Moleculardetection And Identification Of Rhizoctonia Cerealis Response In TaCPK7 And OpIAP-p35 Transgenic Wheats

Wheat sharp eyespot is a soil-borne disease causing mainly by Rhizoctonia cerealis, which would result in great loss in wheat productivity. Wheat sharp eyespot has become a major wheat disease in China during last decades. However, because of lack of immune wheat to the wheat sharp eyespot, progress of the corresponding conventional breeding of anti-R.cerealis is slowly. Majority of cultivars growing extensively in China are susceptible to this disease. Thus, it is important to identify anti-R. cerealis genes, develop effective molecular makers and molecular breeding in wheat against R. cerealis. In our previous work, Gene Ta CPK7 was isolated from a Synthetic wheat CI12633. The Ta CPK7 was transformed into wheat cultivar Yangmai16 via microprojectile bombardment. After regeneration and resistance selection by Gaeumannomyces graminis, resistant plants were grown in greenhouse. Besides,In this study, the full-length coding sequences of Op IAP and p35 genes were synthesized, respectively. The transformation vector p Ubi:p35-RSS1P:Myc-Op IAP containing two gene expression cassettes was constructed. Embryo calli of Yangmai 16 were bombarded by the gold particle containing p Ubi:p35-RSS1P:Myc-Op IAP vector DNA.The main results of Ta CPK7 transgenic wheats are as follows:(1)Transgenic wheat plants in T1~T2 generations were subjected to PCR, RT-PCR, q RT-PCR and Western blot analyses. Three transgenic wheat lines were obtained.(2)q RT-PCR analysis showed that expression of Ta CPK7 was induced strongly by ET. It also was induced by treatment of H2O2. These suggests that Ta CPK7 might be involved in the ET signaling pathway and antioxidant defence.(3)q RT-PCR Analysis severaldisease-related genes in Ta CPK7 overexpression and silencing plants, Respectively.the results indicate that overexpression of Ta CPK7 can improve resistance to external stimuli Diseasesin transgenic wheats.(4)Sharp eyespot severity assessments analysis showed that the transgenic wheat plants expressing Ta CPK7 displayed significantly enhanced resistance to sharp eyespotcompared with non-transgenic wheat Yangmai 16.The main results of Op IAP and p35 transgenic wheats are as follows:(1)The transformation vector p Ubi:p35-RSS1P:Myc-Op IAP containing two gene expression cassettes wasconstructed. In the expression vector p Ubi:p35-RSS1P:Myc-Op IAP, the Op IAP gene is driven by the rice sucrose synthase-1 promoter and the p35 gene is driven by the maize ubiquitin promoter.And we get the transgenic wheats.(2)Transgenic wheat plants in T0~T2 generations were subjected to PCR, RT-PCR, q RT-PCR and Western blot analyses. The results indicated that the introduced Op IAP and p35 genescould beinherited and expressed in four transgenic wheat lines.(3)Rhizoctonia cerealis isolate R0301 or WK207 was used to inoculate T1 and T2 plants for sharp eyespot severity assessments, The results showed that the transgenic wheat plants expressing Op IAP and p35 displayed significantly enhanced resistance to sharp eyespotcompared with non-transgenic wheat Yangmai 16. Thus, the introduced Op IAP and p35 genes could be used in improving wheat resistance to wheatsharp eyespot caused by different Rhizoctonia cerealis isolates.