珊瑚菜GlROP3基因的克隆、生物信息学分析及原核表达

Cloning,Bioinformatics Analysis and Prokaryotic Expression of GlROP3 Gene from Glehnia littoralis

本研究基于珊瑚菜转录组数据,对珊瑚菜抗逆相关基因进行挖掘,筛选鉴定并克隆了GlROP3基因,对其进行生物信息学分析,使用荧光定量PCR方法对GlROP3基因进行定量表达分析,成功构建GlROP3的原核表达载体并进行原核蛋白的表达。结果表明,GlROP3基因的开放阅读框(ORF)为594 bp,编码197个氨基酸,蛋白相对分子量为21.556 kD,理论等电点为9.28,总平均亲水性为-0.089,为稳定的亲水性蛋白。GlROP3蛋白包含2个GTPase区、2个GDP/GTP结合区、1个效应区、1个插入序列和1个C端多变区。系统进化树分析显示GlROP3与胡萝卜的ROP蛋白亲缘关系较近。荧光定量PCR分析表明GlROP3基因在珊瑚菜花中的表达量较高,其次为根。此外,珊瑚菜根的GlROP3基因受干旱胁迫影响后表达量明显增加。本研究为今后探究GlROP3基因的功能,明确其在植物抗逆过程中的作用,为进一步增强珊瑚菜的抗旱抗逆能力,提高珊瑚菜的产量提供基础。

In this study,based on the transcriptome data of Glehnia littoralis,the stress resistance related genes of Glehnia littoralis were explored,the GlROP3 gene was screened,identified and cloned,and its bioinformatics analysis was performed,the quantitative expression analysis of GlROP3 gene was carried out by fluorescence quantitative PCR.The prokaryotic expression vector of GlROP3 was successfully constructed and the prokaryotic protein was expressed.The results showed that the full-length GlROP3 gene had an open reading frame of 594 bp,encoding 197 amino acid residues,the relative molecular weight of the protein was 21.556 kD,the theoretical isoelectric point was 9.28,and the total average hydrophilicity was-0.089,which was a stable hydrophilic protein.The GlROP3 protein included 2 GTPase domains,2 GDP/GTP-binding domains,1 effector domain,1 RHO insert region and 1 C-terminal hypervariable region.According to our phylogenetic analysis,the GlROP3 protein was closely related to the ROP protein of Daucus carota subsp.sativus.Fluorescence quantitative PCR analysis showed that the GlROP3 gene was highly expressed in flower,followed by roots.In addition,the expression of GlROP3 was significantly upregulated in roots of Glehnia littoralis under drought stress.This study provided a basis for fur-ther exploring the function of GlROP3 gene and clarifying its role in plant stress resistance,so as to further enhance the drought resistance and stress resistance of Glehnia littoralis and increasing its yield.