Cloning And Functional Characterization Of MATE1 Gene From Alfalfa(Medicago Sativa L.)

Multidrug and Toxic Compound Effluents(MATE)protein transport secondary metabolites,hormones and metal ions,and participate in a variety of physiological activities in plants.Alfalfa is an important forage crop with strong resistance to stress.MsMATE1 gene and its promoter in alfalfa were cloned in this study.Plant expression vectors pBI121-MsMATE1 and pBI121-MsMATE1pro::GUS were constructed,pBI121-MsMATE1 was transferred into tobacco.The function of MsMATE1 was preliminarily identified.The main research results are as follows:1.Cloning and bioinformatics analysis of MsMATE1The MsMATE1 gene was cloned by RT-PCR.MsMATE1 is 1470 bp,codes for 489 amino acids,and has the Mat E domain.MsMATE1 protein is a hydrophobic protein,with the highest contents of Alpha helix and Random coil in the secondary structure and contains 12 transmembrane domains.Phylogenetic analysis showed that the relationship between MsMATE1 and MtMATE was the closest.2.Expression characteristics of MsMATE1 geneThe relative expression of MsMATE1 in different tissues of alfalfa was detected by qRT-PCR.The results showed that the expression order of MsMATE1 was shoot >root > leaf,and the expression of MsMATE1 in shoot was significantly higher than that in root or leaf(P<0.01).The expression of MsMATE1 was up-regulated in roots,stems and leaves under iron excess or iron deficiency stress,and the expression of MsMATE1 was significantly increased in roots of alfalfa under iron excess.The experimental results showed that MsMATE1 responded to iron stress.3.MsMATE1 promoter cloning and construction of plant expression vectorThe MsMATE1 promoter with 1598 bp,contains multiple cis-acting elements related to abiotic stress and hormones was obtained.pBI121MsMATE1pro::GUS plant expression vector was constructed.4.Vector construction and genetic transformation of MsMATE1 genePlant expression vector of pBI121-MsMATE1 was constructed and transferred to GV3101 by leaf discs method.A total of 14 resistant plants were screened by Km,among which 4 transgenic positive seedlings(T4,T6,T7,T13)were identified by PCR.5.Physiological indicators of MsMATE1 transgenic tobacco under iron stressThe content of chlorophyll was significantly higher in MsMATE1 transgenic tobacco than that of WT under iron deficiency or iron excess.The activities of antioxidant enzyme(SOD,POD,CAT)and soluble protein content were significantly higher in transgenic tobacco than that of WT under iron deficiency or iron excess,the content of malondialdehyde(MDA)was significantly lower than that of WT.It showed that MsMATE1 gene improved tolerance to iron stress of transgenic tobacco.