Construction Of Expression Vector Of The Triticum Aestivun L. TaPHR1 Gene And Research On Genetic Transformation

It is important for agricultural sustainable development to fully exploit the crop genetic potential of nutrients utilize efficiency and cultivate new varieties of efficient nutrients use. In this research, gene TaPHR1 was used to construct efficient gene expression vector for genetic transformation system of mature embryos of common wheat by A.tumefacienen. At the same time, 26 wheat genotypes were used for factors research on wheat mature embryos callus induction and differentiation to further optimize the conditions of tissue culture of wheat mature embryos. The major results were as followed:1. In this study, Triticum aestivun L. gene TaPHR1 was amplified using specific primers containing restriction enzyme site of KpnI and BamH1. PCR products and the binary expression vector pROK2-Ubi were digested by the corresponding restricted enzymes respectively, and linked directionally. The resulting vector with TaPHR1 was named pTaPHR1. The pTaRHR1 was further introduced into Agrobacterium tumefaciens strain LB4404. At the same time, new expression vector pTaRHR1-PT2 were constructed by changing the promoter Ubi of pTaRHR1 to promoter TaPT2. These vectors were used for phosphorus efficiency genetic transformation system of mature embryos of common wheat by A.tumefaciene.2. Genotype, imbibition time, concentration of plant growth regulator and so on were important influence factors for the induction, differentiation and regeneration of wheat mature embryos callus. Some of excellent wheat material including Jimai22 and so on with the high efficiency of induction, differentiation and regeneration of mature embryos callus were selected out from 26 wheat genotypes via embryo-isolated method (EI). The optimal method of induction, differentiation and regeneration of mature embryos callus of these genotypes were determined. The optimized system for induction, differentiation and regeneration of wheat mature embryos callus was established. 3. Density of A. tumefaciens cell and inoculation time, density of sugar in osmotic treatment, density of Ca2+, putting filter paper, different co-culture time were important influence factors for transformation of wheat mature embryos. A. tumefaciens cell density, inoculation time and selection pressure of selection reagent were confirmed; At the sugar density of 75 g·L- 1, the frequency of resistant callus received the maximum rate to 2.90%(Jimai 22) and 2.78%(Tainong 18); At the Ca2+ density of 20 mmol/L, the frequency of resistant callus received the maximum rate to 5.05%; At the condition of putting filter paper, the frequency of resistant callus received the maximum rate to 4.29%; At the condition of training 3 d, the frequency of resistant callus received the maximum rate to 4.52%; The system optimized relatively of genetic transformation of mature embryo of wheat seed mediated by A. tumefacienens was further optimized.4. TaPHR1 gene were transferred into Jimai 22 by high phosphorus efficient expression vector pTaPHR1 and fertile transgenic plants had been gotten. Molecular detection results by PCR analysis of transgenic wheat showed that the useful gene had been transferred successfully.