Studies On T4 Lysozyme Gene Genetic Transformation And Its Potential As A Selection Marker Gene

T4 lysozyme is a phage lysozyme, has the highest lysozyme activity that can decompose gram-positive and gram-negative bacteria. It also can inhibit germination of fungal zoospore,conidia and elongation of mycelium and deactivated virus by a non-enzyme mechanism, is a kind of efficiently broad-spectrum antimicrobial enzyme. In this study,T4 lysozyme gene is cloned, and successfully inserted it into the expression vector pCambia1304, obtained a recombinant expression vector pCT4.An efficient regeneration and Agrobacterium tumefaciens GV3101-mediated genetic transformation system of Paulownia C125 leaf explants was established in this study. The optimum medium for bud induction: MS +12 mg / L 6-BA +0.6 mg / L NAA. The optimum medium for root induction: MS +2.5 mg/L6-BA +0.1 mg / L NAA +0.15 mg / L IBA. The optimum genetic transformation system is as follows:the best Agrobacterium concentration is OD600 = 0.2, infection time is 3min, 4 days for dark culture, and the optimum concentration of Cef is 600mg / L, the optimum selective concentration of Hyg is 8mg / L.Paulownia C125 leaf explants was transformated with expression vector pCT4 by Agrobacterium tumefaciens GV3101-mediated transformation. PCR amplification and GUS dying were used to detect the regenerative plants , three transgenic plants were obtained. The rate of genetic transformation is 20%. The graft tests were performed under the sterile and in vitro cultured conditions using infected plantlets with phytoplasmas and three transgenic plants clones of Paulownia were obtained. PCR with the conserved phytoplasmas 16SrRNA gene-specific primers was taken for the pathogen detection. The result indicates that the graft transmission of phytoplasma from diseased scion to healthy plant is successful.Tobacco, poplar leaf explants and Taxus callus TM3 were transformated with expression vector pCT4 and the positive control vector pCambia1304 by Agrobacterium tumefaciens GV3101-mediated transformation. Cephalexin and antibiotic hygromycinB is not be used in the processes of transformation and regeneration. The result indicates that: all three control leaves or callus are eventually yellowing, browning or dead in positive control; in all three test leaves have differentiated regenerative plants, callus TM3 is slightly browning in the experimental line (expression vector pCT4). PCR amplification and GUS staining were taken for detection the transformed tobacco plants. The rate of genetic transformation is 11.7%. PCR amplification and GUS staining were taken for detectionof the transformed callus TM3. The results indicate that the target gene was successfully transformated into callus TM3. With the different symptoms and molecular detection results of transgenic plantlets and callus, it can concludes that the T4 lysozyme gene can be used as a newly selection marker gene.