Study On Transformation With Gene PF40 And Transcription Factor DREB In Wheat (Triticum Aestivum L.)

The plasmid expression vector Pc3300IS-DREB1A promoted by 35S promoter expression vector, sense(pAHC40) and knockout(pAHCRNAi) PF40 gene promoted by Ubi promoter expression vectors were constructed and used to wheat transformation study.PF40 gene, cloned from the cDNA library of immature seeds.of millet, was assumed to related to the tillering ability of cereals. To gain more information about the function of PF40 gene, sense and knockout PF40 gene with bar gene was transformed into 4000 young embryos of the winter wheat linesY18 and S99-6123 which have different tillering ability by microprojectile bombardment. There were 613 transformed plants regenerated after resistance selection on the MS medium containing 5mg/L Basta and transplant in the greenhouse. PCR amplification resulted in 43 putative transgenic plants containing the target gene PF40, and the frequency of transformation reached 6.61%. Seeds from 26 putative transgenic plants were planted as 26 lines, of which 19 plants were further done Southern blotting. The results showed that progeny of 19 plants integrated PF40 gene and the frequency of transformation reached 5.38%.There were 12 lines inherited on Mendel's Law completely among the 26 lines, indicating the PF40 gene could be transmitted into the next generation.The tillers per spike of Y18 carrying plasmid pAHC40 and S99-6123 carrying plasmid pAHC40 and pAHCRNAi showed significant difference from their negative control except of Y18 carrying plasmid pAHCRNAi.DREB1A gene, a stress tolerance-related transcription factor in the plasmid Pc3300IS-DREB1A was used to transform the apical meristem of the winter wheat variety LX987.The stem apical meristem of wheat seedling was cultured together with Agrobacterium, and 110 transformed seedlings were regenerated. Monitoring the target gene amplified by PCR, 10 plants integrated the DREB1A gene, and the frequency of transformation reached 9.09%. The transgene could be inherited into T, progeny and the Mendel's Law 3:1 segregation ratio was observed in the most transgenic lines.It was concluded that PF40 gene may influence the tillering or spike-bearing ability in wheat. The Agrobacterium- mediated transformation system based on the receptor of the stem apical meristem of seedling could be a high efficiency ways to wheat transformation for its no genotype dependence.