Identification,transformation And Resistance Evaluation Of The MsLEA4-4 Gene From Medicago Sativa

Abiotic stresses such as drought,low temperature and salt were important factors affecting plant growth and development,which directly determine the formation of yield.That’s responsible for 50% of the annual loss of the world’s major crops.Alfalfa(Medicago sativa)is an important forage,which is rich in nutrients and high yield,while the quality and production will be reduced due to abiotic stresses.More resistance genes have been applied in plants respond to abiotic stress,in which LEA plays an important role.In order to study the resistance function of MsLEA4-4,this study successfully cloned the MsLEA4-4 gene from alfalfa and carried out bioinformatics analysis.At the same time,it was transferred into Arabidopsis thaliana to study the resistance function of MsLEA4-4.The main research results were as follows:1.MsLEA4-4 was cloned from Medicago sativa by RACE-PCR.The c DNA sequence contained a 498 bp open reading frame(ORF),encoded 166 amino acids.The DNA sequence was 1001 bp,included 2exons and 503 bp intron sequence.The amino acid sequence of MsLEA4-4 revealed lack of cysteine(W)and tryptophan(C),and contained 5 repeating sequences of TAQAAKEKTQQ.The sequence alignment analysis showed high similarity with Medicago truncatula and Trifolium pratense,and the evolutionary tree analysis showed that MsLEA4-4 was clustered into LEA4-4 with Mt LEA4-4,so it was named MsLEA4-4.There were a large number of α-helix in MsLEA4-4 amino acid,and most amino acid located below 0,which was belonged to hydrophilic protein.2.A 490 bp promoter sequence of MsLEA4-4 was cloned from alfalfa genome by gene walking method.The analysis of promoter sequence structure showed that there were 3 abscisic acid(ABA)responsive elements(ARBE),1 gibberellin(GA)responsive elements(P-box),2 cis-elements(GCN4,Skn-1)involved in endosperm synthesis,10 cis-elements related to light regulation and 1 cis-element related to circadian rhythm in MsLEA4-4 promoter sequence.3.Quantative real-time PCR results showed that MsLEA4-4 expression levels were more abundant in alfalfa seed and pod.The expression vector of MsLEA4-4::pCAMBIA1301 was constructed and transformed into Arabidopsis thaliana by agrobacterium-mediated method.GUS staining showed that MsLEA4-4 promoter was only expressed in transgenic Arabidopsis flower and silique.The results showed that MsLEA4-4 was a seed-specific gene.4.According to the cis-elements in MsLEA4-4 promoter,alfalfa was subjected to low temperature,drought,salt,heavy metal,exogenous hormone and different light stress.The results of q RT-PCR showed that all the stress induced the expression of MsLEA4-4.It suggests that MsLEA4-4 may be involved in multiple resistance regulation of alfalfa,and cooperated with the metabolic pathway of ABA to participate in the adversity and photoperiod regulation of alfalfa.5.The pCAMBIA3301-MsLEA4-4 expression vector was constructed and transformed into Arabidopsis.The overexpressed pants were detected by RT-PCR,glufosinate and separation of 3:1 traits,and 7 positive overexpressed plants were obtained.OE1,OE4 and OE8 three overexpressed plants with high expression of MsLEA4-4 were used for drought,low temperature,NaCl,H2O2 stress and ABA treatment.The germination rate of overexpressed plants under NaCl and ABA stress was 5~12 times higher than that of wild Arabidopsis thaliana(WT).The chlorophyll content and biomass of overexpressed plants under various stresses were significantly higher than WT.The survival rate of overexpressed plants under NaCl stress was 4~5.3 times higher than that of WT,and overexpressed plants have more lateral roots than WT.The results of antioxidant enzyme activity analysis of overexpressed plants after various stress showed that the soluble sugar content,SOD,CAT and POD activity in overexpressed plants significantly increased than WT,proline and malondialdehyde content significantly decreased comparied to WT.6.Expressions of ABA metabolic pathway,proline metabolic pathway and sevel genes related to osmotic stress in overexpressed plants were analyzed.The expressions of ABF3,ABI5,NCED5,NCED9,P5 CS and RD29 A genes in overexpressed plants were significantly increased.It indicated that MsLEA4-4transfer into Arabidopsis could induce the expression of other genes related to stress,and the expression gene may enhance the tolerance of transgenic Arabidopsis to various stress in cooperation with MsLEA4-4.The results indicated that there were a large number of α-helices in the MsLEA4-4 gene,it was hydrophilic amino acids.There were a large number of cis-acting elements related to stress in the promoter sequence,so they may have anti-stress function;MsLEA4-4 is seed-specific gene;involved in a variety of stress control of alfalfa;overexpressing MsLEA4-4 in Arabidopsis thaliana,the growth index and physiological index ofoverexpressed plants under stress were significantly higher than WT,which improved its resistance to stress.