【作者】 邓祥元；

【导师】 秦松；

【作者基本信息】 中国科学院研究生院（海洋研究所） ， 海洋生物学， 2009， 博士

【摘要】 海带具有很高的营养价值和经济社会价值。自20世纪90年代以来,本实验室在借鉴高等植物基因工程原理和方法的基础上,根据海带自身特点,建立了海带遗传转化体系（海带孢子体表达系统）,它的基本原理是利用基因枪法转化海带配子体,经孤雌或受精途径再生幼孢子体后,用氯霉素筛选幼孢子体获得转基因海带,然后进行海上安全栽培和转基因产品的检测与提取。目前该表达系统已成功实现报告基因（β-半乳糖苷酶基因,lacZ）和功能基因（乙肝表面抗原基因,HBsAg）的稳定表达。由于海带孢子体表达系统需经孢子体再生和海上栽培等阶段,周期较长,而且转基因安全性问题也在一定程度上制约其研究与应用。因此,我们在海带孢子体表达系统的基础上又建立和优化了海带配子体表达系统,并成功实现了报告基因（绿色荧光蛋白基因,GFP）的瞬间表达和功能基因（瑞替普酶基因,rt-PA）的稳定表达。虽然海带配子体表达系统能避免转基因安全性问题,周期较短,但在表达量和生物量积累方面,与孢子体表达系统相比还有较大差距。本文首先在海带配子体表达系统中成功实现了人酸性成纤维细胞生长因子基因（hafgf）和鲎素基因（tac）的稳定表达,制备了转基因海带配子体,然后将光生物反应器培养技术应用于转基因海带配子体的高效增殖,以期解决阻碍海带配子体表达系统发展的量的问题,并为转基因海带配子体的大规模培养提供试验依据和技术支持。本文的研究结果为:1、人酸性成纤维细胞生长因子基因和鲎素基因可以稳定整合到海带配子体基因组中,实现转基因产物的表达。2、根据转基因海带配子体的生长特点,研制开发了一套培养体积为300 ml的鼓泡式光生物反应器,它具有操作简便、成本低廉、适合海带配子体生长等特点。随后将培养体系扩大到2.5 L,并研究了光对转基因海带配子体生长的影响,试验结果显示,转基因海带配子体在光强为30μE m^-2 s^-1时即可达到光饱和生长,最优光周期为14:10 LD,而且蓝光可促进转基因海带配子体的生长。3、在前期研究工作的基础上,为改善反应器内的传质条件,我们又设计研制了2.5 L气升式光生物反应器用于转基因海带配子体的高效增殖。研究发现,气升式光生物反应器较鼓泡式光生物反应器能明显地改善反应器内的传质状态,实现转基因海带配子体更高密度的培养(生物量可达到1,990 mg L^-1),是一套高密度悬浮培养转基因海带配子体的有效装置和设备。更多还原

【Abstract】 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.更多还原