一个麻疯树RING型锌指蛋白基因JcRFP1的克隆及表达

严安心, 谢瑜, 吉柔风, 李诗娟, 徐莺, 陈放*
四川大学生命科学学院, 成都610065

通信作者:陈放;E-mail: chenfangbio@gmail.com;Tel: 028-85417281

摘 要:

首次从麻疯树胚乳cDNA文库中克隆得到一个RING型锌指蛋白基因(GenBank登录号为JF920726), 命名为JcRFP1。该 cDNA长度为728 bp, 包含编码JcRFP1蛋白的完整开放阅读框(516 bp)。JcRFP1基因在麻疯树各器官中均检测到表达且表达 量依次为: 叶>茎>花>果实>种子>根。将克隆到的JcRFP1基因的cDNA序列连接到表达载体pET32a (+)上, 导入BL21 (DE3) pLysS菌株, 成功诱导表达相对分子质量为33.2 kDa的可溶性融合蛋白。该融合蛋白免疫新西兰大白兔, 得到效价为1: 6 500 的抗血清。研究表明, JcRFP1蛋白具有体外泛素连接酶E3活性, 在麻疯树体内可能参与油菜素甾醇信号转导途径。

关键词:麻疯树; RING指蛋白; 原核表达; 泛素连接酶; 油菜素甾醇

收稿:2012-01-09   修定:2012-03-26

资助:科技部“十二五”科技支撑课题(2011BAD22B08)和科技部 国际合作重点项目(2006DFB63400)

Cloning and Expression of a RING Finger Protein Gene JcRFP1 from Jatropha curcas L.

YAN An-Xin, XIE Yu, JI Rou-Feng, LI Shi-Juan, XU Ying, CHEN Fang*
School of Life Sciences, Sichuan University, Chengdu 610065, China

Corresponding author: CHEN Fang; E-mail: chenfangbio@gmail.com; Tel: 028-85417281

Abstract:

A novel RING finger protein gene was cloned from Jatropha curcas endosperm cDNA library (GenBank Accession No. JF920726), named JcRFP1. The cDNA length was 728 bp including the complete coding sequence (516 bp) of JcRFP1. JcRFP1 gene expression could be detected in all organs of J. curcas, and the expression levels are different as follows leaf>stem>flower>fruit>seed>root. JcRFP1 cDNA was ligated into the expression plasmid pET-32a (+) and a soluble fusion protein with the molecular weight of 33.2 kDa was expressed successfully in E. coli strain BL21 (DE3) pLysS including the recombinant plasmid pET32a (+)-JcRFP1ΔN. Thereafter, the fusion protein was used to immunize the New Zealand white rabbits and the polyclonal antibody was obtained, which could be diluted into 1: 6 500 during western blot analysis. The study showed JcRFP1 had in vitro ubiquitin ligase (E3) activity and might participate in brassinosteroid signal transduction pathways in J.curcas.

Key words: Jatropha curcas L.; RING finger protein; prokaryotic expression; ubiquitin ligase; brassinosteroid

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