Isolation And Functional Analyses Of A H~+-Pyrophosphatase Gene, EdHP1, From Pasture (Elymus Dahuricus)

H⁺-pyrophosphatase (H⁺-PPase) is a kind of enzyme involved in H ⁺-translocating and PPi-dependent hydrolyzation process. H⁺-PPase hydrolyze PPi to product free energy, which is coupled with H ⁺ transmembrane translocating to drive H ⁺ proton from into from the cytoplasm into the vacuole with H⁺-ATPase, which creates a cross-tonoplast electrochemical gradient. This electrochemical gradient driving inorganic ions and other solute access to the vacuole, which can not only maintain ion balance and infiltration balance of cells but also reduce the poison of inorganic ions (such as Na⁺ and Cl^-). In addition, it creates vacuole's acidification and Cytoplasm's alkalization, which maintain normal physiological and biochemical reactions in plant cytoplasm. The E. dahuricus belonging to Elymus of Gramineae, is a perennial pasture tolerating to drought and high salt stresses. In this research, a novel H⁺-PPase gene (EdHPl) was isolated from E. dahuricus using RACE method (Rapid-Amplification of cDNA Ends). Bioinforrnatics analysis, subcellular localization assay, analysis of expression pattern and function analysis had been completed.(1) Bioinformatics analysis showed that the EdHPl gene contained 2310 bp openreading frame(ORF) and 356 bp 3'Untranslational region(3'UTR), encoding a protein of 770 amino acid residues, which contained 14 trans-membrane domains and three conservative domains (CS1, CS2 and CS3) and was a typical membrane protein. Homology analyses indicated that EdHP1 had high similarity with H⁺-PPases of other plant species containing barley etc, and minimal homology was 86% , and maximal homology was 96 % with barley, suggested that H⁺-PPases is vary conservative in high plant.(2) Subcellular localization assay showed that EdHP1 was located on the plasma membrane of plant cells. (3) Analysis of expression pattern showed that expression of EdHP1 gene expression induced by drought, high salt and low temperature, low phosphorus nutrition, low potassium nutrition and heavy metals Cd²⁺ abiotic stress in E. dahuricus. Expression of EdHP1 increased gradually from 1 to 24 h under drought, high salt, low phosphorus nutrition and low potassium nutrition stress, and maximal expression was at 24 h. Under cold stress expression of EdHP1 declined followed with increasing expression, and maximal expression was at 5 h and began declining after 12 h. Under Cd²⁺ stress expression of EdHP1 increased gradually from 1 to 12 h, and-declined after 24 h. (4) Functional analysis showed that EdHP1 gene can significantly improve the transgenic tobacco tolerance to drought, high-salt, low phosphorus nutrition, low potassium nutrition and heavy metals Cd²⁺ stress. Under various abiotic stresses, the length of above and under ground in transgenic tobacco were longer than wild-type, and the activity of H⁺-PPase in transgenic tobacco were higher than wild-type. The contents of Na⁺and Cd²⁺ in wild-type were higher than transgenic tobacco under high-salt and Cd²⁺ stress, which suggested that EdHP1 gene translocated Na⁺ and Cd²⁺ to outside of plant cell. The contents of K⁺ in transgenic tobacco were higher than wild-type under potassium nutrition stress, suggested that EdHP1 gene promoted K⁺ into inside of plant cell. All results showed that EdHP1 gene could not only significantly enhance the transgenic plants tolerance to drought, high-salt, low phosphorus nutrition, low potassium nutrition and heavy metals Cd²⁺ stress but also promoted plants to absorb nutritional element such as phosphorus and potassium.EdHP1 was isolated from E. dahuricus for the first time.It was a member of H⁺-PPase family, and was localized on the plasma membrane. Expression pattern of EdHP1was analyzed generally through RT-PCR and Northern methods for the first time. Its function was definite by general analysis .Thus, EdHP1 not only was useful to study the function of H⁺-PPase family but also was used to improve crops tolerance to various abiotic stresses.At the same time,our studys offer the methods and theoretics for others mol-biology research.