| Cucumber green mottle mosic virus (CGMMV) is widely distributed in the world. It is the most important causal agent of diseases affecting cucurbitaceae crops,leading to huge economic losses. Despite the seriousess of CGMMV, no effective controlling methods are available at present for this virus. Bruceine D is extracted from the seed of traditional Chinese medicine Brucea, studies have shown that Bruceine D has significant inhibitive effect on TMV,CMV,PVY. In this sudy, the activity affect of Bruceine D on CGMMV was done.In this study, the CGMMV particles were purified from the cucumber leaves infected with CGMMV. The anti-serum of CGMMV was prepared successfully. By indirect ELISA way, the anti-serum titer was 16,000. Total RNA was extracted from leaves of cucumber infected by CGMMV. RT-PCR was then conducted to obtain CGMMV-MP, which was then ligated into the vector pMD18-T The gene was then modulated into pET-29a, creating the recombinant plasmid pET-29a-MP and sequenced. Sequence analysis revealed that the gene was 795 bp long and 100% identical to the sequence of CGMMV-GX-MP reported previously. The maximum amount of the MP was obtained 6 hours after treatment with IPTG. The anti-serum to the movement protein was prepared by immunizing rabbit with the target protein.The titer of the anti-serum was determined to be 8,000 by using indirect ELISA. Western-blot experiments indicated that the anti-serum showed good specificity.Antiviral effect of Bruceine D on CGMMV was investigated. The half-leaf method and the leaf disc method was used to test the inhibition rate of Bruceine D to the infection and proliferation of CGMMV, respectively. .The results showed that the infection and proliferation inhibition rates were 78.99% and 81.05% respectively, at the concentration 10μg/mL. When the concentration is up to 20μg/mL,40μg/mL, the rate of inhibition increased to 90%. However, significant negative effects on the host plant was obsrerved, which include the presence of large area of dry spots in leaves of natural and identification hosts. Observation under electron microscopy showed that treatment with Bruceine D in vitro results in fracture of CGMMV particles.Relative quantitative PCR was emploied to investigate the effects of Bruceine D on the expressions of the Rep, MP and CP of CGMMV. The results indicated that the inhibition rates of Bruceine D to the expressions of these genes were 88.77%, 92.09%, and 98.09% respectively. The effects of Bruceine D on the gene expression of CGMMV was also confirmed by western-blot exprements, which showed that the expressions of MP and CP were affected by Bruceine D. |
