Cloning And Function Identification Of A Dreb-like Gene RcDREB1in Roegneria Ciliar

Abiotic stress of drought, high-salinity and low temperature are the main factorsaffecting plants growth and development and influencing the agriculture production in ourcountry. On resistance abiotic stress, research had been changed from function genes whichencoded some specific product to cloning switch gene, including signal transduction andgene expression regulation genes. Separate and transfer resistance gene from relatives wildplants, it is important that the resistant genes can be expressed in transgenic plants, andhad become a new way to for resistance cultivar breed.In this paper, using a pair of degeneracy primers designed according to the conservedamino acid sequences of AP2transcription factor family, a full-length cDNA of aDREB-like gene was cloned firstly from Roegneria ciliaris through RT-PCR and RACEtechnologies, and named as RcDREB1. The results showed that the full-length cDNA ofRcDREB1is1171bp, with an untranslated region of57bp at5’terminal and a completePolyA tail at the3’ end, with an open reading frame (ORF) of837bp and encoding apolypeptide of278amino acids, no intron, it contains a conserved AP2domain whichcomposed of60amino acids, fourteenth bits and nineteenth bits for valine (V) andglutamate (E). It is suggested that this gene be a new transcription factor member in theAP2superfamily. Phylogenetic tree analysis showed that RcDREB1shares a highsimilarity with the DREB-like genes in Triticum aestivum. Similar to97%in amino acidsequence.Construction of the prokaryotic expression vector pET30a-RcDREB1. TheSDS-PAGE polyacrylamide gel electrophoresis shows that the target gene RcDREB1expressed in Escherichia coli. Calculation according to the amino acid sequence, theprotein molecular weight is about30kD, and plus the expression vector pET30a S-Tag tagprotein, the expression of the fusion protein with molecular weight about34.6kD.Using real-time quantitative PCR, we found that all of the stresses of high salt,drought, heavy metal (CdCl2), and low temperature could up-regulate RcDREB1expression, but only high salt showed more strong induction of RcDREB1.Construction of eukaryotic expression vector pGAD-RcDREB1, DREB DNA binding domain was verified by yeast one-hybrid experiment. The fusion expression vector into theyeast strain, the transformants were selected on the SD/Leu-,Ura-,His-plate with l0mM3-AT, detected the expression of report gene. The results showed that the RcDREB1protein has DNA binding domain function, and possess an ability to specifically bind thedrought-responsive DRE element.In order to further analysis function of the RcDREB1transcription factor gene whichwas cloned, this study also constructed the expression vector pBI121-RcDREB1containingCaMV35S promoter. Through leaf-disc dipping method mediated by Agrobacteriumtumefaciens were transformed into tobacco, has obtained the Kan resistance of tobacco T0generations of RcDREB1transgenic plant.These RcDREB1This DREB-like gene could be useful in laying the foundation forthe analysis of Triticeae evolution and application in transgenic wheat breeding.