摘要
RNA沉默是一种序列特异性的RNA降解过程 ,主要作用于同源性较高的转录产物 ,它广泛存在于各种生物中 ,在植物中称转录后基因沉默 (Post TranscriptionalGeneSilencing ,PTGS)、动物中称为RNA干扰(RNAinterference ,RNAi)、真菌中被称为RNA压制 (RNAquelling)。病毒诱导转录后基因沉默是由携带基因片段的病毒载体感染植物引起相应的寄主基因沉默的现象 ,是一种典型的RNA沉默现象。本论文建立了病毒载体诱导转录后基因沉默系统 ,并运用该体系研究烟草 (Nicotianabenthamiana)的 1,5二磷酸核酮糖羧化酶 /加氧酶小亚基 (Ribulose - 1,5 -bisphosphate (RuBP)carboxylase/oxylasesmallsubunit,rbcS)基因的部分功能。具体包括以下内容 :1、分别用携带与 1,5二磷酸核酮糖羧化酶 /加氧酶小亚基 (rbcS)基因同源的cDNA片段的烟草脆裂病毒载体 (pTV rbcS)和马铃薯X病毒载体 (pgR10 7 rbcS)侵染烟草 (Nico tianabenthamiana) ,诱导内源rbcS基因沉默 ;2、分别用携带与八氢番茄红素脱氢酶 (phytoenedesat urase ,PDS)基因同源的cDNA片段的烟草脆裂病毒载体 (pTV PDS)和马铃薯X病毒载体 (pgR10 7 PDS)侵染烟草 (Nicotianabenthamiana) ,诱导内源PDS基因沉默 ;3、利用携带与绿色荧光蛋白 (GFP)同源的cDNA片段的烟草脆裂?
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 virus X vector (pTV.PDS和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 vector induced post-transcriptional gene silencing system has been established and optimized in which the seedlings at 21-24-day old and Agrobacterium concentration at OD 600=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.
出处
《分子植物育种》
CAS
CSCD
2003年第3期423-424,共2页
Molecular Plant Breeding
基金
国家转基因植物中试及产业化基地(J99-B-001)资助。
关键词
RNA沉默
转录后基因沉默
病毒诱导
核酮糖1
5二磷酸羧化酶/加氧酶小亚基
RbcS基因
烟草脆裂病毒载体
RNA silencing,Post-transcriptional gene silencing(PTGS), Virus-induced gene silencing (VIGS),Ribulose bisphosphate caboxylase/oxydase small subunit(rbcS),Net photosynthetic rate (Pn)
