新月菱形藻DHA/EPA比率的环境影响因素和分子调控机制

DHA/EPA ratio of the feed can not only significantly improve the quality of the eggs and larvae of aquatic animals, but also promote the growth of aquatic animals and enhance their non-specific immune function and disease resistance. Nitzschia closterium is an important microalgae feed in aquatic nursery, DHA/EPA ratio of which often affects quality and health of the larvae. However, the environmental impact factors and molecular regulation mechanism of DHA/EPA ratio are not yet clear. This project first determines the environmental impact factors and maximum environmental impact factor of the DHA/EPA ratio, and then the maximum impact factor is used to treat Nitzschia closterium to get 2 sets of biomass samples with significantly different DHA/EPA ratio. Total RNA of the samples is extracted, and then transcriptome sequencing is carried out. 2 sets of transcriptome data are compared to access to the differentially expressed genes, among which the genes involved in fatty acid synthesis and their transcriptional regulatory factors are screened according to the abundance changes. The full length of these genes is then obtained by rapid-amplification of cDNA ends (RACE) technology, and the structure and function of encoding proteins is speculated. The relationship of the expression amount of these genes and the DHA/EPA ratio is assayed using real-time quantitative PCR. Together, we will finally reveal the molecular regulation mechanisms of DHA/EPA ratio.

饵料中DHA/EPA比率不仅能显著提高水产动物卵和幼体的品质，而且能促进水产动物的生长，增强其非特异性免疫功能和抗病力。新月菱形藻作为一种重要的水产育苗饵料微藻，其DHA/EPA比率的变化常常影响到苗种的品质和健康指标，但对其DHA/EPA比率环境影响因素和分子调控机制尚没有明确的研究。本项目首先测定分析出DHA/EPA比率的主要环境影响因素和最大环境影响因素，然后利用最大影响因素处理新月菱形藻获得两组DHA/EPA比率差异显著的样品，提取总RNA后进行转录组测序，对比两组数据获得差异表达基因，筛选其中丰度变化最大的参与脂肪酸合成的基因及其转录调控因子，利用cDNA末端快速扩增（RACE）技术获得这些基因的全长并推测其蛋白结构和功能，利用实时荧光定量PCR分析这些基因的表达量与DHA/EPA比率变化的关系，揭示DHA/EPA比率的分子调控机制。

饵料微藻中 DHA/EPA 比率不仅能显著提高水产动物卵和幼体的品质，而且能促进水产动物的生长，增强其非特异性免疫功能和抗病力。新月菱形藻作为一种重要的水产育苗饵料微藻，其 DHA/EPA 比率的变化常常影响到苗种的品质和健康指标，本项目对其 DHA/EPA 比率环境影响因素和分子调控机制进行了详细的研究。首先优化了一种测定微藻湿生物质脂肪酸含量的方法，使得后续实验可以使用较小的生物量和简便的操作进行；其次，测定分析了生长阶段、盐度、初始氮源浓度、温度和光照等因素对于新月菱形藻生长、脂肪酸组成和DHA/EPA比率的影响，结果表明在生长过程中DHA/EPA比率呈现出先升高后下降的变化，盐度温度对DHA/EPA比率的影响较小，在缺氮条件下DHA和EPA的含量和比率均显著高于正常组，而强光可显著提高DHA/EPA比率，进一步使用缺氮和强光处理表明DHA/EPA比率可升高至0.064，是正常组的2.5倍，由此明确了缺氮和强光是DHA/EPA比率的最大环境影响因素；最后，提取了缺氮强光处理组和正常组的总RNA进行转录组学分析，获得了8G的数据，覆盖基因组146倍，分别比对获得了12381个和12525个基因的转录本，获得663个显著差异表达基因，对其进行GO富集分析发现差异基因主要集中在氧化还原反应、氮代谢、小分子代谢、膜组分、细胞间质、催化活性、蛋白结合等类目，进行KEGG富集分析发现差异表达基因主要富集在脂肪酸代谢、光合作用、丙酮酸代谢、糖酵解与糖异生等通路，对脂肪酸代谢通路涉及DHA和EPA合成的去饱和酶和延伸酶经荧光定量PCR进一步分析表明，DHA和EPA主要合成路径中的去饱和酶和延伸酶呈现出显著性上调表达，其中高不饱和脂肪酸的起始合成酶△12去饱和酶（PTD12）大幅上调表达，为EPA和DHA的合成提供了充足的底物，EPA合成的关键限速酶△5去饱和酶（PTD5b）与对照组相比上调表达了28.2倍，因此导致了缺氮条件下EPA的大量累积，而EPA又是DHA合成的唯一底物，推测合成DHA的延伸酶和去饱和酶也同样受到上调表达，使得DHA合成速率超过了EPA的合成速率，从而提高了DHA/EPA比率。