›› 2008, Vol. 51 ›› Issue (7): 681-688.

• 研究论文 • 上一篇    下一篇



  • 出版日期:2008-07-20 发布日期:2008-07-20
  • 通讯作者: 郭巍

Cloning, expression and sequence analysis of carboxylesterase gene from Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

ZHANG Xia   

  • Online:2008-07-20 Published:2008-07-20


羧酸酯酶是昆虫体内重要的解毒酶系之一,与昆虫抗药性产生相关。利用粉纹夜蛾Trichoplusia ni (Hübner)肠粘蛋白多克隆抗体免疫筛选甜菜夜蛾Spodoptera exigua (Hübner)中肠cDNA表达文库,得到编码羧酸酯酶的全长cDNA克隆。该cDNA克隆全长1 812 bp(GenBank登录号EF580101)开放阅读框1 605 bp,编码535个氨基酸。该蛋白活性中心包括3个氨基酸残基(催化三联体): Ser186 Glu319His443 3N-联糖基化位点,具备羧酸酯酶的结构特征,属羧酸酯酶家族(EC: 3.1.1.-)。将该基因与pQE30载体重组,经IPTG诱导,spot-blot鉴定,蛋白获得了表达;以α-醋酸萘酯为底物,检测表达的羧酸酯酶活性为1.3 nmol/100 μL酶液。

关键词: 甜菜夜蛾, cDNA表达文库, 羧酸酯酶, 表达, spot-blot鉴定

Abstract: Carboxylesterases are associated with insecticide-resistance and play an important role in the detoxification of many agrochemicals. Using the polyclonal antiserum against Trichoplusia ni (Hübner) invertebrate intestinal mucin (IIM), a carboxylesterase gene SeC1 was screened from the cDNA expression library of Spodoptera exigua (Hübner) midgut. The full-length of SeC1 is 1 812 bp(GenBank accession no. EF580101), and the open reading frame encodes 535 amino acids. The SeC1 possesses a catalytic triad, consisting of a Ser, a His and a Glu residue (Ser186, His443 and Glu319), which is characterized by carboxylic esters (EC: 3.1.1.-). The SeC1 gene has been recombined into pQE30 vector. The spot blot analysis demonstrated that SeC1 protein was expressed at a low level after being induced by IPTG. The activity for Sec1 was 1.3 nmol/100 μL enzyme buffer with the α-NA as substrate.

Key words: Spodoptera exigua, cDNA expression library, carboxylesterase, expression, spot-blot analysis