The Studies On Transgenic Materials Of Processing Tomato Against CMV And ToMV In Xinjiang

In recent years, virus disease is one of the serious diseases endangering the processingtomato in xinjiang. This is due to planting area expand of processing tomato, long cultivationand continuous cropping areas to increase. Cucumber mosaic virus (CMV) and Tomatomosaic virus (ToMV) are two main viruses which causes serious damage to processing tomato.In actual production, processing tomato plants are mixed infection by two viruses, processingtomato branches appear seriously streak necrosis, while it generated brown patches and jiangsmall defects on the fruit surface.The yields and quality of the processing tomato are seriouslyaffected.Over the years, it has been difficult to find a solution to control crop virus diseaseeffectively, and anti-virus breeding becomes the main strategy to solve this problem. Beacusethe traditional breeding method takes long time, the progress of breeding work was limitedgreatly. In recent years, with rapid development of plant gene engineering technology,especially the development of RNAi technology, it provides a new way to solve this difficultproblem.Interefence target fragments were designed based on the full-length sequences of CMVNS0-4and ToMV SCS-2isolated from processing tomato and technology system for breedingprocessing tomato against CMV and ToMV were constructed by RNAi.1. The construction of RNAi expression vectors: The full-length sequence of the isolateof CMV NS0-4and ToMV SCS-2were analysised using the software, respectively. Twointerfence fragments were designed and joined with enzyme (BamHI/BglII), using a266bpnucleotide for introns. Four RNAi vectors (pBi35STC4、pBi35STC9、pBi35STC12andpBi35STC14) were constructed. Through a series of enzyme digestion and sequencing, theRNAi vectors were successfully constructed.2. The research of tobacco genetic transformation: The four recombinant plasmids weretransformed into Agrobacterium GV3101by electric shock respectively and successfullyintroduced by PCR identification, then transformed into Nicotiana tabacum by leaf discmethod. Through kanamycin (50mg/L) screening and PCR detection, the results showed that32plants were transgenic tobacco plants with pBi35STC4in56regenerated tobacco plants,28plants were transgenic tobacco plants with pBi35STC9in44regenerated tobacco plants,35plants were transgenic tobacco plants with pBi35STC12in58regenerated tobacco plantsand30plants were transgenic tobacco plants with pBi35STC14in50regenerated tobaccoplants. A further sign of purpose fragments successfully were introduced to the genome ofNicotiana tabacum. 3. The preliminary evaluation of RNAi effects on tobacco: Transgenic tobacco and wildtype tobacco were processed3kinds of treatments which is inoculation by CMV only, byToMV only and by CMV and ToMV at the same time. The results showed that100%of wildtype tobacco showed a different degree of symptoms of the virus20d after inoculation, nearly50%of transgenic tobacco with pBi35STC9showed different levels of the symptoms30dafter inoculation, while the remaining50%of the tobacco also showed different symptoms40d after inoculation.10%~30%of the transgenic tobacco plants with pBi35STC4,pBi35STC12and pBi35STC14showed symptoms40d after inoculation. This suggests thatamong T0generation of transgenic tobacco with pBi35STC4, pBi35STC12and pBi35STC14can delay the symptoms and antiviral effect is better than pBi35STC9. After a preliminaryevaluation of the effect of RNAi transgenic tobacco has paved the way to transformprocessing tomato.4. The research of processing tomato genetic transformation: Through the preliminaryscreening of tobacco, three recombinant plasmids (pBi35STC4, pBi35STC12andpBi35STC14) of high resistance were introduced to processing tomato Hongfan3byAgrobacterium-mediated transformation. The results showed that27regenerated plants wereobtained from1200callus of processing tomato,14of them were identified to be thetransgenic plants by PCR and the transformational rate was1.16%.33regenerated plantswere obtained from1500callus of processing tomato,19of them were identified to be thetransgenic plants by PCR and the transformational rate was1.26%.23regenerated plantswere obtained from1000callus of processing tomato,13of them were identified to be thetransgenic plants by PCR and the transformational rate was1.30%. A further sign of purposefragments were successfully introduced to the genome of the processing tomato.5. The preliminary evaluation of RNAi effects on processing tomato: The transgenictomato plants and wild type tomato plants were divided into3groups and they wereincoulated by mechanical friction. Then by observation of the symptom and RT-PCRdetection,the results showed that100%of wild type processing tomato plants showed adifferent degree of symptoms25d after inoculation, while0%~20%of the transgenicprocessing tomato plants with pBi35STC4, pBi35STC12and pBi35STC14showed symptoms40d after inoculation.The results showed transgenic processing tomato plants withpBi35STC12and pBi35STC14could delay the happening of the disease20~25d, Whiletransgenic processing tomato plants with pBi35STC4could only delay5~10d. Anyhow, theeffect pBi35STC12and pBi35STC14on virus interference is better than pBi35STC4. Thismethod laid a solid foundation for processing tomato to improve resistance against viruses.