Cloning And Heterogenous Expression Of Plasma Membrane H～+-ATPase Gene PePMA Of Populus Euphratica Oliv.

Populus euphratica Oliv. is a kind of arbor with high salt tolerant. Many investigation have showed that PM H⁺-ATPase is closely related to that character of the tree.Using homology based strategy, the putative PM H⁺-ATPase from Poulus euphratica Oliv. was cloned. The isolated 3 210 bp cDNA contains a single 2 862 bp open reading frame (ORF) which encodes a putative H⁺-ATPase protein of 953 amino acid residues, with the significant homology to plasma membrane H⁺-ATPase of Prunus persica, Phaseolus vulgaris, Sesbania rostrata and Daucus carota. The predicted molecular weight for this protein is 104 553 Da and estimated PI is 8.6. Ther are 10 transmembrane structures. The N-terminal and C—terminal are both in the cytoplast. The copy number analysis revealed multiple copies of the PM H⁺-ATPase in P.euphratica genome.In the presented studies, E.coli expression vector was constructed by subcloning P.euphratica PM H⁺-ATPase cDNA into E.coli expression vector and transformed to E.coli strain BL21. No specific protein band was observed in subsequent SDS-PAGE assay indicating non-expression of the target gene in E.coli cells. Yeast was also used as the host for expressing P.euphratica PM H⁺-ATPase cDNA. To get it, yeast expression vector was constructed by inserting thee target cDNA into a yeast expression vector under the control of GAL1 promoter and transformed into a yeast strain, INVSc1. The taget protein was expressed by induction of galactose and purified by Ni²⁺-nitrilotriacetic acid chromatography. The gene was subcloned into a yeast expression vector AINE181 with constitutive promoter. The expression construct was transformed into a mutative yeast strain, RS-72, devoid of its endogenous H⁺-ATPase PMA1.The result shows that the transformed gene can complement PMA1 of yeast.