Expression Of MsERD15 And Construction Of Expression Vector In Alfalfa

Alfalfa(Medicago sativa L.)is a perennial legume with high quality,high protein content.The characteristics of rich nutritional value make it plays very important role in the development of animal husbandry.However,fall dormancy of alfalfa and changes in the external environment(vegetation degradation and soil salinization),the alfalfa planting is limited.Therefore,to break dormancy and cultivate new varieties of Alfalfa strong tolerance is particularly important for the development of alfalfa industry.In Arabidopsis,early dehydration responsive protein(ERD15)participates in the biotic and abiotic stress responsive,and has a great relationship with the ABA signaling pathway.Although the gene discovered earlier,however,no one has studied the relationship with dormancy and the use of ERD15 improved the resistance of plants is relatively small by means of genetic engineering.There were no researchs on Alfalfa.So to study the effect of ERD15 on alfalfa and its stress tolerance is great for theoretical and economic value.In this experiment,according to the previous research results we got an early dehydration response protein gene named MsERD15 and its c DNA sequence.The expression of MsERD15 gene was detected in a growth period of alfalfa leaves from5-10 months after cutting in the natural environment and different stress conditions by fluorescence quantitative PCR.At the same time the correlation were analyzed between the expression quantity of MsERD15 gene during during the period of plant nutrition growth under natural conditions with temperature,rainfall and sunshine length.The results of stress processing showed that: MsERD15 gene was up induced by Na Cl,PEG6000,cold and ABA.The response of Na Cl stress was the most severe maximum value,and it was 46.7 times than control.The expression of MsERD15 gene in response to cold and Na Cl was the most rapidly that the difference was significant in the 0.5 hour.IAA treatment inhibited the expression of MsERD15 gene.GA3 hormone induced the expression levels of MsERD15 gene,but it did not changesignificantly.It is hypothesized that MsERD15 gene was to be involved in response to abiotic forcing,and induced by ABA.The expression of MsERD15 gene under SA and Me JA stress was not significantly decreased at 0.5h and 1h,after that it was significantly increased,and reached the maximum value in 12 hours,then decreased,but it also was significantly higher than that of control.Which may be related to SA and Me JA levels of intracellular.MsERD15 was in response to SA and Me JA stress,while SA and Me JA can be treated as a factor of biological stress,so we suggest that MsERD15 is involved in the process of biological stress.The fluorescence quantitative PCR results of MsERD15 gene in alfalfa in natural environment showed that the MsERD15 gene basically was increased first and then low trend in every growth period after mowing from May to October,except September.The September had a word "M" trend.However,the overall expression is increased.The physiological process that may be related to the body on September when alfalfa had entered dormancy state,so it had the different expression patterns of usual growth.In general before June 21 st the FD1 and FD2 had a low expression level than FD8 and FD9,after that the result was contrary.And FD1 and FD2 expression was sustained up regulated,then on September it had a little down in Alfalfa.While the the expression level of MsERD15 gene in FD8 and FD9 increased first and then decreased and then increased.But it was lower than in FD1 and FD2.Probably due to the Alfalfa of different fall dormancy level had different sensitive reaction on the environment.Fall dormancy type was more sensitive to the environment,so it had a higher level than non Fall dormancy type.The results of correlation analysis showed that the expression level of MsERD15 gene in FD1 and FD2 alfalfa was positively correlated with temperature,rainfall,and negatively correlated with sunshine time.FD1 had a significant correlation with rainfall and temperature.This result shows that the FD1 and FD2 alfalfa are more sensitive to the external environment,at the same time shows that the MsERD15 in the fall dormancy type Alfalfa plays a more important role in response to changes in the external environment.We speculated that MsERD15 is involved in the regulation of Alfalfa fall dormancy through the light response process.In this study,we constructed the over expression vector of MsERD15 gene and transformed the alfalfa by Agrobacterium mediated transformation.The Bar gene was detected by screening the genomic DNA.At last we got three plants(1,2,7)obtained bar gene.They were positive transgenic alfalfa.