Studies On Gene Transformation In Bryopsis Hypnoides (Bryopsidales, Chlorophyceae)Mediated By Agrobacterium Ti Plasmid

Bryopsis hypnoides Lamouroux is a siphonaceous coenocytic marine green alga belonging to the order Caulerpales. The cell organelles of B. hypnoides could agglutinate in seawater and regenerate into mature individuals. Thus, if the plasmid which contains the:arget gene is added into the protoplasm extruded, the foreign gene will be enclosed with:he formation of aggregates. In this study, we used the protoplasts of B. hypnoides as receptor of foreign gene, the Agrobacterium plasmid Ti as the vector, adding the foreign gene into the protoplasts before aggregation to see if the foreign gene could be integrated and expressed in the regenerated individuals. The study was aimed at establishing an alternative algae recombinant system. The main results obtained as follows:1. The lectin gene of B. hypnoides was selected as interest gene for investigation according to it was the inherent genetic of this alga which can integrate and express successful after transformation. To estimate the potential role of the lectin in the development of B. hypnoides aggregation, real-time quantitative PCR and western-blotting were used to detect its change during its different development stages. The quantity of the lectin gene transcript increased slightly at the five minute aggregation stage and decreased to the lowest level at6h. The immuno-localization via immunogold electron microscopy indicated that the lectin was located in both chloroplast and cytoplasm. We propose that the pivotal role of the lectin is in the agglutination of the cell organelles and maintenance of newly formed aggregates.2. Genome walking was used to get the flanking sequence of lectin gene, and we found a strong promotor in the5’untranslated region. Thereafter, the5’untranslated region,3’ untranslated region, lectin coding sequences and GFP coding sequences were amplified by PCR and used to construct four different Ti vectors p3301SLec5’G3’UTR, p3301SGFP, p3301SGFP5’-3’UTR, p3301SGFP5’S3’UTR. The Ti vectors were first transformed into E.coli DH5a, and then they were transformed into Agrobacterium.3. Cultivated the Agrobacteriums which contain Ti vectors to OD600=0.3, adding acetosyringones(AS) for inducing. Based on the results of relative transcript of lectin during different development stages, we added the Agrobacterium tumefaciens strains which were pre-induced by50μg/mL AS into B. hypnoides protoplasts rightly after they were squeezed into artificial seawater. During the agglutination process was achieved from6h to12h, we added the more strains to protoplasts each hour. When the algae grew about10cm, we collected them to detect foreign genes by PCR. The PCR detection could not amplify the band of the target gene GFP.