蒺藜苜蓿小G蛋白ROP在共生过程中的作用:1.MtROP5的克隆和表达分析

【目的】对蒺藜苜蓿ROP进行克隆与表达分析,为深入研究ROP在共生互作过程中的功能提供理论依据。【方法】通过比较基因组学和RT-PCR方法克隆了蒺藜苜蓿的MtROP5,采用半定量RT-PCR方法检测了MtROP5在蒺藜苜蓿不同组织中的表达水平,采用荧光实时定量PCR方法检测了MtROP5在根瘤菌侵染宿主植物根系后72h内不同时间阶段的表达水平。【结果】从蒺藜苜蓿中克隆了含有MtROP5完整编码区的cDNA序列,长度826bp,编码197个氨基酸。序列分析表明,MtROP5编码的蛋白具有典型的ROP蛋白特征结构域,与其它几种植物中ROP蛋白具有高度的同源性和相似性。半定量RT-PCR分析表明,MtROP5在蒺藜苜蓿的根、茎、叶、花、根瘤中均有表达,茎和花中表达量最高,根和根瘤中次之,在叶中表达最弱。荧光实时定量PCR分析表明,与未接种根瘤菌的对照处理相比较,MtROP5在根瘤菌侵染的72h内不同时间阶段的根系中都增强表达。【结论】克隆的cDNA序列是一个蒺藜苜蓿小G蛋白ROP,推测MtROP5可能在共生互作的早期信号传导过程中行使一定的调控作用。

Role of Small GTP-Binding Protein ROP in M. truncatula in Symbiosis: 1. Isolation and Expression Analysis of MtROP5 Gene

Objective] The objective of this study is to isolate ROP (Rho GTPase of plant) gene from Medicago truncatula and analyze its expression pattern in order to further investigate the function of ROP in symbiotic interaction. Method] Through analysis of comparative genomics, MtROP5 gene in M. truncatula was isolated by RT-PCR. Using semi-quantitative RT-PCR, the expression level of MtROP5 gene was detected in different tissues of M. truncatula, and real-time quantitative RT-PCR was used to detect the expression levels of MtROP5 gene in inoculated root which was infected by Sinorhizobium meliloti Rm1021 during the 72 hours post-inoculation. Result] MtROP5 gene with a complete open reading frame of 826 bp encoding a peptide of 197 amino acid residues was isolated from M. truncatula. Results of sequence analysis showed that MtROP5 contains the typical structures of ROP GTPase family. Homologous comparison with some plant ROPs indicated that MtROP5 shared high similarity with other plant ROPs. Expression analysis of RT-PCR showed that MtROP5 gene expressed in root, stem, leaf, flower, nodule, especially higher expression in stem and flower, next in root and nodule, and the expression level of MtROP5 gene in leaf was lower. Compared with the expression level of MtROP5 gene in uninoculated root, the expression level of MtROP5 gene was up-regulated at the different stages (0 to 72 hours) post-inoculation. Conclusion] A small GTPase MtROP5 gene in M. truncatula was obtained. MtROP5 gene may involve in the regulation of signal transmission that occur at early stage of symbiotic interaction between legume plant and Rhizobium.