›› 2018, Vol. 61 ›› Issue (4): 391-397.doi: 10.16380/j.kcxb.2018.04.001

• 研究论文 •    下一篇

家蚕滞育关联山梨醇脱氢酶基因的启动子活性分析

朱娟1, 2, 谢雨辰1, 2, 陈艳荣1, 2, 唐顺明1, 2, 易咏竹1, 2, 沈兴家1, 2,*   

  1. (1. 江苏科技大学生物技术学院, 江苏省蚕桑生物学与生物技术重点实验室, 江苏镇江 212018; 2. 中国农业科学院蚕业研究所, 农业部蚕桑遗传改良重点实验室, 江苏镇江 212018)
  • 出版日期:2018-04-20 发布日期:2018-04-20

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

ZHU Juan1,2, XIE Yu-Chen1,2, CHEN Yan-Rong1,2, TANG Shun-Ming1,2, YI Yong-Zhu1,2, SHEN Xing-Jia1,2,*   

  1.   (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)
  • Online:2018-04-20 Published:2018-04-20

摘要: 【目的】明确家蚕Bombyx mori滞育关联基因山梨醇脱氢酶基因BmSDHBmSDH-1, BmSDH-2aBmSDH-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。

关键词: 家蚕, 启动子, 山梨醇脱氢酶, 滞育, 转录起始位点, 表达调控

Abstract:
【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.

Key words: Bombyx mori; promoter, sorbitol hydrogenase, diapause, transcription initiation site, expression regulation