DsRNA Mediated Virus Resistance To Tobacco Mosaic Virus (TMV) In Tobacco Plants

In this study, in order to effectively transcribe dsRNA in plants, an inducer of RNA silencing, two copies of Tobacco mosaic virus (TMV) partial movement protein gene (â–³MP) were ligated with soybean intron in inverted repeat manner, the recombinant was then inserted into binary vector pBIN438 under control of the 35S promoter. Fifty transgenic tobacco plants expressing TMVâ–³MP gene were generated by Agrobacterium tumefaciens-mediated transformation, resistance and possible mechanism for resistance of these transgenic plants were evaluated.The specific primers were designed and synthesized according to the published sequence of TMV MP gene, sense and antisence partial â–³MP fragment was obtained by PCR amplification using cDNA clone of pTMP(+) as template, and the amplified fragment was cloned into pGEM-T Vector to produce recombinant clone pGEM-â–³MP Antisense â–³MP fragment was obtained by digesting the clone pUCm-â–³MP with PstI and BamHl and sense â–³MP fragment was obtained by digesting with Sail. Antisense â–³MP fragment was inserted into vector pSK-In containing sense soybean intron, then the fragment containing intron and antisense â–³MP fragment was digested by Sail and BamHl, and subsequently inserted into plant expression vector pBIN438 to produce recombinant clone pBIN-In-â–³MP. Sense â–³MP fragment was inserted into pBIN-Inâ–³MP which had been digested with Sail and dephosphorylated to produce recombinant clone pBIN-TMVâ–³MP(i/r), which contains inverted repeats of TMV â–³MP separated with the intron.pBIN-TMVAMP ( i/r) was transformed into A. tumefaciens EHA105 by tri-parental mating method. The positive clones confirmed by PCR and digestion were used for transformation of tobacco plants. The vector pBIN-TMVâ–³MP (i/r) was transformed into tobacco by A. tumefaciens-mediated method via leaf disc. 50 kanamycin-resistant transgenic plants expressing TMVâ–³MP (i/r) were obtained. PCR and Southern blot analysis of transgenic plants showed that TMVâ–³MP (i/r) had been integrated into tobacco genome. Fifty transgenic plants expressing TMVâ–³MP (i/r) were inoculated with TMV, symptom observation and ELISA detection showed that three kinds of categories be typed: (1) Immune, plants had no symptom throughoutthe entire growing period, and no virus was detected; (2) Resistant, plants had no symptom in early stage, but slight symptoms were observed in late stage, limited virus particles were detected; (3) Susceptible, plants had similar or slight mild symptom as compared with untransformed tobacco plants. Among the 50 transgenic tobacco plants, 5 plants (10%) belonging to immune type, 2 (4%) belonging to resistant type and 43 plants (86%) belonging to susceptible type. Northern blot analysis showed that mRNA accumulation varied among transgenic lines and inverse correlation between transgenic transcript accumulation and virus resistance was found.