Resistance To Cucumber Mosaic Virus (CMV) In The Transgenic Tomatoes Inserted By Ribozyme And Antisense, Sense Genes Of CMV-CP

Cucumber mosaic virus (CMV) is one of the most severe virus pathogens in tomato plant and causes its yield and quality reduced in many countries. In our country the losses in the yield and economy achieve 15% of total output and several billion Yuan every year. Up to now, there is no effective method available for protecting plants from CMV infection and reducing the losses of the yield. Due to the lack of CMV-resistant gene present in natural plants, the traditional plant breeding cannot meet the requirement for improvement of crop production. In recent years the rapid developments of plant genetic engineering and molecular biology have provided new ways for controlling virus infection and the possibility of protecting plants from diseases. Ribozyme technology is one of the new ways developed in current years. Ribozymes (RNA enzymes) are RNAs-like protein enzymes through an intramolecular cleavage of RNAs and can potentially be used to protect plants against virus diseases in terms of that they have the ability to cleave viral RNAs. Gene silencing, which is another technology developed in the past several years, has emerged as a topic of general interest in molecular biology and is a viral defense system which might be one of its original functions in primitive eukaryotes. It can protect plant's genome from transposons and transgene's expression in addition to defending the plant against viruses. Antisense technology can inhibit gene expression and virus replication in transgenic plants. We discussed the mechanisms of ribozyme, gene silencing and antisense RNA in plant protection through the way of gene transformation. The results are follows: Cloning coat protein gene of the Chinese strain of CMV: We obtained the sick leaves with typical symptoms of CMV disease in Langfang, Hebei province and proved that the CMV exist in leaves through DAS-ELISA identification. This virus strain was named as 988LJ. A 1049bp cDNA fragment of the coat protein from this strain obtained contains an open reading frame of 657bp and 218 of deduced amino acid residues, which are high identity with those from the other trains reported. Construction of three plant expression vectors of ribozyme, antisense and sense genes: Four target sites in the cDNA sequence encoded for the CMV coat protein were selected. The desired poly-hammerhead ribozyme RNA sequence containing four catalytic domains was constructed according to the four sites and then inserted into the plant expression vector. Synchronously, the sequence containing the same region of the CMV CP gene as ribozyme, however, without the catalytic domains, was inserted into the vectors via antisense and sense orientations. Attainment of the regenerated plants transformed by three plant expression vectors: three vectors were transformed into three tomato cultivars by Agrobacterium mediated-transformation method. A lot of putative transgenic plants were obtained from the regenerated plants. The marker gene nptII and the ribozyme, antisense, sense genes in these transformants were detected by PCR and they could be amplified in most of the transformants with the correct products. Southern blot was used to analyze some of the transformants, resulting in that the genes were successfully inserted into plant genomes and expressed correctly. CMV infectivity tests using T1 and T2 transgenic plants: In order to test viral resistance of the plants, T1 progeny of the transgenic plants were inoculated with CMV. Five plants screened from 54 independent transgenic tomatoes showed their protections against CMV infection. Among them, one contained the ribozyme gene, three contained the antisense gene, and one contained the sense gene. 47 plants of T2 progeny of the tomato five resistance lines were inoculated again with CMV, indicating that most of them were highly resistant to CMV. This result showed that the resistance is stably transmitted. Measurements of photosynthetic parameters of the transgenic tomato plants with CMV: After the transgenic plants selected with high resistance, middle resistance to CMV and the non-transformed control plants were inoculated with CMV, the measurements of the photosynthetic parameters, such as content of pigments, leaf fluorescent dynamics were conducted. It is revealed that there was no significant difference in chlorophyll content, and Fv/F0,Fv/Fm between the highly resistant plants inoculated with CMV and those not inoculated with CMV, but there was moresignificant difference in these values between the middle resistant plants inoculated with CMV and the non-transformed control plants. Therefore, it is illuminated that the transgenic plants with the resistance of disease showed that the inserted genes played the role in the protection from virus infection and retardation of developing disease.