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| Construction of Virus-induced Gene Silencing Vector Based on Cucumber mosaic virus (CMV) |
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Abstract Virus-induced gene silencing (VIGS) has been widely used in plant gene functional analysis. In the present study, in order to obtain VIGS candidate Cucumber mosaic virus CMV strain, (CMV) pseudo-recombinants between RNA2 from different CMV strains and CMV Fny strain (CMV-Fny) RNA1 and RNA3 were researched. Results of pseudo-recombinants CMV infection indicated that CMV -F1Tsh2F3 consisting of CMV-Fny genomic RNA1, RNA3 and RNA2 of CMV-TshR2 induced slightly mosaic symptom on tabacco (Nicotiana benthamiana), while the other CMV pseudo-recombinants caused host plants to produce stunt or leaf curf at 7 days post-inoculation(dpi). In order to obtain the Agrobacterium-mediated CMV infectious cDNA clones, genomic RNA1~3 of CMV was cloned into binary expression vector pCB301. Agro-infiltration results of CMV infectious clones under the control of duplicated Cauliflower mosaic virus 35S promoter indicated that the pCB301-CMV phenotype on the host plants was identical to that induced by CMV in vitro trancripts RNAs. The seedlings of N. bethamiana could be infected by pCB301-F1Tsh2VIGSF3 systematically and expressed slightly mosaic symptom at 7 dpi, even though F1Tsh2VIGSF3 lacking 3' end 95 nucleotides of 2b gene. Western blot results indicated that coat protein (CP) of pCB301-F1Tsh2VIGSF3 was lower than that of pCB301-F1Tsh2F3. Chlorosis and yellowing symptom appeared on the whole N. benthamiana plants infected by pCB301-F1TshR2VIGS-Su351F3 at 10 dpi, and qRT-PCR analysis showed that the magnesium chelatase subunit gene of Sulphur mRNA levle of host plants was reduced by more than 80% compared with the CMV-infected control. Chlorosis and yellowing symptom on N. benthamiana induced by inoculation of 3 or 5 passages F1TshR2VIGS-Su351F3 was the same as that caused by infection with pCB301-F1TshR2VIGS-Su351F3, and so CMV-F1TshR2VIGS-Su351F3 existed stably in the host plants. In order to confirm whether CMV-VIGS could knock down other plant functional genes, the seedlings of N.tabacum cv. Xanthi NN was infiltrated with pCB301-F1TshR2VIGS-Beclinl337F3. The data suggested that the upper leaves on autophagy related gene of Beclin1-silenced hosts infected by Tobacco mosaic virus(TMV) produced necrosis symptom, and the Beclin1 mRNA levle of hosts infected by pCB301-F1TshR2VIGS-Beclinl337F3 was reduced by 70% compared with the control plants. Agrobacterium-mediated infectious cDNA clones of CMV and its VIGS vector supply a foundation for constructing CMV-VIGS vector in other host plants.
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Received: 15 April 2015
Published: 11 October 2015
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