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

【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973 bp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23.567 ku，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 DaGSTe1 gene and real time PCR was utilized to examine the DaGSTe1 expression patterns at different developmental stages.【Result】Glutathione S-transferase gene with cDNA of 973 bp was isolated from D.armandi and named DaGSTe1 with GenBank accession number of KJ637332.It encoded a protein of 218 amino acid residues with predicted molecular weight of 23.567 ku and isoelectric point of 7.90.DaGSTe1 shared 94% identity with DpGSTe1.The protein homology demonstrated that the predicted protein structure of DaGSTe1 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 DaGSTe1 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】DaGSTe1 was successfully cloned and was predicted to play a dual role in detoxification of host allele chemicals and transport of male-specific hormones