Isolation And Characterization Of MsGSTU8 From Alfalfa(Medicago Sativa L.)

Soil salinization seriously affects the growth,development and production of crops,and it is very important to understand the mechanism of plant resistance to saline-alkaline stress and how to improve plant tolerance to saline-alkaline stress.GST are proteins with multiple functions in plants,it plays an important role in abiotic stress response and in enzymatic reactive oxygen scavenging mechanism.In this study,MsGSTU8 gene was cloned from alfalfa(Medicago sativa L.),and the gene was ligated with pCBM expression vector and transformed into model plant tobacco and Arabidopsis,which laid a foundation for studying the function of MsGSTU8 gene in plant response to saline-alkaline stress.The main findings are as follows:1.Cloning and analysis of MsGSTU8 geneThe full-length cDNA of MsGSTU8 was isolated by RT-PCR.The gene was 675 bp in length and encoded a total of 224 amino acids.The protein has an N-terminal domain and a C-terminal domain.The sequences similarity of MsGSTU8 and MtGSTU8(Medicago truncatula)was found to be 96% when the two sequences were aligned in Blast of NCBI.2.Ttransformation of MsGSTU8 gene in tobacco and Arabidopsis(1)Agrobacterium mediated transformation of tobaccoThe plasmid pCBM-MsGSTU8 was transformed into tobacco by Agrobacterium mediated method.The infected tobacco leaves were screened in 1.1mg/L glyphosate screening medium,then the adventitious buds were transferred to the rooting medium.The total of 43 resistant seedlings was obtained.(2)Agrobacterium mediated transformation of ArabidopsisThe expression vector pCBM-MsGSTU8 was constructed by ligating the MsGSTU8 gene with the expression vector pCBM containing CaMV35 S promoter and bar gene.The MsGSTU8 was transformed into Arabidopsis by Agrobacterium mediated method.The seeds were collected and placed in 40 ?mol/L glyphosate medium for further screening.The total of 20 resistant seedlings was obtained.3.Identification and saline-alkaline tolerance analysis of transgenic plantA total of 16 transgenic tobacco and 13 transgenic Arabidopsis seedlings were detected by PCR.The transgenic tobacco seedlings were subjected to saline-alkaline stress.The chlorophyll,soluble sugar content and antioxidant enzymes(SOD?POD?CAT?GST)activities of transgenic tobacco were higher than that of control,whereas,relative conductivity,content of MDA,superoxide anion and hydrogen peroxide were lower than that of wild type.The transgenic Arabidopsis seedlings were subjected to salt stress,Root length of transgenic Arabidopsis seedlings were significantly longer than that of wild type.The results suggested that transgenic plants had stronger ability to tolerate saline-alkaline stress compared with wild type,indicating that MsGSTU8 gene has function of salt-alkaline resistance.