Research On Using Of Trichoderma And RNAi Technology To Prevent Tobacco Virus Disease

Virus disease is a large class of diseases that widespread and severe damaged in the tobacco cultivation throughout the world. Tobacco mosaic virus (TMV), potato Y virus (PVY) and tobacco cucumber mosaic virus (CMV) are the most widespread and serious virus. There are several kinds of measures to control virus disease; cultivation resistant varieties and application of antiviral pharmaceutical are most direct and effective method. This article from these two-pronged approaches to carry out research work: one is study Trichoderma a biocontrol fungus anti-viral effect and application methods in tobacco; another is achievement tobacco seedlings by RNAi technology, which can resist three kinds of viruses at the same time. Through the study found that:Trichoderma spores and Trichokonin mixed with tobacco mosaic virus respectively, can cause the virus particle fracture, cohesion, significantly reduced the ability of the virus infection, prevention of passivation are 85.81% and 56.11% respectively. Trichoderma spores mixed with TMV, then inoculate NC89, RNA of viral were significantly lower than the control, only 2.05%, disease index was significantly lower than the control. It is indicate that Trichoderma spores can passive TMV in vitro, and inhibit viral RNA accumulation in tobacco strain. Tobacco NC89 inoculation TMV first, 6 hours late, application of Trichoderma spore suspension and Trichokonin respectively, pestle into juice 48 hours later, then inoculated Samsun-NN with friction means, prevention effects were 97.98% and 70.26%, disease index was significantly lower than the control treatment, Virus levels also decreased, showing that Trichoderma and its product have therapeutic effect on viral disease, can inhibit the replication of the virus in plants and accumulation of the virus, that very important in prevention of virus secondary expansion. Trichoderma and Trichokonin can induce resistance of tobacco, genes of resistance are activated, defensive activity increased, especially in the tobacco inoculated virus, the defensive activity further enhanced, which will help to combat viruses, inhibit replication of viral RNA, reduce harm to tobacco, content of MDA was decreased. The maximum leaf length and width increased significantly, the disease index decreased significantly, induced control effects were 55.46% and 57.68% respectively. Trichokonin as Trichoderma product peptaibols, has some function of Trichoderma spores, can passive virus, inhibit the replication, induced resistance of tobacco; moreover the induced concentration is very lower. The pot and field experiments of Trichoderma spores show that leaves of tobacco treated by Trichoderma special fertilizer after water Trichoderma spores have best appearance quality, relative coordination chemical composition, best assessment of quality smoking, combined with this treatment significantly reduced leaf disease, high yield and value of output, has a high application value.Download TMVand CMV different strains'movement protein amino acid sequence and PVY polyprotein enzyme amino acid sequence from GenBank, use DNAMAN systerm to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CTP which contains restriction enzyme, after digestion got gene chips such as CTP, CT, CP, TP, C, T, P, etc. Download TMV, CMV of different strains'replicase amino acid sequence from GenBank, use DNAMAN to comparative analysis, selecte high coincidence sequence, determine their coding sequences, then synthesis gene fragment CrTr which contains restriction enzyme, after digestion got gene chips such as CrTr, Cr, Tr. 10 fragments are plugged in RNAi-pUC respectively, connect to the carrier reversely by Double Digests, pieces are got after carrier dgested by Pstâ… , after removal of phosphorus, insert to pUCC2300-35-ocs a expression vector dgested by Pstâ… first, transform competent Agrobacterium, obtain plasmid enrichment Agrobacterium, via enzyme validation,obtain Agrobacterium containing hpDNA. aseptic tobacco leaf fragments are infected by Agrobacterium, cultivate in MS media culture to grow roots, that is, RNAi tobacco seedlings are got initial, by toxicity, RT-PCR and other means to detect their resistance, get 11 anti-virus tobacco seedlings; 2 RNAi tobacco strains which have single copies and high resistance are picked out by Southern blot studies, get double homozygous transgenic RNAi tobacco seedlings quickly through haploid breeding, tobacco seedlings anti-virus features retained--can resist three virus intrusion at the same time, they and ordinary tobacco are subject to essentially the same chemical composition.