| Alfalfa(Medicago sativa L.)is the most widely planted forage in the world.It has the characteristics of high yield,strong stress tolerance and good palatability.It is extremely valuable for livestock feeding and is known as the "king of forage".However,abiotic stresses such as drought seriously threaten the growth and development and its yield of alfalfa.In this study,the full-length transcriptome database of alfalfa has been constructed by the three-generation Pac Bio full-length transcriptome sequencing technology in the early stage of our laboratory,which includes simulated salt stress(Na Cl),drought stress(Mannitol)and exogenous Abscisic Acid(ABA)treatment data,and then analyzed the effects of each gene under stresses.The expression level of which genes significantly induced by abiotic stresses were screened out.The late embryogenesis abundant(LEA)family which plays an important defense role in abiotic stress was selected for identification,and the MsLEA10 overexpression vector was constructed and transferred into Arabidopsis and alfalfa to further verify its gene function.The main research results are as follows:1.According to the alfalfa full-length transcriptome database constructed in the early stage of our laboratory,the genes induced by stresses were screened.Analyzed the expression level of each gene under drought,salt and ABA treatments,obtained 98 differentially expressed genes induced under abiotic stresses,and the expression patterns of the legume models plant Medicago truncatula and alfalfa were investigated from the alfalfa CADL database,the M.truncatula genome websites,9genes with significant changes in expression abundance were selected as the candidate genes for stress resistance.2.The 30 alfalfa LEA gene family member sequences were screened in the alfalfa whole genome database,and they were divided into 8 subfamilies contained LEA＿1-LEA＿6,Dehydrin and SMP through evolutionary analysis.These genes were named MsLEA1-MsLEA30 after sorted by protein ID number from small to large.The results of expression analysis showed that LEA genes have tissue expression specificity,the expression of different genes is more diverse,and most genes’ expression level is related to stress tolerance.The prediction results of subcellular location show that LEA proteins are widely distributed in sub-cells.According to the results of q RT-PCR verification of LEA gene family genes under abiotic stresses,as well as stress resistant candidate genes analysis,it is planned to select MsLEA10 gene for drought tolerance function analysis.3.The MsLEA10 gene overexpression vector was constructed and transformed into Arabidopsis and alfalfa.The gene function of MsLEA10 overexpression in Arabidopsis was verified.MsLEA10 belongs to the LEA＿3 subfamily of proteins,contains 2 exons and 1 intron,and is subcellularly located in the endoplasmic reticulum,nucleus and plasma membrane.The root length phenotype evaluation on the drought stress plate showed that the root length of MsLEA10 overexpressed Arabidopsis lines was significantly greater than that of the wild type;the survival rate and chlorophyll content statistics after soil drought treatment showed that MsLEA10 overexpressed Arabidopsis has soil drought tolerance;The results of photosynthesis indicators and physiological indicators show that the MsLEA10 gene can improve the drought tolerance of Arabidopsis.MsLEA10 overexpressing alfalfa positive seedlings were obtained through genetic transformation.The drought tolerance of MsLEA10 in alfalfa needs to be further analysis. |
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