Study On The Cultivation Of Transgenic Anti Mosaic Virus Luohanguo By Rnai Technology

Fructus momordicae(Siraitia grosvenorii) mosaic virus seriously affects the yield of fructus momordicae. And the most effective way for preventing and controlling fructus momordicae mosaic virus is to get resistant varieties by transgene. Thus, the maturation of transgenic and RNAi technology lays the foundation for cultivating transgenic fructus momordicae withanti-mosaic virus ability.The main causal viruses of fructus momordicae mosaic virus was Zucchini yellow mosaic virus(ZYMV), and it is wide distribution and great harmfulness.In order to breed antiviral fructus momordicae cultivar and to obtain virus strain that occurs widespread in local fructus momordicae during production activities. The fructus momordicae plants that are planted in the large scale base and had got mosaic virus were chosen as the experimental materials. For improving the replication efficiency of mosaic virus RNAi genes, this study linked together the multiple functional gene fragments from the viruses and as target fragment transferred into fructus momordicae plants. Consequently, a high efficient genetic transformation system for fructus momordicae was established and fructus momordicae plants harboring broad spectrum of mosaic virus resistance were obtained through improving fructus momordicae germplasm using genetic engineering method.The optimal explants for tissue culture of Siraitia grosvenorii was the shoot tip, while the optimum hormone combination of culture medium on bud induction was 6-BA 0.5 mg/L+NAA 0.05 mg/L, the induction rate was 97.5 % in this condition. The optimum hormone combination of culture medium on multiplication was 6-BA 0.5 mg/L+IBA 0.1 mg/L+GA3 0.1 mg/L and the growth rate could up to 9. Through the experiment of the leaves regeneration conditions, it is found that the optimum culture medium on leaves regeneration was MS+6-BA 1.0 mg /L+IBA 0.5 mg /L+TDZ 0.05 mg /L and the rate of budding could up to 10.68. It can provide a large number of materials for the experiment that study on the cultivation of transgenic anti mosaic virus Luohanguo.In this study, the regeneration frequency of three kinds of explants, stem, petiole and leaf-disc of the tissue culture seedlings of fructus momordicae, was compared and found that leaf-disc with strong regeneration ability and high regeneration efficiency was the most suitable explant for fructus momordicae transformation system. At the same time, this paper has been optimized at the conditions of genetic transformation, and the results showed that pre-culture 1 day, bacterial concentration of 0.2, bacterial infection time 15 min, co-culture 4 days, 200 mg/L Carbencillin concentration and 40 mg/L G418 concentration was chosen as stressor could get the better transformation efficiency.The object fragment of RNAi was obtained by series connection Hc-pro, NIa, NIb, CP and 3’UTR multiple genes from ZYMV. The obtained gene fragment was cloned into the intermediate vector pXQ which could form RNAi stem-loop structure and constructed RNAi structure fragment. Finally, through the PCR identification of positive clones, getting plant expression vector pLou- Anti5 with the correct target fragment. The objective gene was introduced into fructus momordicae using agrobacterium tumefaciens AGL0.A total of 89 transgenic fructus momordicae plants were obtained.After infection experiment, it was conformed that the transgenic fructus momordicae had a certain resistance to infection.Resulting in the obtaining of positive fructus momordicae plants that had broad spectrum of mosaic virus and herbicide resistance, which has lots of production application prospect.