Optimization Of Transformation Model Of Laminaria Japonica (Phaeophyceae) And Introduction Of A Vaccine Gene

The original transformation model for the seaweed Laminaria japonica (Phaeophyceae) employed female gametophytes as micro-particle bombardment targets and parthenogenesis as a route to produce transgenic parthenosporophytes. By using the original model, in situ detection for lacZ expression was carried out in this thesis and resulted in low transformation efficiency and chimeric expression pattern. New approaches were developed in this study by transforming female and/or male gametophytes followed by inbreeding process. LacZ expression was detected in zygotic sporophytes generated by both female- or male-targeted transformation. The former showed similar results to parthenogenesis pathway, while the latter led to higher transformation and expression efficiency and 3.5% zygotic sporophytes were stained all-blue, implying the integration of lacZ at one-cell stage.A vaccine gene encoding hepatitis B surface antigen (HBsAg) was bombarded into female gametophytes and transgenic parthenosporophytes were obtained. Results of quantitative ELISA showed that HBsAg in transgenic kelp was 0.529 μg/mg soluble proteins on average and the highest value was 2.497 μg/mg, implying that recombinant HBsAg in 2.25 g fresh weight of transgenic kelp corresponded to 15 μg vaccine required in vein injection for one adult. Furthermore, HBsAg gene expression was obtained in zygotic sporophytes by using male gametophytes-targeted bombardment.This research provided a potential method for seaweed transformation using spore-mediated bombardment followed by normal development process, also reported for the first time the introduction and expression of a target gene in transgenic seaweed.