香蕉乙烯受体基因RNA干扰表达载体的构建  

Construction of expression vector of ethylene receptor genefrom banana for RNA interfering

作　　者：冯斗 张春发[1] 张颖[1] 

机构地区：[1]中国热带农业科学院热带作物生物技术国家重点实验室

出　　处：《广西农业生物科学》2004年第2期149-153,共5页Journal of Guangxi Agricultural and Biological Science

基　　金：国家自然科学基金项目(3006008);广西大学谭锦球青年科学基金项目

摘　　要：设计两对引物,PCR扩增香蕉乙烯受体基因cDNA序列自AUG启始密码子起长538bp区段的正向(记为5'I)、反向(记为AI')DNA区段。构建含该正、反向互补重复序列DNA片段的中间载体,经测序证实连接正确后,克隆到植物表达载体pCAMBIA-1301中的GUS基因位置并经双酶切证实。在所得表达载体pCAMBIA-1301-S5'I-AI'中,该插入正反互补重复片段处于35S启动子之后,由此转录的mRNA因两端序列反向互补而形成发夹式RNA,因此可应用于RNA干扰研究。同时,文中对构建含正、反向重复序列插入片段植物表达载体过程中出现的插入片段链内退火引起连接困难等问题进行了分析讨论。A 538 bp DNA fragments downstream the AUG start codon of the banana ethylene receptor gene was amplified by PCR in both sense and anti-sense orientation. A vector that contained the inverted-repeat DNA fragment was constructed. After sequencing demonstration,the inverted-repeat DNA fragment was cloned into pCAMBIA-1301 vector in which it replaced the GUS gene under control of the 35S promoter. In addition,some questions,such as the linking difficulty between target inserting fragment and cloning vector resulted from intra-strand annealing during constructing vectors including inverted repeat complementary sequence,were discussed.

关 键 词：乙烯受体基因 RNA干扰 植物表达载体 香蕉 

分 类 号：Q75[生物学—分子生物学]