Construction And Genetic Transformation Of Artificial MicroRNAs For Interfering Cucumber Mosaic Virus (CMV)

Cucumber Mosaic Virus (CMV) is the most important and worldwide plant virus disease with a large number of host plants, can be spreaded by aphids in non-permanence manner. Although many methods were used to control CMV, there are very difficult to suceess. To control spread vectors through chemicals lead to enhance costs and bring about agricultural ecosystem pollution, while the vaccine with an unstable effect under different environmental condition. The resistance-breeding with longer cycles and lower efficiency has not been applied yet. Howerver, genetic engineering becomes better way to create new germplasm resources with resistance to CMV, especially double-stranded RNAs (dsRNAs) mediated gene silencing plays an important role in improving crops'CMV resistance. It is a pity that dsRNAs-mediated resistance are only act on the donor CMV strain, and exist resistance-breaking under low temperature conditions, while microRNAs-mediated gene silencing can overcome above shortcomings with resistance to the target plant viruses, stableing resistance under low temperature conditions and minimizing target fragments to prevent the risk of unhomologous viruses recombination. Here, a fragment of highly conservative regions of 2b gene from resistance-breaking strain CMV-AN as target sequences, miRNA319 precursor originates from plant as backbones, both of them were used to construct artifical miRNA319 (amiR319) in order to induce broad-spectrum resistance to CMV on host plant. The results were achieved in this study as following:1. Through primers fusion and overlap PCR,30nt of a highly conservative fragment located on double-helix interval regions of CMV-AN 2b gene was replaced the bulge of miR319 precursor suceessfully, amiR319 was confrimed by sequencing.2. AmiR319 was inserted into a prokaryotic expression vector pLITMUS28i of double T7 promoters by BamHI and HindⅢdigestion, the recombinants was named as pLITMUS28i-amiR319, while a control was named pLITMUS28i-miR319. Then both were transformed into Escherichia coli strain of HT115 with the missing of RNaseⅢ, respectively. Both are used to amiRNA-mediated resistance to CMV in vitro conditions.3. The recombinant fragment containing 2x35S:amiR319:poly(A) from intermediate vector was digested by BamHI and Ncol, then inserted into the plant binary expression vector pCAMBIA1300, the recombinant vector was named as pCAMBIA1300-amiR319, pCAMBIA1300-miR319 as a control. Then both were transformed into Agrobacterium tumefaciens strain LB4404, respectively. The amiR319 was inserted into pCAMBIA1300-amiR319 in forward orientation, pCAMBIA1300-miR319 was did so.4. The recombinant vectors pCAMBIA1300-amiR319 and pCAMBIA1300-miR319 were both transferred into N.tabacum(Samssun-nn) by agrobacteria-mediated leaf disc transformation, respectively. However, the resistance of transgenic plants to CMV need be further analysised.