Cloning, Expression And Function Analysis Of A Vacuolar H~+-Pyrophosphatase Gene OpVP In Olimarabidopsis Pumila

The vacuolar H~+-translocating inorganic pyrophosphatase (V-H~+-PPase) can both acidify vacuoles inplants and provide energy for secondary transit system of vacuoles, which plays an important role inadaptation to abiotic stress of plants.Object: We aimed to clone a V-H~+-pyrophosphatase gene from Olimarabidopsis pumila, and to analyze theexpression pattern of it in Olimarabidopsis pumila’s different tissues and seedlings under stress treatments.We preliminarily explored the function of it through overexpressing it in model plants.Methods: To clone the V-H~+-PPase gene from Olimarabidopsis pumila, RT-PCR and RACE methods wereused using the leaf cDNA as PCR template. Expression pattern of OpVP was analyzed by semi-quantitativeRT-PCR and quantitative Real-time RT-PCR (qRT-PCR). Function of OpVP gene was studied throughAgrobacterium-mediated genetic transformation of the tobacco leaf, and floral dip method for mediatedgenetic transformation of the Arabidopsis thaliana. Salt tolerance and physical charateristics of transgenicplants were analyzed. Subcellular location of OpVP protein was analyzed by confocal laser microscopy todetected GFP protein in the root tip cells of transgenic Arabidopsis thaliana of p35S:OpVP-GFP.Results:(1)We identified of a V-H~+-PPase gene from Olimarabidopsis pumila, which was designated asOpVP. The full-length cDNA of OpVP was2698bp, the open reading frame (ORF) of which was2313bpin length and encoded a putative770amino acids. The deduced amino acid sequence of OpVP sharedhighest similarity with Arabidopsis lyrata and Arabidopsis thaliana, with an identity of98.6%,98.4%,respectively. The phylogenetic analysis indicated that OpVP belonged to classⅠtype vacuolar H~+-inorganicpyrophosphatase gene. The molecular weight of OpVP protein is80745.9Da and the isoelectric point (pI)is5.13. Computional analysis showed OpVP is a typical membrane protein with14transmembranedomains. Three-dimensional structural analysis showed OpVP protein is a dimeric protein, which consistsof two monomers.(2)OpVP was expressed in tissues of root, stem, leaf, flower and fruit, but the expressionlevel in leaf and flower is relatively higher than in other tissues. Expression level of OpVP wassignificantly induced by treatment of500mmol/L NaCl,1μmol/L ABA,20%PEG6000,1μmol/L IAA and2μmol/L GA3. Subcellular location experiment indicated that OpVP located mainly in the vacuolarmembrane. After being stressed by200mmol/L NaCl, the contents of chlorophyll a, chlorophyll b and Na~+in transgenic tobaccos are more than wild type tobacco, and contrary to the content of MDA.Conclusion: OpVP belongings to classⅠtype vacuolar H~+-PPase gene. OpVP protein was confirmed tolocate in the vacuolar membrane. OpVP was expressed in tissues of root, stem, leaf, flower and fruit inOlimarabidopsis pumila seedlings. Expression level of OpVP was significantly induced by ABA, PEG6000,IAA and GA3treatment, including NaCl stress. The OpVP-overexpressed tobaccos accumulated more Na~+in leaves and showed increased tolerance to salt stress. In this study, the cloned OpVP gene wasoverexpressed in tobaccos, resulting in the tobaccos’ adaptation of salt stress certainly.