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RNA silencing (RNA silencing) of the degradation mechanisms in eukaryotic cells to exogenous genetic factor specificity and high efficiency. To build an inverted repeat structure to the viral nucleic acid fragment transgenic induced post-transcriptional gene silencing (post-transcriptional gene silencing, PTGS), will not only be able to turn the gene transcripts degradation, but also be able to invasion of the viral genome homologous transgenic RNA degradation with the disease a high degree of resistance lasting and high biosafety advantages due RNA stage, also known as RNA-mediated resistance. Virus-mediated gene silencing (Virus Induced Gene Silencing, VIGS) is the target gene fragment was inserted into a viral vector to construct recombinant viruses, infested plants by the replication of the virus-induced gene silencing, thereby inhibiting plant highly homologous to the inserted fragment the expression of the endogenous gene, and are often used for the validation of gene function. This study virus CP gene of PVY ~ N inverted repeat sequence in transgenic tobacco K58 plants for study materials, applications the VIGS virus vector technology down RDR1 expression, the test of RNA-mediated resistance; different temperatures at the same time dealing with the anti- PVY ~ N repeat mediated transgenic virus RNA silencing, and thus the creation of multi-resistant virus the plant and prolonged high level of maintenance of RNA-mediated resistance to provide a theoretical basis. Results and conclusion of the study are as follows: the length 373bp the Nt-RDR1 gene partial fragment was obtained from the plants of varieties of tobacco NC89, Nb-RDR1 benthamiana gene is highly conserved, and the homology of 97.32%. Then inserted in the the VIGS vectors TRV and PVX, successful build the TRV-RP and PVX-RP viral vectors to silence RDR1 gene. The viral vector was transformed into Agrobacterium GV3101 into the four-leaf stage benthamiana using Agrobacterium infiltration injection and transfer genes in tobacco K58. Tobacco on light incubator culture conditions were as follows: 20 ° C light, 14 hours dark 10 hours. 20 days after injection of the inoculated leaves and the new leaves of the RNA extracted from tobacco, RT-PCR, the obtained cDNA was to tobacco EF1α reference gene, Design RDR1 fluorescent primers, fluorescent quantitative PCR and Northern Blotting detection. After testing, the TRV-RP and PVX-RP, the inoculated leaves RDR1 content is significantly reduced, been silent, but the new leaves detect less than viral vectors, RDR1 gene did not occur silence, found no systemic infection. Benthamiana, viral vectors can be systemic infection silencing effect is more obvious in the whole plant. Inoculated with the TRV-RP PVY ~ N TRV carrier for 15 days, and SA transgenic tobacco processing K58, 20 ° C light 14 hours dark 10 hours under the conditions of culture, four days after the extraction of RNA, reverse transcription quantitative PCR. Viral RNA accumulation, the accumulation of TRV-RP vector processing tobacco plant virus. PVY ~ N virus primers A, B, C, D, HC and tobacco reference gene EF1α detecting silence to the extension of the upstream direction, found the TRV-RP inoculated leaves of each gene on the whole lower silencing effect, the process of TRV-RP similar new leaf effect and the effect of the untreated control group. Inoculation of PVY ~ N transfer transgenic tobacco K58, respectively, at 15 ° C, 20 ° C, 25 ° C, 30 ° C, 35 ° C, light 14 hours, darkness 10 hours of the conditions of cultivation, effects of temperature on the silence and extends situation. Four days after the extraction of RNA, reverse transcription quantitative PCR. PVY ~ N virus primers A, B, C, D, HC and tobacco reference gene EF1α detection silencing and extending in the upstream direction of the situation, and found that 30 ° C GT; 25 ° C GT; 20 ° C GT; 15 ° C GT ; 35 ℃.
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