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

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-D ü r)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-hNS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1 30个氨基酸残基,N端有一段长1 7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.%[Objective] The objective of this study is to clone a chemosensory protein gene AlucCSPI from Apolygus lucorum, and to test it's tissue-specific expression and binding characteristics. [Method] The AlucCSPI was cloned from A. lucorum by RT-PCR. Using semi-quantitative RT-PCR and Real-time PCR methods, the expression pattern of AlucCSPI in different adult tissues were determined. AlucCSPI was expressed using pGEX-4T-l vector and BL21 (DE3) prokaryotic expression system, then purified using GSTrap FF. The binding characteristics of AlucCSPI 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 AlucCSPI (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 AlucCSPI was characterized by four conservative Cys. It was found that AlucCSPI was dominantly expressed in the antenna of the female. The molecular weight of recombinant protein of pGEX /AlucCSPl was about 39 kD. After purification with affinity chromatography and treatment with thrombin protease to cut off GST, the AlucCSP1 protein was obtained. AlucCSPl had high affinity with gossypol, tannin, quercetin and rutin hydrate, the binding constant was 13.4, 32.7, 19.7 and 38.5 umol·L-1, respectively . [Conclusion] AlucCSPl 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 AlucCSPl.