Production Of Transgenic Tobacco Plants Resistant To Two Viruses Via Inverted Repeat Transgene And It's Flanking Sequence

RNA silencing in plants, also called post-transcriptional gene silencing (PTGS), is a conserved pathway with important antiviral roles in plants. PTGS can make the plant virus genomic RNAs decayed via sequence-specific RNA degradation, and this makes the host plant resistant to the virus. In order to improve the plant resistance to virus, genes from the pathogen (virus) itself were used as the transgenes to create antiviral transgenic plants (pathogen derived resistance, PDR), and more and more documents showed the mechanism of the PDR is post-transcriptional gene silencing. Double-stranded RNA (dsRNA) is most likely the RNA silencing initiation factor.Plant expression vector pRIR202 was constructed by using 202bp gene sequence from potato virus Y coat protein gene, the transgenes arranged in inverted repeats, and high resistant transgenic tobacco plants were obtained via agrobacterium-mediated transformation. The results of the molecular analysis confirmed that the resistance was mediated by the RNA silencing. Some documents have shown that the RNA silencing can transit from the initiator dsRNA to its flanking sequences, resulting in silencing both the initiator dsRNA and its flanking sequences simultaneously.In this research, PVX CP gene was inserted into the upstream of the inverted repeat construct of plant expression vector pRIR202 and generated the new plasmid pRIR202XCP. A longer fragment about 2kb of PVX (including 718bp CP gene and partial NIB gene) was inserted into the downstream of the inverted repeat structure of pRIR202 and created the new binary plasmid pRIR202-P2kII, and transgenic tobacco plants were obtained via agrobacterium-mediated transformation. The purpose of this study conducted here was to elucidate if the RNA silencing can be transited from the initiating region of inverted repeat of the transgene to its flanking sequences, the sequences of inverted region and the flanking region were from different viruses, and multi-resistant transgenic plants can be acquired. Our findings and conclusions were mainly as follows:1. Construction of the plant expression vectors harboring transgene sequences flank with inverted repeat structure718bp PVX CP gene and 2kb of PVX (including 718bp CP gene and partial NIB gene) were cloned and inserted into the upstream and downstream of the plant expression vector pRIR202 inverted repeat structure, and two plant expression vectors pRIR202-P2kII and pRIR202XCP were successfully constructed. Then pRIR202XCP and pRIR202-P2kII were introduced into agrobacterium strains LBA4404 and GV3101 by using liquid nitrogen freezing method.2. Anti-viral transgenic plant lines generation and disease resistance testingThe recombinant plant expression vectors pRIR202XCP and pRIR202-P2kII were transformed into tobacco NC89 and zhongyan100 via agrobacterium-mediated transformation, and transgenic plants lines were obtained.Transgenic plants which possessed the transgene from pRIR202XCP were highly resistant to virus, and the northern blot analysis showed that the high resistance was caused by the RNA silencing, and the RNA silencing triggered by the inverted repeats (PVY sequence) can effectively influence the flanking region (PVX sequence), and lead the flanking sequences to be silenced. Thus multi anti-virus transgenic plant lines resistant to potato virus Y and potato virus X were obtained.156 strains of transgenic plants harboring pRIR202-P2KII transgenes were obtained, and primary test showed 76% transgenic lines presented resistant to virus. 3. The transgenic plants with high resistant to a variety of viral diseases through the co-silencing effects of the inverted repeats and flanking sequence, which do great advantage to reduce economic losses caused by the mixed infections of various virus.