Construction Of Expression Vectors Of Soft Grain Genes From Triticum Monococcum And Transformation To Wheat

Grain hardness is one of the most important characteristics of bread wheat quality and has an important influence on wheat milling and processing quality. Hardness is mainly affected by Puroindoline genes(Pina and Pinb) and Grain Softness Protein-1(Gsp-1). These genes are located on the Ha locus of 5DS. In this study, three genes(Pinam, Pinbm, Gsp-1m) were cloned from Triticum monococcum. Three single-gene overexpression vectors, three single-gene co-transformed expression vectors, three two-gene co-transformed expression vectors and one three-gene co-transformed expression vectors were completed. Immature embryos of common wheat Kenong 199(KN199) were used for genetic transformation which mediated by gene gun. Regenerated plants were obtained after Bialaphos screening, and the positive plants were identified by PCR. The major results were as followed:1. Construction of expression vectors: Pinam, Pinbm and Gspm were cloned using genomic DNA of DV92 as template. Single gene over-expression vectors including PC186-Pinam, PC186-Pinbm and PC186-Gspm were constructed. Expression vector harboring gene of Pina1 m, Pinb1 m and Gsp1m; expression vector harboring tandem genes of Pina2m-Pinb2 m, Pina2m-Gsp2 m and Pinb3m-Gsp2 m and expression vector harboring tandem genes of Pinb2m-Pina2m-Gsp1 m were all constructed in this study.2. Transformation: 5258 calli induced from mature embryos(cultivar Kenong 199) were obtained by the tissue culture. Among them, 617, 610 and 620 calli were bombarded with Pinam, Pinbm and Gspm, respectively. 718, 535 and 937 calli were bombarded with Pina2m-Pinb2 m, Pina2m-Gsp2 m and Pinb3m-Gsp2 m, respectively. 1220 calli were bombarded with Pinb2m-Pina2m-Gsp1 m.3. Resistance selection: Calli were transformed by the gene gun and the resistant plants were selected by Bialaphos screening. 54, 67 and 62 regenerating plants transformed by plasmids PC186-Pinam, PC186-Pinbm and PC186-Gspm were obtained, respectively. 79, 43 and 93 regenerating plants transformed by plasmids pGEM-Pina2m-Pinb2 m, pGEM-Pina2m-Gsp2 m and pGEM-Pinb3m-Gsp2 m were obtained, respectively. 114 regenerating plants transformed by plasmid pGEM-Pinb2m-Pina2m-Gsp1 m were obtained.4. PCR detection: 3, 4 and 3 positive plants with Pinam, Pinbm and Gspm were detected, respectively. 1, 0 and 2 positive plants with Pina2m-Pinb2 m, Pina2m-Gsp2 m and Pinb3m-Gsp2 m were detected, respectively. 4 positive plants with Pinb2m-Pina2m-Gsp1 m were also detected.5. Dection of the genes at transcription level: 2 transgenic plants for Pinam, Pinbm and Gspm, respectively, were verified after reverse transcription detection. The results showed that the three genes in transgenic plants have been transcribed in wheat.In this study, three genes(Pinam, Pinbm, Gspm) were cloned from Triticum monococcum and inserted to expression vectors. Different combinations of genes were successfully transformed into wheat, which would play important role to further study the interaction and function of Pinam, Pinbm, Gspm in the future.