Cloning And Functional Analyses Of AmDREB2C And AmAtpD Genes From Ammopiptanthus Mongolicus

Ammopiptanthus mongolicus is an unique and strong stress-resistant broadleaf shrub in the northwest desert region of Inner Mongolia. Previous expression profile analysis by RNA-Seq technology showed that the expression of AmDREB2C and AmAtpD, encoding a DREB transcription factor and a chloroplast ATP synthase8subunit respectively, were significantly up-regulated under low temperature and drought stresses. In order to study their functions and mechanisms in response and resistance to abiotic stresses, we first cloned these genes and analysed their expression profiles, and then characterized the function of AmDREB2C by transgenic technology. The main results are as follows:(1) The complete coding region cDNA and gDNA of AmDREB2C were cloned by PCR method. Both the cDNA and gDNA regions consist of1191bp, indicating no introns in this gene. The predicted polypeptide of AmDREB2C consists of396amino acids, with a molecular weight of43.80kDa and an isoelectric point of4.72. The polypeptide contains an AP2domain and a nuclear localization signal, and most probably locates in nucleus.(2) The expression of AmDREB2C was significantly induced by cold and drought stresses, and the up-regulation under cold stress was comparatively lasting and strong.(3) The cDNA fragment of AmDREB2C was successfully ligased to plant over-expression vector and inducible expression vector respectively and introduced into Arabidopsis by Agrobacterium-mediated transformation. The phenotypic characterization of the transgenic lines suggested that AmDREB2C functions as a positive regulator of the tolerances to cold, heat and drought stresses. Compared with wild type plants, the transgenic lines were more sensitive to exogenous ABA and had no significant effect on salt tolerance. Moreover, over expression of AmDREB2C did not cause obvious growth retardation, and the over-expression lines showed no significant difference with the inducible-expression lines in the improvement of stress resistance.(4) The complete coding region cDNA and gDNA of AmAtpD were cloned by PCR method. It contains of720bp, therefore no introns. The predicted polypeptide consists of239amino acids and has a molecular weight of27.00kDa and an isoelectric point of9.67. The polypeptide was predicted to be hydrophilic, without transmembrane domain, and localized in chloroplasts.(5) The cDNA fragment of AmAtpD was successfully ligased to plant over expression vector and was introduced into Arabidopsis by Agrobacterium-mediated method. The work laid a foundation for further functional analysis of this gene.