Kelp Gametophyte Nutrition Proliferation System To Achieve Heterologous Gene Expression

An original kelp transformation model (PCT/CN03/00534;Trends in Biotechnology, 2005,23,264-268.) has been established in our lab since 1993. In this model, the gametophytes weretransformation target, young sporophytes regenerated through the routes of parthenogenesis andinbreeding, after undergoing the chloramphenicol screening, those survival young sporophyteswere cultivated in containers and harvested before maturation of sporangia. Reporter gene lacZand target gene HBsAg had been expressed by this model.Considering that present transformation route included sporophytes cultivation on the sea,which is a time-consuming procedure. In this research, a new foreign gene expression platformwas establish based on following improvements and test:1) To find a new selection marker for transgenic kelp gametophytes.2) To establish a set of enclosed photo bioreactor for gametophytes proliferation.3) To develop new reporter gene.4) To clone endogenous promoter.5) To expression target gene rt-PA (encoding Reteplase) in this new system.The results of this dissertation were as follows:1) BASTA? selection procedure was 40 mg/L, screening for 7d×3times.2) Promoter SV40 could drive stable expression of bar (BASTA(?) resistance gene) gene in kelpgametophytes.3) By controlling the fluorescent backgrounds, transient egfp gene expression was observed inkelp gametophytes for the first time.4) Genome sequence of phaeophyte actin gene was firstly cloned, and a 5' flank fragment of about450bp was got by genome walking.5) rt-PA gene was successfully expressed in kelp gametophytes, and the Reteplase ingametophytes was 0.159 μg/mg soluble protein on average.The results showed that it is feasible to express foreign gene in kelp gametophytesproliferation system.