Construction And Genetic Transformation Of Pina~m, Pinb~m, Gsp~m Related Vectors Which Controlling The Grain Texture Of T.monococcum

Grain texture has an important influence on processing quality, and is controlled by thesite of Ha consist of Puroindoline a(Pina-D1)and Puroindoline b(Pinb-D1)which arelocated on short arm of dismal5D chromosome, and also is impressed by the Grain SoftnessProtein-1(Gsp-1). Friabilin is a kind of amino acid which is the product of these genes spreadinto endosperm, and it interacts with lipids at the surface of starch. In this study, the cultivarDV92(T. monococcum)is employed as a donor of genes of Pina~m、Pinb~mand Gsp~m, and fiverelated expression vectors are completed. Totallly,16909mature calli and13199immaturecalli of common wheat cultivar “Kenong199” are respectively used for genetictransformation which is mediated by the gene gun. Regenerated plants are obtained afterBiolaphos screening, and the positive plants are idenfied by PCR. The main results show asbelows:1. Construction of expression vectors: single gene expression vector includingpGEM-pinam, pGEM-pinbmand pGEM-gspm; expression vector harboring tandem genes ofPina~m-Pinb~m(pGEM-Pina~m-Pinb~m); and expression vector harboring tandem genes ofPina~m-Pinb~m-Gsp~m(pGEM-Pina~m-Pinb~m-Gsp~m) were constructed based on the pGEMfinished vectors using genomic DNA of DV92as template.2. Transformation:16909calli induced from mature embryo(scultivar Kenong199)wereobtained by the tissue culture, and out of them,13199calli were bombarded with expressionvector harboring tandem genes of Pina~m-Pinb~m. Out of10130calli induced from immatureembryos(cultivar Kenong199),4522calli were bombarded with expression vector harboringtandem genes of Pina~m-Pinb~m(pGEM-Pina~m-Pinb~m), and5608calli were bombarded withexpression vector harboring tandem genes of Pina~m-Pinb~m-Gsp~m(pGEM-Pina~m-Pinb~m-Gsp~m).3. Resistance selection:5608immture embryos calli were transformation by the genegun, the rersistance was selected by Biolaphos screening, and933regenerating plants were obtained(In our study，some of resistant seedlings were selected by Biolaphos screening).4. PCR detection:11postive plants were detected, the function of grain texture needfurther phenotype identification in next study.In this study, we have constructed expression vector harboring tandem genes ofPina~m-Pinb~m-Gsp~m, which was transformed into cultivar Kenong199. Finally, three genesco-transformation was achieved in comment wheat. This investigation provide a possibility toimprove the grain texture in wheat by the gene gun mediated genetic transformation.