花生病程诱导蛋白基因AhPIP1和启动子的克隆、序列分析及原核表达  

Cloning, Sequence Analysis and Prokaryotic Expression of Pathogenesis-In- duced Protein (PIP) Gene from Peanut

作　　者：谢纯政[1,2] 李万福[1,2] 刘海燕[1] 李玲[2] 梁炫强[1] 

机构地区：[1]广东省农业科学院作物研究所,广州510640 [2]华南师范大学生命科学学院,广州510631

出　　处：《分子植物育种》2009年第1期177-183,共7页Molecular Plant Breeding

基　　金：国家自然科学基金项目(30571179);国家863计划(2006AA10Z156;2006AA10A115);广东省自然科学基金(07117967);广东省农科院作物所所长基金资助

摘　　要：本文采用RACE法克隆了花生病程诱导基因(pathogenesis-induced protein gene)全长,命名为AhPIP1,并在GenBank登录,登录号为EU860093。序列分析表明该基因的开放读码框579bp,编码193个氨基酸。以YY20基因组DNA为模板PCR扩增获得AhPIP1基因组序列长1883bp,包含3个外显子和长度分别为602bp和600bp的2个内含子。同源性比较发现AhPIP1与辣椒病程诱导蛋白CaPIP1(ABF72432,AAT35532)相似性达82%。采用Genome Walking方法获得AhPIP1启动子,序列分析表明该启动子序列长513bp,包含干旱胁迫、病程胁迫应答元件和生长素、赤霉素应答元件。构建pET32a-AhPIP1原核表达载体,转化表达受体菌E.coli BL21,原核表达获得大小约40kD AhPIP1融合蛋白,与推测的AhPIP1基因编码产物的大小一致,表明AhPIP1基因在大肠杆菌中能够表达。To characterize the function of the A hPIP1 （pathogenesis-induced protein gene from A rachis hypogaea L.）, we have isolated AhPIP1 （EU860093） gene by RACE and promoter region by GenomeWalking method from peanut and have been expressed the AhPIP1 in BL21 in this paper. As result, the AhPIP1 gene encodes a 193-amino-acid polypeptide, exhibiting high similarity with other members of PIP, and the open-reading flame is 579 bp. Genome amplification sequence is 1 883 bp, including three exons and two introns, length of 602 bp and 600 bp respectively. The AhPIP1 promoter sequence about 513 bp which were received by Genome Walking method contain DRE,MYB/MYC, GARE, SEBF core sequences or other abiotic-responsive cis-acting elements and the result indicate the A hPIP1 gene may play important role in peanut stress responses and growth and development, we obtained the recombination AhPIP1 protein, expressed in BL21 about 40 kD in SDS-PAGE gel. Cloning the gene and expressing the fusion protein has laid the foundation for the function study ofA hPIP1.

关 键 词：花生 AhPIP1 克隆 原核表达 

分 类 号：S435.652[农业科学—农业昆虫与害虫防治]