Optimization Of A Gene Silencing Vector Based On Cucumber Mosaic Virus

As a typical member of the genus Cucumovirus in the family Bromoviridae, Cucumber mosaic virus (CMV) has the most widespread host range in the world. Virus-induced gene silencing (VIGS) is an efficient tool to study the functions of plant gene. In this study, we constructed a gene silencing vector based on CMV-Tsh RNA2. In order to analyze gene silencing efficency, different reporter genes were cloned into CMV-Tsh RNA2, accumulation of target gene was assessed by semi-RT-PCR, and infectious clones of CMV of Agrobacterium were constructed successfully. Main results in this study followed as:Firstly, pseudo-recombination viruses were constructed, with RNA1and RNA3from CMV-Fny and RNA2from different strains of CMV subgroup I including Cb7RNA2, CNa RNA2and Tsh RNA2. The pseudo-recombination viruses were mechanically inculated onto Nicotiana. benthamiana7days post-inoculation(dpi), syptoms of crimple and twist were obsvered on host plants inoculated with F1F2F3, F1C2F3, F1CNa2F3, but light mosaic apperard on the seedlings of host plant infected by F1Tsh2F3. Thus, CMV-Tsh RNA2was chosen to construct a gene silencing vector.Secondly, three candidates of gene silencing vector based on2b of CMV-Tsh RNA2were constructed. The first one was named Tsh R23/42b whose2b was shorter than normal by95nt; the second one was named Tsh R2A2b whose2b was deleted completely; Tsh R22b was construed by inserting by Mlu Ⅰ and SnaB Ⅰ between stop codon of2b and UTR of RNA2. In vitro transcripts of pTsh R23/42b、pTsh R2Δ2b and pTsh R22b were mixed with CMV-Fny RNA1, Fny RNA3respectively to inoculate onto N. benthamiana, and infectivity of pseu-recombinant virus were dected by RT-PCR.353bp PDS fragment of N. benthamiana(named NbPDS) was inserted into pTsh R23/42b、pTsh R2Δ2b and pTsh R22b respectively to compare photobleaching on N. benthamiana after inoculation. pTsh R23/42b was chosed as a gene silencing vector to do further analysis.Thirdly, the effect of gene silencing of different reporter genes were tested. Fragments of PDS, Su or Green fluorescent protein gene(GFP) were inserted into pTsh R23/42b to analyze phenomenon of photobleaching, yellow leaf table on N. benthamiana or red level on N. benthamiana16c. Besides, semi RT-PCR was used to analyze expression level of target gene in plants. The resluts showed that obviorus photobleaching was observed on N. benthamiana inoculated with F1Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana infected by F1Tsh R23/42b-Su351F3; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; the accumulation of PDS,Su and GFP mRNA all declined. Sap inoculation was adopted to analyze stability of F1Tsh R23/42b-NbPDS354F3in N. benthamiana. The results indicated that F1Tsh R23/42b-NbPDS354F3could exist in N. benthamiana steadily and induce photobleaching in host plants.Fourthly, partial promoters of PDS, GFP of N. benthamiana, fragments of soybean PDS(named proPDS,35s and GmPDS) and part of Beclin were inserted into pTsh R23/42b to analyze photobleaching on N. benthamiana and soybean, red level on16C and symptoms on NN transgene tobacco inoculated with TMV. The resluts showed that:significant photobleaching was observed on N. benthamiana and soybean; the leaf of16C which was inoculated with F1Tsh R23/42b-GFP217F3had turned red under UV lamp; Systemic necrosis emerged on NN transgene tobacco which was inoculated with TMV, after being inoculated with F1Tsh R23/42b-Beclin346F37dpi.Fifthly, CMV infectious clones of Agrobacterium were constructed. Full-length sequences of Tsh R23/42b, Tsh R23/42b-NbPDS354, Tsh R23/42b-Su351, Fny RNA1, RNA2, RNA3were inserted into transient expression vector pCB301to transform into Agrobacterium. Photobleaching and yellow leaf table on N. benthamiana induced by F1Tsh R23/42b-NbPDS354F3and F1Tsh R23/42b-Su351F3were analyzed through inoculating N. benthamiana with Agrobacterium infiltration inoculation. The resluts showed that:symptoms induced by CMV-Fny of Agrobacterium were in keeping with ones induced by in vitro transcripts RNA; significant photobleaching was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-NbPDS354F3; yellow leaf table was observed on N. benthamiana which was inoculated with F1pCB301-Tsh R23/42b-Su351F3.