The Study On The Function Of MsWRKY33 Transcription Factor In Regulating Salt Tolerance Of Medicago Sativa.L

Alfalfa（Medicago sativa.L）is widely planted because of its value in use,however,the problem of soil salinization has seriously affected the yield and quality of alfalfa,therefore,it is imperative to analyze the mechanism of alfalfa in response to salt stress.WRKY transcription factor is an important factor for plants to resist abiotic stress.Although many WRKY transcription factors have been reported in recent years,there are few studies on WRKY transcription factors in alfalfa.The specific molecular mechanism of WRKY transcription factors regulating stress resistance is still unclear.Through this study,the mechanism of the MsWRKY33 transcription factor of alfalfa was explored,and its biological function in regulating alfalfa against salt stress was explored.The main results are as follows:1、In the present study,MsWRKY33 gene was isolated by the method of homologous cloning. Bioinformatics analysis showed that MsWRKY33 has two typical WRKY domains,which belongs to I WRKY transcription factors.Phylogenetics analysis showed that MsWRKY33 was highly similar to Mt WRKY33 and At WRKY33.Subcellular localization found that the MsWRKY33 protein was localized on the nucleus.2、Construct the MsWRKY33 promoter fusion GUS expression vector to transform Arabidopsis thaliana,perform GUS histochemical staining on the transgenic lines,and find that MsWRKY33 is mainly expressed in the roots and leaf veins of the transgenic lines,indicating that the expression of the MsWRKY33 is tissue-specific and salt stress can induce MsWRKY33 gene expression..3、qRT-PCR analysis showed that MsWRKY33 was highly expressed in the root and leaf tissues of alfalfa,and it could be induced by Na Cl,PEG,low temperature,and high temperature stress.In addition,MsWRKY33 could be induced by Me JA,SA,GA3 and ABA four hormone,and found that MsWRKY33 responds more quickly to GA₃and ABA.4、Yeast one-hybrid experiment found that MsWRKY33 can specifically bind to W-box cis-acting elements,and it can also bind to the upstream promoter of ACS2,PAD3 and CYP71A13genes.5、The transcriptional activation test showed that the activation domain of MsWRKY33 was located at the N-terminus（P1 segment）.Though yeast two-hybrid experiment obtain 3 proteins that interact with MsWRKY33.Respectively are Ms CAMBP、Ms Mpv17 and Ms PRPS10.Yeast two-hybrid and Bi FC experiments were performed to analysis the interaction between these three proteins and MsWRKY33,the results shown that MsWRKY33 has a strong interaction with Ms CAMBP in the nucleus,and has weak or no interaction relationship with Ms Mpv17 and Ms PRPS10.6、The MsWRKY33 overexpressing alfalfa transgenic lines were obtained by Agrobacterium-mediated transforming method,It was found that the activities of SOD,POD were significantly higher than that of non-transgenic alfalfa,indicate that MsWRKY33 can improve the salt tolerance of transgenic lines.qRT-PCR analysis found that the expression of CYP71A13,ACS2and PAD3 can be induced by salt and the expression level is much higher than non-transgenic plants,indicating that MsWRKY33 can recognized and combined with the W-box in the promoters of these three genes to regulate their expression,therefore,regulated the salt tolerance of alfalfa.In summary,MsWRKY33 improves the salt tolerance of alfalfa by regulating the expression of the downstream salt tolerance genes ACS2,PAD3 and CYP71A13,and influence the antioxidant enzymes activity in the plant.This study explained the molecular mechanism of MsWRKY33 regulating the salt tolerance of alfalfa,and obtained the transgenic line of overexpressing MsWRKY33.