华山松大小蠹谷胱甘肽S-转移酶基因DaGSTe1的克隆与表达  

Cloning and expression of glutathione S-transferase gene DaGSTe1 of Dendroctonus armandi

作　　者：马俊宁[1] 代鲁鲁 张然然[1] 陈辉[1] 

机构地区：[1]西北农林科技大学林学院,陕西杨凌712100

出　　处：《西北农林科技大学学报（自然科学版）》2015年第8期116-122,132,共8页Journal of Northwest A&F University(Natural Science Edition)

基　　金：国家林业公益性行业科研专项(201004077);国家自然科学基金项目(31170607);教育部长江学者和创新团队发展计划资助项目(IRT1035)

摘　　要：【目的】克隆华山松大小蠹谷胱甘肽S-转移酶基因的全长序列,并对其序列特征及不同发育时期的表达规律进行研究,以揭示谷胱甘肽S-转移酶在华山松大小蠹克服寄主抗性及物质转运过程中的分子调控机制。【方法】采用RT-PCR和RACE克隆华山松大小蠹谷胱甘肽S-转酶基因全长cDNA序列,用实时荧光定量PCR检测该基因在华山松大小蠹幼虫、蛹和雌雄成虫中的表达情况。【结果】获得cDNA全长为973bp的华山松大小蠹谷胱甘肽S-转移酶基因,并命名为DaGSTe1(GenBank登录号:KJ637332),其编码一个由218个氨基酸组成的多肽,分子质量约为23.567ku,理论等电点为7.90。华山松大小蠹DaGSTe1与山松大小蠹DpGSTe1氨基酸序列的相似度最高,达94%。根据对系统发育树的分析推测,DaGSTe1属于Epsilon类谷胱甘肽S-转移酶。DaGSTe1蛋白三维结构包括一个N端结构域和一个C端结构域,其中N端结构域包括典型的4个β片层和3个α螺旋(β1α1β2α2β3β4α3),C端结构域包括5个α螺旋(α4α5α6α7α8)。DaGSTe1基因在华山松大小蠹不同发育期均有表达,其中在雄性成虫中的表达量最大,为幼虫和蛹的8倍,雌性成虫的4倍。【结论】克隆得到了华山松大小蠹谷胱甘肽S-转移酶家族基因DaGSTe1,推测该基因具有降解寄主毒素的作用,而且参与华山松大小蠹雄性特异性激素的转运过程。[Objective] The research was conducted to clone glutathione S-transferase （GST） gene in Dendroctonus armandi and analyzed its sequence features and expression at four developmental stages for demonstrating the molecular mechanism of D. armandi GST in resisting phytotoxins and transporting hormones. [Method] RT-PCR and RACE were employed to clone DaGSTel gene and real-time PCR was utilized to examine the DaGSTel expression patterns at different developmental stages. [Result] Glutathione S-transferase gene with cDNA of 973 bp was isolated from D. armandi and named DaGSTel with GenBank accession number of KJ637332. It encoded a protein of 218 amino acid residues with predicted molecular weight of 23. 567 ku and isoeleetric point of 7.90. DaGSTel shared 94% identity with DpGSTel. The protein homology demonstrated that the predicted protein structure of DaGSTel was similar to GST Epsilon members,with an N domain （β1α1β2α2β3β4α3） and a C domain （α4α5α6α7α8）. Gene expression patterns at different developmental stages illustrated that DαGsSTel was expressed at all developmental stages, but was only expressed at high quantity in male adult, which was eight times of that at larvae and pupae stages and four times of that in female adult. [Conclusion] DaGSTel was successfully cloned and was predicted to play a dual role in detoxification of host allele chemicals and transport of male-specific hormones.

关 键 词：华山松大小蠹 谷胱甘肽S-转移酶 基因克隆 发育阶段 

分 类 号：Q78[生物学—分子生物学] S763.38[农业科学—森林保护学]