RNA Mediated Transgenic Resistance To Potato Virus Y By Transforming Tabacco With The CP Gene Segments And Inverted Repeats

Posttranscriptional gene silencing(PTGS) is due to sequence specific RNA degradation of transgene in the cytoplasm while the transgene transcribed normally in the nucleus.Transformation using untranslatable gene from plant virus can also result posttranscriptional gene silencing specific to virus gene that make transgenic plant resistant to virus, this kind of resistance is termed as RNA-mediated virus resistance and belongs to PTGS. More and more researches show that dsRNA maybe the trigger of PTGS.We showed previously that the full length of untranslatable coat protein (CP) gene of potato virus Y (tobacco veinal necrosis strain, PVYN) could confer PVYN resistance to tobacco (NC89) plants and we have proved that this resistance is RNA-mediated resistence.In the first part of our study, transgenic tobacco (NC89) plants were generated to express different regions of the CP gene of PVYN. The experiments were undertaken to find if shorter transgene could induce RNA-mediated potyvirus resistance in transgenic plants or not. If shorter transgene can also confer PVY resistance to transgenic plants ,we assume that multiple viruses resistance could be obtained by transforming plant with a single chimeric transgene consisting of gene segments from different viruses, in other words we can insert more different genes into one vector which express cassette has a limited space for gene insertion. In the second part of our study we used the same gene fragment of the first part of study to generate an inverted repeats construction, this inverted repeats could produce dsRNA subsquently by transcription, and we want to dissert if this kinds of tansgene construction could make transgenic plant generate highly virus resistance with high frequency.The results and conclusion are as follows:1, Vector pROKII and three gene segments were used to construct plant express vector, one 202bp long gene segment derived from 5'end of PVYN-CP,and one 252bp and another 202bp long gene segments all derived from 3'end of PVYN-CP.These three untranslatable genes were all driven by CaMV35S promoter. And introduced into tobacco (NC89) plants via Agrobacterium tumefaciens-mediated transformation. After tissue culture we got 320, 235 and 195 transgenic plants separatly and found most of them show susceptible phenotype ,only 3 of 320 show resistant to PVY, this tell us 202bp short gene segment can also be used to generate resistant transgenic plant.2, Northern blot analysis of leaf samples showed that all progeny T0 transgenic plants expressed PVYN CP RNA, and the levels of transcript accumulation of transgenic lines were varied. The highly resistant plants had a low-level accumulation of the RNA transcribed from transgene whereas the RNA in susceptible plants was accumulated to higher levels. The results revealed an inverse correlation between transgene transcript accumulation and virus resistance, this indicate that the resistance is RNA- mediated.3. The 202bp fregment from 3'end of PVYN-CP was used as the repeat gene to construct an inverted repeat structure, also a sister structure was made that the two repeat genes have the same orientation. These two non-start code gene weretransformed into tobacco drived by CaMV35S promoter, after tissue culture we gain 220 and 198 transgenic plants. Based on the virus infection assay , amazingly, we found that more than 82.8 percent of the plant transformed with the construct of IR (inverted repeats)were immune to the virus infection in contrast to that of the plants transformed with control structure resulted in none resistant transgenic line. The results showed that induction of PTGS by the structure that can produce double-stranded RNA transcripts is more efficient than structure that contain either single sense and antisense CP gene or repeat sense CP gene fragments.4, Northern blot analysis of leaf samples showed that the RNA in susceptible plants was accumulated to higher levels than that in the highly resistant plants. It indicated that this is a RNA-mediated virus resistanc...