DsRNA-mediated Resistance To Cucumber Mosaic Virus (CMV) And Potato Virus Y (PVY) In Transgenic Tobacco Plants

RNA silencing is a natural antiviral defense reaction in plants, and RNA silencing suppressors coded by most plant viruses can overcome the host defense reactions. Plant expression vectors containing inverted repeats fragments of partial CMV 2b gene, PVY HC-Pro gene, 2b and HC-Pro genes were constructed by utilizing post-transcriptional gene silencing principle. Transgenic tobacco plants Yunyan 85 anti-CMV, PVY, both CMV and PVY were obtained by forming dsRNA in plants to silence target gene 2b and HC-Pro, and the feasibility of anti-virus gene engineering strategy of transgenic plants mediated by dsRNA was ascertained.1. Cloning and sequence analysis of CMV 2b geneThe diseased tobacco samples showing visible CMV symptoms were collected from Bozhou, Anhui Province. CMV RNA2 partial fragment was amplified by RT-PCR, and then it was cloned and sequenced, and 2b gene included in it is 336 nucleotides, encoding 111 amino acids. Nuclear sequence comparing showed that CMV 2b genes derived from Anhui shared the highest sequence similarity (98.8 %) with that of 2b genes from Korea (AF033667). A phylogenetic tree based on alignment of nucleotide sequences of 2b genes was constructed. The results also showed that CMV 2b genes from Anhui had the closest relationship with AF033667.2. Construction of inverted repeat plant expression vector1) Construction of inverted repeat plant expression vector of partial 2b geneApproximately 300 bp sense fragment CMV 2b (r) and antisense fragment CMV 2b (i) were amplified using 2b gene from Anhui as template. Antisense fragment CMV 2b (i) and sense fragment CMV 2b (r) were inserted into the two sides of intron of vector pSK-In in priority order, and the recombinant plasmid pSK-2b (r)-In-2b (i) was constructed. Sense and antisense fragment containing intron was further inserted into plant expression vector pBIN438, and the recombinant vector pBIN-2b (r)-In-2b (i) containing inverted repeats of partial sequence of CMV 2b genes was acquired ultimately.2) Construction of inverted repeat plant expression vector of partial HC-Pro gene (Referring to the Master thesis of Chen Wei)3) Construction of inverted repeat plant expression vector of partial 2b and HC-Pro genes The inverted repeats of HC-Pro gene was inserted into the pBIN-2b (r)-In-2b (i). The recombinant plasmid pBIN-2b (r, i)-HC (r, i) which simultaneously contained sense and antisense fragment of 2b and HC-Pro genes was obtained ultimately.3. The obtaining of positive transgenic tobacco plantsThree plant expression vectors of pBIN-2b (r)-In-2b (i), pBIN-HC (r)-In-HC (i), pBIN-2b (r, i)-HC (r, i) were introduced into Agrobacterium tumefaciens EHA105, and Nicotiana tabacum Yunyan 85 plants were transformed via leaf disc. Three resistance regenerative plants were obtained after precultivating, agrobacterium-mediated infiltrating, differentiation cultivating and rooting cultivating. Resistance regenerative plants were detected by PCR, and T₀ generation positive transgenic plants of transforming inverted repeat fragment containing 2b gene, HC-Pro gene, both 2b and HC-Pro genes were obtained.4. Identification of disease resistance of T₀ generation transgenic tobacco plantsTobacco plants transformed with 2b gene were inoculated with CMV, and tobacco plants transformed with HC-Pro gene were inoculated with PVY, and tobacco plants transformed with 2b and HC-Pro genes were inoculated with CMV and PVY. T₀ generation transgenic tobacco plants anti-CMV, PVY, both CMV and PVY were obtained through observing biological symptoms and indirect-ELISA detecting.