Analysis Of Heavy Metal Detoxification Function Of MsYSL6 Gene Alfalfa

YSL transporters have a wide range of long-distance metal transport activities and have been extensively studied in plant metal homeostasis and detoxification.As high quality forage,metal nutrient status directly affects the safety of alfalfa growth and utilization.In this study,the function of MsYSL6 gene in alfalfa transport regulation under cadmium and high copper stress was preliminarily verified by molecular biological means.The results are as follows:1.Cloning and GUS analysis of MsYSL6 promoter fragmentA 1738 bp MsYSL6 gene promoter fragment was cloned from Medicago sativa Zhaodong.Element analysis showed that the MsYSL6 promoter fragment contained a certain number of core elements and hormone related cis acting elements.GUS staining of alfalfa hairy roots determined the activity of MsYSL6 promoter.After cadmium and excess copper stress,the staining of Alfalfa hairy roots transformed with MsYSL6 promoter became deeper and larger,indicating that MsYSL6 promoter responded to cadmium and excess copper stress.2.Subcellular localization of MsYSL6 proteinThe p BWA(V)HS-MsYSL6--GLosgfp expression vector was constructed and transiently transformed into tobacco leaf by injection.The observation by laser confocal fluorescence microscope showed that MsYSL6 protein might be located in cytoplasm,nucleus and cytoplasmic membrane.3.Analysis of MsYSL6 gene expressionThe results of quantitative RT PCR showed that the expression of MsYSL6 was significantly increased under cadmium or excess copper stress in alfalfa roots,indicating that MsYSL6 was induced by cadmium or excess copper.Abscisic acid induced,while salicylic acid inhibited the expression of MsYSL6.The expression of MsYSL6 was not affected by methyl jasmonate.4.Physiological response of transgenic MsYSL6 tobacco to cadmium or excess copper stressCadmium and high copper stress transformed MsYSL6 tobacco.The contents of chlorophyll and soluble sugar in transgenic tobacco were significantly higher than WT,while the contents of H2O2 and O2-were significantly lower than WT,indicating that MsYSL6 gene enhanced the resistance to cadmium and high copper in tobacco.Under cadmium stress,the cadmium content in roots and shoots of transgenic tobacco was significantly higher than WT,and the iron content in roots and shoots was also extremely significantly higher than WT,indicating that MsYSL6 promoted the absorption and transport of cadmium and iron under cadmium stress.Under high copper stress,copper content in roots and shoots of transgenic tobacco had no significant difference with WT,while iron content in roots was significantly higher than WT.The results showed that MsYSL6 promoted iron absorption under high copper stress.The results showed that MsYSL6 could promote the absorption and transport of cadmium and iron under cadmium stress.Under high copper stress,MsYSL6 may promote iron absorption.