中国农业科学 ›› 2012, Vol. 45 ›› Issue (20): 4187-4196.doi: 10.3864/j.issn.0578-1752.2012.20.008

• 植物保护 • 上一篇    下一篇

绿盲蝽化学感受蛋白基因AlucCSP1的克隆、表达谱分析及结合特征

 滑金锋, 张帅, 崔金杰, 王道杰, 王春义, 雒珺瑜, 吕丽敏   

  1. 1.中国农业科学院棉花研究所棉花生物学国家重点实验室,河南安阳455000
    2.河南大学植物逆境重点实验室,河南开封475000
  • 收稿日期:2011-10-08 出版日期:2012-10-15 发布日期:2012-02-21
  • 通讯作者: 通信作者崔金杰,Tel:0372-2562296;E-mail:cuijinjie@126.com
  • 作者简介:滑金锋,E-mail:heda321@163.com
  • 基金资助:

    农业部公益性行业科研专项(201103012)、中央级公益性科研院所基本科研业务费专项(2012ZL038)

Cloning, Expression Analysis and Binding Characteristics of Chemosensory Protein Gene AlucCSP1 in Apolygus lucorum (Meyer-Dür)

 HUA  Jin-Feng, ZHANG  Shuai, CUI  Jin-Jie, WANG  Dao-Jie, WANG  Chun-Yi, LUO  Jun-Yu, LU Li-Min   

  1. 1.中国农业科学院棉花研究所棉花生物学国家重点实验室,河南安阳455000
    2.河南大学植物逆境重点实验室,河南开封475000
  • Received:2011-10-08 Online:2012-10-15 Published:2012-02-21

摘要: 【目的】克隆、组织特异性表达以及荧光结合特征分析绿盲蝽 (Apolygus lucorum Meyer-Dür)化学感受蛋白(chemosensory protein,CSP)。【方法】设计特异性引物,用RT-PCR技术克隆绿盲蝽化学感受蛋白基因AlucCSP1,使用半定量RT-PCR和Real-time PCR技术对AlucCSP1组织特异性表达进行研究,在BL2(DE3)工程菌株中用pGEX-4T-1载体进行原核表达,并在GSTrap FF柱中纯化和去标签蛋白。使用bis-ANS作为荧光配基,研究AlucCSP1蛋白与58种气味标样和5种棉花次生代谢物质的结合特征。【结果】得到了绿盲蝽化学感受蛋白基因,命名为AlucCSP1(GenBank登录号:JN573217)。该序列阅读框全长393 bp,编码130个氨基酸残基,N端有一段长17个aa的信号肽,具有4个保守的半胱氨酸残基。绿盲蝽AlucCSP1几乎全部在雌虫触角中表达。构建pGEX/AlucCSP1原核表达载体,在BL21(DE3)工程菌株中表达出分子量约为39 kD的融合蛋白,用亲和层析法纯化、经凝血酶作用去除标签蛋白获得纯化的AlucCSP1蛋白,该蛋白与棉酚、单宁、槲皮素、芦丁水合物亲和力较强,结合常数分别为13.4、32.7、19.7、38.5 μmol•L-1。【结论】克隆得到绿盲蝽化学感受蛋白基因AlucCSP1,在雌性触角内特异表达,并进一步得到约13 kD的AlucCSP1纯蛋白。为研究AlucCSP1在绿盲蝽化学感器中的作用奠定了基础。

关键词: 绿盲蝽, 化学感受蛋白, 表达谱, 原核表达, 纯化, 竞争结合

Abstract: 【Objective】The objective of this study is to clone a chemosensory protein gene AlucCSP1 from Apolygus lucorum, and to test it’s tissue-specific expression and binding characteristics.【Method】The AlucCSP1 was cloned from A. lucorum by RT-PCR. Using semi-quantitative RT-PCR and Real-time PCR methods, the expression pattern of AlucCSP1 in different adult tissues were determined. AlucCSP1 was expressed using pGEX-4T-1 vector and BL21 (DE3) prokaryotic expression system, then purified using GSTrap FF. The binding characteristics of AlucCSP1 with fifty-eight standard odorant samples and five cotton secondary metabolites were investigated using bis-ANS as fluorescence ligand.【Result】The open reading frame full length of AlucCSP1 (GenBank accession: JN573217) was 393 bp, encoding 130 amino acids, including 17 aa of signal peptide in the N-terminal. Sequencing and analysis indicated that AlucCSP1 was characterized by four conservative Cys. It was found that AlucCSP1 was dominantly expressed in the antenna of the female. The molecular weight of recombinant protein of pGEX /AlucCSP1 was about 39 kD. After purification with affinity chromatography and treatment with thrombin protease to cut off GST, the AlucCSP1 protein was obtained. AlucCSP1 had high affinity with gossypol, tannin, quercetin and rutin hydrate, the binding constant was 13.4, 32.7, 19.7 and 38.5 μmol•L-1, respectively .【Conclusion】AlucCSP1 was cloned from A. lucorum and it was dominantly expressed in the female antenna. This may be valuable for studying the role in A. lucorum chemical sensors of AlucCSP1.

Key words: Apolygus lucorum, chemosensory protein, expression profiles, prokaryotic expression, purification, competitive binding