裂殖壶菌人工海水配方优化及pks3基因功能的分析-optimization of artificial seawater formula of schizochytrium sp. and analysis of pk s3 gene function.docxVIP

iiii组成，开放阅读框全长为 3693bp，编码 1230 个氨基酸，其氨基酸序列与 GenBank中报道的 pks3 基因的氨基酸序列有 81%的相似性,含有两个保守的活性域，分别 是 3－酮酰－ACP 还原酶酶域（KR 酶域）和脱水/异构酶酶域（DH 酶域）。本文分别以这两个酶域为同源重组位点，以 Zeocin 抗性基因 Ble 基因为抗 性筛选标记基因，构建了基因敲除载体，希望通过基因敲除的方法来研究 pks3 基因的功能。结果表明，电转化同源重组载体后，经过传代、筛选和鉴定得到了 两株稳定遗传的 pks3 基因缺失的裂殖壶菌，分别是 KR 酶域缺失的转化菌株 KR- 和 DH 酶域缺失的转化菌株 DH-。这两株转化菌在菌落形态、菌体大小等方面都 与出发菌株 OUC168 有很大差别，并且生产速度缓慢、生物量低、DHA 积累量 几乎为 0。据推测这种现象是由于裂殖壶菌 pks3 基因被缺失后，其 DHA 的合成 代谢的终止不仅使得 DHA 不能积累，也使得细胞自身的代谢平衡被打破，直接 影响了细胞的正常的生理活动，导致细胞生长迟缓，生物量大幅降低。本文对人工海水配方的优化为提高裂殖壶菌的生物量和 DHA 积累提供了新 的方法；对 pks3 基因的克隆和功能研究为揭示裂殖壶菌的 DHA 合成途径和研究 DHA 合成相关酶的功能打下了基础。关键词：DHA；裂殖壶菌；人工海水；基因敲除；聚酮合酶基因 3iiiiiiformula optimization about artificial seawater andPreliminary function research on pks3 gene ofSchizochytriumAbstractDocosahexaenoic acid (DHA) as an omega-3 polyunsaturated fatty acid (omega-3 PUFA) is widely used because of it’s important physiological functions such as cancer resistanting, immunity enhancing, intelligence increase, eyesight protection and so on. Schizochytrium is a kind of marine fungi with rich DHA, which is becoming the new resource for production of DHA because it is safe, easy to cultivate and proliferate and it contains high content and good quality of lipid. At present, in Japan, the United States and some European countries, Schizochytrium has been applied to industrialized production of DHA. However, in domestic, the technology for DHA production is still in its infancy, and low DHA yield is an urgent problem to be resolved. Therefore, improvement of DHA production of Schizochytrium by using various technology and biological methods is particularly important. This paper carried out the following work on two aspects: one is optimization of ion concentration in the culture medium to improve the DHA production of Schizochytrium, the other is cloning and study of the function of DHA synthesis related genes of Schizochytrium.1, On the basis of substitution of the natural seawater using artificial seawater in the medium of Schizochytrium,