斜纹夜蛾蜕皮响应基因E75D的克隆、 原核表达分析及microRNA作用位点预测

›› 2012, Vol. 55 ›› Issue (5): 510-519.doi:

斜纹夜蛾蜕皮响应基因E75D的克隆、原核表达分析及microRNA作用位点预测

高璐, 左洪亮, 姜春来, 刘海远, 钟国华

华南农业大学昆虫毒理研究室，广州 510642

收稿日期:2011-12-16 修回日期:2012-04-25 出版日期:2012-05-20 发布日期:2012-05-20
通讯作者: 钟国华 E-mail:guohuazhong@scau.edu.cn
作者简介:高璐，女， 1986年生，河南安阳人，硕士，研究方向为昆虫分子生物学， E-mail: gaolu0424@163.com
基金资助:
国家自然科学基金项目(30971944)

Molecular characterization, prokaryotic expression analysis and miRNA binding site prediction of an ecdysone inducible gene E75D from Spodoptera litura (Lepidoptera: Noctuidae)

GAO Lu, ZUO Hong-Liang, JIANG Chun-Lai, LIU Hai-Yuan, ZHONG Guo-Hua

Laboratory of Insect Toxicology, South China Agricultural University, Guangzhou 510642, China

Received:2011-12-16 Revised:2012-04-25 Online:2012-05-20 Published:2012-05-20
Contact: ZHONG Guo-Hua E-mail:guohuazhong@scau.edu.cn
About author:gaolu0424@163.com

摘要/Abstract

摘要： E75是昆虫蜕皮级联反应中的早期转录因子之一。本研究运用RT-PCR和RACE技术，首次获得了斜纹夜蛾Spodoptera litura E75D基因，命名为Sli-E75D (GenBank 登录号： JQ266225)，其开放阅读框全长1 836 bp，编码611个氨基酸残基， 3′非翻译区全长为358 bp，同时获得其5′非翻译区共341 bp。经核苷酸序列比对分析， E75在鳞翅目昆虫间保守性较高，尤其3′非翻译区具有高保守性，但由于启动子不同， E75异构体mRNA 5′端序列存在差异；经氨基酸序列比对， Sli-E75D与棉贪夜蛾Spodoptera littoralis、烟草天蛾Manduca sexta、家蚕Bombyx mori E75D的一致性分别为98.4%， 79.3%和76.5%。基于E75 3′非翻译区的高保守性，利用PITA和RNAhybird程序预测了最有可能调控鳞翅目昆虫E75基因的3种miRNAs： miR-14， miR-33和miR-87。构建pET28a-Sli-E75D表达载体，分别转化BL21(DE3)和Transetta(DE3)菌株，检测大肠杆菌Escherichia coli稀有密码子对E75D原核表达的影响， SDS-PAGE结果显示，转化Transetta(DE3)菌株的pET28a-Sli-E75D可高效表达大小约74.79 kD（含预测的67.19 kD Sli-E75D， 7.6 kD T7·Tag 和His·Tag）的重组蛋白，与其理论分子质量基本吻合，而转化BL21(DE3)菌株的pET28a-Sli-E75D质粒只见微量重组蛋白表达。由于Transetta(DE3)菌株可补充大肠杆菌6种稀有密码子的tRNA，较BL21(DE3)更适合于E75D的外源表达。qPCR检测了斜纹夜蛾从末龄幼虫到成虫发育过程中各时间点Sli-E75的相对表达水平： Sli-E75在6龄幼虫期的表达量较低，从预蛹开始，表达量急剧升高，并在蛹中期达到最高峰，之后迅速下降，但成虫期表达水平又出现回升。这些结果有助于深入研究E75在昆虫蜕皮级联反应中的作用。

关键词: 斜纹夜蛾, E75D, 克隆, 原核表达, microRNA

Abstract: E75D is one of the important early transcription factors in the molting process of insects. The cDNA of E75D was cloned from Spodoptera litura by RT-PCR and RACE technology for the first time in this experiment and named Sli-E75D (GenBank accession no. JQ266225). Sli-E75D consists of a 1 836 bp open reading frame encoding 611 amino acids, with a 341 bp 5′ untranslated regions (UTR) and a 358 bp 3′UTR. E75, especially its 3′UTR, is highly conserved among the Lepidoptera insects; however, it shows significant difference in 5′UTR among four isoforms because of the difference of their promoter. The deduced amino acid sequence of E75D in S. litura share 98.4%, 79.3% and 76.5% identity with the homologues in Spodoptera littoralis, Manduca sexta and Bombyx mori, respectively. The miRNAs miR-14, miR-33 and miR-87, which are the most possible regulator of E75, were predicted by PITA and RNAhybird programs based on the highly conserved 3′UTR of E75. The recombinant vector pET28a-Sli-E75D was constructed and transformed into Escherichia coli BL21(DE3) and Transetta(DE3), respectively, to study the effects of rare codons on E75D expression. SDS-PAGE analysis of prokaryotic protein showed that the Transetta(DE3) which transformed pET28a-Sli-E75D recombinant vector expressed much more prokaryotic recombinant E75D protein (consisting of 67.19 kD Sli-E75D, and 7.6 kD T7·Tag and His·Tag) than BL21(DE3). The result of SDS-PAGE analysis demonstrated that Transetta(DE3) is better for the prokaryotic expression of E75D because it supplies tRNAs corresponding to six rare codons in E. coli. The expression levels of Sli-E75 in developmental stages from the last instar larva to adult were detected by qRT-PCR. The results of qRT-PCR revealed that Sli-E75 was expressed at a low level in 6th instar larva, and its expression increased rapidly from prepupa and reached the peak in the middle pupal stage. Then, the expression level of Sli-E75 decreased quickly in the last stage of pupa and rebounded again in the adult. These results can contribute to the in-depth study of E75 in molting process in insects.

Key words: Spodoptera litura, E75D, cloning, prokaryotic expression, microRNA

高璐, 左洪亮, 姜春来, 刘海远, 钟国华. 斜纹夜蛾蜕皮响应基因E75D的克隆、原核表达分析及microRNA作用位点预测[J]. , 2012, 55(5): 510-519.

GAO Lu, ZUO Hong-Liang, JIANG Chun-Lai, LIU Hai-Yuan, ZHONG Guo-Hua. Molecular characterization, prokaryotic expression analysis and miRNA binding site prediction of an ecdysone inducible gene E75D from Spodoptera litura (Lepidoptera: Noctuidae)[J]. , 2012, 55(5): 510-519.

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