【作者】 张琳；

【导师】 于文功； 潘克厚；

【作者基本信息】 中国海洋大学 ， 生药学， 2011， 硕士

【摘要】 随着石油资源的日益枯竭与能源需求量日益增长这一矛盾的突出,可再生能源的开发及利用受到世界各地的关注。人们迫切寻找一种能够代替化石燃料的可再生资源。然而,原料问题一直是制约生物柴油发展的“瓶颈”。曾经,人们寄希望于传统的油料作物、粮食、动植物油脂、农林废弃物等,但是这些都有各种各样的缺点。微藻可以生长在海洋、河流以及湖泊里,不用占用耕地。它们能十分有效地利用太阳能进行光合自养,富集油脂。微藻还具有生长周期短,可以大规模培养的特点,被认为是制备生物柴油的优良材料。超长链多不饱和脂肪酸(very long-chain polyunsaturated fatty acids, VLCPUFAs)尤其是EPA(eicosapentaenoic acid)和DHA(docosapentaenoic acid)对人体及动植物细胞都是极为重要的,对某些疾病起到明显的预防和治疗作用。但是人类自身只能合成少量的EPA和DHA,远远不能满足自身需要。因此需要摄入一定量的VLCPUFAs以补充人体自身合成的不足。目前,VLCPUFAs的主要来源是深海鱼油,但是由于海洋可捕捞鱼类资源的日益减少和市场需求的迅速增长,该途径已经远远不能满足需要,寻找更为持续、稳定的VLCPUFAs来源已经成为当务之急。许多种类的海洋微藻能够从头合成VLCPUFAs且含量较高,脂肪酸组成稳定,没有难闻的鱼腥味等优点正逐渐成为鱼油的良好替代资源,具有巨大的开发潜力。微拟球藻(Nannochloropsis oculata),属于真眼点藻纲(Eustigmatophyceae),微拟球藻属(Nannochloropsis)。该藻富含EPA,因而被认为有望成为EPA产品生产的生物资源。此外,该藻油脂含量非常高,达到干重的30%以上。很多微藻在获得高油脂含量的同时却造成了生物量减少的现象。目前的研究表明,微拟球藻生物量的积累与油脂含量之间呈现最优的组合,被认为是最适于开发的能源微藻之一。总之,微拟球藻是一类非常重要的应用性经济微藻,但是其分子信息比较匮乏,需要进行深入研究。本实验以微拟球藻为研究对象,分别对其油脂代谢中的脂酰辅酶A合成酶(acyl-coenzyme A synthetase,ACS)和EPA合成中的延伸酶(Elongase)进行了研究。本研究从微拟球藻中克隆得到一个长链脂酰辅酶A合成酶(long acyl-coenzyme A synthetase, LACS)基因的cDNA全长,并命名为NOLACS。NOLACS的ORF为1947bp,预测编码648个氨基酸残基,蛋白相对分子质量为71kDa。NCBI中进行BlastX比对发现,NOLACS基因编码的蛋白质与三角褐指藻、假微型海链藻等的LACS高度相似。根据NOLACS编码的长链脂酰辅酶A合成酶能够互补LACS缺陷型酿酒酵母YB525生长设计酵母互补实验验证其功能,并分析了该酶的底物偏好性。结果显示,NOLACS能够活化饱和脂肪酸和部分不饱和脂肪酸,尤其偏好于长链脂肪酸。另外,进一步研究了限氮处理对微拟球藻中总脂含量以及NOLACS基因转录水平的影响,并探讨总脂含量与NOLACS基因转录水平的关系。运用RACE方法克隆得到了一个延伸酶的cDNA全长序列,其ORF为522个碱基,预测编码173个氨基酸残基。比对结果显示,该序列与三角褐指藻、假微型海链藻、新孢子虫等相似性较高。本实验尝试在酿酒酵母INVSc1中对其进行功能验证,未得到其功能,分析该基因可能是假基因。鉴于微拟球藻重要的生态及经济价值,获取更多的分子生物学信息并对其进行深入研究是必要的。本研究不仅有助于阐明该藻复杂的脂类合成途径以及脂肪酸组成随环境因子变化而变化的机理,也为微拟球藻基因表达调控及遗传转化提供了分子基础,因此具有重要的理论意义和广阔的应用前景。更多还原

【Abstract】 Owing to the prominent contradiction of exhausting fossil fuels and growing energy demands, the development and utilization of renewable energy has attracted worldwide attention. People are eager to find a substitute for fossil fuels. However, raw material has been the "bottleneck" which restricts the development of biodiesel. Once people pinned the hope on traditional oil crops, food, animal fats, farming and forestry residues, etc. However, these materials have various kinds of drawbacks. Microalgae can grow in oceans, rivers and lakes, without occupying any plough land. They can quite effectively utilize solar energy for autotrophic photosynthesis and lipid enrichment. Microalgae have been considered as an excellent choice of biodiesel for high growth and mass cultivation.Very long-chain polyunsaturated fatty acids (VLCPUFAs), especially EPA and DHA, are very important for cells and have been found significantly alleviate the symptoms of many diseases. As human cannot synthesize enough VLCPUFAs, dietary supplementation of VLCPUFAs is indispensable. Currently, the principal source of VLCPUFAs is deep-sea fish. However, it is insufficient to an expanding market. To exploit sustainable and continuable sources is urgent need. Many kinds of marine microalgae can synthesize VLCPUFAs with high contents, stable composition of fatty acids, and without unpleasant odor of fish. They are becoming good alternative resources of fish with great development potential.Genus Nannochloropsis is one of class Eustigmatophyceae. Nannochloropsis are rich in EPA, so that they are expected to become the biological resources for EPA. In addition, Nannochloropsis have very high oil content that is more than 30% of dry weight. Many microalgae could obtain high oil contents with reduced biomass at the same time. Nannochloropsis have been proposed as the excellent candidates for fuel production with the optimal combination of biomass productivity and lipid content. In short, they are important applied economic microalgae with deficient molecular information. They deserve intensive investigation. In this study, Nannochloropsis oculata was chosen for study of acyl-coenzyme A synthetase and fatty acid elongase. The cDNA of LACS gene was isolated from N. oculata and named as NOLACS. Nucleotide sequence analysis identified an open reading frame of 1947 bp in length, which encodes a protein of 648 amino acids with a predicted molecular mass of 71 kDa. The predicted amino acid sequence was highly similar to the LACSs of other species,such as Phaeodactylum tricornutum and T. pseudonana. The NOLACS encodes a long-chain acyl-coenzyme A synthetase; it recovered the function of Saccharomyces cerevisiae YB525, a LACS-deficient yeast strain. The substrate specificity of the enzyme was also assayed in the yeast. It was found that NOLACS can activate saturated and some unsaturated fatty acids with a preference to long-chain fatty acids. The effects of nitrogen deficiency on lipid contents and transcription of NOLACS are also assayed.One elongase from N. oculata was cloned by RACE whose ORF is 522 bp encoding 173 amino acids. The results of alignment indicated that NOLACS exhibited higher homology to Phaeodactylum tricornutum, T. pseudonana, Neospora caninum Liverpool etc. The elongase gene was transformed into yeast strain INVSc1(S. cerevisiae) to testify the function. It has been proved that it is a pseudogene.In the light of the great ecological and commercial value, N. oculata deserves intensive investigation at the molecular level. This study not only makes a significant contribution to understanding the metabolic pathways of lipids, but also sets the molecular basis for regulation of gene expression and genetic transformation. The research has important theoretical significance and broad application prospect.更多还原