烟草HrBP的研究与应用  

Study and Application of HrBP from Tobacco

作　　者：陈卓[1] 于丹丹[1] 郭勤[1] 刘家驹[1] 王贞超[1] 李向阳[1] 毕亮[1] 胡德禹[1] 杨松[1] 宋宝安[1] 

机构地区：[1]贵州大学绿色农药与农业生物工程国家重点实验室培育基地,贵阳550025

出　　处：《生物技术通报》2012年第9期59-68,共10页Biotechnology Bulletin

基　　金：国家重点基础研究发展计划项目("973"计划)(2010CB-126105);贵州省教育厅自然科学研究项目重点项目[黔科教(2009)0132号];贵州省优秀科技教育人才省长专项资金项目[黔省专合字(2009)104号];贵州省科技成果重点推广计划项目[黔科合成字(2010)5034];贵州省科技厅农业攻关项目[黔科合NY字(2011)3052号]

摘　　要：旨在进一步发掘HrBP在新农药创制和抗病品种选育等方面的价值,采用BLAST和Clustal X2软件对HrBP进行序列同源性分析,发现其序列较保守;采用PSIPRED Protein Structure Prediction在线服务器进行烟草HrBP二级结构预测,采用UCLDepartment of Computer Science进行HrBP跨膜结构预测,发现两种可能存在的跨膜方式;采用SWISS-MODEL、Phyre和CPH进行HrBP空间结构预测,发现以膜蛋白为模板进行模建的蛋白具有相似的空间结构;建立基于Real-time PCR的HrBP mRNA定量分析方法,其相关系数为0.999 6;通过ProtScale方法分析HrBP抗原指数,并获得序列特异性较高的多肽,采用9-氟甲氧羰基固相合成法制备多肽,并由此制备多克隆抗体,采用Indirect-ELISA和Western blotting测定其效价和特异性,表明多克隆抗体可特异识别烟草HrBP。采用DIBA和Western blotting试验研究多克隆抗体对常见植物的抗原识别特性,发现多克隆抗体对这些植物的HrBP均有一定识别能力。此外,采用Real-time PCR和Indirect-ELISA等方法进行烟草各组织部位HrBP的表达量的研究,发现HrBP在各组织部位存在差异表达。To further explore the potential value of HrBP in new pesticide creation and disease resistance breeding, the sequences homology was firstly analyzed by BLAST and by Clustal X2 software, and the results indicated that the sequence of HrBP was much conserved, then tobacco HrBP secondary structure was predicted by the server of PSIPRED Protein Structure, the transmembrane structure of HrBP was also analyzed by UCL Department of Computer Science with obtaining to two kinds of feasible transmembrane mode ; SWISS-MODEL, Phyre and CPH were used to predict HrBP spatial structure with obtaining the similar results about HrBP spatial structure at the condition of membrane proteins as a modeling template ; a method of quantitative analysis HrBP mRNA was established by Real-time PCR, and the results indicated that the correlation coefficient of Real-time PCR was reached at 0.999 6 ; Polypeptides with sequence-specific were obtained by ProtScale software, the polypeptide was synthetized by fmoc solid phase synthesis methods for preparation polyclonal antibody, and its titer and specificity were determined by indirect-ELISA and Western blotting. The results shown that polyclonal antibody can detect the specific band in tobacco leaves. The recognition ability of this polyclonal antibody was evaluated against maize, rice, cabbage, radish and Chenopodium amaranticolor by DIBA and Western blotting, and the results showed that the polyclonal antibody possessed recognition ability to these plants to some extent. In addtion, Real-time PCR and Indirect-ELISA method were used to evaluate the differentially expression of HrBP in various tissues in tobacco.

关 键 词：Harpin结合蛋白 生物信息学分析 基因克隆表达 多克隆抗体制备 烟草组织差异表达 

分 类 号：S572[农业科学—烟草工业]