Cloning And Characterization Of Vacuola H~+-PPase Gene AlVP1 From Aeluropus Littoralis

Drought is one of the major environmental factors limiting plant growth and productivity. As a unique proton pump, H+-translocating inorganic pyrophosphatase can energize the transport of inorganic ions across the vacuolar membrane, maintain the osmotic potential of the cell, and enhance the water absorption capacity of plants under water stress, so as to improve the tolerance to drought stress.In this study, the cDNA of H+-translocating inorganic pyrophosphatase(AlFP1) was isolated from a monocotyledonous halophyte Aeluropus littoralis by RT-PCR,3'RACE and 5'anchor PCR technology. The full-length cDNA of AlVP1 was 2802 bp, comprising of a 131 bp 5UTR,370bp 3UTR and 2301 bp ORF encoding 766 amino acids. The deduced amino acid sequence showed a 84%?96% identity to H+-translocating inorganic pyrophosphatase from some higher plants. Hydrophobic analysis showed that the AlVP1 protein has 14 transmembrane domains, and contained three conserved domains of H+-translocating inorganic pyrophosphatase family which are CS1, CS2 and CS3.qPCR result showed that AlVP1 expression was induced by drought, ABA, potassium deficiency, NaCl and cold (4?) stress. Under drought, ABA, potassium deficiency and NaCl stress, AlVP1 transcripts were remarkably up-regulated in roots, but down regulated in leaves. Under cold stress, AlVP1 transcripts in roots were significantly decreased, but remarkably increased in leaves.PTF101- AlVP1 plant expression vector was constructed and transformed into tobacco by Agrobacterium-mediated leaf-disc method. PCR and RT-PCR showed that the AlVP1 gene was integrated into the tobacco genome and expressed. Under drought stress condition, transgenic tobacco plants had a more robust root system, and higher root dry weight comparing with the wild type tobacco plants. Meanwhile, the leaf relative water content, chlorophyll content and superoxide dismutase enzyme activity of transgenic tobacco plants were higher than those of the wild type tobacco plants, and malondialdehyde content was lower than that of the wild type plants. The results indicated that AlVP1 overexpression conferred increased drought tolerance in transgenic tobacco plants.