柠条锦鸡儿木质素合成关键基因CkCOMT的克隆及表达载体构建

Cloning and Expression Vector Construction of the Key Gene CkCOMT Related to Lignin Synthesis in Caragana korshinskii

为了提高柠条锦鸡儿的抗非生物胁迫能力和饲草价值,利用分子生物学和生物信息学方法,克隆并分析柠条锦鸡儿木质素合成关键基因CkCOMT。在获得409 bp中间片段基础上,通过RACE技术,拼接获得基因全长1 378 bp,其中包括75 bp的5’非编码区,205 bp的3’非编码区,polyA (12)尾,ORF为1 098 bp,编码365个氨基酸。经预测分析,CkCOMT蛋白分子量为39.95 k D,属稳定的疏水性蛋白,二级结构以无规则卷曲为主。在该基因与豆科其他植物及模式植物的氨基酸序列进化树分析中,CkCOMT与苜蓿及大豆的亲缘关系最近。在此基础上,CkCOMT基因全长片段与p CanG-HA线性载体连接,成功构建pCanG-CkCOMT-HA重组质粒。该研究为下一步分析CkCOMT基因在木质素合成和代谢途径中的调控作用奠定了基础,同时,也为提高柠条锦鸡儿生存能力和经济价值提供了理论条件。

In order to improve abiotic stress tolerance and forage value of Caragana korshinskii, the lignin synthesis key gene CkCOMT was cloned and analyzed under the help of molecular biological and bioinformatics methods.The core sequence was 409 bp fragment, by means of RACE, and we get 1 378 bp total sequence which included75 bp 5’ noncoding region, 205 bp 3’ noncoding region and polyA(12) sequence. Its ORF was 1 098 bp and it encoded 365 amino acids. After predictive analysis, CkCOMT protein molecular weight was 39.95 kD, which was a stable hydrophobic protein, and most of secondary structures were irregular curls. In the phylogenetic analysis of the amino acid sequence of this gene and other plants of the leguminous family and model plants, it had the closest genetic relationship with Medicago sativa and Glycine max. On this basis, the pCanG-CkCOMT-HA recombinant plasmid was successfully constructed by the CkCOMT full sequence linked to pCanG-HA linear expression vector.This study could lay the foundation for the analysis of the regulation of CkCOMT gene in lignin synthetic and metabolic pathways. At the same time, it could offer theoretical conditions for enhancing the survival and economic value of Caragana korshinskii.