Cloning And Salt Tolerance Function Analysis Of LpMATE1 Gene From Perennial Ryegrass

Perennial ryegrass(Lolium perenne L.)is an important high-quality cold-season turf grass and forage grass,which belongs to Lolium and Gramineae,has the characteristics such as fast growth rate,high nutritional value,high grass yield,barren resistance and extensive management resistance.In China,there is a wide saline-alkali soil area but the salt-alkali tolerance of perennial ryegrass is at a medium level,so its popularization and application are greatly limited.In this study,the LpMATE1 gene fromMATE family significantly induced by salt stress was cloned from perennial ryegrass based on previous RNA-Seq data,and the function of LpMATE1 gene was systematically verified.The results of this study are of great significance for breeding new Perennial ryegrass varieties with salt tolerance,establishing and perfecting the genetic transformation system of perennial ryegrass,and studying the function and molecular regulation mechanism of LpMATE1 gene in plant salt tolerance.The main results are as follows:1.The LpMATE1 gene,which is significantly induced by salt stress in perennial ryegrass was mined and cloned by RNA-Seq technique.The gene has an open reading frame of 1650 bp and encodes a polypeptide chain containing 549 amino acids.Bioinformatic prediction shows that this gene encodes a class of hydrophobic transmembrane proteins with high stability located on cell membranes.Homology analysis of this gene and other species MATE family genes showed that it has high homology with OsMATE34 gene.2.The subcellular localization fusion expression vector pBI121-eGFP:LpMATE1 of perennial ryegrass was constructed,and Agrobacterium mediated transient transformation of tobacco was carried out.The localization of the fusion protein on the cell membrane was observed by laser scanning confocal microscope.3.In order to preliminarily verify the function of LpMATE1 gene in plant salt tolerance,the overexpression vector pRI101-AN35S:LpMATE1 was constructed in Arabidopsis thaliana and transformed by Agrobacterium-mediated flower dipping method.After Kana(kan)resistance screening and PCR identification of positive plant,positive transformants with LpMATE1 overexpression in T3 generation Arabidopsis thaliana were obtained.The salt-tolerant phenotype of transgenic Arabidopsis thaliana was verified.The results showed that the germination rate,root length and fresh weight of transgenic Arabidopsis thaliana were significantly higher than those of wild type under NaCl treatment.4.To further verify the function of LpMATE1 gene in salt tolerance of perennial ryegrass,the ultra-expression vector PANIC-6B:LpMATE1 was constructed,and the callus of perennial ryegrass was infected by Agrobacterium tumefaciens and cultured until differentiation and rooting.The ultra-expression positive plants of perennial ryegrass LpMATE1 were obtained by GUS staining and PCR identification.The results showed that under salt stress,the wilting degree of transgenic perennial ryegrass was lighter than that of wild type,and the relative growth rate,relative growth biomass,relative conductivity and POD activity of transgenic perennial ryegrass were better than those of wild type.Based on the above research results,LpMATE1 gene of perennial ryegrass plays an important role in improving salt tolerance of perennial ryegrass,so as to provide an important theoretical basis for salt-tolerant breeding of perennial ryegrass.