花生病程诱导蛋白基因AhPIP1与启动子的克隆序列分析及原核表达.pdfVIP

分子植物育种，2009 年，第7 卷，第1 期，第177-183 页 Molecular Plant Breeding, 2009, Vol.7, No.1, 177-183 新基因、新种质、新品种 New Gene Germplasm 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 1,2 1,2 1 2 1* 谢纯政 李万福 刘海燕 李玲 梁炫强 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 * 通讯作者, Liang804@ 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579 bp，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 1 883 bp，包含3 个外显子和长度分别为 602 bp 和 600 bp 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 513 bp，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40 kD 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。 关键词 花生, AhPIP1 , 克隆, 原核表达 Cloning, Sequence Analysis and Prokaryotic Expression of Pathogenesis-In- duced Protein (PIP) Gene from Peanut Xie Chunzheng 1,2 Li Wanfu 1,2 Liu Haiyan 1 Li Ling 2 Liang Xuanqiang 1* 1 Crops Research Institute, Guangdong Academy of Agriculture Sciences, Guangzhou, 510640; 2 College of Life Science, South China Normal Univer- sity, Guangzhou, 510631 * Corresponding author, Liang804@ Abstract To characterize the function of the AhPIP1 (pathogenesis-induced protein gene from Arachis 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 elemen