Virus Carrier Induced Post-transcriptional Gene Silencing System To Create And Tobacco (nicotiana Benthamiana Plants) Promoter Rbcs Gene Function Studies

RNA silencing is a nucleotide sequence-specific process of RNA degradation in plants (post-transcriptional gene silencing), animals (RNA interference (RNAi) and fungi (quelling). It leads to the degradation of homologous RNA from among endogenous genes, transgenes or RNA viruses. Virus-induced post-transcriptional gene silencing (VIGS) is a type of RNA silencing that is initiated by virus vectors carrying homologous fragment of host genes.During this study the system of virus-induced post-transcriptional gene silencing for studying gene function has been established and optimized. The function of Ribulose-1 , 5-bisphosphate carboxylase/oxylase small subunit (rbcS) gene has been investigated in detail.This thesis consists of the following parts: 1. The endogenous Ribulose Bisphosphate Carboxylase small subunit (rbcS ) gene silencing with tobacco rattle virus vector and potato virus X vector (pTV.rbcS and pgR107.rbcS) in Nicotiana benthamiana. 2. The endogenous phytoene desaturase (PDS) gene silencing with tobacco rattle virus vector and potato vims X vector (pTV.PDS pgR107.PDS) in Nicotiana benthamiana. 3. Transgene (GFP) silencing with pTV.P in tobacco (Nicotiana benthamiana 16c). 4. The function of rbcS gene by the VIGS system: (1) Phenotypes of rbcS gene silenced plants. (2) Transcription levels of rbcS gene by RT-PCR and Northern analysis. (3) Protein level of rbcS analyzed by the antibodies of RbcS and RbcL. (4) Photosynthetic pigments in rbcS silenced plants by HPLC method. (5) Some key parameters in photosynthesis in rbcS gene silenced plants. (6) Probability analysis of the tobacco rattle virus vector system as a useful and effective technique to study rbcS gene function.The following results have been obtained: (1). The virus vectorinduced post-transcriptional gene silencing system has been established and optimized in which the seedlings at 21-24-day old and Agrobacterium concentration at OD6oo=1-1.5 showed the best results. (2). The gene silencing mechanism revealed was difference between transgenic plant and non-transgenic plant in initiation, conduction and maintenance stages. (3). The TRV-based vector for VIGS was of some advantages compared to potato virus vector. (4). The expression level of rbcL was regulated by rbcS. (5). RbcS gene did not relate to the collection of photosynthetic energy. (6). RbcS gene regulated the activation of Rubisco. (7). RbcS gene regulated the carboxylation function of Rubisco. (8). The tobacco rattle virus vector was superior to potato virus vector in VIGS to study the rbcS gene function indicated by the statistical analysis of the experimental information.