中国农业科学 ›› 2010, Vol. 43 ›› Issue (4): 873-880 .doi: 10.3864/j.issn.0578-1752.2010.04.028

• 研究简报 • 上一篇    

家蚕谷光甘肽-S-转移酶GSTe3基因的鉴定及其真核表达

潘敏慧,许湲,余泉友,刘佳,刘迪,赵丹红,鲁成

  

  1. (西南大学生物技术学院/农业部蚕桑学重点开放实验室)
  • 收稿日期:2009-05-25 修回日期:2009-06-19 出版日期:2010-02-20 发布日期:2010-02-20
  • 通讯作者: 鲁成

Identification and Eukaryotic Expression of GSTe-3 Gene of Silkworm (Bombyx mori)

PAN Min-hui, XU Yuan, YU Quan-you, LIU Jia, LIU Di, ZHAO Dan-hong, LU Cheng
  

  1. (西南大学生物技术学院/农业部蚕桑学重点开放实验室)
  • Received:2009-05-25 Revised:2009-06-19 Online:2010-02-20 Published:2010-02-20
  • Contact: LU Cheng

摘要:

【目的】谷胱甘肽-S-转移酶(Glutathiones S-tansferases,GSTs)是一类由多基因编码的多功能同工酶超家族,在解毒内源和外源性有毒物质中起着重要作用,家蚕GSTs的研究,可为解析家蚕解毒机制奠定基础。【方法】利用生物信息学和分子生物学方法克隆和分析家蚕GSTe3(BmGSTe3),利用RT-PCR分析该基因在家蚕体内的表达情况,利用重组杆状病毒真核表达系统在sf9细胞中真核表达BmGSTe3。【结果】在家蚕中克隆了家蚕的GSTe3,该基因由5个外显子与4个内含子组成,外显子总长度为512 bp,属于昆虫特异的Epsilon家族。BmGSTe3包含N-末端和C-末端2个结构域,N-末端由b-a-b-a-b-b-a共7个结构基序组成,而C-末端则由5个?螺旋构成,在启动子上游2 500 bp区域内共发现了15个可能的转录调控元件。BmGSTe3的表达具有较高的组织特异性,它只在家蚕血液和头部表达。BmGSTe3在sf9细胞中表达的BmGSTe3蛋白具有较好的GSTs酶活性。【结论】BmGSTe3属于昆虫特异的Epsilon家族,其蛋白具有较好的GSTs酶活性。

关键词: 家蚕, BmGSTe3, 真核表达, GSTs酶活

Abstract:

【Objective】 Glutathione S-transferases (GSTs) are a superfamily of multifunctional enzymes found in almost all living organisms. They play important roles in detoxification of endogenous and exogenous toxic substance. It will lay a foundation for the detoxification mechanism of silkworm to study on GSTs of Bombyx mori. 【Method】 BmGSTe3 gene was cloned and analyzed by bioinformation and molecular biology method. The expression of BmGSTe3 was analyzed in silkworm by RT-PCR. BmGSTe3 gene was eukaryotic expressed in sf9 cells by Bac-to-Bac Baculovirus Expression System. 【Result】 The BmGSTe3 is composed of 5 exons and 4 introns and its CDS length is 512 bp. BmGSTe3 contains 15 possible transcription regulatory elements in the upstream of 2 500 bp and two domains, which N-terminal domain is composed of 7 motifs, namely b-a-b-a-b-b-a, while C-terminal domain is composed of 5 ? helixs. BmGSTe3 has a conservative Ser catalyse site, belongs to Epsilon family members and express only in hemolymph and brain. BmGSTe3 protein has good GSTs enzyme activity. 【Conclusion】 BmGSTe3 belongs to Epsilon family members and its protein has good GSTs enzyme activity

Key words: Bombyx mori, BmGSTe3, eukaryotic express, GSTs enzyme activity