雨生红球藻中虾青素合成及几何异构体的分析研究

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	雨生红球藻中虾青素合成及几何异构体的分析研究
	巩风英
学位类型	硕士
导师	刘建国
	2017-04-28
学位授予单位	中国科学院海洋研究所
学位授予地点	山东青岛
学位专业	海洋生物学
关键词	雨生红球藻虾青素几何异构体蛋白
摘要	虾青素因其超强的抗氧化性而具有多种生理功能，如提高动物免疫力、颜色，清除自由基，预防和治疗肿瘤，预防老年痴呆等。此外，天然虾青素在水产、家禽养殖业和保健品、化妆品等领域具有广泛的应用前景和研究价值。雨生红球藻是目前公认的天然虾青素最好的生物来源。结构上，天然虾青素大部分为全反式结构，但存在不同比例的顺式结构，顺式、反式虾青素异构体之间存在功能差异。因此，通过对虾青素的异构体进行分离和分析以及探索异构体的产生原因，具有十分重要的生产和理论意义。本文的主要工作如下： 1、为研究不同藻株生产虾青素的能力及所产虾青素异构体特性，本文以雨生红球藻H₀、H₂、H₃、H₆和T₂藻株为研究对象，测定其细胞生长，光合、呼吸作用强度，叶绿素和虾青素含量及异构体比例等。结果表明：在细胞生长，光合作用、呼吸作用速率，叶绿素、类胡萝卜素含量以及藻细胞抗逆境能力和虾青素含量等各个方面，T₂藻株均显著优于其他四个藻株（H₀、H₂、H₃和H₆）。但是，五株雨生红球藻所产虾青素异构体的比率未发现显著性差异（P>0.05）。 2、为研究雨生红球藻细胞不同阶段（细胞转变阶段和虾青素积累阶段）细胞外部形态和内部组成的变化，本文以雨生红球藻H₆藻株为研究对象，测定其细胞生长，光合、呼吸作用强度，叶绿素和虾青素含量及异构体比例及其蛋白水平等。结果表明：细胞转变阶段（0-3天），雨生红球藻藻细胞内各色素水平和蛋白水平均发生显著的变化，体现为叶绿素急剧下降，类胡萝卜素和虾青素含量迅速升高，各异构体比例呈波动变化。而虾青素积累阶段（3-15天），藻细胞的各项生命活动趋于平缓，对于已经完成细胞形态转变的雨生红球藻细胞，主要生理变化过程体现在虾青素含量的积累，各异构体比例维持相对稳定状态；在此阶段，蛋白的变化也趋于平缓且蛋白表达量较低。 3、为研究虾青素异构体含量，本文采用低温条件下的丙酮研磨法提取虾青素并防止虾青素的异构化，通过胆固醇酯酶酶解法得到游离虾青素，利用高效液相色谱法对虾青素及其几何异构体以及其他类胡萝卜素含量进行分析。结果表明：相同的外界环境下，不同藻株所产的虾青素异构体比率无显著性差异（P>0.05），而不同培养阶段的雨生红球藻，虾青素异构体存在一定的差异性（P<0.05），顺式、反式虾青素含量逐渐升高；虾青素积累阶段后期，虾青素异构体比率无显著差异性（P>0.05）。 4、为研究雨生红球藻藻细胞虾青素积累和异构体产生的原因，结合Western Blot技术研究与顺式、反式异构体差异碳原子形成相关蛋白的表达量。结果表明：法呢基焦磷酸合成酶（FFPS）和牻牛儿基牻牛儿基焦磷酸合成酶（GGPS）在细胞转变阶段表达量较高，在虾青素积累阶段表达量较低，在整个培养周期中呈逐渐下降的趋势，但是上述蛋白为催化反式异戊烯基的酶，至于雨生红球藻中是否存在催化顺式异戊烯基酶以及顺式、反式异戊烯基酶在雨生红球藻中是如何协同作用还需要进一步研究。 5、本文将色素含量变化及细胞外部形态变化及其蛋白含量变化有机结合起来，阐述了光照、色素、活性氧和虾青素的关系，分析了逆境条件下虾青素的积累机制。结果表明：高光等逆境环境破坏叶绿素，降低光合作用能力，表观可见藻细胞绿色减少。此外，由于叶绿素的破化导致相关蛋白D1、PsaA、RBCL等含量降低，使得其介导的电子传递链受损，进一步引起藻细胞内电子剩余，活性氧产生；机体通过增强叶绿体和线粒体呼吸消除体内活性氧，但由于藻细胞呼吸能力不足以清除光抑制产生的活性氧，推测藻细胞通过虾青素等类胡萝卜素大量合成以应对逆境条件，因此细胞呈现红色。 6、将类胡萝卜素/叶绿素，虾青素/类胡萝卜素，虾青素异构体的比例等指标作为虾青素产品的检测指标。结果表明：随着培养时间的增加，类胡萝卜素/叶绿素及虾青素/类胡萝卜素的值逐渐升高，虾青素异构体比例趋于稳定。上述指标的加入有助于标准化雨生红球藻来源的虾青素品质和规范虾青素市场。
其他摘要	Astaxanthin’s powerful antioxidant properties endow it many important physiologic functions, such as, improving animal immunity, being a foe of free radicals, precventing and treating of cancer, preventing Alzheimer's disease, coloring agent and antioxidants. Therefore, the natural astaxanthin has a widespread application and prospect on auaculture and poultry, health-food, osmetics and so on. Furthermore, Haematococcus pluvialis is regard as the best biological source of astaxanthin. Natural astaxanthin mostly was trans-astaxanthin, but exists a little cis-astaxanthin, tran-astaxanthin and cis-astaxanthin have differences on physiologic. It will be very significant that the separation and analysis of the astaxanthin isomers, and explore the reason of astaxanthin isomerization. The work in this dissertation is as follows: 1, to explore the ability of astaxanthin acculumation and the differential characteristic of cis/trans geometrical isomers during different algal strains, different Haematococcus pluvialis stains H₀, H₂, H₃, H₆ and T₂ are taken as research objects and the research is focus on that the cell growth, the variations of photosynthetic rate and respiratory rate, and the content of chlorophyll and astaxanthin, and the ratio of astaxanthin isomers. Results show that strainT₂ was significantly better than the other four strains (H₀, H₂, H₃, H₆) on the cell growth, photosynthesis, respiration rate, content of chlorophyll, carotenoid and algal cells resistance to adversity and astaxanthin content and so on. But five algal strains have no significant difference (P > 0.05) on the ratio of astaxanthin isomer. 2, to explore the change of cells morphology and internal composition during different stages of Haematococcus pluvialis (cell transformation phase and astaxanthin accumulation phase), stain H₆ are taken as research objects and the research is focus on that the cell growth, the variations of photosynthetic rate and respiratory rate, and the content of chlorophyll and astaxanthin, and the ratio of astaxanthin isomers and the content of protein. Results show that cells have a significantly change on the content of pigment and protein during cell transformation phase (0-3days), mainly was the sharply decrease of chlorophyll content and quickly increase of carotenoids and astaxanthin, and fluctuation changes of isomers ratio. All above changes was slowly on the astaxanthin accumulation phase (3-15days), mainly was the increase of astaxanthin content. 3, to explore the content of astaxanthin geometric isomers, this article use the acetone grinding method under low temperature to prevent the isomerization of astaxanthin, by cholesterol esterase enzyme to get free astaxanthin, and use high-performance liquid chromatography to analyze the astaxanthin isomers and other carotenoids from the pigment’s extract of Haematococcus pluvialis. Results show that different strains (H₀, H₂, H₃, H₆ and T₂) have no significant difference on the ratio of astaxanthin isomer under same culture conditions. The ratios of astaxanthin isomer have a significant difference on different cell phases in Haematococcus pluvialis, and the ratio have no difference on the late astaxanthin accumulation phase. 4, to explore the reason of astaxanthin accumulation and geometric isomers, western blot technique is used to analyse the protein expression about catalytic the cis/trans carbon atoms. Results show that FFPS and GGPS have a higher expression on cell transformation phase and lower on astaxanthin accumulation phase. Over the whole culture time, the content of FFPS and GGPS decrease gradually. But the above proteins are trans-prenyl enzyme, there is still need further research on whether Haematococcus pluvialis exists cis-prenyl enzyme, and how the cis-prenyl and trans-prenyl enzymes interact with each other in Haematococcus pluvialis. 5, in this dissertation, the pigment content and cell morphological and protein content are combined, expounds the relationship between light and pigment, ROS and astaxanthin, analyzes the mechanism of astaxanthin accumulation under stress conditions. Results show that: high light destroys chlorophyll, which reduce the ability of photosynthesis causes the green color decline. In addition, the breaken of chlorophyll causes the decline of related protein D1, PsaA and RBCL content, which is mediated the electron transport chain is damaged, further causing the generation of ROS and residual electron. Algal cell enhance the ability of chloroplast and mitochondrial respiratory to eliminate ROS. But the cell’s respiration ability is not enough to scavenging ROS produced by light inhibition. Speculated that the algal cells through synthesize a large number of astaxanthin and other carotenoids to cope with stress conditions. 6, the indexes such as carotenoid/chlorophyll, astaxanthin/carotenoid and the ratio of the isomers of astaxanthin are used as indicators of astaxanthin quality. Results show that: with the culture time increase, the ratios of carotenoid/chlorophyll and astaxanthin/carotenoid gradually are increased, and the proportion of astaxanthin isomers tends to be stable. The addition of the above indicators can help to regulate the quality of astaxanthin and astaxanthin market.
学科领域	生物学其他学科
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136551
专题	海洋生物技术研发中心
作者单位	1.中国科学院海洋研究所 2.中国科学院大学
推荐引用方式 GB/T 7714	巩风英. 雨生红球藻中虾青素合成及几何异构体的分析研究[D]. 山东青岛. 中国科学院海洋研究所,2017.

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