(1. Jiangsu Key Laboratory of Sericutural Biology and Biotechnology, College of Biotechnology, Jiangsu University of Science and Technology, Zhenjiang, Jiangsu 212018, China; 2. Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Sericultural Research Institute, Chinese Academy of Agricultural Sciences, Zhenjiang, Jiangsu 212018, China)
【Aim】 To clarify the transcriptional characteristics of sorbitol dehydrogenase (BmSDH) genes (BmSDH-1, BmSDH-2a and BmSDH-2b) in the silkworm, Bombyx mori. 【Methods】 The transcription initiation sites of three BmSDH genes were determined by 5′RACE technique. The promoters of BmSDH about 1 kb in length and BmSDH-2a in different lengths were cloned by PCR. Then report plasmids with a fruit fly luciferase gene driven by BmSDH promoters in different lengths, pGL3-BmSDH-P-luc, were constructed. Using dual luciferase detection system, the promoter activity of BmSDH was detected by co-transfecting the BmN cells with pGL3-BmSDH-P-luc and pRL-CMV which contains a renin-luciferase reporter gene, and the effects of hormones on the promoter activity of BmSDH-2a was detected by adding juvenile hormone analogue (JHA), ecdysone hormone (20E) and diapause hormone (DH), respectively, to culture media in the gradient concentrations. 【Results】 The transcription initiation sites of BmSDH-1, BmSDH-2a and BmSDH-2b are located at 41, 41 and 40 bp upstream the translation initiation site, respectively. The promoter activity of BmSDH-2a was significantly higher than those of BmSDH-1 and BmSDH-2b. For the BmSDH-2a gene, the promoter activity of the 355 bp fragment was significantly higher than those of the 674 bp and 1 117 bp fragments. In BmN cells, the activity of 1 117 bp promoter of BmSDH-2a increased with the increase of DH concentration; however, when the DH concentration was higher than 100 ng/mL, the promoter activity was decreased to some degree but maintained at a high level. In JHA treated BmN cells, the promoter activity was decreased gradually as the hormone concentration increased. In 20E treated BmN cells, the promoter activity significantly increased when 0.1 ng/mL of 20E was applied, and then decreased gradually with the increase of hormone concentration. 【Conclusion】 The transcription initiation sites of the BmSDH genes were determined. The promoter activity of BmSDH-2a is significantly higher than those of BmSDH-1 and BmSDH-2b. A certain concentration of 20E can significantly enhance the promoter activity of BmSDH-2a. These results will contribute to clarifying the function of BmSDH genes in diapause process in B. mori.
ZHU Juan,XIE Yu-Chen,CHEN Yan-Rong et al. Determination of the promoter activity of diapause-associated sorbitol dehydrogenase genes in the silkworm, Bombyx mori
[J]. ACTA ENTOMOLOGICA SINICA, 2018, 61(4): 391-397.