甘青青兰Dt4CL基因过表达载体构建与生物信息学分析

Overexpression Vector Construction and Bioinformatics Analysis of Dt4CL Gene from Dracocephalum tanguticum Maxim

4香豆酸辅酶A连接酶(4-coumarate:coenzyme A ligase, 4CL)是黄酮类、木质素生物合成的关键酶之一,位于苯丙氨酸代谢途径的主途径向分支途径过渡的关键点上,控制进入不同的次生代谢支路以产生不同的次级代谢产物。本试验以甘青青兰(Dracocephalum tanguticum Maxim)为材料,利用PCR技术从甘青青兰中克隆出该家族成员一个基因,命名为Dt4CL1,利用双酶切及质粒重组法构建过表达载体Dt4CL1-PHB。对该基因编码的蛋白进行理化性质、疏水性、信号肽、二级结构、三级结构及系统进化树等生物信息学分析。结果显示该基因ORF全长1 719 bp,编码572个氨基酸,预测分子量为61.61 kD,理论pI为5.39。系统进化分析表明,Dt4CL1蛋白与丹参、夏枯草及小冠薰的4CL蛋白同源性最高。本研究结果为之后研究Dt4CL1的功能提供科学依据。

4-coumarate(4-coumarate: coenzyme A ligase, 4 CL) is one of the key enzymes in the biosynthesis of flavonoids and lignin. It is located at the key point of the transition from the main pathway to the branch pathway of phenylalanine metabolism, and controls the entry into different secondary metabolic pathways to produce different secondary metabolites. In this experiment, a gene named Dt4 CL1 was cloned from Dracocephalum tanguticum Maxim by PCR. The overexpression vector Dt4 CL1-PHB was constructed by double enzyme digestion and plasmid recombination. The protein encoded by the gene was analyzed by bioinformatics, including physical and chemical properties, hydrophobicity, signal peptide, secondary structure, tertiary structure and phylogenetic tree.The results showed that ORF was 1 719 bp in length and encoded 572 amino acids. The predicted molecular weight was 61.61 kD and the theoretical pI was 5.39. Phylogenetic analysis showed that Dt4 CL1 protein had the highest homology with the 4 CL protein of Salvia miltiorrhiza, Prunella vulgaris and Ocimum tenuiflorum. It provides a scientific basis for the study of the function of Dt4 CL1.