柠条锦鸡儿CkP5CS基因克隆及表达特性分析

doi:10.13560/j.cnki.biotech.bull.1985.2018-0349

摘要/Abstract

摘要： 旨在克隆柠条锦鸡儿Δ¹-吡咯啉-5-羧酸合成酶基因（P5CS）的全长cDNA序列,研究其组织表达特异性,分析CkP5CS基因在低温、高盐和PEG处理下的表达情况,以阐明CkP5CS基因在柠条锦鸡儿中的生物学功能。利用RACE技术克隆P5CS基因cDNA序列,并对其全长基因进行生物信息学分析;构建系统发育树,研究其与相似序列的同源性;利用实时荧光定量PCR方法,分析CkP5CS基因的组织表达特异性以及在低温、高盐和渗透胁迫下的表达情况。结果显示,克隆得到柠条锦鸡儿P5CS基因全长cDNA 序列,命名为CkP5CS,全长2 604 bp,包括5'非翻译区（5'-UTR）102 bp,3'非翻译区（3'-UTR）363 bp,开放阅读框2 139 bp,能编码712个氨基酸,预测蛋白质分子量为76.8 kD,理论等电点为8.6,二级结构主要包括α-螺旋、延伸链和无规则卷曲。多序列比对和系统进化分析表明,柠条锦鸡儿P5CS与白刺花SdP5CS、大豆GmP5CS、豇豆VuP5CS同源性较高,分别为85.1%、84.2%和82.6%。实时荧光定量PCR结果显示,CkP5CS在柠条锦鸡儿的根、茎和叶中均有表达,没有组织特异性;同时,该基因的表达受低温、高盐和渗透胁迫的诱导。克隆得到柠条锦鸡儿CkP5CS基因,其在柠条锦鸡儿适应低温、高盐和渗透胁迫过程中发挥作用。

关键词: 柠条锦鸡儿, CkP5CS基因, 分子克隆, 表达分析

Abstract: The study aims to clone the full-length cDNA sequence of gene P5CS from Caragana korshinskii,analyze its tissue expression specificity and expression under the stresses of low temperature,high salt and PEG,and clarify its biological functions. The full-length cDNA of P5CS from C. korshinskii was isolated by RACE technique and analyzed by bioinformatics. Phylogenetic tree was constructed for studying homology with similar sequences. RT-PCR method was used to analyze the tissue expression specificity and the expression of CkP5CS under low temperature,high salt and osmotic stress. As results,the full-length cDNA sequence of P5CSgene of C. korshinskii was cloned and named as CkP5CS with a length of 2 604 bp,containing a 5'-UTR of 102 bp,a 3'-UTR of 363 bp,and a 2 139 bp opening reading frame,and it encoded 712 amino acids with the molecular weight of 46.8kD and theoretical isoelectric point of 8.6.The main secondary of the protein was a helix,extended strand and random coils. Comparison of amino acid sequences and phylogenetic analysis the CkP5CS showed high identity with those from Sophora davidii SdP5CS（85.1%）,Glycine max GmP5CS（84.2%）and Vigna unguiculata VuP5CS（82.6%）. Real-time PCR analysis revealed that CkP5CS expressed in roots,stems,leaves,indicating that there was almost no tissue specificity. The transcript level of CkP5CS was induced in response to low temperature,high salt and osmotic stress. As conclusion,CkP5CS gene isolated from C. korshinskii plays an important role in the adaptive process under low temperature,high salt and osmotic stress.

Key words: Caragana korshinskii, CkP5CS gene, molecular cloning, expression analysis

张腾国, 史中飞, 寇明刚, 郑晟, 王娟. 柠条锦鸡儿CkP5CS基因克隆及表达特性分析[J]. 生物技术通报, 2018, 34(9): 209-218.

ZHANG Teng-guo, SHI Zhong-fei, KOU Ming-gang, ZHENG Sheng, WANG Juan. Cloning and Expression Analysis of CkP5CS Gene in Caragana korshinskii[J]. Biotechnology Bulletin, 2018, 34(9): 209-218.

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