苜蓿夜蛾保幼激素酯酶cDNA的克隆、序列分析及表达研究
cDNA cloning, sequence analysis and expression of JHE gene in Heliothis viriplaca
张 欢** 郭博智 樊 东***
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DOI：10.7679/j.issn.2095-1353.2015.015
作者单位：东北农业大学农学院，哈尔滨 150030
中文关键词：苜蓿夜蛾，保幼激素酯酶，克隆，序列分析，表达
英文关键词： Heliothis viriplaca, Juvenile hormone esterase, cloning, sequence analysis, expression
中文摘要：
【目的】 研究苜蓿夜蛾Heliothis viriplaca保幼激素酯酶（Juvenile
hormone esterase，JHE）的功能和作用机理。【方法】 本试验提取苜蓿夜蛾的总RNA，并利用RT-PCR和RACE技术，扩增得到苜蓿夜蛾保幼激素酯酶基因的全长cDNA序列，命名为Hvjhe（GenBank登录号：JQ901384）。【结果】 该基因含有3 106 bp，包括一个1 746 bp的开放阅读框，编码一个含581个氨基酸的多肽，分子量约为63.9 ku，多肽的等电点为5.11，且氨基酸序列具含有5个保幼激素酯酶特有的氨基酸保守模块。推导的氨基酸序列与烟芽夜蛾Heliothis virescens和棉铃虫Helicoverpa armigera中获得的保幼激素酯酶相似性较高，分别达到83%和82%。荧光定量PCR结果表明，该基因在苜蓿夜蛾不同发育时期和不同组织中都有mRNA水平的特异性表达，且在预蛹期表达量相对较高，取食期、蛹期期和成虫期表达量相对较低；在中肠和脂肪体内的表达量相对于其它组织较高。该基因在大肠杆菌Escherich coli表达系统中进行了诱导表达，经SDS-PAGE和Western blot检测结果表明，表达出与预测的蛋白分子量相符的融合蛋白。【结论】 研究结果表明本试验获得了苜蓿夜蛾中一个新的保幼激素酯酶基因的cDNA序列。
英文摘要：[Objectives] To investigate Juvenile hormone esterase (JHE) cDNA sequence and function in Heliothis viriplaca. [Methods] Total RNA was isolated from H. viriplaca and the JHE cDNA sequence was cloned by RT-PCR and rapid amplification of cDNA ends(RACE). A full-length cDNA sequence encoding JHE was obtained and designated as Hvjhe (GenBank accession number: JQ901384. [Results] The cDNA sequence, 3 106 base pairs in length, contained an open reading frame of 1 746 base pairs coding for a polypeptide of 581 amino acid residues with a predicted molecular weight of 63.9 ku and pI 5.11. The Hvjhe sequence contained five specific conserved motifs identified in JHEs of other insect species. The deduced amino acid sequence of Hvjhe shared 83% and 82% identity with homologues in H. virescens and Helicoverpa armigera, respectively. Transcript analysis on the Hvjhe sequence during various developmental stages and in different tissues was determined by qRT-PCR. The results of qRT-PCR revealed that Hvjhe mRNA was expressed at a low level in the feeding, pupal and adult stages, but at a high level in the prepupal stage. The Hvjhe transcript was more highly expressed in the fat body and midgut than in other tissues. SDS-PAGE and Western-blot results revealed that Hvjhe was expressed in E. coli. The molecular weight of expressed protein was consistent with the predicted molecular weight of Hvjhe. [Conclusion] The results indicate that a novel juvenile hormone esterase cDNA sequence was successfully obtained.