Cloning And Characterization Of A Novel DREB Transcription Factor Gene MrDREB1 From The Medicago Ruthenica

In this study,primers were designed based on the squences of DREB in the plants of Medicago falcata recorded in the NCBI,778 bp full-length cDNA which named MrDREB1(GeneBank:MF977759)was cloned by RACE and RT-PCR from medicago ruthenica seedlings sterile culture for five weeks.The bioinformatics analysis shows that the protein of MrDREB1 was highly conserved and neither signal peptide structures nor trans-membrane structure were found,instructing them to be non-secretory protein and non-transmembrane protein.The phylogenetic relationship shows that the DREB transcription factors of medicago ruthenica is most close to the factors of medicago truncatula and medicago sativa.To study the function of the MrDREB1,the technology of GateWay was used to construct MrDREB1 overexpression vector and tasiRNA vector,the empty vecter was used as control.The recombinant vector was transformed into Agrobacterium strain Arqua1 via the freeze-thaw method.Two weeks transgenic hairy roots in medicago ruthenica seedlings were subjected with 0??300mM Mannitol?200mM NaCl using Agrobacterium rhizogenes-mediated transformation of Medicago ruthenica roots forten days.Each experiment performed three replications.Then,all of the roots were calculated its length and fresh weight by single plant of root tissue.The results showed that the transgenic roots with overexpression vector are much longer than empty vecter under abiotic stress.In contrast,silencing the gene of MrDREB1 in transgenic roots are shorter than control.It can be concluded that transgenic medicago ruthenica of MrDREB1 exhibited an increased plant tolerance under abiotic stress.The real-time quantitative PCR technique was used to analyse the expression characteristic of MrDREB1 gene.The results showed that relative gene of MrCAS15 A,MrPP2C with overexpression MrDREB1 was increased in the transgenic medicago ruthenica,which were contrary with silencing the gene of MrDREB1.It is also proved that transgenic Medicago ruthenica DREB1 transcription factor can change the expression of downstream gene.