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| RNA Silencing-mediated Resistance to Tomato yellow leaf curl China virus in Transgenic Tobacco Plants |
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Abstract In recent years, Tomato yellow leaf curl China virus (TYLCCNV) is spreading continuously from south to north in China, which caused the yield reduction, even no yield and serious economy loss. We aimed to obtain transgenic tobacco to immune to TYLCCNV. In this study, we constructed inverted-repeat dsRNA (double-strand RNA)(pBIN19-2CP) with TYLCCNV partial of coat protein gene(187 bp) using RNA interference technology to silence the expression of the target gene. In order to get the transgenic tobacco, the dsRNA had been transformed into Nicotiana benthamiana. Restriction endonuclease Kpn I and Xho I were introduced to amplify the reverse fragment, and restriction endonuclease BamHI and Hind Ⅲ were introduced to amplify the forward fragment. pBIN19 was used as plant expression vector. The inverted-repeat dsRNA recombinant was carried out by PCR analysis, restriction enzyme digestion and DNA sequencing, the results were consistent with expected. The recombinant was transferred into Agrobacterium tumefaciens LBA4404 with the freeze-thaw method, then recombinant A. tumefaciens was transformed into tobacco by leaf discs method. And the infection medium was MS, the co-cultivation medium was MS+6-BA (1 mg/L)+NAA (0.1 mg/L), the selection medium was MS+6-BA(1 mg/L)+ NAA(0.1 mg/L)+Kn(50 mg/L)+Cef(250 mg/L), the rooting medium was MS+NAA(0.2 mg/L)+Kn(50 mg/L)+ Cef (250 mg/L). We had obtained the transgenic tobacco plants. To evaluate the transgenic tobacco, PCR screening, Southern blot and Northern blot were carried out. The PCR screening results of the transgenic plants showed that the positive plants were obtained among the transgenic plants. Some positive plants were chose randomly to conduct Southern blot, the result showed that the target gene had been integrated into tobacco genome. When transgenic tobacco plants were inoculated with TYLCCNV, symptom observation and PCR screening demonstrated that among 41 transgenic tobacco plants, 6 plants (14.6%) were immune type, 5 (12.2%) were resistant type and 30 plants (73.2%) were susceptible type. Northern blotting analysis indicated that the levels of target mRNA accumulation varied among transgenic N. benthamiana lines, and inverse correlation between target mRNA accumulation and virus resistance was found. The transcript accumulation in the immune type transgenic tobacco plants was little, but the level of transcript accumulation in the resistant type and the susceptible type transgenic tobacco plants were obviously high. This study has certain theoretical directive significance for controlling TYLCCNV using RNA interference technology.
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Received: 14 November 2012
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