Function Characterization Of Four Stress Genes From Ammopiptanthus Nanus

Ammopiptanthus nanus belongs to Ammopiptanthus genus of the legume family(Fabaceae)together with Ammopiptanthus mongolicus,and is one of relict plants since the disappearance of the ancient Mediterranean in the Tertiary Period.This genus is characterized by its strong tolerance to abiotic stresses such as drought,salinity,poor soil quality,extreme temperatures,and well known as a precious genetic resource for abiotic resistance.Up to date,some literatures are available for cloning and function analysis of resistant genes from A.mongolicus,but few from A.nanus.Betaine aldehyde dehydrogenase(BADH)is a key enzyme in the biosynthesis patyway of glycine betaine,which is an important compatible solute in plant cell.Molybdenum cofactor sulfurase(MCSU)sulfurates and activates molybdenum cofactor,which is involved in the oxidation of abscisic aldehyde into abscisic acid(ABA)catalyzed by abscisic aldehyde oxidase in the last step of ABA biosynthesis.Phospholipase D(PLD)hydrolyzes phospholipids to produce phosphatidic acid,which plays critical roles in growth,development,and stress response of plant.Vacuolar H~+-pyrophosphatase(V-PPase),one of proton pump on the vacuolar membrane,utilizes the energy released by pyrophosphates hydrolysis to activate proton transport into vacuolar.These four enzymes involve in different physiological metabolism pathway and are closely related to growth,developmental and stress response of plant.Some reports are available about gene cloning,function characterization and transgenic application,but few literatures available from A.nanus.Based on the gene cloning by rapid amplification of cDNA ends in our presvious research,the present study was planned to anlysize stress-resistant function of these four genes by comprehensive application of bioinformatics analysis,real-time quantitative PCR,prokaryotic expression,subcellular localization,functional complementation of mutant or overexpression in yeast and Arabidopsis,to provide excellent candidates for transgenic improvement of crop abiotic tolerance.The main results are as follows:(1)The cDNA of the AnBADH gene is 1868 bp in full length,and contains an open reading frame(ORF)of 1512 bp.Its putative protein contains 503 amino acids with molecular weight of 54.71 kDa,isoelectric point of pH5.39,instability index of 28.36,and grand average hydropathicity of-0.015,contains one conserved domian of nicotinamide adenine dinucleotide phosphate-dependent aldehyde dehydrogenase superfamily,but does not contain any transmembrane region or signal peptide.The endogenous expression of the AnBADH gene was induced by high salinity,dehydration,heat,cold stress,and ABA induction in A.nanus.The E.coli line transformed with the AnBADH gene exhibited significantly higher growth rate and survival rate than the untransformed control.Under high salinity and dehydration stress,the heterologous expression the AnBADH gene in Arabidopsis improved root development,their relative water content,betaine and free proline contents were significantly higher,and their relative electrolyte leakage and the malondialdehyde content were significantly lower than the untransformed control.(2)The cDNA of the AnMCSU gene is 2544 bp in full length,and contains an ORF of 2289 bp.Its putative protein contains 762 amino acids with molecular weight of 84.85kDa,isoelectric point of pH6.02,instability index of 37.95,and grand average hydropathicity of-0.225,contains typical MCSU domains,but does not contain any transmembrane region or signal peptide.Bioinformatics prediction and transient expression in tabacoo leaf showed that the AnMCSU protein was localized in the cytoplasm.The endogenous expression of the AnMCSU gene in A.nanus was induced by heat,dehydration,high salinity stress and ABA induction,and inhibited by cold stress.The heterologous expression of the AnMCSU gene in Arabidopsis improved seed germination and root development.Under high salinity and low temperature stress,the transgenic lines accumulated higher ABA and free proline contents,and exhibited higher tolerance to high salinity and chilling stress than the untransformed control.(3)The cDNA of the AnPLD?gene is 2832 bp in full length,and contains an ORF of2427 bp.Its putative protein contains 808 amino acids with molecular weight of 91.58 kDa,isoelectric point of pH5.47,instability index of 43.82,and grand average hydropathicity of-0.418,contains one C2 domain and two HKD motifs,but does not contain any transmembrane region or signal peptide.Bioinformatical prediction and transient expression in tabacoo leaf showed that the AnPLD?protein was localized in the cytoplasm.The endogenous expression of the AnPLD?gene in A.nanus was induced by dehydration,high salinity,cold stress,and ABA induction,and inhibited by heat stress.The heterologous expression of the AnPLD?gene in Arabidopsis improved root development,but inhibited seed germination under the stress conditions.Under high salinity stress and ABA induction,the transgenic lines accumulated lower malondialdehyde content,and exhibited higher tolerance to high salinity than the untransformed control.The endogenous expression of resistance-related genes AtABI,AtNCED,AtRD29A,AtRD29B and AtADH was upregulated.(4)The cDNA of the AnVP1 gene is 2684 bp in full length,and contains an ORF of2298 bp.Its putative protein contains 765 amino acids with molecular weight of 80.13 kDa,isoelectric point of pH5.46,instability index of 23.02,and grand average hydropathicity of0.631,and contains 5 conserved domains and 13 transmembrane regions.The endogenous expression of the AnVP1 gene in A.nanus was induced by high salinity,dehydration stress,and ABA induction,and inhibited by heat and cold stress.The heterologous expression of the AnVP1 gene in the yeast mutant inhibited its sensitiveness to high salinity and osmotic stress.Under high salinity and dehydration stress,the heterologous expression of the AnVP1gene in the Arabidopsis mutant improved root development,and the transgenic lines exhibited significantly higher content of Na~+/K~+,relative water and free porline,and significantly lower malondialdehyde content and relative electrolytic leakage than the untransformed mutant.The above results indicated that the four genes AnBADH,AnMCSU,AnPLD?and AnVP1 play critical roles for abiotic resistance in A.nanus,their heterologous expression enhances resistance of the mode plant,and they are hopeful to be used in transgenic research for crop resistance.