披碱草(Elymus dahuricus)焦磷酸化酶基因EdHP1的克隆及功能鉴定

Isolation and Functional Analyses of a H^+-Pyrophosphatase Gene,EdHP1,from Pasture(Elymus dahuricus)

为了从抗逆性优良的牧草中挖掘新的耐盐、抗旱基因,进而为作物分子育种提供优良的基因资源,采用RACE(Rapid-Amplification of cDNA Ends)方法,克隆了披碱草的焦磷酸化酶基因EdHP1,其全长序列包含2 310 bp,编码770个氨基酸,含有14个跨膜区,是一个典型的膜蛋白,同时包含三个保守区(CS1、CS2和CS3)。氨基酸同源性比较发现,EdHP1与来自大麦等10种高等植物的焦磷酸化酶基因的同源性大于86%。EdHP1基因的表达受干旱、高盐和低温等非生物胁迫的诱导。功能分析证明,EdHP1基因能够显著提高转基因烟草对干旱、高盐胁迫的抗性,可用于作物的抗逆遗传改良。

The E. dahuricus belonging to Elymus of Gramineae, is a perennial pasture and has remarkable tolerance to drought and high salt stresses. The expression of H^＋-Pyrophosphatase（H^＋-PPase） as proton pump leads to H^＋ electrochemical gradient on cell membrane, which provide energy for transport of various ions. In addition, the H^＋-PPase is able to transport Na^＋ into vacuole, which alle-viates Na^＋ toxicity to cells and keep osmotic potential, and enhance plants tolerance to drought and high salt stresses. In this research, to isolate novel stress-relative genes from pasture for improving crop tolerance to high salt and drought stresses in the furture, a novel H^＋-PPase gene （EdHP1） was isolated from E. dahuricus using RACE method （Rapid-Amplification of cDNA Ends）. Sequence analysis revealed that the full length cDNA of theEdHP1 gene was 2 310 bp, encoding a protein of 770 amino acid residues, and was a typical membrane protein with 14 trans-membrane domains and three conservative domains （CS1, CS2 and CS3）, and designated asEdHP1 . Homology analyses indicated thatEdHP1 had high similarity with H^＋-PPases of other plant species including barley etc （minimal homology was 86%）. The expression analyses indicated that the expression of EdHP1 were induced by drought, high salt and low temperature stresses in E. dahuricus. The functional analyses indicated that the over-expression of EdHP1 enhanced tolerance tolerance of transgenic tobacco plants to drought and high salt stresses, suggesting thatEdHP1 might be useful for improving tolerance to environmental stresses in crop plants.