Tomato Mosaic Virus, Attenuated Vaccine (tomv And-k) 98.5kda Replication Enzymes And Movement Protein Transgenic And Their Mutual Relations And Disease Resistance

Transgenic study on 98.5KDa replicase and movement protein gene in attenuated tomato mosaic virus (ToMV-K)Postgradute: Wei Junya Tutor: Prof Zhang Jishu Prof Qiu Bingsheng(College of Life Science ,Northwest Sci-tech University of Agricultura and Forestry, Yangling,Shaanxi,712100)Plant viral disease is one of the vital viral diseases of crops and its infection can make the crops to suffer heavy losses.When plants are inoculated with viable mild strain of some viruses,the plants acquire resistance against closely related severe strains.Attenuated plant virus can protect their hosts from being attacked by related challenge virus.The presentance and development of plant gene-engineering initate a new approach to protect crops from viral diseases.An attenuated vaccine ToMV-K,originally derived from the nitrous acid-treated L-TMV,is an avirulent strain capable of protecting tomato and tobacco against the challenge of other tobamoviruses. This vaccine strain has been demonstrated genetic and immunological stability.The 98.5KDa replicase gene and movement protein gene are exist in ToMV-K genome,98.5KDa replicase gene had a main function and movement protein gene had a subordination function on its protecting effect.Therefore,introducing 98.5KDa replicase gene and movement protein gene.and the combination of the two genes into tobacco plants conferring their resistance to viruses is a new method of establishing the anti-virus transgenic plants.The work on 98.5KDa replicase gene and movement protein gene.and the combination of the two genes cloning,prokaryotic expression in E.coli and the resistance of transgenic plants to virus are described in this thesis.The 98.5KDa replicase gene (Nr) and movement protein gene (Nm) from ToMV-K genome DNA were cloned by means of PCR amplification.The Nr gene and Nm gene were inserted into the prokaryotic expression vector PET-30(a)by the restriction endonuclease EcoRⅤ/SacⅠ.The recombinant vector pET-Nr and pET-Nm were induced by the IPTG in E.coli BL21.The result showed that the Nr gene and Nm gene were overexpressed respectively as inclusion bodies after the cell were lysed by ultrasonic wave. The inclusion bodies were prepared and gained the anti-serum.The Nr gene and Nm gene from the recombinant vector pET-Nr and pET-Nm respectively were inserted into the plant expression vector pRoKⅡ by the restriction endonucleases. The Nm gene from the recombinant vector pRNm was inserted into the plant expression vector pRoKⅡ by a series of the restriction endonucleases. The Nr gene from the recombinant vector pRNr were inserted into the recombinant vector pR35S2-Nm by the restriction endonuclease ScaI/BamHI and gain the recombinant vector pR35S2-Nm-Nr.The Nr gene ,Nm gene and 35S2-Nm-Nr gene were respectively transformed into the tobacco plants by transfection of Agrobaterium. Using PCR, Southern Dot Blot and Western Blot,we detected that the Nr gene ,Nm gene and 35S2-Nm-Nr gene were introduced into genomic DNA of tobacco plants. The transgenic tobacco plants show different resistance to challenging inoculation of TMV and ToMV-K.The results of transgenic plants tested show that all transgenic plants could delay the virus disease development. The symptom of the transgenic tobacco plants with 35S2-Nm-Nr gene is mild and shows the highest resistance to virus infection.The result shows that the new method of anti-virus gene engineering is availability。...