Screening And Identification Of Arabidopsis Factors Interacted With Rice Stripe Virus SP

Rice stripe virus belongs to the Tenuivirus, has repeatedly occurred in great quantities in different regions of our country. Rice stripe disease caused by this virus has a huge influence of rice production in China. RSV is composed of four single-stranded RNA. RNA4 segment code disease specific protein. A lot of research has done in RSV pathology, molecular biology; it was found that SP protein accumulation in the infected leaves is correlated with the mosaic chlorosis symptom. Thus the protein for pathogenesis is speculated, but the pathogenic mechanism about SP proteins is rarely reported.In order to understand the RSV-SP protein pathogenic mechanism, in this thesis we screen arabidopsis yeast two hybrid cDNA libraries with RSV-SP as bait protein and want to find out the key protein factor interactd with SP protein.Firstly we screen and verify Arabidopsis thaliana factors interacted with RSV-SP protein use YTH technology. We construct a YTH bait vector pGBKT7-SP, and turn it into yeast AH109. AH109 cells containing pGBKT7-SP were transfected with Arabidopsis library plasmid, yeast cells grow on SD/-Leu/-Trp/-His medium. Choose colony grew well on SD/-Leu/-Trp/-His/-Ade medium containing X-a-gal. Extract plasmids from yeast cells grew well and showed blue on selected medium, transform bacteria, PCR amplification insertion fragment size, sequence and blast analysis. Get three positive clones, chlorophyll a/b binding protein (LHCB5), hydroxy phenyl pyruvate oxidase (HPPD), glyceraldehyde phosphate dehydrogenase(GAPB). Using yeast two hybrid technology verify these genes. Firstly building pGADT7-HPPD, pGADT7-GAPB and pGADT7-LHCB5 vectors, transform yeast cells and culture on SD/-Leu/-Trp/-His medium. We next choose positive colonies grow on SD/-Leu/-Trp/-His/-Ade medium contains X-a-gal. Finally we confirm that HPPD and GAPB have obvious interactions with SP protein, and LHCB5 protein interactions is weak. Thus, HPPD and GAPB proteins may play an important role in the process of with RSV-SP interactions.In addition, in order to define the the role of HPPD gene in Arabidopsis thaliana with RSV, we constructed the HPPD over-expression vector, silen vector and promoter-drived GUS report vector. For the study of RSV-SP protein pathogenesis laid a solid foundation. These three vectors have transformed into agrobacterium EHA105, and transfect wild-type Arabidopsis using the inflorescence infiltration method. We collect seeds producted by transgenic plants To generation plants. These seeds were screened on resistance medium plates.At last, we have got 33 over-express of transgenic plants,6 transgenic silencing plants, and 6 promoter-drived GUS report plants in the subsequent experiments with different phenotypy.By detecting RSV infection rate in the transgenic plants, the role of HPPD on RSV-SP protein of pathogenic mechanism will identified in further study.