蒙古沙冬青J蛋白基因AmJ20的克隆与表达分析  

Cloning and Expression Analysis of AmJ20 from J Protein Gene in Ammopiptanthus mongolicus

作　　者：殷玉梅[1] 郭慧琴[1] 任美艳 薛敏[1] 王茅雁[1] 

机构地区：[1]内蒙古农业大学生命科学学院,呼和浩特010018

出　　处：《基因组学与应用生物学》2017年第9期3855-3862,共8页Genomics and Applied Biology

基　　金：内蒙古自然科学基金重大项目(2012ZD02);国家自然科学基金(30960158);内蒙古自然基金项目(2014MS0326)共同资助

摘　　要：DNA J蛋白是植物中广泛存在的一类分子伴侣,对于维持正常和逆境条件下蛋白质的稳态具有重要作用。我们在前期通过对强抗逆植物蒙古沙冬青进行转录组分析,发现一个寒旱诱导表达的J蛋白即Am J20编码序列,但其3'端编码区不完整。本研究通过3'RACE获得其c DNA全长序列(800 bp),推测其编码蛋白由179个氨基酸残基组成,含一个典型的J结构域,属于Ⅲ型J蛋白,此外还具有叶绿体和细胞核双定位信号;利用半定量RT-PCR方法检测该基因在野外生长植株中的时空表达模式,发现其在春、夏、秋季只有微量表达,甚至检测不到转录本,而在严冬时节高表达,此外在叶片中的表达量明显高于根、嫩枝、花蕾和幼嫩果荚;利用RT-PCR方法克隆到该基因编码区c DNA并将其插入到植物超表达载体上,为通过转基因植物分析该基因的功能提供了理论依据。DNA J proteins is a widespread molecular chaperone in plants,and plays important roles in maintaining homeostasis of proteins under both normal and stress conditions.In the early stage,we conducted transcriptome analysis on the strong stress resistant plant Ammopiptanthus mongolicus and found a cold and drought induced expression of J protein,namely Am J20 coding sequence,but its 3＇-end coding region was not complete.In this study,we firstly acquired the full-length c DNA sequence（800 bp） of Am J20 by 3＇-RACE method.We predicted that its encoding protein consisted of 179 amino acids,containing a typical J domain,which belonged to the type Ⅲ J protein.In addition,it contained a pair of chloroplast and nuclear localization signal.Then,we adopted semi quantitative RT-PCR method to examine the spatio-temporal expression patterns of Am J20 in A.mongolicus plants grown in the wild.The results showed that in spring,summer and autumn,the expression levels of Am J20 were very low or even undetectable,while in winter,its expression level was markedly high.Furthermore,its expression level in young leaves was significantly higher than those in roots,twigs,flower buds and young pods.We finally cloned the coding region c DNA of Am J20 by RT-PCR and successfully inserted it into plant expression vector,which laid a foundation for further functional analysis of this gene.

关 键 词：沙冬青 J蛋白 表达分析 基因克隆 载体构建 

分 类 号：Q943.2[生物学—植物学] S793.9[农业科学—林木遗传育种]