| Laminaria japonica Aresch (Laminariales, Phaeophyta) is one of the most important seaweeds in China because of its high nutritional value, good economic and social efficiency. It has a heteromorphic life cycle with an alternation between a macroscopic diploid sporophyte generation and a microscopic haploid gametophyte generation. An original kelp transformation model, named Laminaria japonica sporophytic expression system, has been established in our laboratory since 1990s. In this model, gametophytes were transformed using microprojectile bombardment, and young sporophytes were regenerated through the routes of parthenogenesis or inbreeding. After undergoing chloramphenicol screening, those surviving young sporophytes were cultivated in containers and harvested before maturation of sporangia. Then, products were detected and extracted from transgenic kelp. Currently, reporter gene lacZ and functional gene HBsAg have been stably expressed by this model.Considering that present transformation route included sporophytes regeneration and cultivation on the sea, which is a time-consuming procedure. Thus, L. japonica gametophytic expression system has been established and developed to avoid transgenic safety problems and shorten transformation time. Up to now, transient expression of reporter gene GFP and stable expression of functional gene rt-PA have been realized in L. japonica gametophytes. But the expression level and biomass accumulation rate of this new model are much less than that of sporophytic expression system.In this study, transformation, selection, detection and expression of human acidic fibroblast growth factor gene and tachyplesin gene were successfully realized in L. japonica gametophytes. To avoid problems of contamination of culturing transgenic marine plants in open systems, biochemical engineering technology including photobioreactor, an effective alternative to the commercial aquiculture of transgenic kelp, was utilized in the cultivation of transgenic gametophytes for rapid vegetative propagation to resolve problems of quality and quantity of this new model.The results of this dissertation were as follows:1,Human acidic fibroblast growth factor gene and tachyplesin gene were stably integrated into L. japonica gametophytes genome, and successfully expressed.2,A bubble-column photobioreactor with a working volume of 300 ml has been designed, constructed and applied in cultivation of transgenic L. japonica gametophytes. Results showed that the photobioreactor is very favorable to culture the transgenic L. japonica gametophytes, and it is compact, low cost and easy to operate monoseptically. And then, the culture system was scaled up to 2.5 L and effect of light regime on culture growth of these gametophytes was investigated. The culture growth of transgenic L. japonica gametophytes was light saturated when incident light intensity was more than 30μE m-2 s-1, and blue light could promote the culture growth.3,For improving mass transfer conditions of photobioreactor, a draft-tube internal-loop airlift photobioreactor with an effective cultivation volume of 2.5 L has been designed, manufactured and conducted to cultivate transgenic L. japonica gametophytes. Results demonstrated that the airlift photobioreactor could improve mass transfer conditions compared with bubble-column photobioreactor, and a higher dry cell density (1,990 mg L-1) was obtained. Thus, a promising and powerful apparatus (airlift photobioreactor) has been established for cultivation of transgenic L. japonica gametophytes in laboratory.
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