Preliminary Verification Of Medicago Alfalfa MfERF022 Gene For Improving Abiotic Stress Resistance And Promoting Nodulation Function

Medicago falcata(Medicago falcata L.)is a kind of cold-resistant,drought-tolerant and nutrient-rich legume pasture,which can be used as an experimental material for the breeding of radon resistance.Based on the analysis of the Medicago falcata transcriptome in the early stage of the laboratory,using gene cloning technology,a 501 bp long Medicago falcata MfERF022 gene(Gene Bank No.MW715671)c DNA fragment was successfully cloned.RT-q PCR was used to detect the expression level of MfERF022 gene at 4℃,-8℃,250 m M Na Cl,300 m M mannitol for 0h,2h,4h,8h,24 h and 48 h.MfERF022 gene was found in roots,stems and leaves.All of the above abiotic stresses have obvious responses,indicating that the MfERF022 gene is involved in regulating the response of plants to abiotic stress.The chromosome walking technique was used to clone the promoter fragment of the MfERF022 gene with a length of 1070 bp,and the promoter was preliminarily verified by GUS staining to have promoter activity.Construct MfERF022-p KGWRR-OE,MfERF022-p KGWRR-RNAi,MfERF022-p KGWRR-CK expression vectors,use hairy root transformation technology to transform the target gene MfERF022 into Medicago truncatula A17 roots.After two weeks of treatment with 300 m M mannitol and rhizobia,the root length and root weight were counted and the root RNA was extracted.Analysis of the data found that after 4℃,-8℃,200 m M Na Cl and 300 m M mannitol,the root weight of Medicago truncatula A17 in the overexpression group decreased compared with the control group,while the root weight of Medicago truncatula A17 in the interference group was reduced.The heavy increase indicates that the MfERF022 gene can respond to abiotic stress and play a negative regulatory role.However,after rhizobia stress,compared with the control group,the number of nodules of Medicago truncatula A17 in the overexpression group increased,while the number of nodules of Medicago truncatula A17 in the interference group decreased,indicating that MfERF022 can respond to rhizobia stress and promote alfalfa and nodules Symbiosis between bacteria.Using RT-q PCR technology to detect,compared with the control group,the expression of Mt RD2 and Mt CAS15 A downstream of MfERF022 decreased in the overexpression group;compared with the control group,the expression of Mt RD2 and Mt CAS15 A downstream of MfERF022 increased in the interference group,indicating that the MfERF022 gene It may negatively regulate the expression of Mt RD2 and Mt CAS15 A.This study preliminarily verified that Medicago falcata MfERF022 can regulate the resistance of Medicago truncatula to abiotic stress and promote the nodulation function,providing a candidate gene for studying the resistance of Medicago falcata.