螺旋藻、节旋藻系蓝藻门（Cyanophyta），段殖藻目（Hormogonales），颤藻科 （Oscilatoraceae），是光合自养型原核微生物。人们借助于 16S rRNA、16S-23S rRNA （ITS 区）、cpcB、cpcA、hoxY、psbA、cpcE-F 等基因序列分析，发现螺旋藻、节旋 藻在属水平上具有明显差别，认为螺旋藻、节旋藻应为独立的两个属。但关于种定义 的问题仍在争论。本实验室在获得大量新藻株的基础上，欲采用多相分类方法，构建 相对完善的螺旋藻、节旋藻种属分类系统。 课题组成员首先基于 16S rRNA 基因、ITS 区、rpoC1 基因、cpcB 基因、psbA 基 因、hoxY 基因序列分别构建系统发育树，均支持螺旋藻、节旋藻独立为两个不同属的 观点。但是，个别藻株的归属不稳定，且属内藻株的基因序列相似性较高，无法确定 不同种。因此为了验证基于基因序列分类的准确性，进一步寻找种水平分类的靶标分 子，本课题以渤海海滨湿的螺旋藻、节旋藻藻株为研究对象，检测其脂肪酸组成及含 量，通过聚类分析寻找特征脂肪酸，并以脂肪酸为标记进行系统发育分析，丰富螺旋 藻、节旋藻的分类依据和方法，以期获得更能反映藻株亲缘关系的分类结果。 主要研究内容， 1. 藻株的分离纯化，从在渤海海滨湿地及其周边水域采集的水样中，分离纯化获 得 34 株单藻丝纯种，扩大了本研究的样本量，并对其进行形态特征进行了研究； 2. 脂肪酸检测方法的建立，首先对比了 VF-23MS 与 HP-88 两种色谱柱对 37 种 脂肪酸甲酯的分离效果，选取了能将 37 种脂肪酸甲酯有效分离的 HP-88 色谱柱，并 确立了最优色谱条件，即进样口，加热器 250℃，分流比 8:1；控制模式为流速控制， 流速为 0.5mL/min，柱前压 19.783psi；升温程序，初始温度 100℃，保持 4min，以 8℃ /min 上升至 175℃，保持 7min，然后以 1℃/min 上升至 186℃，保持 3min，最后以 5℃ /min 升至 230℃，保持 30min；检测器温度（FID）260℃。其次，在脂质萃取过程中， 考察了不同溶剂的萃取效果，依据脂质提取率和色谱图质量，最终确定正己烷为提取 藻株脂肪酸的萃取溶剂。再次，Central Composite 响应面优化螺旋藻脂肪酸提取条件， 建立回归模型，获得优化方案，即超声时间 21min，水浴时间 93min，催化剂浓度 1.8%， 甲酯化温度70℃，在此优化条件下进行6次平行试验，得到平均峰面积为31008.1667， 与理论值相比，其平均偏差为 5.59%。最后，对整个实验方法进行加标回收率实验， 在最优实验条件下回收率为 94.56%~103.23%，表明该方法回收率比较稳定，为后续 的脂肪酸检测提供了依据； 3. 螺旋藻、节旋藻藻株分类，应用本研究建立的检测方法对 84 株供试藻株进行 脂肪酸提取、气相色谱分析，最后通过聚类分析，寻找可用于螺旋藻（节旋藻）属种 类的脂肪酸生物标记；II 主要结论如下， （1）F-351、F-1070 两藻株不含 C18:3n6，结合基于 16S rRNA 基因、ITS 区等分 子方法的聚类结果，进一步支持节旋藻、螺旋藻两属之间的差异性脂肪酸成分为 γ- 亚麻酸的观点； （2）节旋藻属脂肪酸组分除含有 γ-亚麻酸（C18:3n6）这一特征组分外，均有高 含量的 C16:0；另外，大部分藻株也含有 C16:1、C18:0、C18:1n9c 、C18:2n6c；以 10 个脂肪酸统计参数为指标对节旋藻属藻株进行聚类分析，将 81 个供试藻株划分为 5 个脂肪群；并通过聚类分群分析发现，以脂肪酸为标记的分类结果与基于 psbA 基因 序列的聚类结果存在一定的一致性； （3）依据脂肪酸分析，修正藻株 TJBC3 的分类地位，该藻株应归为螺旋藻属； 针对基于psbA基因序列及联合基因分析提出的藻株F904独立于螺旋藻属、节旋藻属， 与原先对该藻株为 Spirulina platensis 的鉴定结论之间冲突，本实验结果认为该藻株仍 属于节旋藻属。 关键词，螺旋藻；节旋藻；脂肪酸；气相检测；聚类分析III ABSTRACT Spirulina and Arthrospira are photosynthetic procaryote microbiology,belonged to Cyanophyta, Hormogonales, Oscilatoraceae. By analyzing the 16S rRNA, 16S-23S rRNA （ITS region）, cpcB, cpcA, HoxY, psbA,cpcE-F and other gene sequences, it had been found that Spirulina and Arthrospira have obvious difference in genus, so they should be two separate genera.But the question about the definition of species is still controversial. Onthe basis of having a lot of new algae strains, we tried to use multiple methods to build a relatively complete classfication system for differentiatingSpirulina and Arthrospira species. First of all, on the basis of 16SrRNA,ITS region, rpoC1, cpcB, psbA and hoxYgene sequences, we constructed phylogenetic tree respectively, which all support the fact that Spirulinaand Arthrospira are two different separate genera. However, because of the unstable ownership of the individual algae strains and the higher similarity of gene sequence of algae strains, we cannotseparate the species exactly. Therefore, in order to verify the accuracy of the classification bsed on gene sequences, and further to look for the target molecules, we took the Spirulinaand Arthrospiraalgal strains from Bohai Sea coastal wetlands to detect its fatty acid composition and content. Then we used clustering analysis to find characteristic fatty acids, by which we can make phylogenetic analysis to rich the basis of classification and methods in Spirulina and Arthrospira. Furthmore, we hope it could provide potential results to reflect the relationship classification ofalgae strains. The main research contents: 1. Isolation and purification of algae strains: We collected strains from the water samples from Bohai Sea coastal wetlands and its surrounding water. Then we isolated and purified 34 purebred single filaments to enlarge the sample size of this research, and study its morphological characteristics ; 2. The establishment of the fatty acid detection method: First we compared the VF - 23 ms and HP - 88 chromatographic column separation effect of 37 kinds of fatty acid methyl ester, then we chose the HP-88 chromatographic column which can effectively isolate the 37 kinds of fatty acid methyl ester and established the optimal chromatographic conditions, namely inlet: heater 250 ℃, split ratio 8:1; flow rate at 0.5mL / min, column pressure 19.783psi;temperature programming: initial temperature is 100℃ and kept 4 minutes, then raised up to 175 ℃ at the speed of 8℃ / min. Next, we kept 7 minutes and increased temperature to 186℃ at the speeds of 1℃/min.Finally, we kept 3 minutes, then up to 230℃ at the speed of 5 ℃ / min, then stay for 30 minutes; detector temperature （FID）:260℃.Secondly, in the lipid extraction process, we inspect the extraction effect ofIV different solvents, based on lipid extraction yield and quality of chromatogram, ultimately we determined to choose the normal hexane as the extracting solvent to extract the fatty acids in algal strains. Again, we used Central Composite response surface to optimize fatty acid extraction conditions.Morever, we established regression model andobtained the optimization scheme, namely ultrasonic time: 21min, water bath time :93min, catalyst concentration: 1.8%,methyl esterification temperature: 70 ℃ .Under the optimized conditions, six parallel experiments were carried out. The average peak area is 31008.1667.When compared with the theoretical value, the average deviation is 5.59%.Finally, we carried out the recovery experiments for the whole experiment.In the optimal experimental condition the recovery ratio was 94.56% ~ 103.23%, showed that this method is stable, providing the basis for subsequent fatty acid detection; 3. Spirulina and Arthrospira algal strains category:Applying the detection method developed in this study to the 84 strains of algae provided to extract fatty acids and analyzingthe strains by gas chromatography, finally we found the fatty acid biomarkersin virtue of cluster analysis that could be used for distinguishing Spirulina（Arthrospira） species of genus; The main conclusions are as follows: (1)As these two algal strains, F-351 and F-1070, didn’t contain C18: 3n6, we combined the clustering results of molecular methods based on 16SrRNA gene, ITS region and so on. Finally, our results further support the view that the difference of fatty acid composition between the two genera Spirulinaand Arthrospira is the γ-linolenic acid; (2)In addition to containing the feature component γ- linolenic acid （C18: 3n6）, all strains of Arthrospiraalso has high levels of C16: 0. Besides, most of the algal strains also contain C16: 1, C18: 0,C18: 1n9c, C18: 2n6c; with 10 fatty acids statistical parameters as indicators ,clustering analysis is carried out on Arthrospira genus algae strains ,the 81 tested algal strains are divided into five fat flock; Through the analysis of clustering grouping , we found that the classifica