钝顶螺旋藻中β-胡萝卜素转化合成虾青素及其抗氧化功能研究

Astaxanthin is a kind of carotenoid with strong antioxidant ability. β-carotene ketolase (crtO) and β-carotene hydroxylase (crtR) are the critical enzymes in the biosynthesis of astaxanthin. As a precursor in the synthesis of astaxanthin, β-carotene is abundant in Spirulina platensis. However, there is little astaxanthin that can be synthesized in Spirulina platensis. This study intends to adopt genetic engineering technology to clone crtO and crtR genes in Spirulina platensis, build the p215tM-crtO-crtR carrier, and then transform the carrier into Spirulina platensis by electroporation. The engineered strain containing target genes will be obtained by detection of related molecular and physiological parameters. Furthermore, this study will explore antioxidant effects of the transgenosis generated astaxanthin in vitro and in vivo: the anti-oxidative and anti-damage effects of obtained astaxanthin on liver cell LO2 will be investigated by detecting reactive oxygen species and reactive nitrogen levels; D-galactosamine induced liver injury model in ICR mice will be established to explore effect of obtained astaxanthin on glutamate oxaloacetoacetate transaminase and glutamate pyruvate transaminase as well as antioxidant factors such as superoxide dismutase, glutathione peroxidase, catalase and lipid peroxidation in serum and liver. This study would establish the theoretical basis for development of high-astaxanthin-content Spirulina platensis strains and astaxanthin healthcare products.

虾青素是一种高抗氧化活性的类胡萝卜素。β-胡萝卜素酮化酶（crtO）和β-胡萝卜素羟化酶（crtR）是虾青素合成途径中的关键酶。螺旋藻含有丰富的合成虾青素前体物质β-胡萝卜素，但虾青素含量极低。本研究拟克隆钝顶螺旋藻crtO和crtR基因并构建p215tM-crtO-crtR载体，利用电击方法转化到钝顶螺旋藻中，通过对相关分子与生理指标的检测，最终获得含有靶基因工程藻株。其次，对工程藻株中虾青素的体内外抗氧化作用进行研究，以活性氧和活性氮水平为指标，探索转化虾青素对LO2肝细胞的抗氧化损伤机制；基于ICR小鼠D-氨基半乳糖所致肝损伤模型，研究其对血清和肝脏转氨酶GOT、GPT以及SOD、GSH-Px、CAT等抗氧化因子和脂质过氧化的影响，为开发高产虾青素的螺旋藻新品种以及虾青素功能营养制品开发奠定理论基础。

虾青素一种天然类胡萝卜素，因其具有抗氧化、抗肿瘤等生理功能逐渐作为人类的膳食保健品和水产动物的饲料添加剂，商业开发中的虾青素主要来源于红发夫酵母与雨生红球藻。研究表明，β-胡萝卜素是虾青素代谢合成途径中的重要中间产物，本项目采用了两种方法实现钝顶螺旋藻体内β-胡萝卜素向虾青素的转化，同时对虾青素进行提取纯化，检测其对氧化损伤细胞的抗氧化与保护功能，主要内容如下：.1)利用改良后的Zarrouk培养基对新鲜的钝顶螺旋藻株进行液体和平板培养，采用常压室温等离子体（ARTP）诱变技术结合β-紫罗酮筛选剂对螺旋藻定向筛选，确定ARTP照射时间和β-紫罗酮筛选浓度。通过测定生长速率初筛出生长速率较快的诱变藻株，测定比较原始藻株和诱变藻株的基础成分含量及β-胡萝卜素与虾青素含量，获得虾青素高产藻株；设定不同缺氮处理组探究氮含量对虾青素积累速率的影响。.2)以钝顶螺旋藻FACHB-904总基因组为模板，成功克隆了β-胡萝卜素羟化酶crtR基因，并且对CRTR蛋白质理化性质、跨膜性、氨基酸与系统发育等生物信息学分析。构建了钝顶螺旋藻F906 crtR基因的原核表达载体，通过SDS-PAGE与Western Blot蛋白水平定性分析证明了crtR基因编码蛋白在大肠杆菌中表达，利用高效液相色谱（HPLC）检测验证了CRTR蛋白酶可将β-胡萝卜素催化形成玉米黄质。将crtR基因与信号肽相连接，构建S-crtR与pYES2/NT-A重组质粒，将其转化到酿酒酵母，通过SDS-PAGE、Western Blot和RNA转录分析验证了钝顶螺旋藻FACHB-904 crtR基因成功在酿酒酵母中分泌表达，获得重组酿酒酵母菌株S7。.3)利用HPLC检测螺旋藻与重组酿酒酵母S7发酵液中代谢产物，确定了类胡萝卜色谱检测条件，探索了不同的发酵时间和螺旋藻浓度对发酵中累积虾青素的影响，定量分析了发酵前后培养液中角黄素与虾青素浓度。通过有机溶剂的粗提取与硅胶层析的方法，从发酵液中分离纯化得到了虾青素，并作用于巨噬细胞RAW264.7受损细胞，证明了共发酵液中虾青素对巨噬细胞RAW264.7具有抗氧化与保护细胞的作用。