Comparison Of Resistance To Potato Virus Y Mediated By NIa-Vpg, NIa-Pro, NIb And CP Genes In Transgenic Tobacco Plants

RNA silencing is a defense mechanism for plant to resist virus infection, the nature is post-transcriptional gene silencing (PTGS), generally caused by dsRNA. HpRNA constructed in vitro can efficiently induce gene silencing. Virus genome encode a multi-functional protein, different proteins have different functions in virus replication, proliferation and the spread of the process. Interfering the expression of different genes may induce different effects of gene silencingAccording to the published nucleotide sequence of potato virus Ynecrosis stain (PVY~N), the PVY~N NIa-Vpg, NIa-Pro gene was synthesized by reverse transcription-polymerase chain reaction (RT-PCR), NIb and CP genes were provided by the laboratory, and constructed into binary vectors pROKII corresponding expression vectors. The four genes was introduced into tobacco (NC89) plants via Agrobacterium tuefaciens mediated transformation, transformed tissue was selected in the presence of Kanamycin sulfate. Regenerated plants were screened by PCR, and a variety of transgenic plants were obtained. Northern blot showed the levels of transcript accumulation varied among transgenic lines. SiRNA was detected in the presence of disease-resistant plants. The results revealed an inverse correlation between transgenic transcript accumulation and virus resistance. The result will provide a basis on effectively target site selection sequence and the successful application of RNA-mediated virus resistance strategy.1. According to the published nucleotide sequence of potato virusY necrosis stain (PVY~N), the PVY~N NIa-Vpg, NIa-Pro gene was synthesized by reverse transcription-polymerase chain reaction. NIb and CP genes were provided by the laboratory. The inverted repeats DNA sequence of hpRNA derived from 3′ends 400bp and 500bp of PVY~N NIa-Vpg, NIa-Pro, NIb and CP gene, and constructed into binary vectors pROKII corresponding expression vectors.2. Recombinant binary vectors and pROKII were then introduced into tobacco (NC89) plants via Agrobacterium tumefaciens-mediated transform- ation system. PCR and double digestions also confirmed that all the target genes were transferred into Agrobacterium tumefaciens.3. The transformed tissues were selected in the presence of Kanamycin, and the regenerated plants were screened by PCR. 126, 101, 100, 132and 34 plants transformed with NIa-Vpg, NIa-Pro, NIb, CP and pROKII, respectively, were obtained.4. Resistance tests indicated that hpRNA with different regions had different effects on inducing RNA-mediated gene silencing. Plants transformed with NIa-Vpg, NIa-Pro, NIb and CP were resistant to PVYN infection, and the proportion of disease resistant transgenic plants was 65.08%, 58.42%, 43.00% and 63.64%, respectively.5. The total RNA was extracted from the transgenic plants with susceptible or resistant responses to PVY~N, Northern blot showed the levels of transcript accumulation varied among transgenic lines. The results revealed an inverse correlation between transgenic transcript accumulation and virus resistance.6. Northern blot analysis of siRNA showed that siRNA was detected in the resistant plants.