Construction Of OsVIP1 RNAi Transgenic Rice Plants And Screening Of The VirE2 Interacting Proteins In Rice

Rice is one of the most important cereal crops in the world. Its germplasm has long been kept improving through traditional breeding ways. However, owing to various factors, further improvement has reached a bottleneck. With the development of transgenic technology, exogenous genes can be transferred into rice plants and integrated into the rice genome through the Agrobacterium-mediated transformation method, and stably inherited and expressed in plants. The transgenic technology has brought new approaches and prospects to the project of rice germplasm improvement. Nevertheless, because the rice is not the native host to the Agrobacterium, the lower transformation efficiency especially for indica rice is the central issue in the genetic transformation.It is well known that the Agrobacterium-mediated plant transformation involves many proteins from both the bacterium and host plant. Analyzing the proteins will help in deepening our understanding about the transformation course and improving rice transformation efficiency through molecular biology methods.Arabidopsis AtVIP1 (Arabidopsis thaliana VirE2-interacting protein 1) protein has played crucial roles in the Agrobacterium-mediated transformation of tobacco and Arabidopsis, such as the formation and importion of T-complex and the integration of T-DNA strand into the plant chromosome.In this study, we obtained a rice protein sequence homologous to Arabidopsis thaliana AtVIPl by homologous searches, named OsVIP1. We constructed RNAi expression vectors with 2 fragments (R1 and R2) and used them to silence the OsVIPl gene expression of rice Zhonghua 11. The callus growth state was evaluated after infection of Agrobacterium containing the RNAi vectors and the TO generation positive transgenic plants were detected. We also checked the gene expression with semi-quantitative RT-PCR. Meanwhile, in order to obtain other rice proteins that could interact with the bacterial virulence protein VirE2, we screened a rice cDNA yeast two-hybrid library using the VirE2 as the bait. The results of the research were as follows:1 RNAi vectors (pDS1301-2-Rl and pDS1301-2-R2) with 2 fragments (R1 and R2) of the OsVIPl cDNA were successfully constructed and transferred into the rice Zhonghua 11 callus.2 A detailed statistic analysis for the growth state of the resistant transgenic callus and transgenic plants was made, and the result showed that the resistant callus percentage of the RNAi experiment was much more lower than the control;3 The positive RNAi TO generation plants were detected. We obtained 35 and 32 individual transgenic plants for the R1 and R2 fragments, respectively.4 The OsVIPl and PBZI gene expression in some TO generation transgenic plants were detected with semi-quantitative RT-PCR. In some transgenic rice plants, OsVIPl expression was suppressed to some extent; PBZI gene expression also decreased.5 A rice yeast two-hybrid cDNA library was screened using VirE2 as the bait and 142 clones were obtained, among which 61 clones contained fragments lager than 500 bp and 53 of the 62 clones were sequenced. Two candidates were selected for further study.