摘要
用基因工程手段提高裂壶藻生长速度和脂肪酸含量
裂壶藻(Schizochytrium sp.)又称裂殖壶菌,属不等鞭毛门(Stramenopiles),是一种异养微藻。裂壶藻合成丰富的多不饱和脂肪酸,其二十碳五烯酸(timnodonic acid, DPA)和二十二碳六烯酸(docosahexaenoic acid, DHA)占总脂肪酸60%以上,占细胞干重的43%,是用于工业发酵生产长链多不饱和脂肪酸的几种非常理想的异养真核微藻之一。
目前,利用裂壶藻生产长链多不饱和脂肪酸面临的主要问题之一是在高密度发酵过程中,葡萄糖转化后积累出的乙酸会对发酵过程微生物产生负面影响。为解决这一难题,我们拟通过电击转化法将大肠杆菌中的乙酰辅酶A合成酶基因(acetyl-coA synthetase,ACS)引入到海洋裂壶藻Schizochytrium sp. TIO1101中。对转化子进行PCR、Southern和Western免疫印迹检测的结果显示：外源ACS基因转化成功并在传代过程中稳定表达。摇瓶培养及高密度发酵实验的检测结果显示：外}源ACS基因表达可以降低培养基醋酸浓度,增加细胞内乙酰辅酶A库存,显著提高生物量和脂肪酸含量。以上结果表明,过表达ACS促进了裂壶藻三羧酸循环方向的代谢,降低了乙酸的积累,能显著提升DHA的产量。证明引入的ACS对改善裂壶藻代谢方面确实有重要意义。
雨生红球藻遗传转化研究
雨生红球藻Haematococcus pluvialis)是一种单细胞绿藻,新形成细胞体积较小,呈球形或卵圆形,黄绿色,有两根鞭毛。雨生红球藻则是属于绿藻门(Chlorophata),绿藻纲(Chlorophyceae),团藻目(Volvocales),红球藻科Haematococcaceae),红球藻属(Haematococcus)。微藻是天然虾青素重要的来源之一。微藻中虾青素含量最高的是雨生红球藻,也是所有已知能积累虾青素生物体中合成量最高的物种,其合成量最高可达细胞干重的4%。
在转基因裂壶藻的基础上,结合以前的经验教训,分别用农杆菌侵染法和基因枪法对雨生红球藻做了外源基因稳定表达的实验尝试。同时从启动子,报告基因,抗性筛选基因和目的基因的组合方面进行考虑,构建了多个质粒。通过荧光标记验证,抗性筛选,PCR鉴定,结果证明农杆菌侵染法和基因枪法均可将外源基因导入雨生红球藻。由于雨生红球藻自身的生物特性及实验设计的复杂程度,未来将对外源蛋白的定位,功能性验证等方面进行深入研究,并将是实验室未来的一个长期的研究方向。
Increasing the growth rate and fatty acids content of Schizochytrium by genetic engineering
Schizochytrium sp., one of heterotrophic microalgae belongs to phylum Stramenopiles, Schizochytrium synthesizes rich unsaturated fatty acids. It has been found that timnodonic acid (DPA) and docosahexaenoic acid (DHA) account for more than60%of its total fatty acids and43%of its dry cellular weight. Schizochytrium is one of a few desirable heterotrophic microalgae which can be used to produce long chain unsaturated fatty acids through fermentation.
During high density fermentation, transformation of glucose causes accumulation of acetic acid which will affect the fermentation negatively. In this research, the acetyl-coA synthetase gene, ACS, of E. coli was introduced into Schizochytrium sp. TIO1101with electroporation with such an introduction and the expression of the introduced gene verified by immunoblotting and polymerase chain reaction, In rotating bottle culture and high density fermentation, it was found that the acidity of transformant changed and the growth rate and glucose consumption rate of transformant were higher than those of the wild strain. As was evidenced by biomass and metabolite measurement, the biomass and the content of fatty acids were increase significantly in comparison with the wild. These findings indicated that over expression of ACS shifted the metabolism of Schizochytrium towards tricarboxylic acid cycle, and reduced the synthesis of acetic acid. Introduction of ACS gene has significantly improved the metabolism of Schizochytrium.
Genetic transformation of Haematococcus pluvialis
Haematococcus pluvialis is a single cell green alga. Newly formed cells of H. pluvialis appear to be spherical or oviform, and have two flagellates. H. pluvialis belons to genus Haematococcus, family Haematococcaceae, order Volvocales, class Chlorophyceae, phylum Chlorophata. Microalgae are the major source of astaxanthin, of them H. pluvialis contains the richest astaxanthin. H. pluvialis contains the richest astaxanthin among all known species accumulating this chemical, reaching4%of the dry cellular weight.
Basing on the basis of Schizochytrium transformation and taking previous experience into account, Agrobacterium-medizted transformation and particle bombardment (gene gun) were tried in this study in order to express foreign gens stably in this alga. From available promoters, reporter genes, antibiotic resistance genes and target genes, a few recombinant plasmids were constructed. As were proved by fluorescence marker, antibiotic selection, and polymerase chain reaction, both Agrobacterium-mediated transformation and particle bombardment were effective for transferring foreign genes into H. pluvialis. Due to the biological characteristics of H. pluvialis and the complexity of planted experiment, the location and function verification of foreign protein need to be studied further. This will be a long term researching goal of the lab in which I tried to transform Haematococcus.
引文
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