Study On Improving Efficiency Of Cotton Transformation Using Virulence Genes Of Agrobacterium Strains

Cotton is the most important economic crop in Xinjiang .It occupies main station in national economy .In present-day Genetic Breeding ,it is important approach to make use of gene engineering to transfer foreign gene into cotton cell .But at present ,all kinds of transformation methods are set a limit to cotton as far as development of plant transformation technique .The study used virulence protein genes on the Ti plasmid to help transformation .In this process , liposome packed vir genes and target gene ,then transfer them into cotton via pollen-pathway transformation technique .NLSs ,formed by VirD₂ & VirE₂ transiently expressing in vivo ,lead target gene into nucleus .So we would establish high efficiency system of cotton genetic transformation by this method in order to improve transformation efficiency and genetic stability.[Object]to use VirD₁,VirD₂ & VirE₂ ,which play key role during Agrobacterium transformation ,we used Xinluzao No.13 as the receptor of pollen-pathway technique via liposome packing vir genes and target gene out of cotton cell .Transformation efficiency of improved pollen-pathway transformation technique was detected by kanamycin selecting and PCR .At last , feasibility of transformation technique was made sure by evaluating the technique .[Content]1) verified plant express vector pUC-pBin-VirD₁ , pUC-pBin-VirD₂ , pUC-pBin-VirE₂ &pBin-GFP .These vectors were prepared for pollen-tube pathway transformation.2) According different combinations ,we transferred express vector of VirD1 ,VirD2 ,VirE2 &GFP packed by liposome out of cell .3) Reconstructed a new GFP gene to make it be suitable for expressing in plant .we introduced signal peptide at 5 end and specific sequence (KDEL) retaining ER at 3 end.[Result]1) Had triumphantly constructed plant express pBLG-pR1a-GFP and obtained transgene tobacco via Argrobacterium-mediated transformation2) Transformation efficiency was improved obviously with help of three virulence protein genes .More efficiency was obtained by Liposome packing out of plant cell.The study will offer a new pathway for cotton genetic transformation .It will make practical sense for improving efficiency and course of cotton breeding.