Cloning And Functional Analysis Of Drought Tolerance Related Genes (GhTrx, GhGR) On Gossypium Hirsutum L.

Drought，as the main facter influencing cotton yield and quality, has been growing a tranendencydecreasing elements of cotton in China since1980s. Every year, the production amounts of cotton hasreduced by more than30%for drought, especially in Xinjiang province, the main cotton producing region,,where drought has been the main factor to limit cotton production. Drought, as the key factor to affectcotton output in all kinds of biological constraints is second only to the biological stress caused by pest anddisease, directly affecting the normal metabolism of cotton growth and development. With cotton cellsdehydrated due to drought-stress, membrane system and enzyme system was destroyed, the cell functionslost, eventually led to plant metabolism disorders. And then the protein synthesis system broke down andhormone balance caused damaged. Therefore, it provided a good approach for improving cotton droughtresistance capacity and cultivating drought resistance breeds that studying cotton drought resistantmechanism with molecular biology methods, which would open up new path.In this research, the drought resistant material Zhong H177was studied as research material. TwoEST of genes were excavated SSH cDNA librarys, cloned by RACE and RT-PCR, named GhTrx andGhGR separately. Drought resistant related expression analysis studied for the two genes by Real-timePCR. More in-depth researches for GhTrx and GhGR gene were studied by GFP sub-cellularlocalization.Through arabidopsis and gene gun transgenic technologies to analysis genes function.The full length cDNA sequences of GhTrx and GhGR were obtained. The full length of GhTrx is1340bp, including5’-UTR191bp, complete ORF864bp and3’-UTR285bp, which encodes a polypeptide of287amino acids. The full length of GhGR is1035bp, including5’-UTR122bp, complete ORF792bp,3’-UTR121bp, which encodes a polypeptide of263amino acids.Bioinformatics analysis showed that GhTrx protein has the similarity of the70%,72%and76%toPopulus trichocarpa, Ricinus communis and Arabidopsis thaliana, Phylogenetic analysis showed GhTrxwas the closest to Ricinus communis,It forecast that GhTrx protein contains3conserved domains viaconservative structure domain analysis, Secondary structure prediction showed that the protein iscomposed of α-helix, β-sheet, turn and random coil.Bioinformatics analysis showed that GhGR has highsimilarity of90%,91%and91%to Populus trichocarpa,Ricinus communis.and Vitis vinifera respectively.Phylogenetic analysis showed GhGR was much closer to Vitis vinifera.It forecast that GhGR proteincontains1transmembrane domain via transmembrane analysis.Its express condition was analyzed by Real-time PCR at different organizations and different periodsin zhong H177materials. The experimental results demonstrated that GhTrx and GhGR protein could beinduced by drought tolerance. Induced by drought stress, GhTrx and GhGR showed high expression levelsand roots on drought-stress materials Zhong H177especially outstanding higher expression in root and leafthan stem. Thus We speculate that GhTrx and GhGR gene increases of its expression on drought-stresscondition in root and laef for againsting outside drought environment.Tt may plays a role in improvingdrought resistance capacity of Gossypium hirsutum L. Experimental results revealed that GhTrx and GhGRgenes play an important role against drought-stress of Gossypium hirsutum L The Real-time PCR detectionfound it is closely correlated for the genes expression changes and the drought-stress.Plant fluorescence expression vector GFP-GhTrx and GFP-GhGR were built and transformated intoonions endepidermis cell by particle gun. The result showed that the GhTrx protein was located at cellmembrance, GhGR protein was located at cell membrance and cell nucleus. Eukaryon expression vector pBI121-GhTrx was built and transformated into Arabidopsisthaliana.which was acquired transgenic T3arabidopsis seeds. The gene function analysis resultsShowed that the transgenic plants can improve arabidopsis drought-stress.The GhTrx genes thatoverexpression in arabidopsis can improve the ability of fighting drought.Eukaryon expression vector pBI121-GhTrx and pBI121-GhGR were built and transformated intocottons of drought resistant materials and drought sensitive material which were acquired geneticallyengineered cotton bolls and seeds which were78bolls and1128seeds. This is the foundation of droughtstress function analysis of GhTrx and GhGR genes in the cotton.