Surya, Yamane, Chhaya, Sudipta, Gaurav, and so on who helped directly or indirectly during my study. I am thankful to M/s Anup Computers, Dharwad for their meticulous typing of the manuscript. I apologise for any omission, which of course not deliberate.
DHARWAD DECEMBER, 2009
(Atul Kumar Upadhyay)
vii
CONTENTS Sl. No.
1. 2.
3.
4.
5. 6.
Chapter Particulars
Page No.
CERTIFICATE ACKNOWLEDGEMENT LIST OF TABLES LIST OF FIGURES LIST OF PLATES LIST OF APPENDICES INTRODUCTION REVIEW OF LITERATURE 2.1 Importance of B. thuringiensis. 2.2 Classification and diversity of B. thuringiensis 2.3 Structure of B. thuringiensis endotoxins. 2.4 Mode of action of three-domain Cry toxins. 2.5 Structure-function analysis of δ-endotoxins MATERIAL AND METHODS 3.1 Confirmation of clones having cry1Ac and cry2. 3.2 Creation of random mutation using GeneMorph Random Mutagenesis kit. 3.3 Cloning of mutated amplicons of cry1Ac and cry2 into pET32C+. 3.4 Transformation of E. coli BL21 (DE3)pLysS 3.5 Cloning of cry1Ac mutant amplicons in yeast expression vector (pYES2/CT). 3.6 Yeast (Saccharomyces cerevisiae) Transformation. EXPERIMENTAL RESULTS 4.1 Confirmation of clones and bioassay for insecticidal activity. 4.2 Random mutagenesis and expression analysis. 4.3 PCR based cloning of mutated amplicons of cry1Ac and cry2. 4.4 Bioassay results. 4.5 Sequence analysis. 4.6 Cloning and expression of mutated amplicons of cry1Ac into the yeast expression vector. DISCUSSION SUMMARY AND CONCLUSIONS REFERENCES APPENDICES
iii v viii ix x xi 1-3 4-18 4 5 8 9 11 19-33 19 26 27 29 31 31 34-52 34 37 44 48 48 48 53-58 59-60 61-73 74-78
viii
LIST OF TABLES
Table No.
Title
Page No.
1.
Bioassay results of cry1Ac mutants in E. coli
39
2.
Bioassay results of cry2 mutant clones in E. coli
42
3.
Mutation in cry1Ac and the observed effect on mortality of P. xylostella
50
4
Bioassay results of cry1Ac mutants expressed in yeast
52
5
List of some of the earlier noticed mutations and their effect on the toxicity
57
ix
LIST OF FIGURES
Figure No.
Title
Page No.
1
Complete nucleotide sequence of native cry1Ac (3.5 kb) from Bacillus thuringiensis.
21
2
Full length amino acid sequences of native cry1Ac gene.
22
3
Full length nucleotide sequence of native cry2 gene.
23
4
Map of construct pAK125 having mutant of cry1Ac in pET32C+
45
5
Map of construct pAK220 containg mutant of cry2 in pET32C+
45
6
Map construct of pYAK110 containing full length mutant of cry1Ac in pYES2/CT
45
7
Alignment of native and mutated cry1Ac showing position of altered amino acids.
49
x
LIST OF PLATES
Plate No.
Title
Page No.
1
PCR confirmation of cry1Ac in pET32C+
35
2
Restriction analysis of cry1Ac in pET32C+
35
3
Expression analysis of cry1Ac in E. coli
35
4
PCR confirmation of cry2 in pET32C+
36
5
Restriction analysis of cry2 in pET32C+
36
6
Expression analysis of cry2 in E. coli
36
7
Confirmation of cry1Ac mutants amplicons
38
8
Confirmation of cry1Ac mutants in pET32C+ by colony PCR
38
9
Confirmation of cry2 mutants amplicons
38
10
Confirmation of cry2 mutants in pET32C+ by colony PCR
46
11
Confirmation of mutagenic cry1Ac clones in pYES2/CT by colony PCR
46
12
Leaves treated with M25 of cry1Ac
47
13
Putative structure of 3-domain Cry proteins
47
xi
LIST OF APPENDICES
Appendix No.
Title
Page No.
I
Extraction/Lysis Buffers and Solutions for Plasmid preparation
74
II
Buffers and solutions for Agarose Gel Electrophoresis
75
III
Media for Bacterial Culture
75
IV
Media for yeast culture
76
V
Yeast transformation Reagents
77
VI
Yeast total DNA extraction buffer
77
VII
Binding Buffer for Isolation of Protein (In Non-Denaturing Form)
77
VIII
Buffers and solutions for SDS PAGE
78
1
1. INTRODUCTION Agriculture is the backbone of the Indian economy. Successful crop production in many cases is highly dependent on effective control of insect pests. The diversity of agriculture, horticulture and forest species leads to the evolution of a wide variety of insect pests. Mono-cropping, crop luxuriance due to application of higher doses of nitrogen, reduction in the population of natural enemies of pests and continued availability of alternative hosts due to increase of irrigated area have all added to cause certain species constraints insect as the main problem in cultivation of many crops. The use of chemical substances to control pests started in the mid-1800s. Early insecticides were inorganic chemicals and organic arsenic compounds. Organochloride
compounds,
organophosphates,
carbamates,
pyrethroids
and
formamides followed them. Many of these chemicals which are still being used today have long residual action and toxicity to a wide spectrum of organisms that has made them attractive for use. However, the use of synthetic organic insecticides developed during the last half of this century may pose risks to human health and can cause environmental problems (Kuo et al., 2000). Consequently, research has been channelized on the development of alternative strategies for safe insect pest management. One contemporary approach that has received attention is the development of Bacillus thuringiensis (Bt) toxins as insecticides. B. thuringiensis is a gram positive bacterium which is entamopathogenic (Schnepf et al., 1998). The larvicidal activity of B. thuringiensis is attributed to the parasporal crystals it produces. Because of the crystalline structure of these endotoxins, they are called as crystal proteins (Cry). The genes encoding for the crystal proteins are named as cry genes, and their common characteristic is the expression of the genes during the stationary phase. These crystal proteins accumulate in the cell and are released upon completion of sporulation. These Cry proteins are classified into different categories according to the homology of their amino acid sequences. Over 300 cry genes have been classified into 47 groups and the 22 cyt genes so far discovered have been divided into two classes. The number and type of
2
δ-endotoxins produced determine the bioactivity of a Bt strain (Crickmore et al., 1998, Schnepf et al., 1998, Hofte and Whiteley, 1989). To date, 40 divisions of primary rank have been named and 116 unique sequences in the cry gene super family. The molecular mass of Cry proteins or insecticidal crystal proteins (ICP) B. thuringiensis ranges from 25 kDa to 140 kDa. The morphology, size and number of parasporal inclusions vary among different B. thuringiensis strains. The toxic protein against lepidopteran for instance, belongs to the cry1, cry2, cry8, cry9 or cry20 groups. Within the Cry1 protein group, there are around ten different subclasses, and each subclass has a specific range of activity against different lepidopteran insects Additionally, in certain strains a cytolytic protein, cytolysin (Cyt), is found in the crystal inclusions of the strains active against diptreous strains (Smith and Couch, 1991). The B. thuringiensis insecticidal δ-endotoxins have three-domain structure, with the seven amphipathic helices which comprises domain I being essential for toxicity (Li et al., 1991; Grochulski et al., 1995)). δ-endotoxins are characterized by their narrow range of specificity towards selected group of insects and the specificity is attributed to domain II. Domain II is made of three antiparallel β-sheets. Domain III is made of two antiparallel β-sheets into β-sandwich structure (Grochulski et al., 1995). Intermolecular interactions through salt bridges and hydrogen bonding between domains III and I have been identified through X-ray crystallographic studies. It is assumed that domain III β- sandwich of δ-endotoxins can take part in other functions such as stability, receptor binding, specificity determination and ion channel gating (Schnepf et al., 1998). In addition to traditional bioassays on larvae of insect species, advancements in molecular biology paved the way for a thorough understanding of structurefunction relationships of δ-endotoxins. Protein engineering studies are based on methods for introducing mutations in the genes that encode proteins of interest and for producing the proteins in large amount in bacteria for further analysis.
3
Numerous modifications can be created in δ-endotoxins using various protein engineering techniques such as (a) Single or multiple amino acid change in variable and conserved regions through site directed mutagenesis. (b) Restriction fragment exchange between closely related Cry toxin genes or with other bacterial toxin genes and (c) Exchange of domains between toxin genes through PCR mediated cloning or in vivo recombination in recA+ (recombinant proficient) Eschericha coli Strains. The effect can be studied on 1) Stability of proteins using proteases like trypsin and larval midgut juice. 2) In vivo toxicity analysis on larvae. 3) In vitro toxicity analysis in insect cell lines-lawn assay. 4) Prediction of secondary structural changes using CD spectrometry.
Mutation studies will not only reveal the mechanism by which δ-endotoxins work, but it can generate toxins with enhanced toxicity with or without new brush border membrane (BBMV) binding properties (Manojkumar, and Aronson, 1999). These toxins could be used in resistant management as alternatives for the toxins already in use to which insects may become resistant by losing receptors. Keeping these in view, the present investigation was undertaken with the following objectives. 1. To create random mutations in cry1Ac and cry2. 2. To analyze the effect of altered nucleotides on the toxicity to a chosen insect.
4
2. REVIEW OF LITERATURE Bacillus thuringiensis is an aerobic, Gram-positive, spore forming soil bacterium; first time isolated from diseased silkworm larvae (Ishiwata, 1901) and was subsequently named as Bacillus sotto (Aoki and Chigasaki, 1915). A decade later, Berliner (1915) from Germany isolated another strain that killed grain moth larvae in stored grain. Since the latter strain was discovered in the province of Thuringen, it was subsequently named as B. thuringiensis, (Knowles, 1994). This bacterium under sub optimal or stressed condition makes a dormant spore and one or more large crystalline inclusions. The crystal inclusion is popularly known as insecticidal crystal protein (ICP) and the successful generation of transgenic insect resistance crop plants by transferring the ICP gene of Bacillus thuringiensis (Perlak, 1990) is one of the major breakthroughs in crop science that the new genetics has provided. The present research envisaged native cry1Ac and cry2 from Bacillus thuringiensis to create random mutations and analyze the effect of altered nucleotide on their toxicity. The relevant literature on these aspects is briefly reviewed here. 2.1 Importance of B. thuringiensis During sporulation Bacillus thuringiensis (Bt) produces insecticidal crystal (Cry) proteins. Each of these proteins has a unique spectrum of toxicity (Uawithya, 1998). Because of rapidly expanding use of Bt toxins in pest control, The knowledge of mode of action of Bt toxins is becoming increasingly important for proper deployment of this valuable biopesticide and avoidance of insect resistance (Tabashnik, 1994). B. thuringiensis accounts for 90 per cent of the biopesticides used to combat insect pest (Ferre et al., 1995). Being a bio-agent, it has many advantages viz., eco-friendly, specific to certain order of harmful insect pests while not being harmful to predators, beneficial insects, mammals and birds (Ferre et al., 1995) The crystal produced by B. thuringiensis are populary referred to as δendotoxins or Cry proteins which are found to be effective against lepidopterans,
5
(Caterpillars), dipterans(flies) and coleopteran beetles (Hofte and Whitely, 1989). The ICP’s initially were grouped into four classes on the basis of their host range and sequence homology (Crickmore et al., 2002) as cry 1 and 2 against lepidopterans, cry 3 against coleopterans and cry 4 against dipterans. Some Crystal proteins are also related to be nematicidal (Bravo et al., 1998). A few Crystal toxins have specific action on dipteran insects and are called ‘cyt’ (Knowles, 1994). However with several cry genes being reported the classification has undergone changes. Development of better strains of Bacillus thuringiensis as microbial insecticide, increased efficiency in production and quality control lead to the development of formulations with high activity and improved spray charecteristics (Vanfrankenhuyen, 1993). However, some constrains like specificity, narrow host range, low persistence on the plant, and high costs of production have limited their penetration into major crop markets. In spite of these drawbacks, B. thuringiensis formulations are some of the most eco-friendly insecticides ever used (Gelernter and Evans, 1999). 2.2 Classification and diversity of B. thuringiensis B. thuringiensis started out as an independent species distinguishable from other bacilli, such as Bacillus cereus Frankland. It is very close to B. cereus except that it produces a crystal at sporulation (sometimes more than one) which is usually bipyramidal in shape (sometimes square, flat or amorphous) and is toxic to lepidopteran, dipteran, or coleopteran insect larvae. This has resulted in a taxonomic problem. Some classical bacterial taxonomists felt that B. thuringeinsis should be a subspecies of B. cereus (Smith and Couch, 1991; Gordon et al., 1973). It was not possible to separate B. cereus and B. thuringeinsis by crossed immuno electrophoresis of ultrasonic extract of spore free-grown vegetative cells (Krieg et al., 1987) and by fatty acid patterns. Most importantly DNA homologies of 80-101% were found. As B. thuringenesis became increasingly commercially important and different isolates were discovered, the need for a method to identify and classify B. thuringiensis subspecies became apparent. Bone and Ellar (1989) and de Barjac and Franchan (1981)
6
developed an identification and classification scheme based on serological analysis of vegetative cell flagellar (H) antigens plus biochemical characteristics. Over 3500 strains of B. thuringenesis available in 1999 were divided into 82 serovars and registered at International Entomopathogenic Bacillus Centre at Pasteur institute, Paris (Lecadet et al., 1999). The techniques that have been applied to the identification/classification of B. thuringenesis are high performance liquid chromatography (HPLC), plasmid mapping, cloning and sequencing of the crystal toxin genes (Berliner, 1915). B. thuringiensis soil isolates are distributed globally (Chak, 1990). Meadows (1993) analyzed three prevailing hypothetical niches of B. thuringiensis in the environment: as an entomopathogen, as a phylloplane inhabitant, and as a soil microorganism. Available data are still insufficient to choose among these and other possibilities, although B. thuringiensis seems to have been more readily isolated from insect cadavers or stored product dusts than from soil. To obtain novel B. thuringiensis strains for discovering new cry genes, isolation of numerous new B. thuringiensis strains is becoming a routine activity in many industries. The characterizations done for most of the collections were based on bioassays against different insect larvae without identification of the cry genes present in the B. thuringiensis strains. In the last few years, some PCR based methodologies have been proposed to identify different cry genes in B. thuringiensis strains (BenDov et al., 1997). B. thuringiensis was present in dust from grain storages than in other habitats. Sodium
dodecyl
sulfate-polyacrylamide
gel
electrophoresis
(SDS-PAGE)
differentiated 92 distinct protein profiles of B. thuringiensis. Serological identification also showed great diversity among the Spanish isolates which were distributed among 38 of the 58 known serovars. The most frequently found serovars were aizawai, kurstaki, konkukian, morrisoni, and thuringiensis, which together represented more than 50% of the serotyped isolates. Significant insecticidal activity was observed by toxicity assays against the lepidopterans, Heliothis armigera (76.1% of the assayed
7
isolates), Spodoptera exigua (50.5%), and Plutella xylostella (19.7%), (Iriarte et al., 2002). Armengol et al. (2007) studied crystal morphology of 445 isolates from all the natural regions of Columbia and the most abundant type were found to be bipyramidal (60%) Ten per cent of isolates were toxic to S. frugiperda and five per cent against Culex quinquefasciatus larvae. Similarly, Ferre et al. obtained 202 B. thuringiensis isolates from coffee plantation that showed diverse crystal morphology viz., oval (37%), bipyramidal (3%),
bipyramidal and oval (12%), pleomorphic (35%),
bipyramidal, oval and pleomorphic (10%), bipyramidal, oval and cubic (3%). The flagellar antigen serotypes, cry genes and crystal proteins of 570 B. thuringiensis isolates of China were determined, and the pesticidal activity was assayed against the insects, P. xylostella, Heliothis armigera, Phaedon brassicae and Locusta migratoria manilensis, and the snail, Oncomelania hupensis. The assayed isolates showed high mortality of the assayed pests with 14.9%, 6%, 1.6%, 1.1%, and 0.2% of the isolates killing more than 90% of P. xylostella, H. armigera, P. brassicae, O. hupensis, and L. migratoria manilensis, respectively. These B. thuringiensis isolates were distributed within 35 H-serotypes, in which isolates of H3 were the most abundant (20%) followed by H5 (13%), H7 (9%) and H4 (8.7%). Two hundred and sixty five isolates did not show any amplification product for the genes cry1Aa, cry1Ac, cry1C, cry2, cry3, cry4, cry7Aa (Meiying et al., 2008). A total of 146 B. thuringiensis strains were obtained from environmental soil samples which exhibited a diverse number, size and morphology of parasporal inclusion bodies: irregular (47%), oval (20%), bipyramidal (3%), bipyramidal and cubic (1%), bipyramidal, oval and irregular (5%) and bipyramidal, oval and cubic crystals (2%). Fifty six percent of the strains amplified with the cry2 primer, 54% with vip3, 20% with cry1, 9% with cry3-cry7 and 8% with cry8 (Arrieta et al., 2006). Finally it can be said that with increasing number of isolates being isolated, poricity of availability of antisera, its necessary to device newer form of classification at the earliest.
8
2.3 Structure of B. thuringiensis (Bt) Endotoins B. thuringiensis (Bt) δ-endotoxins are globular protein molecules, which accumulate as protoxins in crystalline form during late stage of the sporulation. Protoxins are liberated in the midgut after solubilization and is cleaved off at Cterminal part to release ~66 kDa active N-terminal toxic molecule (Bravo et al., 2004). The protoxin contains well-conserved cysteine residues (as many as 16 in Cry1Ac), which helps in bridging the protoxin molecules through intermolecular disulphide bonds and thereby crystal formation. Currently, 3-dimensional protein structures have been determined for three Bt toxins through X-ray crystallography. Among them two are crystals forming (Cry) proteins or δ-endotoxins viz. Cry1Aa (Lepidoptera-specific; Grochulski et al., 1995) and Cry3A (Coleoptera-specific; Li et al., 1991). Since primary amino acid composition determines the final structure of a protein, closely related proteins, Cry1Aa and Cry3A, with 36% amino acid sequence identity showed superimposable structure with similar mode of action, whereas Cyt2A protein, which shares less than 20% amino acid sequence identity, is made of single domain with different functional properties (Schnepf et al., 1998). The tertiary structure of δ-endotoxins is comprised of three distinct functional domains connected by a short conserved sequence. Each domain of δ-endotoxins has independent and inter-related functions in the larval midgut, which brings out colloid osmotic lysis (Knowles, 1994). Phylogenetic analysis on the domains of δ-endotoxins revealed that domain I is the most conserved and domain II is hyper variable. (Bravo, 1997). To date, the tertiary structures of six different three-domain Cry proteins, Cry1Aa, Cry2Aa, Cry3Bb, Cry4Aa and Cry4Ba have been determined by X-ray crystallography (Li et al., 1991; Grochulski et al., 1995; Morse et al.; 2001; Galitsky et al., 2001; Boonsrrm et al., 2005.). All these structures display a high degree of similarity with a three-domain organization, suggesting a similar mode of action of the Cry three-domain protein family. The N-terminal domain (domain I) is a bundle of seven α-helices in which the central helix-α5 is hydrophobic and is encircled by six other amphipathic helices; and thus helical domain is responsible for membrane
9
insertion and pore-formation. Domain II consists of three anti-parallel β-sheets with exposed loop regions, and domain III is a β-sandwich (Li et al., 1991; Grochulski et al., 1995; Morse et al.; 2001; Galitsky et al., 2001; Boonsrrm et al., 2005.). The exposed regions in domains II and III are involved in receptor binding (Bravo et al., 2005). Domain I shares structural similarity with other PFT like colicin Ia and N and diphtheria toxin, supporting the role of this domain in pore-formation. In case of domain II, structural similarities with several carbohydrate-binding proteins like vitelline, lectin jacalin, lectin Mpa have been reported (de Maagd et al., 2003). Domain III, shares structural similarities with other carbohydrate-binding proteins such as the cellulose binding domain of 1,4-β-glucanase C, galactose oxidase, sialidase, β-glucoronidase, the carbohydrate-binding domain of xylanase U and βgalactosidase (de Maagd et al., 2003). These similarities suggest that carbohydrate moieties could have an important role in the mode of action of three-domain Cry toxins. In the nematode C. elegans mutations in bre genes involved in the synthesis of certain glycolipids lead to Cry5 resistance showing that glycolipids are important receptor molecules of Cry5 (Griffits et al., 2005). The independent evolution of the three structural domains and domain III swapping among different toxins generated proteins with similar mode of action but with very different specicities (Bravo, 1997; de Maagd et al., 2001). 2.4 Mode of action of three-domain Cry toxins. The mode of action of Cry toxins has been characterized principally in lepidopteran insects. The primary action of Cry toxins is to lyse midgut epithelial cells in the target insect by forming pores in the apical microvilli membrane of the cells (Aronson and Shai, 2001; de Maagd et al., 2001, Bravo et al., 2005). Nevertheless, it has been recently suggested that toxicity could be related to G-protein mediated apoptosis following receptor binding (Zhang et al., 2006). Cry proteins pass from crystal inclusion protoxins into membrane-inserted oligomers that cause ion leakage and cell lysis. The crystal inclusions ingested by susceptible larvae dissolve in the alkaline environment of the gut, and the solubilized inactive protoxins are cleaved by
10
midgut proteases yielding 60-70 kDa protease resistance protein (Bravo et al., 2005). Toxin activation involves the proteolytic removal of an N-terminal peptide (25-30 amino acids for Cry1 toxins, 58 residues for Cry3A and 49 for Cry 2Aa) and approximately half of the remaining protein from the C-terminus in the case of long Cry protoxins. The activated toxin then binds to specific receptors on the brush border membrane of the midgut epithelium columnar cells (de Maagd et al., 2001; Bravo et al., 2005) before inserting into the membrane. Toxin insertion leads to the formation of lytic pores in microvilli of apical membranes (Aronson and Shai, 2001; Bravo et al., 2005). The three-dimensional structure of Cry2Aa protoxin showed that two αhelices of the N-terminal region occlude a region of the toxin involved in the interaction with the receptor (Morse et al., 2001). A Cry1Ac mutant that retained the N-terminus end after trypsin treatment binds nonspecifically to Manduca sexta membranes and was unable to form pores on M. sexta brush border membrane vesicles (BBMV) (Bravo et al., 2002) For Cry1A toxins, at least four different binding-proteins have been described in
different
lepidopteran
insects;
a
cadherin-like
protein
(CADR),
a
glycosylphosphatidyl-inositol (GPI)-anchored aminopeptidase-N (APN), a GPIanchored alkaline phosphatase (ALP) and a 270 kDa glycoconjugate (Vadlamudi et al., 1995; Knight et al., 1994; Jurat-Fuentes and Adang, 2004; Valaitis et al., 2001). Oligomeric structures of Cry1Ab and Cry1Ac increase 100-200-fold their binding affinity to the APN receptor, showing apparent dissociation constants of 0.75-1.0 nM (Gomez et al., 2003; Pardo-Lopez et al., 2006). Using tryptophan fluorescence analysis, structural changes observed after binding of the oligomeric Cry1Ac toxin to the APN recptor were studied by analyzing the binding of N-acetylgalactosamine (GalNAc) to the Cry1Ac toxin, since GalNAc is a binding determinant in the Cry1AcAPN interaction (Pardo-Lopez et al., 2006). The in vitro interaction of GalNAc with oligomeric Cry1Ac induced a conformational change in the toxin and enhanced its insertion into lipid membranes indicating that the interaction of the pre-pore oligomer of Cry1A toxins with APN is important for facilitating membrane insertion (PardoLopez et al., 2006).
11
4.5 Structure-function analysis of δ- Endotoxins and mutagenesis. N-terminal part of the toxic fragment comprising of six amphipathic helices (α- 1, 2, 3, 4, 6, 7) with a central hydrophobic helix (α-5) makes the domain I of δendotoxins (Li et al., 1991; Grochulski et al., 1995). Although domain I of δendotoxins shares little sequence homology with other bacterial toxins like Colicin A, Diphtheria B subunit and Pseudomonas exotoxin, it works similar to other bacterial toxins by forming pores in the cell membranes (Parker and Pattus, 1993). pH dependent ion channel formation is a common feature of bacterial toxins such as colicin, botulinum toxin and diphtheria toxin (Slatin et al. 1990). pH dependent conformational change in δ-endotoxins provided evidence for assuming a similar mode of action for Bt toxins (Convents et al., 1990). Two alternative models viz. "penknife model" (Hodgman and Ellar, 1990) and "umbrella model" (Li et al. 1991) were proposed to explain the pore forming mechanism of domain I of δ-endotoxins , both of them previously proposed for the colicin toxin (Parker and Pattus, 1993). Ion channels formed by the δ-endotoxins appear to have more than one channel due to aggregation of toxins. Co-operative gating of more than one identical channel is observed lending support to the co-operative gating hypothesis (Slatin et al., 1990). Model artificial membranes like liposomes, planar lipid bilayers (PLB) and phosphotidylcholine vesicles are used to assess the pore forming ability of toxins under in vitro conditions. Mutations in the helix region along with synthetic peptide mimicking studies has shown clear evidence for the ion channel formation by domain I. Studies on orientation of the membrane-bound state of the seven α- helices comprising of the pore forming domain of Cry3A δ-endotoxins showed that α4 -α5 helix loop inserts into the membrane in a hairpin-like manner, leaving all other helices on the surface of the membrane in a bound state (Gazit et al., 1998). This observation supports the "umbrella model" proposed by Li et al., (1991). Following insertion of the toxin, helix 1 is removed due to protease digestion and it is the only helix, which did not bind to BBMV vesicles. Therefore, it is clear that the N-terminal fragment comprising of
12
domain I alone is enough to form ion channel in PLB. Synthetic peptide mimicking studies showed that α5 helix and α4-α5-helix loop is important for toxin aggregation and ion channel formation (Gerber and Shai, 2000). Specific mutations within the α4α5 loop of Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170 (Kanintrokul et al., 2003). Mutations were created in the regions of the cry1Ac1 gene encoding residues within three helices, i.e., 2, 5, and 6, and found that helix 5 was the only one in which many of the mutations abolished toxicity (Aronson and Zhang, 1995.; Wu and Aronson, 1992). Thirty-nucleotide mutagenic oligonucleotides were used to obtain random mutations in regions of cry1Ac1 gene encoding residues in helix α-4 and the loop. Four site-specific mutations which should have altered the hydrophobic properties of helix α-3 were examined. Many mutations in helix α-4 resulted in either the loss of toxicity or toxin instability, and one mutant toxin had enhanced activity. Mutations in the loop connecting helices α-4 and α-5 or within helix α-3, had little effect on stability or toxicity. (Kumar and Aronso, 1999). δ-endotoxins are characterized by their narrow range of specificity towards selected group of insects and the specificity is attributed to domain II. Domain II is made of three antiparallel β-sheets, oriented parallel to the α-helices of domain I. Apex of domain II is formed by three surface exposed loops of variable length and the tips of these hairpins are comprised of residues 310 to 313, 367 to 379 and 438 to 456 from sheets 1, 2 and 3 respectively (Grochulski et al., 1995). These surface exposed loops located in the hypervariable blocks of domain II of the δ-endotoxins are identified as specificity determining regions. Protoxins (~130 kDa) are converted into toxic fragment (~ 66 kDa) by gut proteases. In vitro experiments like BBMV binding and immunoblotting with labelled toxins showed the direct correlation between toxicity and binding of toxins to the midgut receptors. Binding site heterogeneity (binding of different toxins to different receptor sites or partially overlapping sites in different insects) was identified as a major specificity-determining factor for insecticidal activity of δ-endotoxins.
13
Ligand blot analysis of SDS-PAGE separated Heliothis virescens BBMV proteins with labeled δ-endotoxins sharing high homology in the domain II loops revealed that the receptor A (170 kDa) was recognized by the all the tested toxins (Cry1Aa, Cry1Ab, Cry1Ac, Cry1Fa, and Cry1Ja), whereas receptor B (130 kDa) was recognized by Cry1Ab and Cry1Ac and the third receptor C (110 kDa) was recognized only by Cry1Ac (Jurat-Fuentes and Adang, 2001). Three toxins (Bt2, Bt3 and Bt73) with varying toxicity levels towards H. virescens were tested for their kinetic properties. Concentration of binding sites and equilibrium dissociation constants of these toxins showed no significant differences in binding affinity, rather considerable differences in the concentration of binding sites were observed (Van Rie et al., 1989). Hence, there is a complexity in toxin-membrane interactions in the insect midgut environment. Binding of toxin to the receptor is mediated by a two-step mechanism involving initial reversible binding followed by irreversible binding and membrane insertion. Several studies showed that toxicity did not depend on initial reversible binding rather it was correlated well with irreversible binding (Garczynski et al., 1991). These results support the earlier idea that the postbinding events seem to be integration of δ-endotoxins into the membrane and formation of pores. Receptor-bound toxin molecule could facilitate additional toxintoxin interactions. Thus, the toxins insert themselves into the membrane as oligomers. This gives the idea that domain II is involved in other processes like toxin-toxin interactions apart from receptor binding. Extensive mutagenesis followed by real time receptor binding analysis using an optical biosensor (BIAcore) on wild type and Cry1Ac toxins revealed the sequential binding of toxins through domain III and II to site 1 and site 2 of gypsy moth APN receptor. Based on this study bivalent sequential binding model is proposed to δ-endotoxins binding (Jinkins et al., 2000). Receptor binding models constructed based on the BBMV binding studies showed that toxins sharing high homology in the loops of domain II recognize same receptor molecules in larval midgut, which results in cross resistance. Thus, selecting toxins with less homology in
14
the domain II will be a better alternative to delay the resistance development against Bt toxins. Difference in activity of the toxins is due to difference in affinity for a single binding site and also difference in the concentration of binding sites. δendotoxins binding receptors in the larval midgut are identified as glycoprotein molecules. It has been supported by X-ray crystallographic data that domain II loops showed immunoglobulin like structural folds (Li et al., 1991). Structural similarity is observed between δ-endotoxins folds and other known carbohydrate binding proteins like plant lectin jacalin (Machura pomifora), outer layer protein I from hen's egg. Carbohydrates are used as recognition epitopes by these folds. So far, three kinds of glycoproteins (Amino peptidase N, Cadherin-like proteins and anionic glycoconjugates) have been identified as receptor molecules for different insect species (Agarwal et al., 2002). Genetic engineering studies involving exchange of fragments of C-terminal region of toxic fragment between closely related toxins but having different specificity to tested insects showed that the specificity determining regions are essentially located in domain II (Widner and Whiteley, 1989; Caramori et al., 1991; Schnepf et al., 1990; Ge et al., 1991). Hydrophobic interactions between δ-endotoxins loops and insect midgut receptor molecules were tested by substituting hydrophobic residues with alanine residue or replacing positively charged residues with negatively charged residues. Mutations were created in the domain II-loop 2 residues of Cry1Ab toxin. Alanine substitution in
368
RRP
370
residues abolished the toxicity toward Manduca
sexta and H. virescens due to reduced binding affinity to BBMV. Positively charged residues in the domain II might help in orientation of the toxin to midgut receptor molecules. Hydrophobic aromatic side chain residue at the position 371 is important for the irreversible binding. When phenylalanine at 371 position was replaced by hydrophilic aliphatic and smaller side chain residues such as Cysteine, Valine, and Serine amino acids toxicity was reduced but not by Tyrosine or Trytophan amino acid substitution (Rajamohan et al., 1995). Alanine substitution in loop 3 residues in
15
Cry1Ab toxin (G439A and F440A) substantially reduced the toxicity toward M. sexta and H. virescens. The loss of toxicity was correlated with reduced initial binding (Rajamohan et al. 1996). Mutants generated using site-directed mutagenesis in the loops of Cry3A toxin were tested against Tenebrio molitor. Alanine substitution in the loop 1 (Y350A, Y351A, N353A and D354A) residues resulted in loss of toxicity due to reduced receptor binding. Loop 2 mutants (P412A and S413A) did not show any effect on receptor binding and toxicity. Thus, loop 2 is not involved in toxicity determination against Tenebrio molitor. However, loop 3-block mutant (Alanine substitution of
481
QGSRG
486
residues) showed enhanced toxicity due to increased
irreversible binding (Wu and Dean, 1996). Recently Gomez et al. (2003) have demonstrated that loops α-8 and 2 of Cry1Ab domain II interact with Manduca sexta Bt-R1 receptor. From these studies, it is clear that all loops of domain II are not involved in binding of toxins to the receptor molecules of single insect species. Therefore, a toxin, which became ineffective due to loss of receptor recognition, need not be ineffective on other susceptible insects. High homology in the domain II of toxins results in cross-resistance due to sharing of the receptor molecules. It is established that loops in the domain II affect irreversible or reversible binding through hydrophobic interactions with receptor molecules. Domain III is made of two antiparallel β-sheets into β-sandwich structure. Intermolecular interactions through salt bridges and hydrogen bonding between domains III and I have been identified through X-ray crystallographic studies. Initially it was proposed that maintaining the stability of the protein is the major function of this domain. From the studies on β-strand structure of other protein molecules, it could be assumed that domain III β-sandwich of δ-endotoxins can take part in other functions such stability as receptor binding, specificity determination and ion channel gating (Schnepf et al., 1998). Arginine rich block in the domain III of δ-endotoxins is called "arg face", through which domain III makes contacts with domain I and regulates the ion channel
16
conductance. Recent studies involving site-directed mutagenesis of conserved regions of the domain III and domain III exchange between cry genes demonstrated the above-mentioned functions for domain III of δ-endotoxins. Three substitution mutants were created in the "arg face" of Cry1Aa domain III with negatively charged amino acids (R528G, R530G and R530K). One mutant was poorly expressed in E. coli due to instability of the protein molecule. This may be due to the disturbance of salt bridges between domains III and I as predicted from X-ray crystallographic studies. Other two mutants showed reduced toxicity towards Bombyx mori, without any alteration in protein structure (predicted from CD spectroscopy and trypsin digestion assay on SDS-PAGE) and binding to BBMV of B. mori. But these mutants showed reduced conductance in PLB membranes. Mutations created in the highly conserved region of Cry1Ac toxin (R 525G or R525A and R529G or R529A) resulted in 4-to 12fold and 3-fold reduction in toxicity, respectively. These mutants displayed one quarter of the maximum conductance recorded for the native Cry1Ac protein. This result is consistent with earlier observation that domain III "arg face" influences the ion channel formation through domain I interactions (Chen et al., 1993 and Masson et al., 2002). Site directed mutagenesis in highly conserved region of domain III in the Cry1Ac toxin of Bacillus thuringiensis was done to probe the function of four alternating arginines located at amino acid positions 525, 527, 529, 531. Ten mutants were created: eight single mutants, with each arginine (R) replaced by either glycine (G) or aspartic acid (D), and two double mutants (R525G/R527G and R529G/R531G). In lawn assays of the 10 mutants with a cultured Choristoneura fumiferana insect cell lines (Cf1), replacement of a single arginine by either glycine or aspartic acid at position 525 or 529 decreased toxicity 4 to 12-fold relative to native Cry1Ac toxin, whereas replacement at position 527 or 531 decreased toxicity only 3fold. The reduction in toxicity seen with double mutants was 8-fold for R525G/R527G and 25-fold for R529G/R531G. (Masson et al., 2002)
17
All the three domains of δ-endotoxins are closely packed together with the largest number of interdomain contacts found between domains I and II (Li et al., 1991 and Grochulski et al., 1995). In Cry1Aa, domains I and II are linked by four salt bridges: Asp222-Arg281, Arg233-Glu288, Arg234-Glu274, and Asp242-Arg265 (Grochulski et al., 1995). Three salt bridges, structurally equivalent to Arg233-Glu288, Arg234-Glu274, and Asp242-Arg265, are also found in Cry3A (Li et al., 1991 and Grochulski et al., 1995). In addition, for all four salt bridges at least one of the amino acid residues forming the bridge is located within block 2, a sequence that is highly conserved among Cry toxins (Hofte et al., 1998; Schnepf et al., 1998 and Bravo 1997). However, only in the case of the Asp242-Arg265 salt bridge are both amino acids located within block 2. Salt bridges appear to play an important role in toxin stability and function. Mutations preventing the formation of the Asp242-Arg265 (Meza et al., 1996 and Dean et al., 1996), Arg233-Glu288, or Arg234-Glu274 salt bridges in Cry1Ab resulted in substantial losses of stability or activity. The importance of these salt bridges is further supported by the observation that domain I exchanges between different Cry1 proteins can lead to inactive recombinant proteins (Rang et al., 1999), possibly caused by the absence of one or more essential salt bridges. In contrast, disulfide bond engineering experiments with Cry1Aa have demonstrated that pore formation requires domain I to swing away from the rest of the molecule (Schwartz et al., 1997), implying that the interdomain salt bridges must be broken during toxin insertion into the membrane. Binding studies using reciprocal hybrids made by exchanging a fragment between 451-623 amino acids of Cry1Aa with that of Cry1Ac on BBMV from Lymantria dispar showed the location of receptor binding in the third domain (Lee et al., 1995). Therefore, first direct evidence for domain III binding to receptor was established. Interestingly, hybrid with Cry1Aa third domain resulted in binding of 210-kDa receptor molecule, which is not recognized either by Cry1Aa or Cry1Ac, showing that the domain III also influences the receptor binding. Since loss of receptor binding is attributed as a major reason for the resistance development towards existing toxins, hybrid toxins with differential binding capacity can be used.
18
Chimeric protein constructed by exchanging domain III of Cry1E (inactive on Spodoptera exiqua) with that of Cry1C (most active on Spodoptera exigua) showed toxicity level equal to most active toxin, Cry1C. In heterlogous binding assay, it was demonstrated that hybrid toxin was bound to the receptor that is recognized by Cry1E toxin. Since Cry1E is already capable of binding to S. exigua BBMV without showing any appreciable toxicity, replacement of domain III with Cry1C might have helped in stabilizing the domain II interaction with the receptor (Bosch et al., 1994). Hybrid toxin comprising of domains I and II from Cry1Ab and domain III from Cry1C showed more toxicity than Cry1C towards S. exiqua (De Maagd et al., 1996). BBMV Binding studies showed that the chimeric toxin with domains I and II from Cry1Ab and domain III from Cry1C was failed to bind to 200-kda receptor, which is recognized by parental toxin Cry1Ab and another reciprocal hybrid with domains I and II Cry1C and domain III from Cry1Ab bound to 200-kDa proteins. This shows the involvement of the domain III in receptor binding. These studies suggest that the Cry1C domain III substitution in previously weak inactive toxins like Cry1Ab and Cry1E makes them toxic towards S. exigua. Therefore, domain III exchange can be followed for the other weak toxins to make them more active on agronomically important pests.
19
3. MATERIAL AND METHODS The present study was conducted to create random mutations in cry1Ac and cry2 and to analyze the effect of altered nucleotides on the toxicity to a chosen insect, Plutella xylostella (diamond back moth). The material and methods employed for achieving the objectives are detailed below. 3.1 Confirmation of clones having cry1Ac and cry2. Clones were picked up and revived on ampicillin agar plates and confirmation of the clones for the presence of cry1Ac and cry2 in different vector was done by following different methods. 1. PCR amplification and 2. Restriction confirmation. For PCR amplification and restriction analysis first plasmid was isolated by following method. Plasmid isolation: The alkaline lysis protocol of Sambrook et al., 2001, with certain modifications was used for isolation of plasmids. Colonies were inoculated to 10 ml LB with ampicillin (100 μg/ml) and incubated over night at 37°C with 175 rpm. Overnight grown culture was centrifuged at 5000 rpm for 2 min in 2.0 ml micro centrifuge tubes. The supernatant was removed and pellet was washed with STE buffer (0.25 volume of original culture) and then it was centrifuged at 5000 rpm for 2 min. The pellet was resuspended in 200 μl of icecold alkaline lysis solution I by vigorous vortexing. Later, 400 μl of freshly prepared alkaline lysis solution II was added to each tube and the contents were mixed by inverting the tubes for 4 to 5 times and kept in ice for about 5 min.
To this
suspension, 300 μl of alkaline lysis solution III was added and again mixed thoroughly by gently inverting the tubes 4-5 times. The tubes were stored on ice for 5 minutes and centrifuged at 13,000 for 8 min at 40C. The supernatant was transferred to fresh tubes and equal volume of phenol: chloroform: isoamyl alcohol (25:24:1) was added to precipitate proteins and mixed well. It was centrifuged at 13,000 rpm for 10 min at 4°C. The aqueous layer was transferred to a fresh tube and two volumes of
20
isopropanol was added. The contents were mixed and allowed to stand for 2 min. at room temperature. The solution was later centrifuged at 13,000 rpm for 5 min. The supernatant was discarded and the pellet was washed with 70 per cent ethanol and spun for 1 min at 13,000 rpm to recover the plasmid. The supernatant was discarded, pellet dried completely and dispensed into 30 μl of T10E1 (pH 8.0) containing 3 μl of RNase A (10 mg/ml). The solution was kept at 50°C for 15 min and then stored at 20°C (Appendix I). 3.1.1 Confirmation by PCR The confirmation of the presence of a native cry1Ac and cry2 (Fig. 1, 2 and 3) in cloning and expression vectors, cloned earlier at the Institute of AgriBiotechnology, University of Agricultural Sciences, Dharwad, India, (Kumaraswamy, 2005 and Sangmesh, 2004) respectively was done by PCR amplification of clones with the following cry1Ac and cry2 gene’s specific primers. 3.1.2 Specific primer designing for cry1Ac and cry2 To amplify full length native cry1Ac and cry2 genes, specific full length primers were designed, having BamHI – XhoI and BamHI – XbaI as forward and reverse restricrion sites for cry1Ac and cry2 respectively, from the sequences deposited in GenBank, NCBI, using offline softwares BIOEDIT and GENETOOL. The primers were synthesized at MWG NDRR Complex, Gandhi Circle, Girinagar, Bangalore. 3.1.3 PCR primers employed for amplification of given cry genes. Primer Gene Cry1Ac cry2A Cry2b
Name
Sequence
F
5’agagatggaggtaacttattctaga3’
R
5’ggatcctccttatcagagtacgttt3’
F
5’ggtaccatatgaatagtgtattgaat3’
R
5’gtacggatcctactcaaaccttaataa3’
F
5’ tctagagggaggagcatgatggatggcta3’
R
5’ ggatcctaaagaggttgccccagagta3’
Tm (°C)
Amplicon size (bp)
51.6
~ 3500
60
~ 2000
52.5
~ 2200
21 ATGGATAACA GTAGAAGTAT TCGCTAACGC GTTGATATAA GAACAGTTAA GAAGGACTAA CCTACTAATC CTTACAACCG TATGTTCAAG AGGTGGGGAT GGCAACTATA CCGGATTCTA TTAAATATCG TCCCAATTAA CGTGGAATGG AATAGTATAA ATAACAGCTT GCGGGGAATG TTATCTTCAC TTTGTCCTTG ATATATAGAC GTACCACCTC GCTGGAGCAG TTTAATAATA CTTGGCTCTG AGAACTTCAC AGATATCGGG TACCGGGGAC CAGTCCGGAA AGGGTATTTA ATTGAATTTG AAGGCGGTGA GATTATCATA GATGAAAAAC AATTTACTTC GGAAGTACGG CTATTGGGTA AAATTAAAAG AAAATCTATT TCCTTATGCC CCGCCACACC GCCCATCATT GACCTAGGTG AATCTAGAGT GCGGAGAAAA AAAGAGGCAA GCGGATACGA GCTTATCTGC GAAGGGCGTA GATTTTAATA AACAACCAAC CGTGTCTGTC GAAGGTTGCG TGCGTAGAAG CAAGAAGAAT GTACCAGCTG AATCCTTGTG ACAAAAGAAT GAAGGAACAT
ATCCGAACAT TAGGTGGAGA AATTTCTTTT TATGGGGAAT TTAACCAAAG GCAATCTTTA CAGCATTAAG CTATTCCTCT CTGCAAATTT TTGATGCCGC CAGATTATGC GAGATTGGGT TTGCTCTATT CAAGAGAAAT CTCAGAGAAT CCATTTATAC CTCCTGTAGG CAGCTCCACC CTTTATATAG ATGGAACGGA AAAGGGGTAC GTGCGGGATT TTTACACCTT TAATTCCTTC GAACTTCTGT CTGGCCAGAT TAAGAATTCG CTATTAATCA GCTTTAGGAC CGTTAAGTGC TTCCGGCAGA ATGAGCTGTT TTGATCAAGT AAGAATTGTC AAGATCCAAA ATATTACCCT CCTTTGATGA CCTATACCCG TATTTCCCTA CGCTTTTCCC TTGAATGGAA CGCATCATTT TATGGGTGAT TTCTCGAAGA AATGGAGAGA AAGAATCTGT ATATTGCCAT CTGAGCTGTC TTTTCAATGC ATGGCTTATC GTTCGGTCCT CGGGTCGTGG TAACCATTCA AGGAAATCTA ACGGAGGTGC ATTATGCGTC AATTTAACAG TAGAATACTT TTATCGTGGA
CAATGAATGC AAGAATAGAA GAGTGAATTT TTTTGGTCCC AATAGAAGAA TCAAATTTAC AGAAGAGATG TTTGGCAGTT ACATTTATCA GACTATCAAT TGTGCGCTGG AAGGTATAAT CTCAAATTAT TTATACGAAC AGAACAGAAT TGATGTGCAT GTTTTCAGGA CGTACTTGTC AAGAATTATA GTTTTCTTTT AGTCGATTCA TAGCCATCGA GAGAGCTCCA ATCACAAATT CGTTAAAGGA TTCAACCTTA CTACGCTTCT GGGTAATTTT TGTAGGTTTT CCGTTTCTTC AGTAACCTTT TACTTCTTCC ATCCAATTTA CGAGAAAGTC CTTCAGAGGG CCAAGGAGGC GTGCTATCCA TTATCAATTA CAATGCAAAA CCAAAGTCCA TCCTGACTTA CTCCTTAGAC CTTTAAGATT GAATCCATTA CAAACGTGAA GGTTGCTTTC GATTCATGCG TGTGATTCCG ATTCTCCGTA CTGCTGGAAC TGTTGTTCCG CTATATCCTT TGAGATCGAG TCCAAATAAC GTACACTTCT AGTCTATGAA AGGGTATAGG CCCAGAAACC CAGCGTGGAA
ATTCCTTATA ACTGGTTACA GTTCCCGGTG TCTCAATGGG TTCGCTAGGA GCAGAATCTT CGTATTCAAT CAAAATTATC GTTTTGAGAG AGTCGTTATA TACAATACGG CATTTTACAA GATAGTCGAA CCAGTATTAG ATTAGGCAAC AGAGGCTTTA CCAGAATTCG TCATTAACTG CTTGGTTCAG GCCTCCCTAA CTAGATGTAA TTGAGTCATG ACGTTTTCTT ACACAAATAC CCAGGATTTA AGAGTAAATA ACTACAAATT TCAGCAACTA ACTACTCCGT AATTCCGGAA GAGGCAGAAT AATCAAATCG GTTGAGTGTT AAACATGCGA ATCAATAGAC GATGACGTAT ACGTATTTAT AGAGGGTATA CATGAAACAT GTCGGAAAGG CATTGTTCGT ATTGATGTAG AAGACGCAAG GTCGGAGAAG AAATTGGAAT TTTGTAAACT GCAGATAAAC GGTGTCAATG TATGATGCGA GTGAAAGGGC GAATGGGAAG CGTGTCACAG AACAATACAG ACGGTAACGT CGTAATCGAG GAAAAATCGT GATTACACGC GATAAGGTAT TTACTCCTTA
ATTGTTTAAG CCCCAATCGA CTGGATTTGT ACGCATTTCT ACCAAGCCAT TTAGAGAGTG TCAATGACAT AAGTTCCTCT ATGTTTCAGT ATGATTTAAC GATTAGAGCG GAGAGCTAAC GGTATCCAAT AAAATTTTGA CACATCTTAT ATTATTGGTC CATTCCCTTT GTTTGGGGAT GCCCAAATAA CGACCAACTT TACCGCCACA TTACAATGCT GGCAGCATCG CTTTAACAAA CAGGAGGAGA TTACTGCACC TACAATTCCC TGAGTAGTGG TTAACTTTTC ATGAATTTTA ATGATTTAGA GGTTAAAAAC TATCAGATGA AGCGACTTAG AACTAGACCG TCAAAGAGAA ATCAAAAAAT TCGAAAATAG TAAGTGTGCC GTGAAAAGCC GTAGGGATGG GATGTACAGA ATGGGCACGC CGGTACCTCG GGGAAACAAA CTCACTATGA GTGTTCATAG CGGCTATTTT GAAATGTCAT ATGTAGATGT CAGAAGTGTC CGTACAAGGA ACGAACTGAA GTAATGATTA GATATAACGA ATACAGATGG CACTACCAGT GGATTGAGAT TGGAGGAATA
TAACCCTGAA TATTTCCTTG GTTAGGACTA TGTACAAATT TTCTAGATTA GGAAGCAGAT GAACAGTGCC TTTATCAGTA GTTTGGACAA TAGGCTTATT TGTATGGGGA ACTTACTGTA TCGAACAGTT TGGTAGTTTT GGATATCCTT AGGGCATCAA ATTTGGGAAT TTTTAGAACA TCAGGAACTG GCCTTCCACT GGATAATAGT GAGCCAAGCA CAGTGCTGAA ATCTACTAAT TATTCTTCGA ATTATCACAA TACAAAATGT GAGTAATTTA AAATGGATCA TATAGATCGA AAGAGCACAA AGATGTGACG ATTTTGTCTG TGATGAGCGG TGGCTGGAGA TTACGTTACG ACATGAGTCC TCAACACTTA GGGCACGGGT AAATTGCCCC AGAAAAGTGT CTTAAATGAG AAGACTAGGG TGTGAGAAGG TATCGTTTAT TCAATTACAA CATTCGAGAA TGAAGAATTA TAAAAATGGT AGAAGAACAA ACAAGAAGTT GGGATATGGA GTTTAGCAAC TACTGTAAAT AGCTCCTTCC ACGAAGAGAG TGGTTATGTG TGGAGAAACG GTCTCATGCA
Fig. 1. Full length sequence of cry1Ac gene
60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160 2220 2280 2340 2400 2460 2520 2580 2640 2700 2760 2820 2880 2940 3000 3060 3120 3180 3240 3300 3360 3420 3480 3540
22
MDNNPNINECIPYNCLSNPEVEVLGGERIETGYTPIDISLSLTQFLLSEFVPGAGFVLG LVDIIWGIFGPSQWDAFLVQIEQLINQRIEEFARNQAISRLEGLSNLYQIYAESFREWE ADPTNPALREEMRIQFNDMNSALTTAIPLLAVQNYQVPLLSVYVQAANLHLSVLRD VSVFGQRWGFDAATINSRYNDLTRLIGNYTDYAVRWYNTGLERVWGPDSRDWVR YNQFRRELTLTVLDIVALFSNYDSRRYPIRTVSQLTREIYTNPVLENFDGSFRGMAQ RIEQNIRQPHLMDILNSITIYTDVHRGFNYWSGHQITASPVGFSGPEFAFPLFGNAGN AAPPVLVSLTGLGIFRTLSSPLYRRIILGSGPNNQELFVLDGTEFSFASLTTNLPSTIYR QRGTVDSLDVIPPQDNSVPPRAGFSHRLSHVTMLSQAAGAVYTLRAPTFSWQHRSA EFNNIIPSSQITQIPLTKSTNLGSGTSVVKGPGFTGGDILRRTSPGQISTLRVNITAPLS QRYRVRIRYASTTNLQFHTSIDGRPINQGNFSATMSSGSNLQSGSFRTVGFTTPFNFS NGSSVFTLSAHVFNSGNEVYIDRIEFVPAEVTFEAEYDLERAQKAVNELFTSSNQIG LKTDVTDYHIDQVSNLVECLSDEFCLDEKQELSEKVKHAKRLSDERNLLQDPNFRG INRQLDRGWRGSTDITIQGGDDVFKENYVTLLGTFDECYPTYLYQKIDESKLKAYT RYQLRGYIEDSQDLEIYLIRYNAKHETVNVPGTGSLWPLSAQSPIGKCGEPNRCAPH LEWNPDLDCSCRDGEKCAHHSHHFSLDIDVGCTDLNEDLGVWVIFKIKTQDGHAR LGNLEFLEEKPLVGEALARVKRAEKKWRDKREKLEWETNIVYKEAKESVDALFVN SQYDQLQADTNIAMIHAADKRVHSIREAYLPELSVIPGVNAAIFEELEGRIFTAFSLY DARNVIKNGDFNNGLSCWNVKGHVDVEEQNNQRSVLVVPEWEAEVSQEVRVCPG RGYILRVTAYKEGYGEGCVTIHEIENNTDELKFSNCVEEEIYPNNTVTCNDYTVNQE EYGGAYTSRNRGYNEAPSVPADYASVYEEKSYTDGRRENPCEFNRGYRDYTPLPV GYVTKELEYFPETDKVWIEIGETEGTFIVDSVELLLMEE*
Fig. 2. Complete Amino Acid sequence of cry1Ac
23
AGGGAGGAGC TATACCATAT TAATTAATAA AAAAATATTC TTTAAGGAGG GCGTATAATG CAAAAGGAAT GGAACTGTGG AGTGAGTTAC AGAGAGACAG GCGGAAACAG AACCCTAACC TTATTTCTAA TTATTTGCAC GATGAATGGG ACAAGAGATT ACTCGTTTAC GTATCTATCT TATGCAAGTG TTATATCCTC CTTTCTAATA CTTGCTGCAA TTTAATCAAA AGTTGGCTAG GAATCCTTTG AACTATTTCC GAAGATTTAA ACACCTGGTG GCTGTTCATG ATTTCGCCGA TTTGGAAATC AGAGGGAATG ATTCGAGTTA AACGATGGAG GCAAGTAGTA TTTGATCTTA AGGTTCTTAT
ATGATGGATG CACAAATTAT TTATAATCAA GTTATTATCA AATTTTATAT TAGCGGCTCA GGACGGAGTG CTAGTTTTCT GGAATTTAAT AAAAATTCCT GCATGCAAGC GAAACGCTGT ATAGATTACC AGGCAGCCAA GAATTTCAGC ACTCTAACTA ACGATATGTT GGTCGTTGTT GTAGTGGACC TTTTCCAAGT CCTTCCCTAA GGGTTAATTA ATTTTAATTG ATTCAGGTTC AGACAACTTT CAGATTATTT GAAGACCGTT GAGCACGAGC AAAATGGTTC TACATGCAAC AAGGTGATCT GAAATAGTTA CTATAAACGG TTAATGATAA ATTCTGATGT TGAATATTAT GT 2172
GCTAAACTGT GCGTATAACA AGTTAGAGTT GGCTAATTTA GAATAGTGTA TGATCCATTT GAAAAAAAAT GTTAAAGAAA ATTTCCTAGT GAATCAAAGA AAATGTAGAA TCCTTTATCA CCAGTTTCAG TTTACATCTT AGCAACATTA TTGTATAAAT AGAATTTAGA TAAATATCAA ACAGCAGACC TAATTCAAAT TATAGTTGGT CAGTGGAGGA TAGCACATTT AGATCGGGAG AGGGTTAAGG TATTCGTAAT ACACTATAAT TTATATGGTA TATGATTCAT TCAAGTGAAT TGTTTTTGAA CAATCTTTAT TAGGGTATAT TGGAGCTCGT ACCATTAGAT GCTTGTACCA
AGGCTTTCAT AAAGTGAGAA GTAATTGTGG GTATCTTTAA TTGAATAGCG AGTTTTCAAC AATCATAGTT GTGGGGAGTC GGTAGTACAA CTTAATACAG GAGTTTAATC ATAACTTCTT ATGCAAGGAT TCTTTTATTA CGTACGTATC ACGTATCAAA ACATATATGT AGTCTTCTAG CAATCATTTA TATGTGTTAA TTACCTGGTT ATTTCGTCTG CTCCCCCCAT GGCGTTGCCA AGTGGTGCTT ATTTCTGGAG GAAATAAGAA TCTGTGCATA TTAGCGCCAA AATCAAACAC CAAAACAACA TTAAGAGTTT ACTGCTACAA TTTTCAGATA ATAAATGTAA ACTAATATTT
GTTTAAAGTA TGATTCCTAT TTGTAAATAA TTTTAATATA GAAGAACTAC ACAAATCATT TATACCTAGA TTGTTGGAAA ATCTAATGCA ACACTCTTGC GACAAGTAGA CAGTTAATAC ACCAACTGTT GAGATGTTAT GAGATTACTT GTGCGTTTAA TTTTAAATGT TATCTTCCGG CTTCACAAGA ATGGATTTAG CTACTACAAC GTGATATAGG TGTTAACGCC CCGTTACAAA TTACAGCTCG TTCCTTTAGT ATATAGCAAG ACAGAAAAAA ATGACTATAC GAACATTTAT CGACAGCTCG CTTCAATAGG ATGTTAATAC TTAATATCGG CATTAAACTC CACCACTTTA
TGATCCTTCC GTTTAAGACT GCACTTTCTT TTACTTAATA TATTTGTGAT AGATACCGTA TCCTATTGTT AAGGATACTA AGATATTTTA CCGTGTAAAT TAATTTTTTG AATGCAACAA ATTATTACCT TCTAAATGCA GAAAAATTAT AGGTTTAAAC ATTTGAGTAT TGCTAATTTA CTGGCCATTT TGGTGCTAGG TCACGCATTG TGCATCTCCG ATTTGTTAGG TTGGCAAACA CGGTAATTCA TGTTAGAAAT TCCTTCAGGA TAATATCCAT AGGATTTACT TTCTGAAAAA TTATACGCTT AAATTCCACT TACTACAAAT TAATGTAGTA GAGTACTCAA TTAAAGTTTG
Fig. 3. Full length sequence of cry2 gene
60 120 180 240 300 360 420 480 540 600 660 720 780 840 900 960 1020 1080 1140 1200 1260 1320 1380 1440 1500 1560 1620 1680 1740 1800 1860 1920 1980 2040 2100 2160
24
3.1.3 Requirements Template DNA: The plasmid DNA obtained from clones were diluted to 125ng and used as template DNA for further studies. Taq DNA polymerase (3 u/μl) 10X assay buffer, MgCl2 and dNTPs were obtained from M/S Bangalore Genei Pvt.Ltd., Bangalore. Primers: - Three set of specific primers were used in this study. Gene sequences available in NCBI (http://www.ncbi.nlm.nih.gov) database were aligned using GENETOOL and BIOEDIT software. The details of primers used are listed in (section 3.2.4).The primers were synthesized at MWG NDRR Complex, Gandhi Circle,Girinagar, Banglore. Thermal cycler: Eppendorf Master cycler (5331) was used to run the PCR programme. Standardization of temprature for designed primers Specific primers of 5pM concentration were standardized by keeping a gradient PCR for each set of primer along with the plasmid DNA of reference clones and the temperature at which amplicons of good intensity of expected size were selected as a reference clones for cry1Ac, and cry2 genes, for mutation studies. 3.1.4 Contents of PCR reaction mixture Reagents
Volume/tube(μl)
Taq assay buffer(10x)(with Mgcl2)
2.0
dNTPs(1mM)
2.0
Forward primer (5 pM)
0.5
Reverse primer (5 pM)
0.5
XT-5 Polymerase (30 U/ μl)
0.2
Template (DNA 125 ng) Sterile distilled water Total
1 13.8 20
25
3.1.5 PCR programmes for amplification of native cry genes. Steps
cry1Ac
cry2A
cry2b
Temp
Time
Temp
Time
Temp
Time
Initial denaturation
94.0°C
8.0 min
94.0°C
8.0 min
95.0°C
8.0 min
Denaturation
94.0°C
1.0 min
94.0°C
1.0 min
94.0°C
1.0 min
Annealing
51.6°C
1.0 min
60°C
1.0 min
52.5°C
1.0 min
Extension
72.0°C
3.0 min
72.0°C
2.0 min
72.0°C
3.0 min
72.0°C
8.0 min
Repeat Final extension
35 cycles 72.0°C
8.0 min
Hold
72.0°C
5.0 min
4.0°C
After completion, the samples were stored at 4 oC in refrigerator for further use. 3.1.5 Electrophoresis About 15µl of the amplified products from tube along with 3µl of loading dye (Appendix) was loaded onto 1 percent Agrose gel (Appendix II) along with λ HindIII digest as DNA molecular weight marker. Electrophoresis was done at 70v for 2 hrs. the buffer used was 1XTAE at p H 8.0 (Append II). After separation, the bands in gels were visualized under UV light and documented using gel documented system, Syngen, Germany. 3.1.6 Confirmation by restriction analysis Confirmation was done through comparative restriction analysis of the plasmid of selected clones and control vector to ensure the insert size using BamHI for cry1Ac and BamHI – XbaI for cry2. 3.1.7 Confirmation of expression by SDS-PAGE analysis Induction and isolation of recombinant protein was done as per the procedure outlined in pET system manual, 11th edition provided by Novagen with minor modifications. A single isolated colony of E. coli BL21(DE3)pLysS containing pSK102 (pET32C+ containing cry1Ac gene) and pSK202 (pET32C+ containing cry2
26
gene) separately was inoculated in 1 ml of LB + 34 μg/ml chloromphenicol + 100 μg/ml Amp and incubated overnight at 37oC with shaking at 200 rpm. The overnight grown culture was diluted in 10 ml of fresh LB in 1:100 ratio and incubated till the OD600 reached 0.5-1.0 (approximately 3 hours). The log phase cells were induced by adding 1mM IPTG and incubated for additional 5 hours. The vectors pSK102 and pSK202 in E. coli BL21 (DE3)pLysS containing plain pET32C+ were inoculated and induced. A batch of uninduced culture was maintained in each case. Following the induction cells were pelleted at 13,200 rpm for 10 minute. Resuspend the pellet in 100 μl of 100 mM Tris (pH 8.0). 100 μl of 2X SDS loading dye was added to 100 μl of sample heated at 95o C for 10 min. and the supernatant was collected after spinning at 10,000 rpm for 5 min. To confirm the expression of cry1Ac and cry2 and to fractionate the expressed protein from crude extract, SDSPAGE was done as per the protocol given in Sambrook and Russell (2001). The crude extract was stacked in 5 per cent stacking gel and separated in 12 per cent resolving gel. The gel was run till the relative front reached the lower end, stained by comassie brilliant blue (CBB) with methanol and glacial acetic acid as fixative and finally destained in methanol, acetic acid and water to remove background dye. 3.2 Creation of random mutation using GeneMorph II Random Mutagenesis Kit Using GeneMorphII Random Mutagenesis kit from Stratagene – An Alient Technologies Company, random mutations were created in cry1Ac and cry2. GeneMorph II Random Mutagenesis contains Mutazyme II DNA polymerase – 2.5 U/µl 10X Mutazyme II reaction buffer – 10X 40mM dNTP mix – 10mM each dNTP 1.1-kb Gel standard- 20ng/µl 3.2.1 Additional materials required Thermocycler Eppendorf master cycler gradient was used to run the PCR programme.
27
PCR Primers Various concentrations of primer, (explained in section 3.1.3) including 5, 10, 20, 30, 40, 50 pm were used to optimize the amplification of cry1Ac and cry2 gene using plasmid DNA of the expressed clones as template. For cry1Ac yeast total DNA having yeast plasmid with native cry1Ac was also used as template to create random mutations. Thin walled PCR tubes Thin walled PCR tubes were used as these PCR tubes are optimized to ensure ideal contact with the multiblock design to permit more efficient heat transfer and to maximize thermal-cycling performance.
3.2.2 Contents of PCR reaction mixture Reagents
Volume/tube(μl)
Mutazyme II reaction buffer (10X)
5.0
dNTPs (10mM each dNTP)
1.0
Forward primer (20 pM)
0.5
Reverse primer (20 pM)
0.5
Mutazyme II DNA Polymerase (2.5 U/ μl)
1.0
Template (Plasmid/DNA 1 to500 ng)
1.0
Sterile distilled water
41.0
Total
50
3.2.3 PCR Programme For the creation of mutations the PCR programmes earlier standardised were used. 3.3 Cloning of mutated amplicons of cry1Ac and cry2 into pET32C+ vector. 3.3.1 Separation of PCR amplified products by agarose gel electrophoresis About 80μl of the amplified product from each tube along with 8μl of loading dye were loaded onto 0.7% agarose gel along with 1 kb ladder (Bangalore Genie
28
Bangalore) as DNA molecular weight marker. Electrophoresis was done at 50V for initial 15 minutes and then 70V for 45 minutes. The buffer used was TAE (pH-8.0). The DNA bands in the gel were visualized on a UV-transilluminater and documented using a gel documentation system (Uvitech Cambridge, England). 3.3.2 Gel elution of PCR product Expected amplicons (3.5kb, 1.8kb and 2.2kb) were set-apart by carrying out electrophoresis. Amplicons were eluted out using a sharp sterile scalpel by keeping the gel at low intensity UV transilluminator. The gel pieces were collected in sterile preweighed 2.0 ml micro centrifuge tubes. Eppendorf Gel extraction kit (QIAprep spin Miniprep Kit) was used to elute the DNA from agarose gel as described in user’s manual. 3.3.3 Restriction of pET32C+ and mutated amplicons with respective restriction enzymes pET32 series of expression vectors and expression host, E. coli. BL21 (DE3)pLysS were purchased from Novagen, USA. pET32C+ and mutagenic amplicons (cry1Ac and cry2) were linearised by BamHI and XhoI. 3.3.4 Vector Dephosphorylation The vector pET32C+ was dephosphorylated using antaratic phosphatase (New England Biolabs, USA). The vector pET32C+ was dephosphorylated at the rate of 1 µl of enzyme per 1 µg of vector DNA at 37°C for 15 min. and the inactivation of the enzyme was done at 65°C for 5 min. 3.3.5 Ligation and transformation Equimolar concentration, approximately 0.54 pmoles of free ends of insert and vector backbone was used for ligation under 1X cohesive end ligation buffer with 1 U of T4 DNA ligase, incubated over night at 16oC. Around 15 µl of ligation mixture was used for transforming 100 µl of competent E. coli cells. Preparation of competent cells and transformation was done as per the protocol mentioned by Sambrook and Russel, 2001. All the chemicals (except mentioned specifically) required for media
29
preparation for bacterial and yeast culture and other molecular work were purchased from Hi Media, Mumbai, India. 3.3.6 Preparation of competent cells The competent cells of E. coli BL21 (DE3)pLysS were prepared by following the protocol mentioned in Sambrook and Russell (2001) with minor modifications. An isolated colony from E. coli BL21 (DE3)pLysS plate was inoculated in 5 ml Luria broth with appropriate selection pressure (ampicilline 100 µg/ml) and incubated at 37°C overnight at 200 rpm. Next day, the culture was diluted to 1:100 using Luria broth i.e., 0.5 ml of culture was added to 50 ml of Luria broth. It was incubated for 2 to 3 hr till an OD of 0.3 to 0.4 at 600 nm was attained. The culture was chilled on ice for 30 min and 25 ml of culture was dispensed into two sterile centrifuge tubes. The cells were pelleted at 6000 rpm for 5 min at 4°C, the supernatant was discarded and the pellet was suspended in 12.5 ml ice-cold 0.1 M CaCl2. The centrifuge tubes were again kept in ice for 30 min and later centrifuged at 5000 rpm for 5 min, the pellet dispensed in 1 ml of ice-cold 0.1 M CaCl2 and to this 88 μl of dimethyl sulfoxide (DMSO) was added. About 100 μl of the competent cells were distributed to pre-chilled 1.5ml microcentrifuge tubes and stored at –20°C or used immediately for transformation. 3.4 Transformation of E. coli BL21 (DE3)pLysS About 100 μl of freshly prepared competent cells were taken in a chilled micro centrifuge tube and 10 μl of ligation mixture was added, mixed gently and chilled on ice for 30 min. Heat shock was given by shifting the chilled mixture to 42°C water bath for exactly 2 min, and immediately chilled on ice for 5 min. To this 900 μl of Luria broth was added and incubated at 37°C at 200 rpm for 45 min to allow bacteria to recover and express the antibiotic marker encoded by the plasmid. The cells were pelleted at 13,000 rpm for 1 min, 900 μl of supernatant discarded and the pellet was dissolved in the remaining 100 μl of supernatant and spread on Luria agar plates having ampicillin @100 μg/ml and incubated overnight at 37°C.
30
3.4.1 Confirmation of transformed clones The transformants grown on Luria-Bertani agar containing 100 µg/ml ampicilin were screened for the presence of recombinant plasmids by colony PCR using gene specific primers with the conditions mentioned in section 3.3.2. Original plasmid was used as positive control and total DNA from E coli BL21 (DE3)pLysS as negative control. PCR positive clones were further confirmed by restriction digestion in one hour at 37oC with BamHI and XhoI . 3.4.2 Expression and purification of His tagged protein The transformed E.coli. colony was inoculated in 5 ml of Luria-Bertani agar containing 100 µg/ml ampicillin and incubated overnight at 37oC . Next day, the culture was diluted in 1:100 ratio without selection pressure and again incubated at 37oC with shaking condition until it reaches the log phase of growth. Later the induction of expression of target protein was done by adding optimal concentration of IPTG (1mM) and incubated for 5 hrs at 37oC. After that the cells were harvested by centrifugation at 5000g for 15 min at 4oC and resuspended in 4ml of binding buffer pH 7.8. Lysozyme of final concentration of 1mg/ml was added and
the cell
suspension was incubated in ice for 30 minutes.TritonX-100, DNase, and RNase were added to the final concentration of 1 per cent, 5µg/ml and 5µg/ml respectively and incubated at 4oC on rocking platform for 10 min. Finally the suspension was centrifuged at 3000g for 30 min at 4oC and the supernant was collected as raw active protein in fresh tube. 3.4.3 Bioassay The toxicity of mutated clones was tested through bioassay studies. The active protein isolated from the clones was tested against the insect P. xylostella. 27 μg of raw protein was spread on the cabbage leaf along with the control (E. coli. along with plain pET32C+) and reference as native cry1Ac already cloned in prokaryotic expression vector, and air dried for 1 hour. A single disk was placed on moist Whatman paper in each petridish. Ten third instar P. xylostella larvae were released on each leaf disk separately with a paint brush and incubated at 28 ± 2ºC, 60 ± 5 per
31
cent relative humidity and 14 hour photoperiod. The insect mortality with active crude protein from recombinant E. coli. mutant clones were recorded at an interval of 24, 48 and 72 hours, and compared with the control. Each treatment was replicated three times. 3.4.4 Sequencing of clones For the sequencing, the clones were selected on the basis of toxicity against Plutella xylostella. The toxic domain of full length mutant genes of cry1Ac cloned in pET32C+ a prokaryotic expression vector were sequenced using gene specific forward primer described earlier and further as primer walking by designing internal primers, at Ocimum Biosolutions, Hyderabad. The sequences were subjected to analysis using CLUSTALW algorithm available offline. 3.5 Cloning of mutant amplicons of cry1Ac in yeast expression vector (pYES2/CT) Mutated amplicons of cry1Ac full length were cloned and used for the expression in yeast. The pYES2/CT (Invitrogen) and Saccharomyces cerevisiae INVSc1 were used for this purpose. The genotype and phenotype of S. cerevisiae INVSc1 host are given below: Genotype: his3Δ1/his3Δ1 leu2/leu2 trp1-289/trp1-289 ura3-52/ura3-52 Phenotype: His-, Leu- , Trp- and UraINVSc1 is a diploid strain auxotrophic for histidine, leucine, tryptophan and uracil, i.e., the strain cannot grow on SC minimal medium deficient in histidine, leucine, tryptophan and uracil. Error prone PCR product of cry1Ac was cloned in to pYES2/CT using BamHI and XhoI. These clones were transferred into E. coli as described in 3.4. The presence of insert in clones was confirmed by colony PCR using respective primers 3.6 Yeast (Saccharomyces cerevisiae) Transformation Saccharomyces cerevisiae INVSc1 was inoculated in 10 ml of YPD broth for overnight at 30oC under shaking condition at 200 rpm. After overnight growth it was
32
diluted to get 0.4 OD at 600nm in 50ml of YPD broth and incubated for additional three to four hours. Then it was centrifuged at 15,000g and resuspended in 40ml of 1X TE centrifuged at 1,500g for 10 min and dissolved in 2ml of 1X LiAc/0.5X TE and incubated at room temperature. For transformation, 1μg of plasmid DNA and 100 μg of denatured sheared salmon sperm DNA with 100 μl of yeast suspension were mixed. To this mixture 700μl of 1X LiAc/40% PEG-3350/1X TE was added and thoroughly mixed. It was incubated for 30 minutes at 30oC and 88 μl of DMSO was added and thoroughly mixed. Heat shock was given at 42oC for 7 minute and centrifuged at 13,000 rpm for 10 seconds. Supernatant was removed, pellet was dissolved in 1ml 1X TE centrifuged at 13,000 rpm for 10 seconds and supernatant was removed. The pellet was re-dissolved in 50-100μl of TE and spread on SC minimal medium lacking uracil (Appendix-IV and V). 3.6.1 Isolation of Total DNA from Yeast and confirmation of clones by PCR Transformed yeast cells were grown on SC-U minimal broth overnight at 30OC under shaking condition at 200 rpm. Cells were harvested by centrifugation for 1 min at 13,000 rpm. The pellet was washed with distilled water and again centrifuged for 1 minute at 13,000 rpm. The pellet was re-suspended in lysis buffer (AppendixIV) and 200mg of acid washed glass beads (Sigma) were added. This mixture was vortexed for 5 minu. Protein was removed by adding equal volume of phenol: chloroform: IAA (25:24:1) and centrifuged at 13,000 rpm for 10 min. The supernatant was collected and the DNA was recovered by isopropanol precipitation. The DNA was washed with 70 per cent alcohol and air dried. Pellet was re-dissolved in 50μl TE buffer. Total DNA was used to confirm yeast clones using gene specific primers. 3.6.2 Induction and expression of cry1Ac gene in yeast. The pYES2/CT contains GAL 1 promoter which is induced by galactose. Induction of cry1Ac gene was carried out as per the protocol given in user manual of Invitrogen Company. The transformed S. cervisiae INVSc1 cells harboring the expression plasmid pYAP1, pYAP2 to pYAP10 were grown on SC-U minimal medium. Isolated colonies
33
were inoculated in 200ml of SC-U minimal medium having 10mg/l adenine and 2 per cent raffinose. It was grown at 30OC for three days under shaking conditions. These cells were harvested by centrifugation at 1,500g for 5 min and used to inoculate the 5 SC -U minimal medium (50 ml) having 2 per cent galactose. These cells were grown at 30oC for three days under shaking condition at 200 rpm and the cells were collected by centrifugation at 1,500g for 5 min at 4OC. Lysate was prepared by dissolving the pellet in the breaking buffer (Appendix IV). Equal volume of acid washed glass beads were added and vortexed for 30 seconds followed by holding for 30 seconds on ice. This step was repeated for 4-5 times for a total of four min. It was later centrifuged for 13,000 rpm for 1 min at 4OC. The supernatant was collected in clean pre-cooled centrifuge tube. The total protein of the supernatant was stored at -20OC for further use. 3.6.3 Bioassay The toxicity of mutated colnes was tested through bioassay studies. The active protein isolated from the clones was tested against the insect P. xylostella. 32 μg of raw protein was spread on the cabbage leaf along with the control (S.cervisiae INVSc 1 with plain pYES2/CT) and referene as native cry1Ac already cloned in yeast, and air dried for 1 hour. A single disk was placed on moist Whatman paper in each petridish. Ten third instar P. xylostella larvae were released on each leaf disk separately with a paint brush and incubated at 28 ± 2ºC, 60 ± 5 per cent relative humidity and 14 hrs photoperiod. The insect mortality with active raw protein from recombinant S.cervisiae INVSc 1 mutant clones were recorded at an interval of 24, 48 and 72 hrs, and compared with the control. Each treatment was replicated three times. 3.6.4 Sequencing of clones For the sequencing, the clones were selected on the basis of toxicity against Plutella xylostella. Clones with different efficacy were selected for the sequencing. The toxic domain of full length mutant genes of cry1Ac cloned in pYES2/CT were sequenced using gene specific forward primer described earlier and further as primer walking by designing internal primers, at Ocimum Biosolutions, Hyderabad. The sequences were subjected to analysis using CLUSTALW algorithm available offline.
34
4. EXPERIMENTAL RESULTS The present study was conducted to create random mutations in cry1Ac and cry2 and analyze the effect of altered nucleotide on the toxicity to Plutella xylostella. The results of various experiments conducted towards achieving the defined objectives are presented herein. 4.1 Confirmation of clones 4.1.1 PCR amplification of cry1Ac and cry2 and analysis The PCR was carried out with a set of cry1Ac and cry2 specific primers on the plasmid DNA isolated from clones already cloned and expressed in the laboratory (Fig. 3). The amplicons obtained and separated on 0.8 per cent Agrose gel are presented in Plate 1 and 4. From gel, it is clear that the clones pKK2009 and pSK202 were positive for cry1Ac and cry2 showing 3.5kb and 2.2 kb amplicons respectively. 4.1.2 Confirmation of clones by restriction digestion The PCR positive clones were further confirmed by restriction digestion. The release of 3.5 kb fragment from pKK2009 and 2.2 kb from pSK202 and, the absence of such fragment in pET32C+ plain vector when restricted with BamHI and XhoI confirmed that the clones were recombinant. The release of approximately 3.5 kb (cry1Ac) and 2.2 kb (cry2) fragment from pKK2009 and pSK202 confirmed the presence of cry1Ac and cry2 respectively in pET32C+. The results of restriction digestion are presented in (Plate 2 and 5). 4.1.3 Expression studies in E coli BL21(DE3)pLysS To check the expression of the cloned gene in pSK102 and pSK202 clones, the total protein from IPTG induced pSK102 and pSK202 in E. coli BL21(DE3)pLysS having plain pET32C+ (as a control) were subjected to SDS-PAGE. The protein band corresponding to approximately 130 kDa and 65 kDa (Plate 3 and 6) indicated the expression of cry1Ac and cry2 gene respectively in E. coli BL21(DE3)pLysS and absence of such band in control when loaded at equal amount of crude protein (≈100 ng). For further confirmation of clones expressed, bioassay was employed by feeding
35
36
37
the total protein expressed, in E coli BL21 (DE3)pLysS to P. xylostella and the per cent mortality was recorded. The active protein isolated from the clones was tested against the insect P. xylostella. 27 μg of raw protein was spread on the cabbage leaf along with the control (E. coli. along with plain pET32C+) and reference as native cry1Ac already cloned in prokaryotic expression vector, and air dried for 1 hour. A single disk was placed on moist Whatman paper in each petridish. Ten third instar P. xylostella larvae were released on each leaf disk separately with a paint brush and incubated at 28 ± 2ºC, 60 ± 5 per cent relative humidity and 14 hour photoperiod. The insect mortality with active raw protein from recombinant E. coli. mutant clones were recorded at an interval of 24, 48 and 72 hours, and compared with the control. Each treatment was replicated three times. 4.1.4 Bioassay The insect mortality observed with lysates and supernatant of recombinant E. coli BL21(DE3)pLysS clones (pKK2009and pSK202) that fed to P. xylostella was recorded at an interval of 24, 48 and 72 hrs, with control as extract from plain pET32C+. The bioassay results are given in tabular form (Table 1 and 2). 4.2 Random mutagenesis and expression analysis 4.2.1 Error prone amplification of cry1Ac and cry2 The error prone PCR was carried out with the specific primers of cry1Ac and cry2 and plasmid DNA of expressed clones as template. The amplicons obtained and separated on 1.0 per cent Agrose gel (Plate 7 and 9). From the gel, it is clear that amplification of 3.5 kb for cry1Ac and 2.2 kb for cry2 are the desired bands as mutated amplicons. 4.3 Cloning of mutated amplicons of cry1Ac and cry2 4.3.1 Preparation of inserts Gel elution of the preparative electrophoresis of amplicons of cry1Ac (3.5 kb) and cry2 (2.2 kb) was obtained on PCR amplification of cry1Ac and cry2 gene using
38
39
Table 1. Bioassay results of cry1Ac mutants in E. coli S. No.
Reference
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45
Cry1Ac control M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41 M42 M43
Total larvae
24hrs
Percent mortality
48hrs
Percent mortality
72 hrs
Percent mortality
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
5 0 2 0 2 2 1 1 2 0 2 8 0 4 4 1 2 5 0 0 3 3 6 1 5 5 15 2 5 4 4 9 5 4 2 2 2 0 3 2 0 3 2 2 3
17 0 7 0 7 7 3 3 7 0 7 27 0 13 13 3 7 17 0 0 10 10 20 3 17 17 50 7 17 13 13 30 17 13 7 7 7 0 10 7 0 10 7 7 10
15 0 6 1 5 4 3 6 7 4 5 10 4 5 6 5 12 13 4 13 11 12 12 7 15 14 18 7 12 10 12 10 10 10 3 11 6 0 6 9 6 8 7 7 6
50 0 20 3 27 13 10 20 23 13 17 33 10 17 20 17 40 43 13 43 37 40 40 23 50 47 60 23 40 33 40 33 33 33 10 37 20 0 20 30 20 27 23 23 20
21 0 11 3 9 4 5 10 12 8 10 15 5 6 11 6 14 17 8 17 16 18 18 12 23 20 24 14 19 20 19 17 14 15 3 18 9 14 15 23 15 17 19 19 19
70 0 37 10 30 13 17 33 40 27 40 50 17 20 37 20 47 57 27 57 53 60 60 40 75 67 80 47 63 67 63 57 47 50 10 60 30 47 50 77 50 57 63 63 63
40 S. No.
Reference
46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91
M44 M45 M46 M47 M48 M49 M50 M51 M52 M53 M54 M55 M56 M57 M58 M59 M60 M61 M62 M63 M64 M65 M66 M67 M68 M69 M70 M71 M72 M73 M74 M75 M76 M77 M78 M79 M80 M81 M82 M83 M84 M85 M86 M87 M88 M89
Total larvae
24hrs
Percent mortality
48hrs
Percent mortality
72 hrs
Percent mortality
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
3 1 2 3 3 2 4 5 4 3 8 3 5 1 3 3 7 5 3 6 6 3 6 6 6 6 4 5 6 6 1 1 1 1 6 3 3 6 1 3 5 3 1 8 6 3
10 3 7 10 10 7 13 17 13 10 27 10 17 3 10 10 23 17 10 20 20 10 20 20 20 20 13 17 20 20 3 3 3 3 20 10 10 20 3 9 17 10 3 27 20 10
6 5 7 7 6 7 8 10 10 8 13 8 9 7 6 9 13 13 9 13 14 8 16 15 15 16 9 11 12 15 9 3 8 10 13 14 9 12 8 9 12 12 8 16 15 9
20 17 23 23 20 23 27 33 33 27 43 27 30 23 20 30 43 43 30 43 46 27 53 50 50 53 30 37 40 50 30 10 27 33 43 46 30 40 27 30 40 40 27 53 50 30
14 15 17 13 17 18 20 19 15 17 19 20 16 16 17 20 21 22 18 22 23 13 22 21 21 22 11 17 19 22 15 4 14 18 21 20 15 18 14 15 17 21 13 22 21 15
47 50 57 43 57 60 67 63 50 57 63 67 53 53 57 67 70 73 60 73 77 43 73 70 70 73 37 57 63 73 50 13 46 60 70 67 50 60 46 50 57 70 43 73 70 50
41 S. No.
Reference
92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 111
M90 M91 M92 M93 M94 M95 M96 M97 M98 M99 M100 M101 M102 M103 M104 M105 M106 M107 M108 M109
Total larvae
24hrs
Percent mortality
48hrs
Percent mortality
72 hrs
Percent mortality
30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30 30
0 5 2 7 1 3 1 3 3 3 3 4 2 1 8 2 6 5 2 2
0 17 7 23 3 10 10 10 10 10 10 13 7 3 27 7 20 17 7 7
9 14 7 14 7 12 9 12 11 13 13 12 8 9 17 9 12 12 10 8
30 46 23 46 23 40 30 40 37 43 43 40 27 30 57 30 40 40 33 27
12 20 9 21 12 18 14 21 18 14 14 20 15 18 22 13 21 20 14 14
40 67 30 70 40 60 46 70 60 46 46 67 50 60 73 43 70 67 46 46
42
Table 2. Bioassay results of cry2 mutant clones in E. coli S. No. 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44
Reference Cry2 control M1 M2 M3 M4 M5 M6 M7 M8 M9 M10 M11 M12 M13 M14 M15 M16 M17 M18 M19 M20 M21 M22 M23 M24 M25 M26 M27 M28 M29 M30 M31 M32 M33 M34 M35 M36 M37 M38 M39 M40 M41 M42
Total 24hrs Percent 48hrs Percent 72hrs Percent larvae mortality mortality mortality 30 5 17 18 60 21 70 30 0 0 0 0 0 0 30 4 13 10 33 15 50 30 0 0 5 17 18 60 30 2 7 5 17 19 63 30 4 13 6 20 17 57 30 1 3 3 10 9 30 30 1 3 6 20 12 40 30 2 7 7 23 14 47 30 0 0 4 13 8 27 30 2 7 5 17 10 33 30 8 27 10 33 18 60 30 0 0 5 17 14 47 30 7 23 14 47 20 67 30 4 13 6 20 16 53 30 1 3 7 23 14 47 30 4 13 12 40 14 47 30 8 27 13 43 17 57 30 4 13 7 23 8 27 30 3 10 13 43 17 57 30 3 10 11 37 16 53 30 3 10 12 40 18 60 30 6 20 12 40 18 60 30 1 7 7 23 12 40 30 5 17 15 50 22 73 30 5 17 12 40 20 67 30 12 40 18 60 22 73 30 5 17 11 37 14 47 30 5 17 15 50 21 70 30 4 13 11 37 20 67 30 4 13 12 40 19 63 30 9 30 15 50 19 63 30 5 17 10 33 14 47 30 3 10 11 37 14 47 30 1 3 9 30 19 63 30 3 10 12 40 21 70 30 4 13 8 27 18 60 30 4 13 10 33 21 70 30 4 13 8 27 15 50 30 3 10 9 30 17 57 30 3 10 8 27 18 60 30 5 17 10 33 20 67 30 4 13 8 27 21 70 30 7 23 13 43 22 73
43
S. No. 45 46 47 48 49 50 51 52 53 54 55 56 57
Reference M43 M44 M45 M46 M47 M48 M49 M50 M51 M52 M53 M54 M55
Total 24hrs Percent 48hrs Percent 72hrs Percent larvae mortality mortality mortality 30 4 13 8 27 19 63 30 3 10 10 33 18 60 30 6 20 11 37 19 63 30 4 13 8 27 21 70 30 5 17 9 30 21 70 30 3 10 6 20 18 60 30 3 10 9 30 18 60 30 5 17 10 33 20 67 30 6 20 11 37 20 67 30 6 20 9 30 19 63 30 4 13 10 33 21 70 30 9 30 13 43 18 60 30 5 17 12 40 20 67
44
GeneMorph II Random Mutagenesis Kit as mentioned in section 3.2. These amplicons gave a yield of 500 ng per µl. 4.3.2 Ligation The amplified fragments were ligated to pET32C+ expression vector. The recombinant products of vector pET32C+ and cry1Ac (3.5 kb) and cry2 (2.2 kb) were transformed into E. coli. BL21 (DE3)pLysS using 10 µl of ligation mixture. Super coiled plasmid DNA of pET32C+ was used as positive control. The transformation efficacy was found to be 0.26×104 CFU/µg of the recombinant. 4.3.3 Confirmation of clones The transformed cells were picked up, streaked on Luria agar ampicillin 100 mg per ml plate. The clones were confirmed by colony PCR (same as PCR condition mentioned in section 3.1.1 except template DNA here direct cell are used as template DNA) (Plate 8 and 10). 4.4 Bioassay results The mutation induced and confirmed clones of cry1Ac and cry2 were induced with IPTG and the active protein was isolated. The isolated proteins from different clones, pAK125 and pAK220 (Fig. 4 and 5) were subjected for bioassay against P. xylostella and insect mortality was recorded at an interval of 24, 48, and 72 hours. Each treatment was kept in three replicas. On the basis of mortality per cent after given intervals it was found the 40% clones showed 65 to 75 % mortality that means no effect of mutation in these clones. Almost 41% clones showed little decrease in toxicity that is these clones cause 40 to 60 % mortality to the chosen insect. Only 5% clones showed mortality in the range of 70 to 80% which is slightly more than reference i.e., native cry1Ac. Approximately 9% clones showed mortality less than 40%. One mutant clone (M25), is showing enhanced mortality of 80% which is 10% more than native (Plate 12 and 13). In case of cry2 mutants it was found that majority of mutants i.e., 56% were showing mortality ranging from 51% to 65%. 14 (25%) clones out of 55 were showed
45
46
47
48
mortality less than 50%. Only 10 clones showed mortality in the range of 70 to 75% (Table 1 and 2). 4.5 Sequence analysis Some of the clones which were showing toxicity less than 20% mortality and few which were showing more mortality than reference were sequenced (Fig. 7). Primer walking was done up to 2 kb (toxic region) of the mutated clones and the fragments were assembled as contigs by using BioEdit off line software. The sequenced clones were translated and aligned with reference protein and amino acid changes were recorded which resulted in altered protein toxicity. It was found that mutations in domain I resulted in loss or reduced toxicity towards the insect used for bioassay. Enhanced toxicity was reported in the clones were mutation occurred in domain II. Domain II being hypervariable region confers differential specificity and differential receptor binding in their target cells so, mutation in domain II resulted enhanced or decreased specificity and altered receptor binding. Mutations found in different clones and their effects are listed in Table 3. 4.6 Cloning of mutated amplicons of cry1Ac into the yeast expression vector and expression analysis The 3.5 kb mutated amplicons of cry1Ac was inserted into BamHI and XhoI site of the pYES2/CT vector, generating pYAK1-10 (Fig. 6). These constructs were transferred to S. cerevisiae IVNs 1 and transformants were confirmed by PCR and restriction analysis. The transformants were picked and streaked on SC-U minimal medium lacking uracil. The presence of recombinant vectors in yeast total DNA was confirmed through PCR amplification using gene specific primers. As expected About 3.5 kb amplicons were obtained in yeast clones having pYAK1-10 (Plate 11). 4.7 Expression of cry1Ac in yeast The mutated clones having cry1Ac gene in yeast were induced by galactose in SC-U minimal medium. Pellet was resuspended in sample 500µl of protein extraction buffer and vortexed for five minutes with 200mg of acid washed glass beads (Sigma
49
50
Table 3. Mutation in cry1Ac and the observed effect on mortality of P. xylostella Sl. No.
Clones and changes observed
Effect on Bioassay
1
M2: (G259V, Y277N, C496W and G596A)
Only 10% of mortality
2
M33: (A142P, S145E, F150A, Q165H, Only 10% of mortality L480F, P497T, G452E, S515P and F537S)
3
M34: (L269T, R270Q, E277N and G426R)
Mortality of 50%
4
M38: (G319R and G610 A)
Enhanced mortality of 77%
5
M25: (P393S)
Enhanced mortality of 80%
6
M20: (L352T)
Mortality of 73%
51
Aldrich). Centrifuged for maximum speed (13000 rpm) for two minutes and supernatant was collected in fresh eppendorf tube. The protein of 130kD was confirmed through SDS-PAGE and further toxicity was tested by bioassay. 4.6.2 Bioassay results The isolated proteins from different clones were subjected to bioassay against P. xylostella and insect mortality was recorded at an interval of 24, 48, and 72 hours. Each treatment was kept in three replicas. Only one clone (M8) out of ten mutated clones showed mortality of 73% i.e., more than reference (70%). Two clones were showing mortality less than 40% and rest of 7 clones were showing mortality on an average equal to the reference clone. The bioassay results of clones are presented in Table 4.
52
Table 4. Bioassay results of cry1Ac mutants expressed in yeast S.No.
Reference
Total larvae
24hrs
Percent 48hrs mortality
Percent mortality
72hrs
Percent mortality
1
Cry1Ac
30
5
17
15
50
21
70
2
control
30
0
0
0
0
0
0
3
M1
30
4
13
10
33
21
70
4
M2
30
3
10
12
40
19
70
5
M3
30
1
3
8
27
16
53
6
M4
30
4
13
16
40
21
70
7
M5
30
2
7
10
33
16
53
8
M6
30
2
7
9
30
15
50
9
M7
30
2
7
3
10
10
33
10
M8
30
3
10
10
33
22
73
11
M9
30
4
13
16
40
20
67
12
M10
30
1
3
6
20
8
27
53
5. DISCUSSION Increasing the production and productivity of crop plants is the need in the context of increase in population and decreasing farmland area due to urbanization. The problem of insect pests and diseases among other factors is a hindrance in achieving higher yield and productivity. For, the control of insect pests heavily depended on the use of toxic chemical insecticides which has resulted in the accumulation of toxic compounds in the environment and buildup of resistance to insecticides in pest populations. In order to tackle these problems, effective alternatives to reduce or eliminate the use of toxic chemicals are being investigated, which includes bio pesticides and transgenics as components of Integrated Pest Management (IPM). Amongst the components of IPM is Bacillus thuringiensis which is a microbial insecticide that been used for many decades though it is not ideally suited for all pests. It is known to produce insecticidal proteins viz., insecticidal crystal proteins (Cry) and vegetative insecticidal proteins (Vip) (Schnepf et al., 1998). However, it has been recently used as an excellent source of gene for insect resistance in the development of transgenic crops. The first transgenic plants using cry genes were developed in 1987. The transgenic tobacco plants engineered with truncated genes encoding Cry1Aa and Cry1Ab proteins were found to be resistant to tobacco hornworm (Jouanin et al., 1998). Expression of adequate levels of the Cry proteins in crop plants is necessary for effective control of the target pest. Plants transformed with the full length cry sequences encoding the pro-toxin had extremely low levels of the toxic protein. Vaeck et al., (1987), with a truncated version of the cry1Ab gene, found that the toxin detected in the leaves of transgenic tobacco plants represented up to 0.07 percent of the total leaf protein, while it was only 0.0001 percent when full length gene was employed. Many studies have demonstrated that the native Bacillus thuringiensis genes were very poorly expressed in transgenic plants and that modification of their nucleotide sequence significantly enhanced the expression level of these genes (Perlak et al., 1991 and Gleave et al., 1998). It has been suggested that
54
the low expression of crystal protein genes in plants is a consequence of both poor transcription and translation (Perlak et al., 1990 and Murray et al., 1990). Conventional use of Bt insecticides is faced with some limitations, such as a narrow spectrum, a short shelf life, and development of pest insect resistance. To overcome these problems, many researchers have tried to modify the insecticidal crystal protein-encoding genes of B. thuringiensis strains using genetic manipulation for enhancement of insecticidal activity, faster effects, and delay of resistance development (Aronson et al., 1995 and Kalman et al., 1995). In the present investigation native cry1Ac and cry2 were subjected to mutagenesis through GeneMorph Random Mutagenesis Kit and expressed in prokaryotic expression vector to analyze the effect of altered nucleotide on the chosen insect. Random mutagenesis is a powerful tool for elucidating protein structurefunction relationships and for modifying proteins to improve or alter their characteristics. Error prone PCR is a random mutagenesis technique for generating amino acid substitutions in proteins by introducing mutations into a gene during PCR. Mutations are deliberately introduced through the use of error prone DNA polymerases and/or reaction conditions (Lin-Goerke et al., 1997 and Shafikhani et al., 1997) The mutated PCR products are then cloned into an expression vector and the resulting mutant library can be screened for changes in protein activity. Random mutagenesis and strategies that allow gene recombination in vitro have become important tools for exploring and optimizing protein properties. In vitro random mutagenesis is mostly the result of an EP-PCR using low-fidelity buffer conditions in combination with a DNA polymerase lacking exonuclease proofreading activity. Because Taq DNA polymerase has a high intrinsic error rate between 8 x 10-6 and 2 x 10-4 errors/nucleotide synthesized depending on the reaction conditions (Eckert & Kunkel, 1990 and Cline et al., 1996), it is the preferential choice for most EP-PCR mutagenesis studies. The rate of single base substitutions during DNA polymerase action can be deliberately increased by elevated Mg2+ concentrations
55
relative to the total dNTP concentration, by adding Mn2+ up to 5 mM to the reaction buffer, or by changing the ratio of the different dNTPs (Eckert & Kunkel, 1990). Lower template concentrations or higher numbers of PCR cycles on the other hand result in more target duplications and consequently higher mutation frequencies in the amplified DNA. Using this set of altered conditions, mutation frequencies ranging from 0.01 to 10% have been reported (Leung et al., 1989; Cadwell & Joyce, 1992; Fromant et al., 1995; Vartanian et al., 1996; Lin-Goerke et al., 1997 and Shafikhani et al., 1997). Although Taq polymerase has been widely used for EP-PCR experiments, it has the major drawback that certain types of basepair changes are preferred, independent of the MnCl2 concentration used. It is well documented that mutations generated by this enzyme are dominated by AT in place of GC transitions and AT in place of TA transversions (Lin-Goerke et al., 1997 and Shafikhani et al., 1997; Casson & Manser, 1995). The overall result is a strong bias toward dAMP and dTMP substitutions, making these nucleotides two- to four fold more likely to be mutated compared to G and C residues. Consequently, all types of mutations are not equally represented. To reduce mutational bias in random mutagenesis Mutazyme DNA polymerase has been used, which has a high error rate of ± 3.3 x 10-4 errors/nucleotide synthesized and a general predisposition for the generation of transitions. In addition to its strong preference for GC in place of AT transitions, GC in place of TA transversions are introduced at a higher rate, which explains its overall tendency to mutate G and C residues nearly three times more frequently (Cline & Hogrefe, 2000). The complementary mutational spectrum of this polymerase makes it an ideal candidate to offset the mutational bias inherent to randomization with Taq DNA polymerase which may result in a library with a more balanced mutational profile as suggested by Patrick et al., (2003) and Neylon (2004). In this investigation, an attempt was made to create random mutations in cry1Ac and cry2 and analyze the effect of altered nucleotide, resulting in changed
56
amino acids on the toxicity to Plutella xylostella. Totally 109 mutants of cry1Ac and 55 mutants of cry2 were picked and subjected to bioassay against P. xylostella. Insect mortality was recorded at an interval of 24, 48, and 72 hours. Each treatment was kept in three replicas. On the basis of mortality per cent after given intervals it was found the 40% clones showed 65 to 75 % mortality that means no effect of mutation in these clones. Almost 41% clones showed little decrease in toxicity that is these clones cause 40 to 60 % mortality to the chosen insect. Only 5% clones showed mortality in the range of 70 to 80% which is slightly more than reference i.e., native cry1Ac. Approximately 9% clones showed mortality less than 40%. One mutant clone (M25), is showing enhanced mortality of 80% which is 10% more than native. It is found that enhanced toxicity resulted in M25 mutant may be because of replacement of serine by phenylalanine at 393rd position of amino acid sequence which falls in domain II. In case of M33 nine alterations occurred (A142P, S145E, F150A, Q165H, L480F, P497T, G452E, S515P, and F537S) resulting total loss of toxicity. In case of cry2 mutants it was found that majority of mutants i.e., 56% were showing mortality ranging from 51% to 65%. 14 (25%) clones out of 55 were showed mortality less than 50%. Only 10 clones showed mortality in the range of 70 to 75%. Few clones of cry2 are also sequenced but there is no change in the amino acid resulting mortality equivalent to native. Some of the earlier noticed mutations and their effects are shown in Table 5. Domain I being most conserved among three domains and is involved in the basic function of δ-endotoxins viz., ion channel formation so, most of the mutants in this region resulted in low or no toxicity on tested insect. Mutation created in the highly conserved region of cry1Ac toxin i.e.,( R525G, R525A, R529G, or R529A) resulted in 4 to 12 fold reduced toxicity (Chen et al., 1993 and Masson et al., 2002). Mutants having altered nucleotides in conserved region of cry1Ac and cry2 showed reduced toxicity up to 50% as compared to the respective native genes.
57
Table 5. List of some of the earlier noticed mutations and their effect on the toxicity Sl. No.
Mutations noticed/induced/created
Effect
Reference
1
cry1Ab (F50K)
Loss of toxicity due to impaired pore formation
Ahmed and Ellar, 1990
2
cry1Ac (R93H, R93G, R93A, or R93A)
3-10 folds reduced toxicity due to loss of positive charge
Wu and Aronson, 1992
3
cry1Ac (R209A,P, T213A, No change in toxicity W210L, V218N, Y211N,R,D)
Aronson et al., 1995
4
cry1Ac (α-helix7 Enhanced toxicity due to substituted with large pore formation. Diphtheria toxin fragment)
Chandra et al., 1999
5
cry1Ac1 (N135Q)
Reduced toxicity due to impaired pore formation
Tigue et al., 2001
6
cry1Aa (R526K and N135K)
Specifically reduces the toxicity.
Chen et al., 1993
7
cry1Ab (N372A, A282G and L283S)
36-folds increase in toxicity to gypsy moth.
Rajamohan et al., 1996
58
Domain II being hypervariable region confers differential specificity and differential receptor binding in their target cells so, mutation in domain II resulted enhanced or decreased specificity and altered receptor binding. In several mutants there is complete failure of expression of mutant protein in E.coli. This instability of protein might be due to exposure of proteolytic cleavage sites because of conformational changes in the mutant toxins. Mutants which altered the hydrophobicity of domain I resulted in complete loss of toxicity (Wu et al., 1992). Netural or positively charged amino acids substitution has little effect on domain I function than negatively charged amino acids, which completely abolishes the toxicity. Domain I inserts itself into the lipid bilayer of the membrane. Therefore, hydrophobicity in this domain is important. Modifications can be created in δ-endotoxins using various protein engineering techniques such as single or multiple amino acid changes in variable and conserved regions through site directed mutagenesis, which will be helpful in designing of protein with novel features and prediction of secondary structural changes using CD spectrometry. Restriction fragment exchange between closely related toxin genes or with other bacterial toxin genes can be helpful in the analysis of structure-function relationships of the toxins and catalytic center of toxin.
59
6. SUMMARY AND CONCLUSIONS In the present study, an attempt was made to create random mutations in cry1Ac and cry2 genes from Bacillus thuriengiensis to analyze the effect of altered nucleotides on the toxicity to a chosen insect. The mutated cry1Ac was analyzed for its effect on toxicity by expressing in yeast also. The results obtained are summarized here: Both the native genes i.e., cry1Ac and cry2 were confirmed by PCR amplification and checked for expression by SDS-PAGE analysis. Random mutations were created in the full length genes by using GeneMorph II Random Mutagenesis Kit and taking plasmid DNA of expressed genes as target for mutation. Mutation in domain I of both cry1Ac and cry2 at several residues resulted in total loss of toxicity confirmed by bioassay results against Plutella xylostella. Domain I being most conserved among three domains and is involved in the basic function of δ-endotoxins viz., ion channel formation so, most of the mutants in this region resulted in low or no toxicity on tested insect. Domain II being hypervariable region confers differential specificity and differential receptor binding in their target cells so, mutation in domain II resulted enhanced or decreased specificity and altered receptor binding. Few mutations at the α-helices of domain II of cry1Ac resulted in somewhat better toxicity as compared to native this may be because of large pore formation in the mid gut of insect by this mutated protein as earlier also reported by Chandra et al. 1999. Mutated and native cry1Ac were expressed in Saccharomyces cerevisiae INVSc1, and expression was confirmed by using SDS-PAGE and bioassay. The mutation induced confirmed clones of cry1Ac and cry2 were induced with IPTG and active protein was isolated. The isolated proteins from different clones were subjected for bioassay against P. xylostella and insect mortality was recorded at an interval of 24, 48, and 72 hrs. On the basis of mortality per cent after given intervals it
60
was found the 40% clones showed 65 to 75 % mortality that means no effect of mutation in these clones. Almost 41% clones showed little decrease in toxicity that is these clones cause 40 to 60 % mortality to the chosen insect. Only 5% clones showed mortality in the range of 70 to 80% which is slightly more than reference i.e., native cry1Ac. Approximately 9% clones showed mortality less than 40%. One mutant clone (M25), is showing enhanced mortality of 80% which is 10% more than native cry1Ac. In case of cry2 mutants it was found that majority of mutants i.e., 56% were showing mortality ranging from 51% to 65%. 14 (25%) clones out of 55 were showed mortality less than 50%. Only 10 clones showed mortality in the range of 70 to 75%.
61
REFERENCES Agrawal, N., Malhotra, P. and Bhatnagar, R.K., 2002, Introduction of gene-cloned and insect cell-expressed aminopeptidase N of Spodoptera litura with insecticidal
crystal
protein
Cry1c.
Applied
and
environmental
microbiology, 68: 4583-4592. Ahmed, W. and Ellar, D.J., 1990, Directed mutagenesis of selected regions of Bacillus thuringiensis entomocidal proteins. FEMS Microbiology letters, 68: 97-104. Aoki, K. and Chigasaki, Y., 1915, Uber die pathogenitat dersog, Sotto-Bacillen (Ishiwata) bei seidenraupen, Mitt. Med Fak. Kaiser Univ. Tokyo, 13: 419440 Armengol, G., Escobar, M.C., Maldonado, M.E. and Orduz, S., 2007, Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects. J. Appl. Microbiol., 102(12): 7788. Aronson, A.I., Shai, Y., 2001. Why Bacillus thuringeinsis insecticidal toxins are so effective: unique features of their mode of action. FEMS Microbiol. Letter. 195: 1-8. Aronson, A.I., Wu, D., Zhang C., 1995, Mutagenesis of specificity and toxicity regions of a Bacillus thuringiensis protoxin gene. Journal of Bacteriology. 177: 4059-4065. Arrieta, G., Hernandez, A. and Espinoza, A.M., 2006, Diversity of Bacillus thuringiensis strains isolated from coffee plantations infested with the coffee borer, Hypothenemus hampei. Scientific Electronic Library Online, 52: 1-9.
62
Ben-Dov, E., Zaritsky, A., Dahan, E., Barak, Z., Sinai, R., Manasherob, R., Khamraev, A., Triotskaya, E., Dubitsky, A., Berezina, N. and Margalith, Y., 1997, Extended screening by PCR for seven cry group genes from field collected strains of Bacillus thuringiensis. Appl. Environ. Microbiol., 63: 4883-4890. Berliner, E., 1915, Uber die sehlaffsucht der mehlmottenraupe (Ephestia kuhniella Zell.) und thren Erreger Bacillus thuringiensis sp. Zhurnal Ang. Entomo., 2: 29-56. Bone, E. J., and D. J. Ellar., 1989, Transformation of Bacillus thuringiensisby electroporation. FEMS Microbiol. Lett. 58: 171–178. Boonserm, P., Davis, P., and Ellar., 2005, Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications. J. Mol. Biol. 348: 363–382 Boonserm, P., Mo, M., Angsuthanasombat, Ch., Lescar, J., 2006. Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-Å resolution. Journal of Bacteriol. 188: 3391-3401. Bosch, D., Schipper, B., vab der Kleij, H., de Maagd, R.A., and Stiekema, W.J., 1994, Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management. Biotechnology, 12:915-918. Bravo, A., 1997, Phylogenetic relationships of Bacillus thuringiensis delta-endotoxin family proteins and their functional domains. Journal of Bacteriolog, 179: 2793-2801. Bravo, A., Gomez, I., Conde, J., Munoz-Garay, C., Sanchez, J., Miranda, R., Zhuang, M., Gill, S.S. and Soberon, M., 2004, Oligomerization triggers binding of Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochim. Biophys. Acta, 1667:38-46. Bravo, A., Sa nchez, J., Kouskoura, T., Crickmore, N., 2002. N-terminal activation is an essential early step in the mechanism of action of the B. thuringiensis Cry1Ac
insecticidal toxin. J. Biol. Chem. 277:23985–23987.
63
Bravo, A., Sarabio, S., Lopez, L., Ontiveros, H., Abarca, C., Ortiz, A., Ortiz, M., Lina, L., Villalobos, F. J., Pena, G., Munez-Valdez, M., Soberon, M. and Uintero, R., 1998, Characterization of cry genes in a Mexican Bacillus thuringiensis strain collection. Appl. Environ. Microbiol., 64: 4965-4972. Bravo, A., Gill, S.S. and Soberon, M., 2005, Bacillus thuringiensis Mechanism and uses in comprehensive molecular insectscience. Elsevier BV, Amsterdam, pp.175-206. Cadwell, R.C., and Joyce, G.F., 1992. Randomization of genes by PCR mutagenesis. PCR Methods Appl., 2, 28-33. Caramori, T., Albertini, A. M. and Galzzi, A., 1991, In vivo generation of hybrids between two Bacillus thuringiensis insect-toxin encoding genes. Gene. 98: 37-44. Casson, L.P. & Manser, T. (1995). Evaluation of loss and change of specificity resulting from random mutagenesis of an antibody Vh region. J. Immunol. Methods, 155: 5647-5654. Chak, K. 1990. Identification of novel cry type genes from Bacillus thuringiensis strains. Appl. Environ. Microbiol. 62: 1369–1377. Chandra, A., Ghosh, P., Mandoakar, A.D., Bera, A.K., Sharma, R.P., Das, S. and Kumar P.A., 1999, Amino acid substitution in α-helix7 Cry1Ac δendotoxins of Bacillus thuringiensis leads to enhanced toxicity to Helicoverpa armigera Hubner. FEBS Letters. 458: 174-179. Chen, X. J., Lee, M. K., and Dean, D. H., 1993, Site-directed mutations in a highly conserved region of Bacillus thuringiensis δ-endotoxins affected inhibition of short circuit current across Bombyx mori midgets. Proceedings of National Academy of Sciences, 90: 9041-9045. Cline, J., Braman J.C. and Hogrefe, H.H., 1996, PCR fidelity of Pfu DNA polymerase and other thermostable DNA polymerases. Nucleic Acids Res., 24: 35463551.
64
Cline, J., and Hogrefe, H., 2000, Randomize gene sequences with new PCR mutagenesis kit. Strategies, 13: 157-162. Convents, D., Houssier, C., Lasters, I., and Lauwereys, M., 1990, The Bacillus thuringiensis δ-endotoxins evidences for a two-domain structure of the minimal toxin fragment. Journal of Biologcal Chemistry, 256:1369-1375. Crickmore, N., Zeigler, D. R., Feitelson, J., Schnepf, E., Van Rie, J., Lereclus, D., Baum, J. and Dean, D. H., 2002, Bacillus thuringiensis toxin nomenclature, http:// www. Boils. susx.ac.uk/Home Neil_Crickmore Bt/index. Html (21062002) Crickmore, N., Zeigler, D. R., Feitelson, J., Schnepf, E., Vanrie, J., Lereclus, D., Baum, J. and Dean, D. H., 1998, Revision of the nomenaclature for the Bacillus thuringiensis pesticidal crystal proteins. Microbiol. Rev., 62: 807813. de Barjac, H. and Franchan, E., 1981, Classification of Bacillus thuringiensis strains. Entomophaga, 35: 233-240 de Maagd, R. A., Bravo, A. and Crickmor, N., 2001, How Bacillus thuringiensis has evolved specific toxins to colonize the insect world. Trends in Genetics, 17: 193-199 de Maagd, R.A., Bravo, A., Berry, C., Crickmore, N. and Schnepf, H.E., 2003, Structure, diversity, and evolution of protein toxins from spore-forming entomopathogenic bacteria. Annu. Rev. Genet., 37: 409-433. de Maagd, R. A., Kwa, M. S., Van Der, K. H., Yamamto, T., Shipper, B., Valk, J. M., Shiekema, W. J. and Bosch, D., 1996, Domain III substitution in Bacillus thuringiensis δ-endotoxins Cry1A (b) results in superior toxicity for Spodoptera exiqua and altered membrane protein recognition. Applied and Environmental Microbiology, 62: 1537-1543.
65
Dean, D. H., Rajamohan, F., Lee, M.K., Wu, S.J., Chen, X.J., Alcantara, E. and Hussain, S.R., 1996, Probing the mechanism of action of Bacillus thuringiensis insecticidal proteins
by
site
directed
mutagenesis:
a
minireview. Gene 179:111–117. Eckert, K.A., and Kunkel, T.A., 1990, High fidelity DNA synthesis by the Thermus aquaticus DNA polymerase. Nucleic Acids Res., 18: 3739-3744. Ferre, J., Escriche, B., Bel, Y. And Van Rie, J., 1995, Biochemistry and genetics of insect resistance to B. thuringiensis insecticidal crystal protein. FEMS Microbiol. Lett. 132: 1-7. Fromant, M., Blanquet, S., and Plateau, P., 1995, Direct random mutagenesis of genesized DNA fragments using polymerase chain reaction. Anal. Biochem., 224: 347-353. Galitsky, N., Cody, V., Wojtczak, A., Ghosh, D., Luft, J.R., Pangborn, W., English, L., 2001, Structure of the insecticidal bacterial δ-endotoxins CryBb1 of Bacillus thuringiensis. Acta Cryst. Allogr D., 57: 1101-1109. Garczynski, S. F., Crim, J.W., and Adang, M. J., 1991, Identification of putative insect brush border membrane-binding molecules specific to Bacillus thuringiensis δ-endotoxins by protein blot analysis. Applied and Environmental Microbiology, 57: 2816-2820. Gazit, E. P., Rocca, L., Sansom, M.S.P. and Shai, Y., 1998, The structure and organization within the membrane of the helices composing the poreforming domains of Bacillus thuringiensis δ-endotoxins are consistent with an “umbrella-like” structure of the pore. Proceedings of the National Academy of Sciences. 95: 12289-12294. Ge, A.Z., Rivers, D., Miline, R. and Dean, D.H., 1991, Functional domains of Bacillus thurongiensis insecticidal crystal proteins. Refinement of Heliothis virescens and Trichoplusia ni specificity domains on Cry1A(c). Journal of Biological Chemistry. 266:17954-17958.
66
Gelernter, W. D. and Evans, H. F., 1999, Factors in the Success and Failure of Microbial Insecticides. Integrated Pest Management Rev. 4: 279-316. Gerber, D. and Shai, Y., 2000, Insertion and organization within membranes of the δendotoxins pore-forming domain, helix4-loop- helix5, and inhibition of its activity by a mutant helix 4 peptide. Journal of Biological Chemistry. 275: 23602-23607. Gleave, A.P, Mitra, D.S., Markwick, N.P., Morris, BAM., Beuning, L.L,.1998, Enhanced expression of the Bacillus thuringiensis cry9Aa2 gene in transgenic plants by nucleotide sequence modification confers resistance to potato tuber moth. Mol Breeding, 4: 459–472. Gomez, I., Dean, D. H., Bravo, A. and Soberon, M., 2003, Molecular basis for Bacillus
thuringeinsis
Cry1Ab
toxin
specificity:
two
structural
determinants in the Manduca sexta Bt-R1 receptor interaction with loops α8 and α-8 and 2 in domain II of Cry1Ab toxin. Biochemistry., 42: 1048210489. Gordon, R. E., W. C. Haynes, and C. H.-N. Pang. 1973. The genus Bacillus. U.S. Department of Agriculture, Washington, D.C., 1124-1135. Griffits, J.S., Haslam, S.M., Yang, T., Garczynski, S.F., Mulloy, B., Morris, H., Cremer, P.S., Dell, A., Adang, M.J., Aroian, R.V., 2005, Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science., 307: 922-925. Grochulski, P., Masson, L., Borisova, S., Pusztai-Carey, M., Schwartz, J. L., Brousseu, R. and Cygler, M., 1995, Bacillus thuringeinsis Cry1A(a) insecticidal toxin: Crystal structure and channel formation. Journal of Molecular Biology., 256: 447-464. Hodgman, T.C., and Ellar, D.J., 1990, Models for the structure and function analysis of the Bacillus thuringiensis d-endotoxins determined by compilation analysis. DNA Sequence, 1:97-106
67
Hofte, H. and Whiteley, H.R., 1989, Insecticidal crystal protein of Bacillus thuringiensis. Microbiol. Rev., 53: 242-255. Iriarte, J., Ferrandis, M. D., and Andrew, R., 2002, Letters in Applied Microbiol., 28: 440-444. Ishiwata, S., 1901, On a kind of severe flacherie. Dalnihon Sanshi Kaiho, 9: 1-5. Jinkins, J.L., Lee, M. K., Valaitis, A.P., Curtiss, A.P. and Dean, D. H., 2000, Bivalent sequential model of Bacillus thuringiensis toxin to Gypsy Moth aminopeptidase N receptor. Journal Biological Chemistry, 275: 1442314431. Jouanin, L., Bonade-Bottino, M., Girard, C., Morrot, G. and Gibaud, M., 1998, Plant Sci., 131: 1–11. Jurat-Fuentes, J.L. and Adang, M.J., 2004. Characterization of a Cry1Ac-receptor alkaline phosphatise in susceptible and resistant Heliothis virescens larvae. Eur. J. Biochem.,271: 3127-3135. Jurat-Fuentes, J.L. and Adang, M.J., 2001, Importance of Cry1_Endotoxin Domain II Loops for Binding Specificity in Heliothis virescences (L). Appl. Environ. Microb., 67(1): 323-329. Kalman, S., Kiehne, K. L., Cooper, N., Reynoso, M. S., Yamamoto, T., 1995, Enhanced production of insecticidal proteins in Bacillus thuringiensis strain carrying an additional protein gene in their chromosomes. Applied and Environmental Microbiology.61:3063-3068. Kanintrokul, Y., Sramala, I., Katzenmeir G., Panyim S. and Angsuthanasombat, C., 2003, Specific mutations within the α4-α5 loop of the Bacillus thuringeinsis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170. Molecular Biotechnology., 24: 11-20. Knight, P., Crickmore, N., Ellar, D.J., 1994. The receptor for Bacillus thuringiensis Cry1A(c) d-endotoxin in the brush border membrane of the lepidopteran Manduca sexta is aminopeptidase N. Mol. Microbial., 11: 429-436.
68
Knowles, B.H., 1994, Mechanism of action of Bacillus thuringiensis insecticidal dendotoxins. Adv. Insect. Physiol.24:275-308. Krieg, A., Higer, A. M., Langenbrugh, G. A. and Schnetter, W., 1987, Bacillus thuringiensis Var. tenebrionis. Ein neure, gegenuber larven von coleopteran wirksamer pathotyp. Zeitschrift fur Angewandte Entomologie, 96: 500-508. Kumar, A. S. and Aronson, A. I. 1999, Analysis of mutations in the pore-forming region
essential for insecticidal activity of a Bacillus thuringiensis
delta-endotoxin. J. Bacteriol., 181:6103- 6107. Kumarswamy, G.K., 2005, Expression of cry1Ac from native Bacillus thuringiensis D1 IN Escherichia coli. M. Sc. (Agri.) Thesis, Uni. Agri. Sci. Dharwad (India). Kuo, W.S., J.H. Lin, C.C. Tzeng, S.S. Kao and K.F. Chak, 2000, Cloning of two new cry genes from B. thuringiensis subsp. wuhanensis strain. Curr. Microbiol., 40: 227-232. Lecadet, M. M., Frachon, E., Cosmao Dumanoir, V. and Thiery, I., 1999, Updating the H-antigen classification of Bacillus thuringiensis. J. Appl. Microbiol. 86: 660-672. Lee, M. K., Young, B. A. and Dean, D. H., 1995, Domain III exchanges of Bacillus thuringeinsis Cry1A toxins affect binding to different gypsy moth midgut receptors. Biochemical and Biophysical Research Communications., 216: 306-312. Leung, D., Chen, E., and Goeddel, D., 1989, A method for random mutagenesis of a defined DNA segment using a modified polymerase chain reaction. Technique, 1: 11-15. Li, J., Carroll, J. and Ellar, D. J., 1991, Crystal structure of insecticidal d-endotoxin from Bacillus thuringiensis at 2.5 Å resolutions. Nature., 253: 815-821. Lin-Goerke, J.L., Robbins, D.J., and Burczak, J.D., 1997, PCR-based random mutagenesis
using
manganese
BioTechniques, 23: 409-412
and
reduced
dNTP
concentration.
69
Manoj kumar, A. S., and Aronson, A. I., 1999, Analysis of mutations in the poreforming regions essential for insecticidal activity of a Bacillus thuringiensis d-endotoxin. Journal of Bacteriology., 181: 6103-6107. Masson, L., Tabashinik, B. E., Mazza, A., Prefontaine, G., Potvin, L., Rossea, R. and Schwartz, J.L.,2002, Mutagenic analysis of a conserved region of domain III in the Cry1Ac toxin of Bacillus thuringeinsis. Applied and Environmental Microbiology., 68: 194-200. Meadows, P. M., Ellis, D. J., Butt, J., Jarret, P. and Burges, H. D., 1993, Distribution, frequency and diversity of Bacillus thuringiensis in animal feed mill. Appl. Environ. Microbiol., 58: 1344-1350. Meiying, G., Yan Wu and Shunying, D., 2008, Investigation of the cyt gene in Bacillus thuringiensis and the biological activities of Bt isolates from the soil of China. Biol. Control, 47: 335-339 Meza, R., Nunez-Valdez, M.E., Sanchez, J., and Bravo, A., 1996, Isolated domain II and III from the Bacillus thuringiensis Cry1Ab d-endotoxin binds to lepidopteran midgut membranes. Morse, R., Yamamoto, T., and Stroud, R., 2001 Structure of Cry2Aa Suggests an Unexpected Receptor Binding Epitope. Structure 2001, 9: 409-417. Murray EE, Rocheleau T, Eberle M, Stock C, Sekar V, Adang M. Analysis of unstable RNA transcripts of insecticidal crystal protein genes of Bacillus thuringiensis in transgenic plants and electroporated protoplasts. Plant Mol Biol, 1991, 16: 1035–1050. Neylon, C., 2004, Chemical and Biochemical strategies for the randomization of protein encoding DNA sequences: library construction methods for directed evolution. Nucleic Acids Res., 32:1448-1459. Ogunjimi, A. A., John Mchander, George, O. Gbenle, Daniel, K. O. and Ezekiel, O. A., 2002, Heretologous expression of cry2 gene from a local strain of Bacillus thuringiensis isolates in Nigeria. 36(P+3): 241-256.
70
Pardo-Lopez, L., Gomez, I., Rausell, C., Sanchez, J., Soberon, M., Bravo, A., 2006, Structural changes of the Cry1Ac oligomeric pre-pore from Bacillus thuringiensis induced by N-acetylgalactosamine facilitates toxin membrane insertion. Biochemistry, 45: 10329-10336. Parker, M.W. and Pattus, F., 1993, Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxins. Trends Biochemical Sciences., 18: 391-395 Patrick, W.M., Firth, A.E., and Blackburn, J.M., 2003, User-friendly algorithms for estimating completeness and diversity in randomized protein-encoding libraries. Protein Eng., 16: 451-457.. Perlak FJ, Deaton RW, Armstrong TA, Fuchs RL, Sims SR, Greenplate JT, Fischhoff DA, 1990, Insect resistant cotton plants. Biotechnology, 8: 939– 943. Rajamohan, F., Cotrill, J.A., Gould, F. and Dean, D.H., 1996a, Role of domain II, loop 2 residues of Bacillus thuringiensis Cry1Ab d-endotoxin in reversible and irreversible binding to Manduca sexta and Heliothis virescens. Journal of Biological Chemistry. 271: 2390-2396. Rajamohan, F., Alcantara, Lee, M.K., Chen, X.J., Curtiss, A. and Dean, D.H., 1995, Single amino acid changes in domain II of Bacillus thuringiensis Cry1Ab d- endotoxin affect irreversible binding to Manduca sexta midgut membrane vesicles. Journal of Bacteriology. 177: 2276-2282. Rang, C., Vachon, V., de Maagd, R.A., Villalon, M., Schwartz, J.L., Bosch, D., Frutos, R. and Laprade, R., 1999, Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins. Appl. Environ. Microbiol., 65: 2918-2925. Sambrook, J. and Russel, D. W., 2001, Molecular Cloning: A laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, New York. 2.54-4.0 Sangmesh, S,P., 2004, Cloning and expression of cry2A(a) from native Bacillus thuringiensis isolates. M. Sc. (Agri.) Thesis., Uni. Agri. Sci. Dharwad (India).
71
Schnepf, E., Crickmore, N., Vanrie, J., Lereclus, D., Baum, J., Feitelson, J., Ziegler, D. R. and Dean, D. H., 1998, Bacillus thuringiensis and its pesticidal crystal proteins. Microbiol. Molec. Bio. Rev., 62: 775-806. Schnepf, H.E., Tomczak, K., Ortega, J.P. and Whiteley, H.R., 1990, Specificitydetermining regions of a Lepidoptera-specific insecticidal protein produced by Bacillus thuringiensis. Journal of Biological Chemistry. 265: 2092320930. Schwartz, J.L., Juteau, M., Grochulski, P., Cygler, M., Prefontaine, G., Brousseau, R. and Masson, L., 1997, Restriction of intramolecular movements within the Cry1Aa toxin molecule of Bacillus thuringiensis through disulfide bond engineering. FEBS Letters., 410: 397-402. Shafikhani, S., Siegel, R.A., Ferrari, E. and Schellenberger, V., 1997, Generation of large libraries of random mutants in Bacillus subtilis by PCR-based plasmid multimerization. BioTechniques, 23: 304-310 Slatin, S.L., Abrams, C.K. and English, L., 1990, Delta-endotoxins form cationselective channels in planar lipid bilayers. Biochemical Biophysics Research Communication. 169: 765-772. Smith, R. A. and Couch, G. A., 1991, The phylloplane as a source of Bacillus thuringiensis. Appl. Environ. Microbiol., 57: 311-315. Tabashnik, B.E.., 1994, Evaluation of resistance to Bacillus thuriengiensis. Annual review of Entomology, 39: 47-79. Tigue, N.J., Jacopy, J. and Ellar, D.J., 2001, The α-helix 4 residues, Asn 135, is involved in oligomerization of Cry1Ac and Cry1Ab5 Bacillus thuringiensis toxins. Applied and Environmental Microbiology, 67: 5715-5720. Uawithya,
P.,
Tuntitippawan,
T.,
Katzenmeier,
G.,
Panyim,
S.,
and
Angsuthanasombat C., 1998 Effects of larvicidal activity of single proline substitutions in α3 or α4 of the Bacillus thuringiensis Cry4B toxin. Biochem. Mol. Bio. Int. 44: 825-832.
72
Vadlamudi, R.K., Weber, E., Ji, I., Ji, TH., Bulla Jr., L.A.,1995, Cloning and expression of a receptor for an insecticidal toxin of Bacillus thuringiensis. J. Bio. Chem.270:5490-5494. Vaeck M, Reynaerts A, Hofte H, Jansens S, De Beukeleer M, Dean C, Zabeau M, Van Montagu M,Leemans J., 1987, Transgenic plants protected from insect attack. Nature, 28:33-37. Valaitis, A.P., Jenkins, J.L., Lee, M.K., Dean, D.H., Garner, K.J., 2001, Isolation and partial charecterization of Gypsy moth BTR-270 an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity. Arch. Ins. Biochem. Physiol.46:186-200. Van Rie, J., Jansens, S., Hofte, H., Degheele, D. and Van Mellaert, H., 1989, Specificity of Bacillus thuringiensis d-endotoxin: Importance of specific receptors on the brush border membrane of the mid-gut of target insects. European Journal of Biochemistry, 186: 239-247. Vanfrankenhuyzez, K., 1993, The challenge of Bacillus thuringiensis. In: Bcillus thuringiensis, an environmental biopesticide. Theory and Practice (Eds, P.F. Entwistle, J.S. Cory, M.J. Bailey and Higgs), John Wiley and Sons Ltd., Chichester, UK, pp.311. Vartanian, J-P., Henry, M., and Wain-Hobson, S., 1996, Hypermutagenic PCR involving all four transitions and a sizeable proportion of transversions. Nucleic Acids Res., 24:2627- 2631. Widner, W.R. and Whiteley, H.R., 1989, Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. Kurstaki possess different host range specificities. Journal of Bacteriology, 171: 965-974. Wu, D. and Aronson, A.I., 1992, Localized mutagenesis defines regions of the Bacillus thuringiensis d-endotoxin involved in toxicity and specificity. Journal of Biological Chemistry, 267: 2311-2317.
73
Wu, S.J., and Dean D.H., 1996, Functional significance of loops in the receptor binding domain of Bacillus thuringiensis CryIIIA -endotoxin. Journal of Molecular Biolology,255:628-640. Zhang, X., Candas, M., Griko, N. B., Taussing R., Bulla, L. A., Jr., 2006, A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis Proc. Natl. Acad. Sci. USA.103: 9897-9902. http://www. lifesci. sussex. ac. uk/home/Neil-Crickmore/Bt/vip. html http://www. ncbi. nlm. nih. Gov
74
APPENDIX I Extraction/Lysis Buffers and Solutions for Plasmid preparation: A. STET Buffer Tris-Cl (pH 8.0)
10 mM
NaCl
0.1 M
EDTA (pH 8.0)
1.0 mM
Triton X-100
5 per cent (v/v)
Water
Make up the volume
Make sure the final pH should be 8.0 B. Alkaline Lysis Solution I Glucose
50 mM
Tris-Cl (pH 8.0)
25 mM
EDTA (pH 8.0)
25 mM
Water
Make up the volume
Autoclave and store at 40C C. Alkaline Lysis Solution II NaOH
0.2 N
SDS
1 per cent (w/v)
Water
Make up the volume
Prepare fresh and use at room temperature D. Alkaline Lysis Solution III Potassium Acetate (5M)
60 ml
Glacial Acetic Acid
11.5 ml
Water
28.5 ml
Autoclave and store at 40C
75
APPENDIX II Buffers and solutions for Agarose Gel Electrophoresis A. Recipe for 0.7 per cent Agarose Gel (40 ml) Agarose
280 mg
1 X TAE
40 ml
EtBr (10mg/ml)
0.5 mg
B. 50 X TAE Buffer Tris
242 g
Glacial acetic acid
57.1 ml
0.5 M EDTA (pH 8.0)
100 ml
Water
Make up the volume to 1000 ml
C. 6X Loading Dye Bromophenol Blue
0.25 per cent
Sucrose
40.0 per cent
Water
Make up the volume
APPENDIX III Media for Bacterial Culture A. Luria-Bartani (LB) Tryptone
1.0 g
Yeast Extract
0.5 g
Sodium Chloride
0.5 g
Water
Make up the volume to 100 ml
Agar (for preparing plates)
1.6-1.8 g
76
APPENDIX IV Media for yeast culture A. Yeast peptone dextrose Yeast extracts
10 g
Peptone
20 g
Dextrose
20 g
Water
makes up the volume to 1000 ml
Agar (for preparing plates)
1.6-1.8 g
Note: For preparing plates, dextrose to be added while pouring media. Donot auto clave dextrose with agar B. synthetic complete media (lacking uracil) Yeast nitrogen base with ammonium sulphate
6.7 g
Carbon source (dextrose or raffinose or galactose)
20 g
Amino acids: Adenine, arginine, cystein, leucine, Lysine, threonine, tryptophan
0.1 g
Aspartic acid, histidine, isolucine, Methionine, phenylalanine, praline, Serine, tyrosine, valine
0.05 g
Water
Make up the volume to 100 ml
Agar (for preparing plates)
1.6-1.8 g
77
APPENDIX V A) Yeast transformation a) 10X TE buffer Tris-Cl (pH 7.5) EDTA
100mM 10Mm
b) 10X Lithium acetate (LiAc) For 100 ml, dissolve 10.2g of lithium acetate in 90ml of deionized water. Then adjust the pH to 7.5 with dilute glacial acetic acid and made up the volume to 100 ml. Filter sterilizes it and stored at room temperature. c) 1X LiAc/0.5 X TE Lithium acetate (pH 7.5) 100mM Tris-Cl (pH 7.5) 5mM EDTA 0.5mM Filter sterilized and stored at room temperature. d) 1X LiAc/40 % PEG-3350/1X TE Lithium acetate (pH 7.5) 100mM PEG 3350 40% Tris-Cl (pH 7.5) 10mM EDTA 1Mm Appendix VI B) Yeast Total DNA extraction buffer/ Lysis buffer Tris-Cl 10mM Sodium Chloride 100mM Triton X-100 2% EDTA 1mM SDS 1% C) Breaking buffer for yeast cell lysis Sodium phosphate buffer (pH 7.4) 50mM EDTA 1mM Glycerol 5% PMSF 1mM
APPENDIX VII Binding Buffer for Isolation of Protein (In Non-Denaturing Form) Sodium phosphate
20 mM
NaCl
500 mM
78
APPENDIX VIII Buffers and solutions for SDS PAGE A. 1X SDS gel loading buffer Tris-Cl (pH 6.8)
50 mM
Dithiothreitol
100 mM
SDS
2 per cent (w/v)
Bromophenol blue
0.1 per cent
Glycerol
10 per cent
B.1X Tris-Glycine Buffer Tris
25 mM
Glycine
250 mM
SDS
0.1 per cent (w/v)
C. 12 per cent Resolving Gel (SDS-PAGE) - 40ml Water 30 per cent acrylamide mix 1.5 M Tris (pH 8.8) 10 per cent SDS 10 per cent ammonium persulphate TEMED
13.2 ml 16.0 ml 10.0 ml 00.4 ml 00.4 ml 00.016 ml
D. 5 per cent Stacking Gel (SDS-PAGE) – 10 ml Water 30 per cent acrylamide mix 1.0 M Tris (pH 6.8) 10 per cent SDS 10 per cent ammonium persulphate TEMED
6.8 ml 1.7 ml 1.25 ml 0.1 ml 0.1 ml 0.011 ml
E. SDS Polyacrylamide Gel Staining Solution (1000 ml) Coomassie Brilliant Blue Methanol Glacial acetic acid Water
0.25 g 500 ml 100 ml 400 ml
F. SDS Polyacrylamide Gel Destaining Solution (1000 ml) Methanol Glacial acetic acid Water
500 ml 100 ml 400 ml
