长春碱生物合成途径中关键酶基因的克隆与生物信息学分析

Clonging and Bioinformatics Analysis of Key Enzymes in Biosynthesis of Vinblastine

以长春花幼苗的叶片为材料,通过RT-PCR克隆关键酶基因CDS序列,利用在线软件ExPASy ProtParam、SignalP 4.1、TMHMM V.2.0、TargetP 1.1分析蛋白理化性质、蛋白信号肽、蛋白跨膜区以及亚细胞定位,在线工具Conserved Domain、SOPMA预测蛋白保守结构域和二级结构,MEGA6.0软件构建蛋白系统进化树,为后续的TIA合成提供理论研究依据。结果表明,STR、TDC、和ORCA3关键酶基因的CDS序列大小分别为1 059、1 503、612 bp;生物信息学分析表明,STR、TDC、和ORCA3关键酶定位于细胞不同区室,为酸性蛋白,无信号肽,除STR外无跨膜区,由α-螺旋、延伸链、β-转角和无规则卷曲等二级结构组成;除TDC外均包含高度保守的结构域;聚类分析表明其与小蔓长春花、罗芙木和中果咖啡具有较高的相似性,较近的亲缘关系和遗传距离,该结果可为明确该基因在TIA合成代谢途径中的重要作用奠定基础。

The leaves of Catharanthus rose us L.were used as materials to clone the gene CDS sequence by RT-PCR.The online software ExPASy ProtParam,SignalP 4」,TMHMM V.2.0,TargetP 1.1 were used to analyze the physical and chemical properties of proteins,protein signal peptides and proteins transmembrane region and subcellular localization.Online tool Conserved Domain,SOPMA predicted protein conserved domain and secondary structure, MEGA6.0 software constructed protein system phylogenetic tree, in order to provide theoretical basis for the subsequent TIA synthesis.The results showed that the CDS sequences of STR , TDC and ORCA 3 key enzyme genes were obtained ‘fragment sizes were 1 059 bp, 1 503 bp, and 612 bp, respectively.Bioinformatics analysis showed that the key enzymes of STR, TDC and ORCA3 were localized in different compartments of cells.lt is an acidic protein, no signal peptide, and has no transmembrane region except STR.lt consists of secondary structures such as a-helix, extended chain,P~ tum and random coil, all of them contain highly conserved domains except TDC. Cluster analysis showed that it had higher similarity with the Vinca minor L.,Rauvolfiaverticillata(Lour) Baill and Coffee canephora,and the closer genetic relationship and genetic distance,these results may lay a foundation for the important role of the gene in TIA biosynthesis pathway.