RT-PCR And Dot-Blot Hybridization Detection Of Lily Symptomless Carlavirus And The CP Gene Expression In Escherichia Coli

Lily Symptomless Carlavirus(LSV) is one of the most serious virus infecting lilies. It presents in several places around the world but the ingress is forbidden in our country, for it causes large spoilage in lily plant cultivation. Serology method usually used in virus quarantine and plant tissue culture, especially ELISA, but for extracting LSV as antigen is difficult, most detections have to depend on the imported examine boxes. Molecule biology method has high sensitivity, but not used widely. In this paper, both molecule biology method for detecting LSV and prokaryotic expression were studied for quarantine and antibody making, respectively. Different serology methods were used to detect the infectiveness of LSV in lilies. The results showed that the sensitivity of detection in leaves is about 320μg by ELISA , 160μg by DOT-ELISA and I-ELISA, and 80μg by DAS-ELISA. The percent of detection were 30%,35%,45%and 55%, respectively. The 412bp fragments was amplified from the extracted RNA of LSV infected lily leaves,using the specific primers designed according to the nucleotide sequences of the virus, and then cloned into pUCm-T vector. The cDNA probe were synthesized by digoxigenin random labeling the fragments that recuperated from the recombinant vector, and used in dot-blot hybridization detection. The sensitive level of RT-PCR and dot-blot hybridization detection of the total RNA of infected lily leaves by LSV was 1:10^-6 and 1:10^-2 respectively. LSV coat protein gene was amplified by RT-PCR from the extracted RNA of infected lily, and ligated to the expression vector pUC19. The plasmid was transformed into E.coli DH5a and then induced by IPTG. The gene was highly and especially expressed by SDS-PAGE and Western blot analysis. The molecular weight of the recombinant protein was about 35kD. The discussion was mainly focused on the application of these detection methods, the problem result in the virus extraction progress, and the construction of expression plasmid having LSV CP gene. The results presented in this study made a standard to detect LSV by RT-PCR and dot-blot hybridization, and had Escherichia Coli strain expressing LSV CP gene, which would be helpful for further application.