Study On Agrobacterium Mediated R-Fom-2 Gene Transformation Of Xinjiang Melon

Melon(Cucumis melo L.)in Xinjiang is famous for its unique quality in domestic and foreign. But in recent years, melon diseases especially Fusarium wilt of fungi diseases had a negative effect on melon's products and quality. The main methods to prevent Fusarium wilt are using chemical pesticide which brings about potential danger to health of people and environment. Traditional breeding methods are time consuming and inefficient, it is difficult to breed resistant lines which are fit for planting. The technique of gene engineering to realize disease resistance molecular breeding is a new approach and will has a broad application in the future.We selected'Japan annong-2'as material to clone sequence relating to resisting Fusarium wilt(R-Fom-2 DQ287965) by PCR. Homology analysis indicated the R-Fom-2 obtained shared high similarity with Fom-2. Northern blotting was carried out to analyze the characters of X-Fom-2 which is the core sequence of R-Fom-2. The expression level was very low in susceptible melon either infected by Fusarium axysporum f.sp.melonis or not. The results of Northern blotting preliminarily showed that R-Fom-2 maybe play an important role in the process of resistance to fusarium wilt.Melon cultivars"huanghou"is used in our experiment. To use Agrobacterium -mediated transformation, the optimal conditions for R-Fom-2 gene transferred into Xinjiang melon were studied.The fragment of aimed gene was purified after enzymic digestion plasmid pMD18-T/R-Fom-2-2 with Sac I and Sal I and cloned into pCAMBIA13011 to obtain the recombinant plant expression vector pCA-Fom-2.The regeneration system of melon was achieved. Cotyledons were used as explants. The explants were cultured on MS with 6-BA 1.0 mg/L to form shoot by direct–differentiation.The expression vector pCA-Fom-2 was introduced into Agrobacterium tumefaciens LBA4404 by freeze-thaw method. The Agrobacterium mediated gene transformation procedure was established. The suitable Agrobacterium density (OD₆₀₀=0.5), infection time (10-12 min) and co-cultivation period (3-4d) were necessary to improve transformation frequency. Carb strength was 500 mg/L and Hyg was 10mg/L.Analyzing hygromycin-resistant plants by PCR and RT-PCR method confirmed that R-Fom-2 gene was integrated into melon genome, the transformation frequency was 3%, 85% rooting frequency was obtained.The reaction of detached leaves of transgenic plants to Fusarium oxysporum infection was examined. The results indicated that transgenic plants gained certain resistance. It is further demonsticated that R-Fom-2 was a functional gene of Fusarium wilt resistant genes.To use Agrobacterium-mediated transformation, the R-Fom-2 gene was transferred into Xinjiang melon. The successful integration and expression of the gene was identified by PCR and RT-PCR. This research will not only make preparations on studying disease resistance molecular mechanism of melon, but also provide reference values for selecting excellent melon resistance genes by the means of disease resistance molecular breeding in Xinjiang.