Cloning And Functional Analysis Of The Legume Specific Gene Mtwrp1 In Medicago Truncatula

WRKY transcription factor is a one of the largest families of transcriptional regulators in plants and very important in various biological processes of plants.As a specific protein related WRKY in legumes,WRP has the similar but unique sequence structure with WRKY.Therefore,biological function of WRP is not only similar to WRKY,but also novel.However,the understanding of the function and mechanism of WRP regulation is very limited at present in leguminous forage.So the function and the mechanism of MtWRP1 related WRKY in Medicago truncatula was investigated in this study.The main results are as follows:1.MtWRPl gene was cloned from Medicago truncatula by homologous cloning.The coding region was 984 bp and encoded 327 amino acids.The results of amino acid sequence analysis and homology comparison showed that the core sequence of MtWRP1 was WKKYEEK which was more variable than the core sequences(WRKYGQK)of the most WRKY,indicating that the sequence of amino acid was mutated.The MtWRPl domain was most similar to the typical class I WRKY proteins contained two WRKY domains.However,MtWRP1 had a WRKY domain and a newly discovered N terminal TM domain instead of the two WRKY domains.It was found that the TM domain with five transmembrane structure was highly homologous to the eukaryotic cytochrome b561 protein.In addition,the results of subcellular localization showed that MtWRP1 was localized to Golgi due to TM domain of N,indicating the specificity of legume.2.The expression characteristics of MtWRP1 gene contained the specific TM domain were analyzed by fluorescence quantitative PCR.The results showed that MtWRPl were expressed in root,stem,leaf,flower,fruit and seed of Medicago truncatula.Moreover,the expression levels were diverse in different tissues,and were the highest in the roots.In addition,the results of gene expression induced by rhizobium showed that the expression of MtWRPl was significantly enhanced with the increase of the nodule number,speculating that the formation of root nodules was regulated by MtWRP1 or nitrogen fixation promoted MtWRPl expression.the expression of MtWRPl was enhanced with the increase of age in leaves,and was very low in young leaves.3.The phenotype of MtWRP1 transgenic and over-expressed Arabidopsis lines produced was analyzed.The results showed that the flowering time of transgenic Arabidopsis delayed compared with wild type Arabidopsis,and the phenotype was more obvious in the palnts with higher expression of MtWRPl.Furthermore,compared with wild type Arabidopsis,the phenotype of leaf which turned yellow in transgenic Arabidopsis was less obvious,and the aging rate of transgenic Arabidopsis was slower than the wild type.4.The compelet transgenic Medicago truncatula plants were obtained via somatic embryogenesis with the explants of leaves in this study.The results of the over expression analysis of transgenic plants by fluorescence quantitative PCR showed that the expression of MtWRPl were different in different transgenic lines.In summary,the WRKY domain in MtWRPl gene of Medicago truncatula was similar to the N terminal TM domain with five transmembrane structures of typical class I WRKY proteins,and localized to Golgi due to TM domain of N,indicating the specificity of legume.The founctional results indicated that MtWRPl can delay leaf senescence,while positively regulate the nodule formation.