Different Virus-gene Orders Affect Virus Resistance Mediated By RNA Silencing In Transgenic Tobacco

Potato Virus Y(PVY), tobacco mosaic virus(TMV) and cucumber mosaic virus(CMV)are serious threat to tobacco production and serious impediment to plant growth, causing significant economic losses, so it is imperative to develop resistant varieties. RNA mediated virus resistance is a strategy for the prevention and treatment of viral diseases in recent years,which has been widely used in antiviral research for its advantages, such as a high degree of resistance(almost immune); sustaining resistance; biological safety and so on. In this study,coat protein genes(CP) of PVY, CMVand TMV, suppressor genes of PVY,CMV and TMV(PVY HC-pro、CMV 2b and TMV126) were served as the target sequences. We designed different chimeric-gene hairpin RNA(hpRNA) constructs based on the target sequences and generated transgenic plants, introducing electric shock method by Agrobacterium LBA4404,transformed tobacco leaf discs using NC89.Transgenic tobacco were acquired resistance identification, comparison of different chimeric genes affect the order of RNA-mediated virus resistance, and provide the basis for the cultivation of genetically modified plants and more virus. The main results and conclusions of the study were as follows:(1) According to the laboratory has cloned genes and 2b CMV CP gene, PVY CP gene and the complete sequence of HC-pro gene, TMV CP gene and p126 gene primers were designed and synthesized by PCR amplification length of 300 bp the same size fragments,fragments constructed chimeric genes were inserted into the binary vector pROKⅡ construct a plant expression vector pPTC-CP, pCPT-CP, pTCP-CP, pPTC-2b, pCPT-126 and pTCP-HC.(2) The constructed plant expression vector pPTC-CP, pCPT-CP, pTCP-CP, pPTC-2b,pCPT-126 and pTCP-HC by electroporation into Agrobacterium LBA4404, using tobacco leaf disk transformation NC89. After identification kanamycin and PCR screening, and ultimately transgenic pPTC-CP, pCPT-CP, pTCP-CP, pPTC-2b, pCPT-126 and pTCP-HC of T0 generation plants 102, 103, 116, 114, 105, 108.(3) Transgenic plants by Southern blot analysis showed that foreign gene has been successfully transferred into the tobacco genome, and smooth expression, combined withresistance analysis showed antiviral efficiency and there is no significant relationship between the number of copies.(4) Transgenic plants Northern blot analysis showed that the transgenic RNA level has been expressed in transgenic tobacco accumulation of total RNA inoculation of susceptible inoculation significantly higher disease resistance in transgenic tobacco, that is the purpose of gene transcription accumulation products and resistance capability negative correlation, which also proved to produce virus-resistant transgenic plants resistant to the virus-mediated RNA produced.(5) SiRNA Transgenic Plants Northern blot analysis showed a primary RNA was expressed in vivo, secondary RNA in the transcription and translation occur simultaneously transmitting signals. Quantitative PCR results showed different expression levels of transgenic plants Total RNA of different genes of the order.