Study On Cloning And Transgenic Wheat Of Anti-Rhizoct Onia Cerealis Genes

In this study, plant defense genes TaLTP4 and PvPGIP were cloned and transformed, and tissue-specific promoter RSS1P was researched. Anti-Rhizoctonia cerealis transgenic wheats were obtained. The main results were as follows:(1) A LTP encoding gene in Triticum aestivum(TaLTP4) were successfully cloned by using homologous gene cloning strategy and RT-PCR technique. By taking pAHC25 as frame, we constructed pA25- Ubi::TaLTP4 expression vector . pA25- Ubi::TaLTP4 transformed into"Yangmai 18". The Ubi::TaLTP4 transgenic plants in T0 and T2 generations were subjected to PCR , Q-RT-PCR , Rhizoctonia cerealis resistance tests. The molecular detection results showed that the transgene Ubi::TaLTP4was introduced into Yangmai 18 and was inheritable.The expressive quantity of TaLTP4 in transgenosis wheat was higher than receptor compared wheat"yangmai 18". Resistance to Rhizoctonia cerealis identification were applied on wheat that transformed TaLTP4 genes, we obtained the wheat material that resistance to R. cerealis. Therefore, over expression of TaLTP4 can improve the resistance to R. cerealis. The results that anti-Rhizoctonia cerealis analysis of strain T2 transformed TaLTP4 were in agreement with the expression of experiment on whole alive plant. So, we found a faster and easier method to identify Rhizoctonia cerealis, and prove that TaLTP4 gene had defense function to Rhizoctonia cerealis.(2) A PGIP encoding gene in Phaseouls vulgaris(PvPGIP2) was successfully cloned by using homologous gene cloning strategy and RT-PCR technique; By taking pAHC25 as frame, we constructed PvPGIP2 and TaLTP4 binary expression vector as they have synergistic reaction in function. Then the binary expression vector was through preliminary screening by colony PCR and restriction enzyme digest identification and sequencing analysis. The result showed that we successfully constructed PvPGIP2 and TaLTP4 binary expression vector. In this transformation vector, the PvPGIP2 and TaLTP4 genes were respectively driven by the maize ubiquitin (Ubi) promoter and terminated by the 3′non-transcribed region of the Agrobacterium tumefaciens nopaline synthase gene (Tnos).. Using microprojectile-mediated bombardment to 2,000 mature callus of"Yangmai 18", and through twice Bialaphos screening, we finally obtained 112 plant regeneration. Using specific primers of expression vector gene to conduct PCR detection for the living transformed plants, we obtained 12 plants that PvPGIP2 and TaLTP4 presented positive, and the conversion rate was 0.6%.(3) The RSS1P::TiERF1 transgenic plants in T0 and T1 generations were subjected to PCR, PCR-Southern, RT-PCR, and Q-RT-PCR analyses, and Rhizoctonia cerealis resistance tests. The molecular detection results showed that the transgene RSS1P::TiERF1 was introduced into Yangmai 12 and was inheritable. In the RSS1P::TiERF1 transgenic wheat plants, TiERF1 expressed in root, stem, and leaf except seed. The highest expression level was observed in root. Compared to the untransformated Yangmai 12, resistance to R. cerealis in the RSS1P::TiERF1 transgenic wheat plants was obviously improved, which was similar to that in the Ubi::TiERF1 transgenic wheat plants. The agronomic traits of RSS1P::TiERF1 transgenic plants had no obviously changed compared to untransformated Yangmai 12. These results suggest that RSS1P promoter is feasible to be used for developing new transgenic wheat germplasm.(4) Through analysis and verification on several constructed promoters by GUS staining technique, it showed that Intron had an enhance effect on promoter RSS1P, and expression quantity of RSS1P2897 and RSS1P1715 in callus was extremely low as well as sole Intron possessed the ability of promoter. PCR detection was carried on DNA of callus, and it showed that bombardment expression vector had been into callus.