Cloning And Function Identification Of MfERF009 And MfERF072 Genes In Medicago Falcat

Medicago falcata is a considerable germplasm resource in the breeding of legume plants having high resistance to abiotic stress,the ERF family are transcriptional factors that play crucial roles in plants' whole life as well as response to adversity.However,the practional application value of ERF family in breeding ne w cultivars remains to be further explored.In this study,Medicago falcata was used as the experimental material to clone and analyze the two ERF gene family members on the basis of the sequencing data of transcriptome acquired by the research group 2years ago,the main results as following:1.Two genes that play crucial roles in plant responsing to stress were isolated from alfalfa by RT-PCR(Transcription-Polymerase Chain Reaction),According to bioinformatics analysis,the two genes are members of the ERF family,named MfERF009(GeneBank No.MH817428)and MfERF072(GeneBank No.MN970144).2.To study the function of MfERF009 and MfERF072,using the GateWay technology to construct three expression vectors of each gene based on pKGWRR vector.The plasmids consist of super-overexpression,control and tasiRNA interference vector.3.MfERF009 and MfERF072 were transferred into truncated alfalfa A17 by Agrobacterium rhizogenes Arqua1-mediated hair root transformation,simulating abiotic stress test.The results showed that the plants' which were interferenced MfERF009 gene growth level under drought and high concentration of ABA was significantly better than that of the control group.That means this gene plays an important role in responsing to drought stress,to a great extent,accrossingg the ABA signaling pathway.the plants overexpressing MfERF072 gene explain a strong ability to resist cold,indicating this gene have regulatory effect in plants' response to cold stress.Meanwhile,the levels of SOD,MDA and Pro were detected,which further indicated that these two genes could regulate the growth state of plants in adversity.The expression levels of MtLEA14,MtRD22 and MtCAS15,which play important roles in plants' response to diversity,were analyzed by qRT-PCR.The results showed the expression of MtLEA14 and MtRD22 were up-regulated by MfERF009,indicating that MfERF009 gene can positively regulate the expression of MtLEA14 and MtRD22 to improve the drought resistance of plants.MtLEA14 and MtCAS15 were up-regulated by MfERF072,indicating that MfERF072 can promote the plant's low temperature tolerance by positively regulating the expression of MtLEA14 and MtCAS15 genes.4.MfERF072 and MfERF009 were successfully transferred into truncated alfalfa R108 through the genetic transformation system mediated by Agrobacterium in blade disc.One regeneration seedling transferred MfERF009 and three independent positiveR108 regeneration seedlings transferred MfERF072 were obtained,which lays a foundation for further research on genes function.