| Potato Y virus (Potata virus Y, PVY) is one of the typical species of the potato virus Y family, causing yield decrease of the tobacco and other important economic crops, especially PVY strain of tobacco vein necrosis (PVYN) can cause early death. RNA-mediated virus resistance is a post- transcriptional gene silencing (PTGS). Recently, it has developed an effective control strategy for plant virus diseases. It is based on the virus nucleic acid fragment transgenic to induced gene silencing, not only can degradate the transcripts of transgene, but also degradate the invading virus genome RNA homologousing with the transgene, with advantage of a high degree of resistance (almost immune), resistance persistent and bio-safety, etc. The products of the gene encoding the various sections of Potato virus Y perform different functions in viral replication and proliferation process, so we guess that different functional gene may create different effectiveness of the virus RNA-mediated resistance. At present, such as the virus capsid protein gene, replicase gene and the movement protein gene of RNA-mediated resistance have been reported, but there was no comprehensive and systematic studies.In this study, the P1, HC-Pro, P3, CI gene of PVYN were cloned by RT-PCR technology. Then, the cDNA derived from 3'end of P1, HC-Pro, P3,CI gene were subcloned .Four hpRNA of plant expression vector with the stem length of 400bp and 100bp-loop were constructed,and were transformed into tobacco.The assay of virus resistance showed that the potato Y virus P1, HC-Pro, P3, CI gene of RNA-mediated virus resistance existed obvious differences. The main results presented as follows:1.According to the publishd nucleotide sequence of PVYN, the genome of P1,HC-Pro,P3,CI was synthesized by reverse transcription-polymerase chain reaction (RT-PCR).The sequence has Logined in GenBank, accession number as follows: EU182576.2. PCR amplification of the 3 'end 400bp and 500bp cDNA section of P1, HC-Pro, P3, CI gene, respectively, were taken into pROKII. Four hpRNA of plant expression vector pROKâ…¡-P1,pROKâ…¡- HC-Pro, pROKâ…¡-P3 and pROKâ…¡-CI with the stem length of 400bp-stem and 100bp-loop were constructed.3.Recombinant binary vectors of pROKâ…¡-P1,pROKâ…¡- HC-Pro , pROKâ…¡-P3and pROKâ…¡-CI were then introduced into tobacco(NC89) plants via Agrobacterium tumefaciens-mediated transformation system. PCR and double digestions also confirmed that all the target genes were transferred into Agrobacterium tumefaciens.4. The transformed tissues were selected in the presence of Kanamycin, and the regenerated plants were screened by PCR. 90, 94, 108, 96 and 40 plants transformed with pROKâ…¡-P1, pROKâ…¡- HC-Pro, pROKâ…¡-P3, pROKâ…¡-CI and pROKII(comparison), respectively, were obtained.5. Resistance tests indicated that all the plants transformed with pROKII were susceptible to PVYN infection, while plants transformed with pROKâ…¡-P1,pROKâ…¡- HC-Pro ,pROKâ…¡-P3 ,pROKâ…¡-CI were resistant to PVYN infection, and the proportion of disease resistant transgenic plants was 60.00%,57.45% ,33.33% ,53.13%, respectively.6. The total RNA was extracted from the transgenic plants with susceptible or resistant responses to PVYN, Northern blot showed the levels of transcripts accumulation varied among transgenic lines.Whereas, the RNA in susceptible plants was accumulated to higher levels than that in the resistant plants. The results revealed an inverse correlation between transgenic transcript accumulation and virus resistance. Northern blot of siRNA showed that resistant transgenic plants have special hybridization signal and pROKâ…¡-transgenic plants don't have signal. It is knowable that the resistance of transgenic plants is the result of the RNA-mediated virus resistance. |
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