The Study On Cattleya And Chinese Cymbidium Varieties By Genetic Transformation With CiDREB1,PeDREB2,CyMV-CP Gene

Orchidaceae is one of the largest families in the flowering plants with high ornamental value. As one of important ornamental plants, orchids have an important commercial importance in world flower industry, and distribute mainly in the tropical, subtropical, liking warm and humid, with poor ability to cold and drought tolerance. In recent years, orchids were seriously infected by Cymbidium Mosaic Virus, leadingto declining of ornamental value. Simultaneously, with the world trade widely expanding, kinds of virus diseases spread in plants, and seriously affect the cultivation and production of orchids.Therefore, antiviral, drought and cold resistant new orchid varieties have become the main direction of orchids breeding.In this research, Kanamycin was the antibiotic selection of orchids. Eight species of orchids (Cymbidium hybridium, Cattleya, Chinese Cymbidium, etc.) were transformed by bombardment or Agrobacterium mediated transformation, and protocorm-like bodies (PLBs) were used as target explants, with CiDREB1 gene from Cymbidium insigne and PeDREB2 gene from Phyllostachys edulis. Results showed that the germinat rates of transgenic PLBs were between 1.7-92.2% under the antibiotic selection in the MS culture medium with appropriate Kanamycin concentration.The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was applied to quantitative detect transgenic Cymbidium with CyMV-CP gene. By using RT-PCR method, partial sequence of Actin gene was cloned from Cymbidium. On the base of Actin gene sequence, the primers of Actin-S/A and CyMV-S/A were synthesized, which were suitable for quantitative real-time fluorescent PCR. The plasmid containing the target sequence was constructed to prepare for the standard curve and detect the sensitivity.The melt curve and standard curve analysis results showed that the regression equation of Actin-S/A primers was y = - 3.271X+19.008, and related coefficient: R2= 0.994, ampliative efficiency E=102.2%; and the regression equation of CyMV-S/A primers was y = -3.731X+11.993,and related coefficient: R2= 0.997, ampliative efficiency E=85.4%.It indicated that the primers can be used for detecting the expression of CyMV-CP gene in the transgenic Cymbidium.The assay showed that the exogenous CyMV-CP genes in transgenic Cymbidium PB, PC plants had higher levels of expression, and the expression level obviously higher than control plants, and the separate relatively expression were 14.8 times and 100 times comparing to the negative Cymbidium.