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

Author: DongJianHui
Tutor: ZhangXiaoKe
School: Northwest University of Science and Technology
Course: Crop Genetics and Breeding
Keywords: Elymus EdHP1 gene Abiotic stress Subcellular localization Functional identification
CLC: S54
Type: Master's thesis
Year: 2008
Downloads: 66
Quote: 1
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Abstract

Pyrophosphorylase hydrogen ions (H ^{-PPase) is a class H ^{transport enzyme, but it is also to pyrophosphate (PPi) as substrate hydrolase. H ^{-PPase PPi hydrolysis produces free energy with H ^{uncoupling membrane transport phase, the formation of proton motive force, and H ^{-ATPase with the cytoplasmic H ^{pumped into the vacuole, you can create the electrochemical gradient across the vacuolar membrane, inorganic ions and other solutes out of the vacuole provides the driving force, both to maintain cellular ion balance and osmotic balance, but also to reduce Some inorganic ions (such as Na ^{) on the cytoplasmic poison; same time, you can make vacuolar acidification and cytoplasmic alkalization, is conducive to the cytoplasm of physiological and biochemical reactions carried out smoothly. Elymus is a perennial grass Elymus quality forage, with a strong drought, salinity capability. RACE method used in this study, was cloned from Elymus hydrogen ions pyrophosphorylase gene EdHP1, analyze biological information of the gene, expression patterns and functions of identification and to determine the hydrogen ion pyrophosphorylase protein subcellular localization . (1) The cloned gene EdHP1 Elymus cDNA sequence which sequence comprises 2310 bp open reading frame and the 356 bp 3 'untranslated region. Bioinformatics analysis showed that the gene encoded 770 amino acids; their protein contains 14 transmembrane domains, is a typical membrane protein, and contains three conserved region (CS1, CS2, and CS3). Amino acid homology found, EdHP1 and from barley and other 10 kinds of higher plants pyrophosphorylase greater than 86% homology with barley pyrophosphorylase homology of up to 98%, showing that H ^{-PPase in higher plants are highly conserved. (2) subcellular localization analysis showed that, EdHP1 located in the plasma membrane. (3) EdHP1 gene expression pattern analysis shows that the gene expression is regulated by drought, high salt, low temperature, low phosphorus, potassium and heavy metals Cd ^{2 and other abiotic stress induction. In drought, high salt, low phosphorus, low potassium conditions, EdHP1 gene expression began to increase from 1 h at 24 h maximum; under low temperature stress, EdHP1 expression first increased and then decreased, 5 h the highest expression, 12 h expression began to decline; heavy metal Cd ^{2 conditions, EdHP1 expression began to increase from 1 h at 12 h maximum, 24 h after weakened. (4) EdHP1 gene function analysis shows that in drought, high salt, low temperature, low phosphorus, potassium and heavy metals Cd ^{2 and other stress conditions, the growth of transgenic tobacco were significantly better than the wild-type tobacco; its aboveground growth was significantly greater than the wild-type control; roots also significantly higher than the wild-type controls developed; against tobacco H ^{-PPase activity was measured discovered transgenic tobacco activity was significantly high compared to the wild-type controls . In the high salt and heavy metals Cd ^{2 stress conditions, transgenic tobacco and wild-type control of Na ^{and Cd ^{2 were determined found wild-type control in Na ^{and Cd ^{2 were significantly high than the transgenic tobacco plants, can be seen EdHP1 Na ^{and Cd ^{2 pumped to the extracellular. Phosphorus and potassium in the conditions, transgenic tobacco phosphorus and potassium content was significantly higher than the control, showing EdHP1 can promote transgenic tobacco phosphorus and potassium absorption. This proves that not only can improve EdHP1 gene in transgenic tobacco resist drought, high salt and heavy metals Cd ^{2 ion stress capacity, and promote plant phosphorus and potassium absorption. This study is the first from Elymus EdHP1 gene was cloned by subcellular localization method proved EdHP1 is H ^{-PPase family of a novel plasma membrane protein, by RT-PCR and Northern methods more comprehensive analysis of the EdHP1 gene response to abiotic stresses rhyme pattern comparison system its function were identified, clear membrane type EdHP1 function. This study not only enriched H ^{-PPase family, but also for the genetic improvement of crop resilience provides a new genetic resources, has a certain theoretical and practical significance.}}}}}}}}}}}}}}}}}}}}}}

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

Abstract

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