Expression Vectors’ Construction And Genetic Transformation Of Mutant HMW-GS1Ax1

Processing quality of wheat is largely due to the elasticity and viscosity of wheatgluten, the elasticity of which is mainly determined by the quantity and size of polymericgluten, comprised of high-molecular-weight and low-molecular-weight glutenin subuintscross-linked by inter-chain disulphide bonds. Both of them are major components ofwheat grain protein storaged in the starchy endosperm. The former is highly related toprocessing quality of wheat, especially, the high quality subunits1Ax1and1Dx5+1Dy10.Compared with1Ax1subunit, the effect of1Dx5subunit on flour processing quality ismore prominent.That is because the transgenic lines expressing subunit1Dx5has strongermixing properties than the transgenic lines expressing subunit1Ax1on comparison of theMixograph parameters and gluten protein compositions. It is speculated that this is due toan extra cysteine residue in the repetitive domain of1Dx5subunit.In this study, the1Ax1~*subunit was obtained by using the site-directed mutagenesisstrategy, leading to the serine residue in the repetitive domain of1Ax1subunit replaced bya cysteine residue. And we constructed an eukaryotic expression vector of1Ax1~*gene,which was then successfully transformed into immuture embryos of wheat line L88-31byparticle bombardment. After tissue culture, transgenic plants were obtained, as thenecessary materials for further elucidating the relationship between the cysteine residue inthe repetitive domain of HMW-GS and gluten elasticity. The main results are as follows:(1) The construction of recombinant plasmid pLRPT-1Ax1~*with1Ax1~*gene whichwas cloned by using PCR for site-directed mutagenesis and1Dx5promoter which drovethe specific expression of target gene in the endosperm, was successfully finished.(2) After identification by restriction endonuclease and sequencing analyses, theplasmid pLRPT-1Ax1~*encoding1Ax1~*protein and plasmid pAHC25conferringbialaphos resistance were used for transformation of the wheat variety L88-31.(3) In DNA level, PCR amplifications for the Gus gene and the CaMV35Sterminator were used for the molecular confirmation of the transgenic plants, whoseefficiency of transformation were0.71%and0.48%, respectively. And theco-transformation frequency of the two genes was26.67%. In protein level, SDS-PAGE analysis was performed to detect the expression of target gene, producing4transgeniclines.The results of the present study provided bases for further studying on the function of1Ax1~*gene.