柠条锦鸡儿CkLEA4-1基因的克隆及表达分析

Clone and Expression Analysis of CkLEA4-1 from Caragana korshinskii Kom.

为研究胚胎发育晚期蛋白(LEA)在柠条中的生物学功能,从已构建的柠条锦鸡儿干旱胁迫抑制削减杂交文库中筛选到一条LEA蛋白编码基因,并采用RT-PCR法进行克隆。所得LEA基因的开放阅读框(ORF)长1 119 bp,编码373个氨基酸的蛋白质,命名为CkLEA4-1。结合序列比对与系统进化分析结果,推断CkLEA4-1属于5族LEA蛋白。利用实时荧光定量PCR技术对CkLEA4-1在干旱、高盐等逆境胁迫条件下的表达情况进行初步研究,结果表明,CkLEA4-1受到不同程度的诱导,推测该基因可能与柠条锦鸡儿响应逆境胁迫有关。研究还成功构建了CkLEA4-1的过表达载体p Can G-CkLEA4-1,得到转基因纯合体株系,为进一步研究柠条锦鸡儿CkLEA4-1基因的功能提供了材料。

In order to research the biological function of the LEA protein,a gene encoding LEA protein from the suppression subtractive hybridization library of Caragana korshinskii Kom.was screened and cloned by using RT-PCR method.The open reading frame（ORF）of the gene length was 1 119 bp,encoded a protein of 373 amino acids,named CkLEA4-1.Based on sequence alignment and phylogenetic analysis,it was inferred that CkLEA4-1 belonged to group 5 late embryogenesis abundant protein（LEA）.The expression of CkLEA4-1 under high salt and drought conditions were detected by Real-time quantitative PCR,the results showed that CkLEA4-1 could be induced at different levels,which suggested that the gene might be involved in stress response of Caragana korshinskii Kom..The over-expression vector pCanG-CkLEA4-1 of CkLEA4-1 was constructed successfully,and the transgenic homozygous lines were obtained,which provided materials for the further study of CkLEA4-1 gene function in Caragana korshinskii Kom..