Journal of Apicultural Research and Bee World 47(2): 169–170 (2008)
© IBRA 2008
N OT E S A N D C O M M E N T S
Detection of promoter activity in honey bee (Apis mellifera) embryos by dual luciferase assay. Jun-ichi Takahashi1,2,*, Yuuki Kato2, Masami Sasaki2, Mitsuo Matsuka2, Isamu Shimizu1 1 2
Center for Ecological Research, Kyoto University, Otsu, Shiga 520-2113 Japan. Honey bee Science Research Center, Tamagawa University, Machida, Tokyo 194-8610 Japan.
Received 6 November 2007, accepted subject to revision 11 December 2007, accepted for publication 10 March 2008. Corresponding author. Email:
[email protected] or
[email protected]
*
Keywords: promoter, Apis mellifera, hr3-ie1, luciferase assay
The honey bee Apis mellifera is an important model organism for several disciplines including agriculture, social behaviour, evolutionary ecology, caste determination, education, and behavioural genetics (Robinson, 2002), so has been analyzed to elucidate its genome sequence (The Honey Bee Genome Sequencing Consortium, 2006). The promoter is a region which regulates gene transcription. In reporter assays, the luciferase gene, which can be used as a reporter gene to measure the activity of promoters in organisms or cells, has contributed greatly to the study of gene expression and regulation. Although the information of effective promoters is of great importance for the expression of exogenous genes, they are insufficient for the honey bee. The purpose of this study was to determine the transcriptional activities of eight different promoters within A. mellifera embryos for the development of a foreign gene expression system. The dual luciferase reporter assay system provides an efficient means of performing two reporter assays. In this assay, the activity of a promoter within the honey bee embryo can be measured from expression levels of the firefly Photinus pyralis and Renilla reniformis luciferases (Luc) gene. In this study, plasmids for the dual luciferase assay were constructed essentially as described by Mohammed and Coates (2004). A control plasmid was designed to express the Renilla luciferase protein under the regulation of the hsp82 promoter from Drosophila. Phsp82RenillaLuc was created by inserting a 1-kb KpnI-BamHI fragment from pKhsp82 into the corresponding sites of pBCKS+ (Stratagene) and then inserting the KpnI-PstI fragment from this plasmid into the corresponding site of pRLSV40 (Promega). Eight different experimental plasmids were constructed to drive the expression of the firefly luciferase protein under the regulation of the cytoplasmic A3 actin (BmA3) promoter derived from Bombyx mori, actin5C (act5C), heat shock protein 70 (hsp70), heat shock protein 82 (hsp82), and polyubiquitin (pUB) promoters derived from Drosophila, and
immediate early 1 gene (ie1) promoter derived from virus. A 2.7kb HindII-SalI fragment from the pGL2-Basic plasmid (Promega) containing the firefly luciferase coding region and the SV40 polyadenylation signal was inserted into the SmaI-SalI sites of pSLfa1180fa to create pSLLuc. The act5C promoter was excised from pBSII-Act5c by KpnI-BamHI digestion and inserted into the corresponding sites of pSLLuc to create pSLAct5CLuc. The BmA3 promoter was amplified by polymerase chain reaction (PCR) from the pPIGA3GFP plasmid using the primers 5’AAGCTTGCGCGTTACCATATATGGTG-3’ and 5’CCGCGGAGGGCGGTAGTCCCGCTATAG-3’, cleaved by digestion with HindII-SacII, and inserted into the corresponding sites of pSLLuc to create pSLA3Luc. The heat shock promoter hsp70 was excised from pHSHH1.9 by XhoI-NotI digestion and then ligated into the corresponding site of pBCKS+ to create pBC-hsp70. The DNA fragment containing the heat shock promoter hsp70 was released from pBChsp70 by KpnI-HindIII digestion and then ligated into pSLLuc digested using the same enzymes, thereby creating pSLhsp70Luc. The hsp82 promoter from pBC-hsp82 was excised using the SacI-SalI restriction enzymes and then ligated into the corresponding sites of pBCKS+ to create pBC-hsp82. The DNA fragment containing the heat shock promoter hsp82 was released from pBChsp82 by KpnIHindIII digestion and then ligated into pSLLuc digested with the same enzymes, thereby creating pSLhsp82Luc. The pGL2-IE1 plasmid was constructed to express the firefly luciferase protein under the regulation of the ie1 promoter with an upstream hr3 enhancer element. Both pIE1-3 (Novagen) and pBCKS+ were digested with BamHI and BglII. The DNA fragment containing the hr3-ie1 regulatory sequences was ligated into linearized pBCKS+ to create pSLIE1Luc. To construct pSLHr3IE1Lcu, the hr3-ie1 fragment was released from pBC-IE1 by KpnI and BglII digestion and ligated into the corresponding sites of pSLLuc. A 2-kb KpnIBamHI fragment from pB[pUB-nls-EGFP] containing the
Luciferase assay of the honey bee
Drosophila pUB promoter was inserted into the corresponding sites of pSLLuc to create pSLpUbLuc. Apis mellifera embryos were collected from apiaries at Kyoto and Tamagawa Universities, Honshu, Japan. Eggs oviposited in cells were checked and collected every 30 min for plasmid injection. The eggs were lined up and fixed on warmed glass microscope slides according to the method described by Beye et al. (2002). The plasmid solution was injected into each egg using Transferman NK2 (Eppendorf). For dual luciferase assays, each test plasmid solution was injected into 75 embryos with the internal control plasmid pSLhsp82RenillaLuc. The test plasmid concentration was 300 ng/µl and the concentration of test plasmid and internal control plasmid pSLhsp82RenillaLuc was 1:1 ratios. The injected embryos were incubated for 36 h at 31°C in 90 % humidity in an oxygen saturated chamber. A total of 750 embryos were divided into five groups of 15 embryos per promoter, and each group was homogenized in a 1.5 ml centrifuge tube containing 300 µl of 1x passivelysis buffer (Promega). The dual luciferase activity of Photinus pyralis (Pp) and Renilla reniformis (Rr) in a homogenized solution was detected with the dual luciferase assay system (Promega) and a 20/20n luminometer (Promega) according to the manufacturers protocols (Promega). This measure assay per tube was replicated three times, and we calculated the mean ± S.D. of the results of five tubes per each promoter. With the exception of the ie1 and CMV promoters, which were derived from viruses, insect derived promoters were used for the construction of all promoter-reporter plasmids. The results of the activity assays for the eight promoters (act5C, BmA3, pUB, hr3-ie1, ie1, and CMV, and the heat shock promoters hsp70 and hsp82 with and without 1-h heat shock at 37°C) are presented in Fig. 1.
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(mean ± S.D.). The luciferase activity of the CMV promoter in the embryos was the second highest activity of the promoters tested. The CMV promoter had an activity ratio of 25.18 ± 2.87 (mean ± S.D.). The hsp70 and hsp82 promoters had activity ratios of 1.13 ± 0.10 (mean ± S.D.) and 1.41 ± 0.07 (mean ± S.D.), respectively; these increased slightly to 1.42 ± 0.06 (mean ± S.E.) and 2.27 ± 0.04 (mean ± S.D.), respectively, following heat induction. The activity of the pUB promoter within the embryos was higher 8.19 ± 0.73 (mean ± S.D.) than that of the act5C promoter 3.83 ± 0.56 (mean ± S.D.). Of the promoters tested, the highest level of firefly luciferase expression was obtained with the hr3-ie1 enhancer-promoter in the promoter activity assay. Similarly, a high level of firefly luciferase expression was obtained with the CMV promoter. The ie1 and the CMV promoters of the gene derived from viruses elicited a high level of firefly luciferase expression in the A. mellifera embryos. Kunieda and Kubo (2004) reported that the CMV promoter produced high gene expression in the brain of A. mellifera. These results reveal that the virus ie1 and CMV promoters are efficacious in the regulation of gene expression in A. mellifera. The transcriptional expression of two heat shock protein promoters in A. mellifera embryos increased with heat induction. The effect of heat shock induction on both hsp70 and hsp82 promoter activity was not, however, significantly different from that in the absence of heat induction. This result suggests that the up-regulation of heat shock protein promoter expression in A. mellifera may be caused by stress during embryo manipulation or plasmid induction by microinjection. In the honey bee embryos, the promoters derived from viruses produced a higher level of luciferase protein than that of promoters derived from insects. The pUB, act5C, BmA3, hsp70, and hsp82 promoters produced a relatively low level of expression, and both hsp70 and hsp82 slightly increased the gene expression levels during heat shock. Although these promoters had different levels of luciferase gene expression, all can potentially be used to induce foreign gene expression in A. mellifera embryos.
Acknowledgments We thank Dr. K. Kimura, Dr. Hatakeyama and Dr. M. Sumitani for their useful advice on the experiment of this study. We are also grateful to Prof. C.J. Coates, Prof. M.J. Fraser, Prof. D.A. O’Brochta, and Dr. M. Hatakeyama, who helped us in providing the plasmids. Fig 1. Firefly luciferase expression from various promoter plasmids in Apis mellifera embryos. The average of five replicates is reported and error bars represent 1 Standard Deviation. The (+) represents a heat shock treatment for that promoter. pSL is negative control of promoter less. The results are shown as the ratio of the promoter–firefly luciferase activity to the hsp82–Renilla luciferase activity. The highest 32.49 ± 1.55 (mean ± S.D.) and lowest 1.13 ± 0.10 (mean ± S.D.) luciferase activities were observed for the hr3-ie1 enhancer-promoter and the hsp70 promoter, respectively. The expression ratio observed with the ie1 promoter when induced by a lack of the hr3 enhancer; 2.52 ± 0.17 (mean ± S.D.), was similar to that observed with the BmA3 promoter; 2.22 ± 0.10
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