紫苏caleosin 基因克隆及表达分析

Cloning and Expression Analysis of caleosin Gene from Perilla frutescens

油体钙蛋白(caleosin)是参与油体合成的一类蛋白质。本研究从紫苏转录组数据库中筛选获得caleosin的PfClo1和PfClo2两个基因片段,通过表达特性分析发现,PfClo2基因在紫苏不同组织中几乎不表达,故选定PfClo1基因进行克隆及实时荧光定量聚合酶链式反应(qRT-PCR)分析,探究基因PfClo1在紫苏不同品种中的表达特性及对干旱胁迫的响应。紫苏caleosin的PfClo1基因序列分析结果表明,PfClo1基因的开放阅读框(ORF)序列为609 bp,共编码202个氨基酸,理论等电点为5.19,亲水性系数为0.678,PSORT软件预测PfClo1蛋白定位于细胞质;BLAST同源序列比对表明,PfClo1与丹参和芝麻的caleosin序列相似性较高,属于典型的caleosin家族成员;qRT-PCR分析结果表明,PfClo1基因在‘晋紫苏1号’的花和种子中均有表达,且在开花后30 d的种子中表达量最高;通过分析不同紫苏品种的差异表达发现,PfClo1基因的表达与紫苏油脂合成存在一定的正相关关系;用20%聚乙二醇(PEG)对‘晋紫苏1号’幼苗进行干旱胁迫处理后发现,PfClo1基因在胁迫处理12 h时表达量达到最高,为对照的6.78倍,推测该基因可能在紫苏干旱胁迫诱导中发挥调控作用。该研究结果为深入探究PfClo1基因在紫苏种子发育及干旱胁迫应答中的功能提供了理论依据。

Caleosin is a kind of protein involved in oil body synthesis.In this study,the PfClo1 and PfClo2 gene fragments were obtained from perilla transcriptome database.The PfClo2 gene was almost not expressed in various perilla tissues,hence the PfClo1 gene was cloned and analyzed by qRT-PCR to investigate the expression characteristics of the gene PfClo1 in different perilla varieties and its response to drought stress.The results of PfClo1 gene sequence analysis showed that the full-length ORF sequence of PfClo1 gene was 609 bp,encoding 202 amino acids,the theoretical isoelectric point was 5.19 and the hydrophilicity coefficient was 0.678.The PfClo1 protein was located in the cytoplasm from our prediction by using PSORT software.The PfClo1 protein had high similarity with caleosin sequence of Salvia miltiorrhiza and Sesamum indicum by blast homologous sequence alignment analysis,and belonged to a typical caleosin family member.The results of qRT-PCR analysis showed that PfClo1 gene was expressed in flowers and seeds of‘Jinzisu 1’,and the expression level reached the highest in seeds 30 days after flowering.By analyzing the differential expression of different perilla varieties,it was found that the PfClo1 gene expression was positively correlated with the oil formation of perilla seeds.After the‘Jinzisu 1’seedlings were treated with 20%PEG,it was found that the expression level of PfClo1 gene reached the highest at 12 h of stress treatment,which was 6.78 times that of the control.It is speculated that this gene may play a regulatory role in the induction of drought stress in perilla.The results provide a theoretical basis for further exploring the function of PfClo1 gene during perilla seed development and response to drought stress.