Application Of RNAi Technique In Breeding Processing Tomato Against ToMV And CMV

Tomato mosaic virus and cucumber mosaic virus are two main viruses in the tomato production areasof world and they also seriously affect on the yields and quality in northwest of our country. It is difficult tocontrol the two viruses which jointly infect processing tomato. Breeding resistant variety is the mainmeasure to control the diseases in production. But because of there are many limitations for breedingresistant variety, for example, lacking of a narrow resistant resource and taking a long time. It is noteffective to control the mixed infection which induces damages to processing tomato. As development ofthe molecular biology technology, RNAi technology provides a new way to improve plant resistance.It is suggested that mixed infection of ToMV and CMV could be solved though RNAi technology.Nicotiana occidentalis and tomato are used as model plants. ToMV and CMV coat protein gene sequenceas templates, double-gene fusion RNAi target sequences (TC-cp) are designed. According to RNAi vectorcloning sites and the same restriction sites in the sequence, the double-gene fusion RNAi target sequencesis inserted into piRDG12vector by the counter-repeat way. RNAi expression vector pBi35STC-cp issuccessfully constructed by digestion and connection. RNAi binary vector system is successfullyconstructed by PCR and digestion identification. Then the expression vector pBi35STC-cp plasmid istransformed to Agrobacterium tumefaciens GV3101. Twenty-four transgenic tobaccos and six transgenictomatos are obtained by Agrobacterium infection and they are confirmed to be resistant to mixed infectionof ToMV and CMV by PCR and virus inoculation. It is obtained three tobacco plants and three tomatoplants which are highly resistant to ToMV and CMV mixed infection, and four tobacco plants which arehighly resistant with ToMV infection, and five tobacco plants which are highly resistant with CMVinfection. The results preliminarily showed that RNAi target sequences from two viruses are successfullyintroduced into the genome of tobacco and tomato and they are confirmed to be resistant to ToMV andCMV, respectively. The approach provides theoretical basis in multiple infection for RNAi technology, andlays the foundation for the field trials.