Genetic Transformation And Detection Of The Cymbidium Hybridum With The CymMV Or ORSV Coat Protein Gene

Cymbidium hybridum is a hybrided group which crossed between with Orchidaceae Cymbidium plants, has been cultivated extensively for commercial production. As one with high ornamental value, the orchid has commercial importance in world flower industry. It's also one of the five potted orchids and the important cut-flower species. In recent years, the most seriously damaged disease is orchid virus diseases. Cymbidium mosaic virus(Cym MV) and Odontoglossum ringspot virus(ORSV) are the two major viruses, which caused the severest damage to cultivation and production in the worldwide. They can lead to stunted plants, reduced flowering, and decline of flower quantity and quality, which would seriously affect the ornamental value of flowers, limiting the development of the industry. Therefore, anti-virus orchid variety cultivation has become the main breeding direction. Due to long breeding cycle and lack of antiviral germplasm resources, the orchid anti-virus breeding has been at a standstill. The application of genetic engineering technology has supplied a new strategy for it.In this research, the coat protein genes(CP) of Cym MV and ORSV were cloned and constructed plant expression vectors with p BI121. We transformed the coat protein genes into protocorm-like bodies(PLBs) of Cymbidium hybridum(PC, PB) by Agrobacterium tumefaciens-mediated, and than obtained transgenic plant regenerations. At the same time, a sensitive and accurate method for the detection of transgenic plants was established. These offer references for the orchid anti-virus breeding by gene engineering technology, and provide germplasm resources for training new orchid varieties.1. The coat protein genes(CP) of Cym MV and ORSV were cloned. Sequence of the Cym MV CP gene was 672 bp, the similarity degree of this gene and another gene on NCBI(accession no. AB197937) was 99%. Likewise, Sequence of the ORSV CP gene was 477 bp, the similarity degree of this gene and anther gene on NCBI(accession no. DQ915440) was 99%.2. Plant expression vectors of p BI121-Cym MVCP and p BI121-ORSVCP were constructed. Recombinant plasmids were transformed into Agrobacterium tumefaciens strain LBA4404. A optimization transformation method of orchids protocorm-like bodies(PLBs) was developed.3. The detection methods of common PCR, Nest-PCR and loop mediated isothermal amplification(LAMP), which were established with expression vectors p BI121-Cym MVCP and p BI121-ORSVCP. Moreover, the conditions of LAMP system were optimized, obtained the optimum d NTPs concentration, internal and external primer concentration ratio and the amount of Bst DNA polymerase. The optimized reaction conditions as follows: 0.5 mmol·L-1 d NTPs,0.8 ?mol·L-1 inner primers,0.2 ?mol·L-1 outer primers,4 U Bst DNA polymerase,2.5 ?L 10×Thermo Pol Buffer and 10 ng recombinant plasmid DNA?4. The minimum detection limit of the common PCR, Nest-PCR and loop mediated isothermal amplification(LAMP) respectively were 5.0×10-4ng·?L-1, 5.0×10-7ng·?L-1 and 5.0×10-10 ng·?L-1. So, the sensitivity of Nest-PCR is about 1000 times higher than common PCR; and the sensitivity of LAMP is 1000 times higher than the Nest-PCR. The sensitivity of LAMP is the highest.5. The presence of the introduced gene in the 213 transformation orchid plants individuals was confirmed by common PCR and Nest-PCR. Twenty-nine positive plants were detected by common PCR, and the positive rate was 13.68%. By Nest-PCR, there are 54 positive individuals, which contain all of the common PCR detected individuals. The positive rate of Nest-PCR was 25.35%. The Nest-PCR is more sensitive and accurate for detection of transgeneic orchids.