The Roles Of High-level Expressed Aberrant Homologous Sequence And Virus Encoded Proteins In Gene Silencing

RNA silencing is a common phenomenon of RNA degradation that is induced by homologous sequences. Virus and transposon invasions and various kinds of aberrant RNAs can provoke RNA silencing. In order to understand the influence of the structure of homologous RNA molecules and expression dosage on plant gene silencing, and the inhibitory and general mechanistic effects of some plant virus encoded proteins on silencing, I have carried out the research below.I have used low copy pBIN19 and single copy pMW755I5J binary vectors as backbone plasmids, to create a gene targeting insertion vector designated GFP Tnos. After agro-infiltration into transgenic Nicotiana benthamiana 16C, progeny were analyzed genetically for phenotypic changes, siRNA accumulation, and DNA methylation. The time of silencing, and the percentage and state of silenced plants were recorded, and it was noted that the efficiency of induction of homologous dependent gene silencing (HDGS) by the GFP Tnos vector was markedly higher than mat resulting from other vectors with intact structure. HDGS induction by progeny containing a low copy number of the vector was also dramatically higher than induction by single copy vectors. The reduction of GFP protein and mRNA abundance, and the presence of 21-26 nt siRNAs could be simultaneously detected in silenced plants and the GFP coding region of these plants was methylated extensively. Post-transcriptional gene silencing (PTGS) spread systemically in the first transgenic generation, but was not transmited to the offspring. RT-PCR showed that the GFP transcripts from wild type Nicotiana benthamiana inoculated with the Tnos deletion vector lacked a normal poly(A) tail. The above results showed that two key factors influencing the occurrence and state of PTGS were the structure of homologous products expressed and expression dosage in the plants. Some transgenic plants harboring the GFP Tnos vector also exhibited inhibited expression of GFP, and Northern blots also revealed that most GFP mRNAs transcribed from Tnos accumulated in the nucleus and was not transported into the cytoplasm.Screens of transgenic tobacco transformed with single GFP and three directly repeated GFP copies resulted in recovery of homozygous lines of GFP1 and GFP3 that exhibited green and red phenotypes under UV light respectively. Virus experiments also revealed that the resistance of the GFP3 lines to PVX-GFP was more effective than those of GFP 1. In addition, molecular experiments indicated that resistance was not relevant to copy number or to insertion sites, but instead to the repeated GFP structure and to methylation of genomic DNA.PVX vectors were constructed that contained the S6 gene of RBSDV VI and the 14 kD fragment of BNYVV RNA2. Phenotypic observations and molecular detection indicated that both the RBSDV and 14 kD vectors were effective to some extent in suppressing gene silencing, and that the S6 protein could eliminate methylated sites from the target regions of the genome. The S6 and 14 kD proteins could also suppress GFP silencing when PVX vectors harboring the two suppressors were infiltrated into Nicotiana benthamiana whose GFP silencing had been induced by the Tnos-deleted construct, and the suppression activity of the S6 protein was the strongest. In additional experiments, two RBSDV proteins encoded by S7 and one protein encoded by S8 appeared to suppress gene silencing.