【作者】 马婷；

【导师】 周文礼； 贾旭颖；

【作者基本信息】 天津农学院 ， 渔业资源， 2020， 硕士

【摘要】 渤海湾卤虫是一种营养丰富的生物活饵料,广泛应用于水产经济动物的仔稚期。随着水产经济动物的高质量发展,卤虫体内的不饱和脂肪酸含量已无法满足多数海水鱼、虾苗种发育的需要。因此研究不同环境条件组合对渤海湾卤虫生理生态的影响及饵料对卤虫不饱和脂肪酸积累的分子机制,对优化卤虫培养条件,促进营养强化技术的发展具有重要的指导意义。本文立足于渤海湾卤虫不饱和脂肪酸生物合成代谢的调控机制开展以下研究:环境条件对渤海湾卤虫无节幼体生长、体脂肪酸、抗氧化指标的影响,优化渤海湾卤虫培养条件;分别从代谢组与转录组水平研究优化饵料对渤海湾卤虫不饱和脂肪酸积累的影响,并联合两组学数据分析优化饵料对渤海湾卤虫不饱和脂肪酸合成代谢的调控机制。试验结果如下:1.环境组合对卤虫的存活、生长、体脂肪酸积累和抗氧化能力影响不同,主效应检验中pH对卤虫存活和生长的影响效果明显,饵料种类对卤虫体脂肪酸积累影响最大,盐度对卤虫总超氧化物歧化酶(T-SOD)、过氧化物酶(POD)活力影响较大。筛选出最佳的饲养条件是:光照强度为72μmol/(m~2·s)、盐度为25、pH为8.0、饵料种类为球等鞭金藻,在此环境下渤海湾卤虫存活率最高,体长体重增长明显,体内脂肪酸种类最多,组成成分最高;卤虫的T-SOD、POD、过氧化氢酶(CAT)等抗氧化酶活力较高,丙二醛(MDA)含量较低。2.采用前期优化饵料球等鞭金藻进行养殖试验,并与酵母进行对比,研究优化饵料对卤虫不饱和脂肪酸积累的影响,结果表明球等鞭金藻(IG-group)组卤虫总脂肪酸、多不饱和脂肪酸、二十碳五烯酸(EPA)、二十二碳六烯酸(DHA)、花生四烯酸(ARA)含量均显著高于酵母(Y-group)组。基于LC-MS/MS等代谢组学技术手段构建不同饵料条件下渤海湾卤虫的代谢物差异图谱,得到渤海湾卤虫有13399种代谢物,筛选出5399种上调差异代谢物和4427种下调差异代谢物,通过KEGG通路注释及富集分析发现与脂肪酸相关代谢显著的通路为脂肪酸生物合成通路和不饱和脂肪酸生物合成通路。两条代谢通路中富集在IG-group组卤虫的α-亚麻酸(ALA)、EPA、DHA、油酸、辛酸、肉豆蔻酸、硬脂酸,和富集在Y-group卤虫体内的ARA、棕榈油酸均为渤海湾卤虫不饱和脂肪酸代谢的重要调控代谢物;上述显著差异代谢物可能为影响渤海湾卤虫不饱和脂肪酸的合成代谢的潜在生物标志物。3.利用Illumia高通量测序平台对优化饵料养殖的渤海湾卤虫样本进行转录组测序,转录本拼接后得到总Unigene序列248932条,成功注释到NR库的基因数目为35009条;成功注释到SwissProt为23768条;成功注释到PFAM库的有16382条;GO库成功注释到11445条;KO数据库成功注释到10641条。在基因表达水平上有401个下调基因和148个上调基因,共计9条富集到与脂类代谢相关的通路上,其中与脂肪酸相关通路只有一条,为Y-group组中的ARA代谢通路,该通路上差异基因CD224/GGT1-5高度表达,基因表达的转运蛋白γ-谷氨酰转肽酶、谷胱甘肽水解酶和白三烯-C4合酶高度合成,IG-group组卤虫体内不饱和脂肪酸相关通路中差异基因无明显变化。因此,CD224/GGT1-5基因在该组学角度上可能为酵母组卤虫不饱和脂肪酸生物合成的调控基因。4.转录组学与代谢组学联合分析共筛出46条共同参与两组学的代谢通路,与不饱和脂肪酸相关的通路一共两条,为ARA代谢通路和不饱和脂肪酸生物合成通路。在两通路中Y-group卤虫的CD224/GGT1-5基因、desC基因与ARA呈正相关,即CD224/GGT1-5基因在ARA代谢通路上,加快了Y-group卤虫体内ARA代谢,不断将ARA向脂类物质的转化,降低了Y-group卤虫体内不饱和脂肪酸等物质的合成与积累;Y-group中desC基因在不饱和脂肪酸生物合成通路上促进了卤虫体内的ARA积累,同时抑制了卤虫体内ALA、EPA、DHA、油酸的生物合成。与Y-group组比,IG-group组中desC基因与CD224/GGT1-5基因的正常表达,促进了卤虫体内ALA、EPA、DHA、油酸等不饱和脂肪酸的生物合成,降低了ARA代谢与积累,促进了IG-group组的卤虫脂质的代谢与吸收速率。更多还原

【Abstract】 Bohai Bay parthenogentica Artemia is a nutrient-rich biological live bait,which is widely used in the infantile period of aquatic economic animals.With the high-quality development of aquatic economic animals,the content of unsaturated fatty acids in Artemia becomes unable to meet the developmental needs of most marine fish and shrimp species.Therefore,it is of important guiding significance to study the effects of different combinations of environmental conditions on the physiological and ecological of Artemia and the molecular mechanism of baits on the accumulation of unsaturated fatty acids in Artemia for optimizing Artemia culture conditions and promoting the development of nutrition fortification technology.Based on the regulation mechanism of unsaturated fatty acid biosynthesis and metabolism in Bohai Bay parthenogentica Artemia,the following researches was carried out: Effects of environmental conditions on the growth,body fatty acids and antioxidant indexes of the Bohai Bay parthenogentica Artemia were analyzed to optimize the culture conditions of Bohai Bay parthenogentica Artemia.The metabolome and transcriptome levels were studied to optimize the accumulation of unsaturated fatty acids in Bohai Bay parthenogentica Artemia The effects were combined with the analysis of two sets of scientific data to optimize the regulation mechanism of diets on the metabolism of unsaturated fatty acids in Bohai Bay parthenogentica Artemia.The test results are as follows:1.The combination of environment has different effects on the survival,growth,body fatty acid accumulation and antioxidant capacity of Bohai Bay parthenogentica Artemia.In the main effect test,p H has a significant effect on the survival and growth of Artemia.The type of baits has the largest effect on the fatty acid accumulation of Artemia,Salinity has a significant effect on the activity of Total Superoxide Dismutase(T-SOD)and Peroxidase(POD)of Artemia.The best breeding conditions were screened as follows: light intensity was 72 μmol /(m2 · s),salinity was 25,p H was 8.0,and the type of bait was Isochrysis galbana.The survival rate of Bohai Bay parthenogentica Artemia was the highest in this environment.The body length and weight increase significantly,the body has the most fatty acids and the highest composition;Artemia T-SOD,POD,Catalase(CAT)and other antioxidant enzymes have higher activity and lower Malondialdehyde(MDA)content.2.The culture experiment was carried out by using an optimized bait Isochrysis galbana in the early stage to study the effect of the optimized bait on the accumulation of unsaturated fatty acids in Artemia,while the Yeast group was set as the comparison group.The results showed that the contents of total fatty acids,polyunsaturated fatty acids,Eicosapentaenoic acid(EPA),Docosahexaenoic acid(DHA),and Arachidonic acid(ARA)in Isochrysis galbana(IG-group)group were significantly higher than those in the Yeast(Y-group)group.Based on metabolomics techniques such as LC-MS/MS,the metabolite differential maps of Bohai Bay parthenogentica Artemia under different baits conditions were constructed.There were 13,399 metabolites in Bohai Bay parthenogentica Artemia.5,399 up-regulated differential metabolites and 4,427 down-regulated differential metabolites were selected.Through KEGG pathway annotation and enrichment analysis,it was found that the significant fatty acid-related differential metabolites pathways were the fatty acid biosynthetic metabolic pathway and unsaturated fatty acid biosynthetic pathway.In these two metabolic pathways,α-linolenic acid(ALA),EPA,DHA,oleic acid,caprylic acid,myristic acid,stearic acid enriched in the IG-group,ARA and palmitoleic acid in Y-group Artemia,are all important metabolites for the regulation of unsaturated fatty acid metabolism in Bohai Bay parthenogentica Artemia;the above-mentioned significant differential metabolites may be potential biomarkers affecting the anabolic metabolism of unsaturated fatty acids in Bohai Bay parthenogentica Artemia.3.The Illumia high-throughput sequencing platform was used to sequence the transcriptome of Bohai Bay parthenogentica Artemia samples optimized for baits breeding.After the transcripts were spliced,a total of 248,932 Unigene sequences were obtained.The number of genes successfully annotated to the NR library was 35,009;The Swiss Prot library had 23,768 entries;The PFAM library had 16,382 entries;11,445 entries were successfully annotated in the GO library;and 10,641 entries were successfully annotated in the KO database.At the gene expression level,there are 401 down-regulated genes and 148 up-regulated genes,a total of 9 are enriched to the pathways related to lipid metabolism,of which only one is related to the fatty acid pathways,which is the ARA metabolic pathway in the Y-group.The differential genes CD224/GGT1-5 are highly expressed on this pathway,and the gene-expressing transporters γ-glutamyl transpeptidase,glutathione hydrolase,and leukotriene-C4 synthase are highly synthesized.There were no significant changes in differential genes in unsaturated fatty acid-related pathways in the IG-group Artemia.Therefore,the CD224/GGT1-5 gene may be a regulatory gene for the biosynthesis of unsaturated fatty acids in the Y-group Artemia in this omics perspective.4.The combined analysis of transcriptomics and metabolomics screened a total of 46 metabolic pathways that participated in the two groups.There are two pathways related to unsaturated fatty acids,which are the ARA metabolic pathway and unsaturated fatty acid biosynthetic pathway.In the two pathways,the CD224/GGT1-5 gene and des C gene of Y-group Artemia are positively correlated with ARA,that is,the CD224/GGT1-5 gene is on the ARA metabolic pathway,which promotes the ARA metabolism in Y-group Artemia and accelerates the conversion of ARA to lipids,reducing the synthesis and accumulation of unsaturated fatty acids and other substances in Y-group Artemia;The des C gene in Y-group promotes ARA accumulation in Artemia in the unsaturated fatty acid biosynthesis pathway,and inhibits the biosynthesis of ALA,EPA,DHA,and oleic acid in Artemia.Compared with the Y-group,the normal expression of the des C gene and the CD224/GGT1-5 gene in the IG-group promoted the biosynthesis of unsaturated fatty acids such as ALA,EPA,DHA,and oleic acid in Artemia,which reduced ARA metabolism and accumulation,and promoted the metabolism and absorption of Artemia lipids in the IG-group.更多还原