Cloning and characterization of vacuolar H+-pyrophosphatase gene (AnVP1) from Ammopiptanthus nanus and its heterologous expression enhances osmotic tolerance in yeast and Arabidopsis thaliana

• DOI: 10.1007/s10725-016-0215-6
• Publication Date: 2016-09-12T13:11:11Z
• Authors: Hao Qiang Yu; Nan Han; Yuan Yuan Zhang; Yi Tao; Lei Chen; Yan Ping Liu; Shu Feng Zhou; Feng Ling Fu; Wan Chen Li
• Keywords: Heterologous expression; Osmotic shock; Pyrophosphatases

Vacuolar H+-pyrophosphatase (H+-PPase) is one of the proton pumps involved in the response of plants to osmotic stress. In the present study, the vacuolar H+-PPase gene (AnVP1) was cloned from an epibiotic super-xerophytic plant, Ammopiptanthus nanus, by using rapid amplification of cDNA ends. The full-length cDNA was 2684 bp long, containing an open reading frame of 2298 bp that encoded 765 amino acids with 13 transmembrane regions. The putative protein shared high homology, conserved domains and amino acid residues with the H+-PPase proteins from other plants. The result of quantitative real-time PCR showed that the expression of the AnVP1 gene was strongly induced by salt, dehydration and abscisic acid, and inhibited by cold and heat. Complementary of the AnVP1 gene in the yeast mutant ena1 improved its salt and osmotic tolerance. Heterologous expression of the AnVP1 gene in the Arabidopsis mutant significantly restored its salt and drought tolerance. Under salt and drought stress, the transgenic plants exhibited more vigorous growth, more developed root system, higher proline content and relative water content, and lower malondialdehyde content and relative electrolytic leakage, as well as more Na+ and K+ accumulation in leaf, than the untransformed mutant plants. All these results demonstrate that the AnVP1 gene encodes a functional vacuolar H+-pyrophosphatase that plays a crucial role in adaptation of A. nanus to abiotic stress. After further evaluation for its tolerant function, this gene is hopeful to be used to genetic engineering for improving abiotic tolerance of commercial crops.