Studies Of OsAIP RNAi And Procaryotic Expression

The plant cytoskeleton is a high dynamic system and has crucial functions in plant growth and development. Actin dynamics, or the rapid turnover of actin filaments, play acentral role in numerous processes, such as cell division, cell expansion, cell differentiation, cell-to-cell communication, morphogenesis, organogenesis and development. In this study, to analyze the function of OsAIP in vivo, a RNAi vector was constructed and transformed in rice. Then we got some genetics data which proved its importance. Moreover, we constructed a procaryotic expression vector about OsAIP and successfully expressed in tuner bacteria. Finally, we confirmed the purifying conditions and harvested much highly-pure protein sample which applied to bio-chemical study and testified functions of OsAIP in vitro.A novel polymerase chain reaction (PCR)-based RNAi vector pTCK303 with a maize ubiquitin promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector, only 1 PCR product—a target DNA sequence of 800bp—amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct, which shortened the time span before being transformed into rice.Studying expression of OsAIP in different bacterium, it concluded that tuner bacteria was more useful for OsAIP expression. When testing the effect of inducing temperature, it testified that the lower temperature induction is helpful for the expression of OsAIP, and induction at 16℃was the best. After confirming the inducing temperature of OsAIP expression, we purified OsAIP in large scale through Glutathione-Sepherose and Q-Sepherose respectively. Obtained OsAIP was pure enough and can be applied to bio-chemical study.At the same time, for making it adapt to high-speed and high-efficiency transformation in zhonghua11 and the japonica rice cultivar 'Nipponbare', and accelerating the process of rice research, we improve a high-speed transformation method on seeds pro-culture and time of infection. It demonstrated that the high-speed transformation of rice by infecting early calli with Agrobacterium is practicable. With this method, we could regenerate transformants within 40 days from the beginning of seeds steri'ization to T₀ transgenic planlets. Moreover, the transformation ratio and the regeneration ratio were 85% and 60% respectively.