Targeting 2a Artificial MicroRNA-Mediated CMV Resistance In Lycopersicon

The great damage is caused by Cucumber mosaic virus (CMV) in Lycopersicon esculentum, which resulted in a diminished output and degraded quality. In the experiment, artificial microRNA (miRNA) could confer on transformed Lycopersicon effective resistance to CMV infection. It has been not reported that artificial miRNA could mediated CMV resistance in Lycopersicon in China. It is shown that the transformed Lycopersicon is of great significance to the research and application on the mechanism of miRNA and the breeding methods of Lycopersicon to anti-CMV.In the experiment, according to the expression level of endogenous miRNAs in Lycopersicon, we selected a higher level expression of miRNA¹⁵⁹ as the backbone. The siRNA targeting the gene of 2a in CMV is designed with the program of siRNA Target Finder. Comparison of the homology between siRNA and Lycopersicon by BLAST, an artificial miRNA was constructed, choosing the siRNA to replace the mature sequece of pre-miRNA¹⁵⁹, and then was connected to the vector pBI121. Accordingly, the recombinant vector was transferred into Lycopersicon cells by the Agrobacterium-mediated method. In order to improve the efficacy of transformation, we design a set of experiments to select the proper factors on the transformation system and establish a highly efficient transformation. After the selection of kanamycin, the regenerated Lycopersicon plants have been identified with the molecular methods of PCR and Southern blotting. Diseases-resistant experiment was proceeding on the transformed Lycopersicon after molecular identification. It reveals that the transformed Lycopersicon is superior to the non-transformed Lycopersicon when CMV infected. The transformed Lycopersicon could resist the CMV infection.Together, in the experiment, the pBI121-amiRNA¹⁵⁹-2a was constructed, and then was transformed into Lycopersicon. The results demonstrate that the expression of amiRNA¹⁵⁹-2a targeting the 2a gene of CMV is a new approach to engineering resistance to CMV.