家蚕滞育关联山梨醇脱氢酶基因的启动子活性分析

Determination of the promoter activity of diapause-associated sorbitol dehydrogenase genes in the silkworm,Bombyx mori

【目的】明确家蚕Bombyx mori滞育关联基因山梨醇脱氢酶基因BmSDH(BmSDH-1,BmSDH-2a和BmSDH-2b)的转录特性。【方法】用5'RACE技术确定家蚕3个BmSDH基因的转录起始位点。利用PCR技术克隆3个BmSDH基因约1 kb及BmSDH-2a不同长度的启动子区序列,分别构建带有萤火虫荧光素酶报告基因的载体pGL3-BmSDH-P-luc,并与pRL-CMV报告质粒(含海肾荧光素酶报告基因)共转染家蚕BmN细胞,通过双荧光素酶检测系统检测BmSDH基因启动子活性;分别在BmN细胞培养基中添加昆虫保幼激素、蜕皮激素和滞育激素,通过双荧光素酶检测系统检测不同浓度激素处理对BmSDH-2a基因启动子活性的影响。【结果】BmSDH-1的转录起始位点为A(-41),BmSDH-2a的转录起始位点为C(-41),BmSDH-2b的转录起始位点为A(-40)(翻译起始位点为+1)。双荧光素酶检测结果表明,BmSDH-2a启动子活性极显著高于BmSDH-1和BmSDH-2b启动子,BmSDH-2a 355 bp长度片段的启动子活性极显著高于674 bp和1 117 bp长度片段。用不同浓度滞育激素处理BmN细胞后,BmSDH-2a的1 117 bp启动子活性随着滞育激素浓度的升高有上升的趋势,当浓度高于100 ng/mL时启动子活性有所降低但仍保持在较高水平;用保幼激素进行处理后,随着激素浓度的升高启动子活性逐渐降低;蜕皮激素处理后,0.1 ng/mL激素显著增强启动子活性,当激素浓度继续升高启动子活性逐渐降低。【结论】确定了BmSDH基因的转录起始位点。BmSDH-2a启动子活性显著高于BmSDH-1和BmSDH-2b启动子,一定浓度的蜕皮激素能显著提高BmSDH-2a启动子活性。研究结果有助于阐明BmSDH基因在家蚕滞育中的功能。

【Aim】To clarify the transcriptional characteristics of sorbitol dehydrogenase（ BmSDH） genes（ BmSDH-1,BmSDH-2 a and BmSDH-2 b） 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-2 a 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,wereconstructed. Using dual luciferase detection system,the promoter activity of BmSDH was detected by cotransfecting 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-2 a was detected by adding juvenile hormone analogue（ JHA）, ecdysone hormone（ 20 E） and diapause hormone（ DH）,respectively,to culture media in the gradient concentrations. 【Results】The transcription initiation sites of BmSDH-1,BmSDH-2 a and BmSDH-2 b are located at 41,41 and 40 bp upstream the translation initiation site,respectively. The promoter activity of BmSDH-2 a was significantly higher than those of BmSDH-1 and BmSDH-2 b. For the BmSDH-2 a 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 of1 117 bp promoter of BmSDH-2 a 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 20 E treated BmN cells,the promoter activity significantly increased when 0. 1 ng/mL of 20 E 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-2 a is significantly higher than those of BmSDH-1 and BmSDH-2 b. A certain concentration of 20 E can significantly enhance the promoter activity of BmSDH-2 a.These results will contribute to clarifying the function of BmSDH genes in diapause process in B. mori.