蓖麻PDCT基因启动子的克隆及生物信息学分析

Cloning and Bioinformatics Analysis of the Promoter of Castor PDCT Gene

磷脂酰胆碱甘油二酯胆碱磷酸转移酶(phosphatidylcholine diglyceride choline phosphotransferase,PDCT)是种子中甘油三酯(triglyceride,TAG)积累过程中不饱和脂肪酸合成所必须的酶,因此在油脂合成和积累中起着重要的作用。本研究以‘通蓖5号’蓖麻品种幼嫩叶片为实验材料,根据美国国立生物技术信息中心(National Center for Biotechnology Information,NCBI)上已知蓖麻PDCT基因(LOC8282344)及其基因组(NW-002994322)序列设计引物,通过聚合酶链式反应(polymerase chain reaction,PCR)扩增得到PDCT基因启动子片段。生物信息学分析PDCT蛋白的理化性质以及PDCT基因启动子序列。结果表明克隆到PDCT基因启动子序列3 741 bp,分析预测有多个转录起始位点及大量的顺式作用元件;PDCT基因共编码285个氨基酸,存在跨膜结构,为非分泌蛋白;对蛋白质编码的氨基酸进行同源性比对,并用MEGA 7.0构建了进化树,发现蓖麻和麻疯树,巴西橡胶树,木薯聚类到一起,这一分类与生物学分类一致,均为大戟科植物。对蓖麻PDCT基因及其启动子和蓖麻油脂合成代谢途径的进一步研究提供参考。

Phosphatidylcholine diglyceride choline phosphotransferase(PDCT)is an essential enzyme for the synthesis of unsaturated fatty acids in the accumulation process of TAG in seed,and therefore it plays an important role in oil synthesis and accumulation.In this study,the young leaves of the'Tongbi 5'castor variety were used as experimental materials,according to the National Center for Biotechnology Information(NCBI),design primers based on known castor PDCT gene(LOC8282344)and its genome(NW-002994322)sequence,the PDCT gene promoter fragment was obtained by polymerase chain reaction(PCR)amplification.We analysed the physicochemical properties of PDCT proteins and PDCT gene promoter sequences by bioinformatics.The results showed that the promoter region of the PDCT gene was 3 741 bp,and it was predicted that there were multiple transcription initiation sites and a large number of cis-acting elements.PDCT gene encoded a total of 285 amino acids,and there was a transmembrane structure with non-secretory protein.The encoded amino acids were homologously compared;The phylogenetic tree was constructed with MEGA 7.0.The castor and jatropha,the Brazilian rubber tree,and the cassava were clustered together.This classification is consistent with the biological classification,and all of them are Euphorbia pulcherriaceae.This study concentrated mainly on the research on the PDCT gene and its promoter of castor,to provide reference for the further study in the anabolic pathway of castor oil.