Pathogenic Mechanism Of RSV Coat Protein Via Rubisco SSU Leader Peptide Of Rice Chloroplast

Rice stripe disease is one of the most serious rice diseases and occurs mainly in temperate and subtropical areas. It causes 20%~30% yields lose in japonica rice-growing regions in China. The coat protein(CP) encoded by Rice stripe virus (RSV) is one of the pathogenic protein. In this paper, the Rubisco SSU leader peptide and RSV CP genes were amplified by PCR. The fusion genes PR-CP and PR-S-CP were constructed into the expression vectors pGEX-4T-1 and pET-29a. In order to adapt to the transformation system mediated by Agrobacterium tumefaciens, RSV CP and PR-CP were ligated to the rice transformation vector pTCK303, respectively to investigate the effects of chloroplast located CP on the normal physiology of rice plants and pave the way for further researches on RSV pathogenesis.Plant expression vectors were transformed to callus derived from mature embryo or rice Oryza sativa L. subsp. japonica cv. Nipponbare using A.tumefaciens EHA105. It was shown that NB medium with 2.3mg/L of 2, 4-D promoted the callus'meristrmatic capacity. The efficiency of callus'defferentiation was greatly improved by the adding 10mg/L of KT and 0.5mg/L of NAA in the regeneration medium, and the addition of 100μM As (acetosyringone) in agrobacterium culture medium effectiently induced the activation of theVir gene and thus promoted the integration of foreign genes and transformation efficiency. The target gene was detected by PCR to confirm the success of transformation. Finally, 27 T0 transgenic plants were preliminarily obtained, in which there were 12 ones for CP gene and 15 ones for PR-CP gene. Seeds were harvested after planting of the T0 and germinated on hygromycin (30mg/L) containg NB medium. After 20 days of germination T1 plants showed a 3:1 ratio of resistant to susceptible plants, indicating that the transgenes were stabely integrated into the plant genome. Phenotypes of transgenic rice plants were then oberseved. While the CP transgenic plants showed little phenotypic differences with the control, the plant height of the transgenic plants expressing PR-CP were significantly reduced. This suggested that CP could have an significant impact on the physiology of rice plants when targeted into the chloroplasts.