Establishment Of A Genetic Transformation Model For Ulva Prolifera

Ulva prolifera is multicellular green seaweed. It is the only dominant species ofgreen tides in the Yellow Sea, which can accumulate large amount of biomass in ashort period. Of all the possible reasons, its own biological traits are in the importantposition. Now, much research has been conducted in depth and a large number ofgene sequences were obtained and annotated from U. prolifera, but effectivemethods of verifying these genes‘function at the individual level are not available.Establishing the techniques of genetic transformation and stable expression canprovide a powerful tool for related research and lay the foundation for constructingrecombinant protein expression system of U. prolifera. In addition, fast growth speed,high protein content and closed culture are advantages of U. prolifera.In this paper, we establish the transformation system of U. prolifera at fouraspects, including transgenic receptors, regeneration pathway, transformationmethod and screening method.In terms of transgenic receptors, we invented a new method of eliminating wildalgae from seedlings of U. prolifera with Tigriopus japonicus, obtained a series of U.prolifera strains with genetical uniformity, and proliferated U. prolifera in a closedsuspending culture device. These works can ensure material supplies to genetictransformation and create conditions for safe expression system.As for regeneration pathway, three methods were established, includinginduction and release of reproductive cell, in situ germination of vegetative cell andprotoplast preparation and regeneration. A single cell can develop into new thallus byall three regeneration way. Moreover, the physiobiochemic parameters correlatingwith protoplasts yield was found.Two transformation methods, particle delivery system and glass-bead agitation,were compared. Transient expression of GUS and lacZ gene was detected afterparticle bombardment. It suggested that the parameters of gene gun and vector elements are suitable for U. prolifera.For screening transformants, the sensitivity of protoplast-regenerated seedlingof U. prolifera to phosphinothricin and six kinds of antibiotics was tested. Seedlingswere found not sensitive to ampicillin, kanamycin, streptomycin sulphate and zeocin,less sensitive to chloramphenicol and hygromycin, but very sensitive tophosphinothricin. Besides, the sensitivity to phosphinothricin was significantlycorrelated with photoperiod. LD50and95%confidence range of phosphinothricinwere calculated.In a word, we obtained the experimental materials, established the regenerationpathway, achieved the GUS gene expression and determined the screening process.The above four aspects were systematically studied and the results can providemethodology basis for stable expression system of U. prolifera.