百脉根小G蛋白Rac1基因的克隆与功能分析

小G蛋白Rop在植物细胞信号转导中发挥着重要的分子开关功能。该实验通过RT-PCR方法克隆了百脉根的一个Rop编码基因LjRac1,并对LjRac1基因序列进行生物信息学分析,然后采用半定量RT-PCR检测LjRac1基因在百脉根不同组织中的表达,用荧光实时定量PCR方法检测百脉根接种根瘤菌后LjRac1基因在不同阶段根系中的表达,构建过表达重组质粒,利用发根农杆菌介导的遗传转化法对LjRac1基因功能进行分析。结果表明:(1)序列分析显示,LjRac1完整编码区的cDNA序列长度为594bp,编码197个氨基酸,其编码蛋白具有典型的Rop家族保守结构域;同源分析显示,百脉根LjRac1与大豆GmRac1、野大豆GsRac1的一致性最高(94.42%)。(2)LjRac1基因在百脉根的根、茎、叶、根瘤和花中均有表达,且在根和根瘤中的表达水平较高;接种根瘤菌0.5h后,LjRac1基因在根系中的表达量呈显著升高趋势。(3)过表达转基因植株中LjRac1mRNA的表达水平为对照植株的14.3倍,且过表达植株的结瘤数目较对照明显增加。研究认为,LjRac1基因是一个受根瘤菌诱导增强表达的基因,过表达LjRac1基因可以引起植株结瘤数目的增加,说明LjRac1基因可能参与早期结瘤信号转导途径,从而在根瘤的发育中发挥一定作用。

Functions of Small GTPase Rac1 in Nodulation Process in Lotus japonicus

Small G protein Ropplays an important role as molecular switch during signal transduction in plant cells.A Rop coding gene LjRac1 in Lotus japonicus was cloned by RT-PCR.The gene sequence analysis was carried out by the bioinformation method firstly.Then the expression profile of LjRac1 was detected in different tissues by semi-quantitative RT-PCR and in inoculated roots on different stage of post-inoculation using real-time RT-PCR,respectively.Moreover,over-expression recombinant plasmid was constructed and hairy root transformation method was used to investigate the over-expression effect of LjRac1 mRNA on the early nodulation process.The study revealed that:(1)LjRac1 gene sequence analysis showed that the length of LjRac1 genewas 594 bp,which encoded a peptide with 197 amino acids.LjRac1 contained conserved domains of Rop GTP ase proteins.Homology analysis showed that the deduced amino acid sequence exhibited high similarity with Glycine max GmRac1 and Glycine soja GsRac1(94.42%).(2)LjRac1 was expressed in root,stem,leaf,nodule and flower of L.japonicus,and preferentially expressed in root and nodule.The expression level of LjRac1 was elevated in root 0.5 h after inoculation with Mesorhizobium loti,as compared with that in uninoculated control root.(3)The expression level of LjRac1 mRNA in over-expression transgenic plants was 14.3 times higher than that of the control plants,and the number of nodules in over-expression transgenic plants significantly increased compared with the control plants.The data showed that LjRac1 was the gene of enhancement expression by the induction of M.loti and over-expression LjRac1 could increase the number of nodules,which indicated that LjRac1 gene might be involved in the early stage of symbiosis nodulation and regulated nodule development in L.japonicus.