分离自象山港的15种海洋微藻脂肪酸比较研究

在160μmol.m-2.s-1光强、（20±2）℃条件下对分离自象山港的15种海洋微藻进行培养,在稳定期离心收集,冷冻干燥后用Bligh-Dyer法提取总脂,皂化衍生化后用气相色谱-质谱联用分析系统对其含有的脂肪酸进行定量和定性的分析。结果表明,这15种微藻总脂含量均较高,其中有9种微藻的总脂含量超过干重的10%,中心硅藻纲中共有的含量相对较高的主要脂肪酸为C14∶0、C16∶0、C16∶1（n-7）脂肪酸和EPA,针胞藻纲的赤潮异湾藻则含有高比例的C16∶0、C18∶4（n-3）和EPA,纵裂甲藻纲和甲藻纲中共有的含量相对较高的主要脂肪酸为C16∶0、C18∶4（n-3）、C18∶5（n-3）和DHA,而隶属于定鞭藻门颗石藻纲的颗石藻含量相对较高的主要脂肪酸分别为C16∶0、C18∶4（n-3）和DHA。这些藻是否可以作为生物饵料还需实际养殖投喂效果决定。     

硬脂酰-辅酶A脱氢酶基因在食品级乳酸菌中的表达

为了实现硬脂酰-辅酶A脱氢酶1编码基因在乳酸乳球菌中的表达,采用PCR技术扩增获得人类scd1的编码序列。Nco I和Xba I双酶切后定向插入到食品级表达载体pNZ8149中,构建表达载体pNZ8149-scd1。电转化乳酸乳球菌NZ3900,经菌落PCR和测序鉴定scd1基因成功插入到乳酸乳球菌中。在乳链菌肽诱导下进行scd1的表达,转化株提取脂肪酸,进行脂肪酸含量的气相色谱分析。结果显示,SCD1转化菌株中的C16∶1n-7和C18∶1n-7脂肪酸组分比转化pNZ8149的对照组乳酸菌分别提高了92%~169%和53%~127%。文中以scd1基因为例,尝试并证明了脂肪酸脱氢酶类基因能够在食品级乳酸菌中有效表达,为后续研究奠定了基础。     

海洋破囊壶菌Δ~4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6kb的Δ4-脂肪酸脱饱和酶基因(FAD4)。将FAD4酶切后连接到HindⅢ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22:5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),Δ4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。     

冷冻干燥条件下酿酒酵母细胞脂肪酸组成变化的研究

以气相色谱法测定酿酒酵母(Saccharomyces cereuisiae) F159细胞膜类脂中脂肪酸的组成。结果表明,在低温干燥条件下,酿酒酵母细胞能自身调节脂肪酸的不饱和度,出现C_(16:1)脂肪酸含量增加,C_(16:0)脂肪酸含量略有所增,而C_(18:0)脂肪酸含量却有下降,不饱和直链脂肪酸之和与饱和直链脂肪酸之和的比值增加。说明这些变化使酿酒酵母细胞膜保持流动状态而维持细胞的稳定性和活性。初步探讨了冷冻干燥对酿酒酵母细胞脂肪酸组成的影响,为进一步探索和研究微生物抗冷冻干燥生理生化机制提供了依据。     

海洋破囊壶菌△4-脂肪酸脱饱和酶基因在酿酒母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1.6 kb的△4-脂肪酸脱饱和酶基因(FAD4).将FAD4酶切后连接到Hond Ⅲ/XbaⅠ处理过的pYES2.0载体,构建重组表达质粒pYFAD4.转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子.添加外源脂肪酸C22:5底物,半乳糖诱导表达.气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22:6(占酵母总脂肪含量的41.13%),△4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达.     

不同生态条件对绿色巴夫藻生长与脂肪酸组成的影响

研究不同温度、盐度、光照条件对绿色巴夫藻生长和脂肪酸组成的影响。实验结果表明:绿色巴夫藻的适温范围在5—30℃之间,最适为25℃;该藻为低盐度种类,在试验范围内都能生长,其最适盐度为6‰;生长较适宜的光照强度为3000—5000lx。巴夫藻的主要脂肪酸为C14∶0、C16∶0、C16∶1(n-7)、C18∶1(n-9)、C18∶3(n-3)、C18∶4(n-3)、C22∶6(n-3),其中在20℃时C22∶6(n-3)的含量达到最高,且PUFA(n-3)的含量也较高;在低盐度条件下有利于PUFA(n-3)的合成,盐度为6‰时C20∶5(n-3)的含量最高为总脂的6.02%,在盐度为15‰时C22∶6(n-3)的含量达到最高(总脂的17.79%);光照强度对C22∶6(n-3)的影响不大,在3000lx时C20∶5(n-3)的含量较高。结果表明低光照强度下利于PUFA(n-3)的合成,且C22∶6(n-3)的含量较高。     

海洋破囊壶菌Δ4-脂肪酸脱饱和酶基因在酿酒酵母中的表达

以质粒pGEM-TFAD4为模板,扩增获得1．6kb的△^4-脂肪酸脱饱和酶基因（FAD4）。将FAD4酶切后连接到hin dⅢ,Xba I处理过的pYEs2．0载体,构建重组表达质粒pYFAD4。转化酿酒酵母缺陷型菌INVScl,通过SC-U选择性培养基筛选阳性克隆子。添加外源脂肪酸C22：5底物,半乳糖诱导表达。气相色谱分析表明阳性克隆子总脂肪酸中出现了二十二碳六烯酸C22：6（占酵母总脂肪含量的41．13％）,△^4-脂肪酸脱饱和酶基因在酿酒酵母中得到了表达。     

14种微藻总脂含量和脂肪酸组成研究

比较分析了14种微藻的总脂含量和脂肪酸组成,结果表明：除小球藻、亚心形扁藻、极微小环藻、微绿球藻外,其他微藻的总脂含量均超过其干重的10％。每一纲的微藻脂肪酸组成都有各自特点,绿藻纲中16：0、16：1(n-7)、18：1(n-9)含量较高,但微绿球藻中16：1(n-7)、20：5(n-3)(EPA)含量远高于其他绿藻;金藻纲中含大量14：0、16：0、18：3(n-3)、22：6(n-3)(DHA);硅藻纲中14：0、16：0、16：1(n-7)、EPA含量较高;黄藻纲的异胶藻富含16：0、16：1(n-7)和EPA。     

西南大西洋春夏季阿根廷无须鳕脂肪酸组分的时空特性

阿根廷无须鳕(Merluccius hubbsi)是西南大西洋较为重要的鱼种,具较高的商业价值,为底拖网渔业的主要捕捞对象。本研究利用气相色谱质谱联用仪测定了阿根廷无须鳕肌肉组织脂肪酸组成,探究各脂肪酸含量,包括饱和脂肪酸(SFA)、单不饱和脂肪酸(MUFA)和多不饱和脂肪酸(PUFA)随体长、月份和纬度的变化。结果表明:各类脂肪酸含量高低次序依次为∑PUFA∑SFA∑MUFA;主要脂肪酸包括C22:6n3(DHA)、C16:0、C18:1n9c、C20:5n3(EPA)和C18:0,其中DHA的含量最高,占总脂肪酸含量的35.27%;高PUFA/SFA表明阿根廷无须鳕肌肉可作为PUFA的补充原料;∑SFA和∑PUFA在小体长组个体中(20.1~30.0 cm)的含量较高,特别是DHA和EPA,使得n-3/n-6增加,说明相对较大个体而言,小个体阿根廷无须鳕的营养价值更高;除n-3/n-6外,其余脂肪酸含量均无显著的月份差异;1月最高的n-3/n-6说明肌肉组织在该月份具有较高的营养价值。鱼体各脂肪酸含量随纬度和表温变化不明显。     

九种海洋微藻总脂含量及脂肪酸组成分析

从广东沿海分离出9 种海洋微藻(包括6 种绿藻, 2 种金藻, 1 种硅藻), 在低氮并持续通入二氧化碳(1% CO2)条件下测试其总脂含量, 对其中总脂含量超过其干重45%的海洋微藻进行脂肪酸组成分析并优化反应条件。结果表明,9 种海洋微藻的总脂含量存在着明显差异, 介于8.9%-55.77%之间。其中, 海洋小球藻Chlorella sp.、眼点拟微绿球藻Nannochloropsis oculata、绿色巴夫藻Pavlova viridis 和微拟球藻Nannochloropsis sp.的总脂含量超过干重的45%, 而且, 这4 种富油海洋微藻的C16 和C18 脂肪酸含量丰富, C16:0、C16:1(n-7)、C18:1(n-9)含量较高, C14-C18 长链脂肪酸的含量超过总脂肪酸含量的80%。4 种高脂微藻在30 ℃高温培养条件下比生长速率均超过1.0 d−1。研究结果可为高温富油海洋微藻的工业化培养提供参考依据。     

Deletion of ELOVL5 leads to fatty liver through activation of SREBP-1c in mice

Elongation of very long chain fatty acids (ELOVL)5 is one of seven mammalian fatty acid condensing enzymes involved in microsomal fatty acid elongation. To determine the in vivo substrates and function of ELOVL5, we generated Elovl5(-/-) mice. Studies using liver microsomal protein from wild-type and knockout mice demonstrated that the elongation of gamma-linolenic (C18:3, n-6) to dihomo-gamma-linolenic (C20:3, n-6) and stearidonic (C18:4, n-3) to omega3-arachidonic acid (C20:4, n-3) required ELOVL5 activity. Tissues of Elovl5(-/-) mice accumulated the C18 substrates of ELOVL5 and the levels of the downstream products, arachidonic acid (C20:4, n-6) and docosahexaenoic acid (DHA, C22:6, n-3), were decreased. A consequence of decreased cellular arachidonic acid and DHA concentrations was the activation of sterol regulatory element-binding protein (SREBP)-1c and its target genes involved in fatty acid and triglyceride synthesis, which culminated in the development of hepatic steatosis in Elovl5(-/-) mice. The molecular and metabolic changes in fatty acid metabolism in Elovl5(-/-) mice were reversed by dietary supplementation with arachidonic acid and DHA. These studies demonstrate that reduced ELOVL5 activity leads to hepatic steatosis, and endogenously synthesized PUFAs are key regulators of SREBP-1c activation and fatty acid synthesis in livers of mice.     

ELOVL2 overexpression enhances triacylglycerol synthesis in 3T3-L1 and F442A cells

Elongation of very long-chain fatty acids (ELOVL) members were overexpressed in two preadipocyte cell lines, ELOVL2 and ELOVL3 in 3T3-L1 cells, and ELOVL1-3 in F442A cells. Cells overexpressing ELOVL2, whose preferred substrates are arachidonic acid (AA, C20:4n-6) and eicosapentaenoic acid (EPA, C20:5n-3), showed an enhanced triacylglycerol (TAG) synthesis and subsequent accumulation of lipid droplets. Incorporation of fatty acid (FA) but not of glucose into TAG was enhanced by ELOVL2-overexpression. Two lipogenic genes encoding diacylglycerol acyltransferase-2 (DGAT2) and fatty acid-binding protein-4 (FABP4, aP2) were induced in ELOVL2-overexpressing cells, whereas no such effect was seen on the fatty acid synthase (FAS) gene.     

Depression and adipose polyunsaturated fatty acids in an adolescent group

The purpose of the present study was to investigate the relation between adipose tissue polyunsaturated fatty acids, an index of long-term or habitual fatty acid dietary intake and depression. The sample consisted of 90 adolescents from the island of Crete. There were 54 girls and 36 boys, aged 13-18. The mean age was 15.2 years. Subjects were examined by the Preventive Medicine and Nutrition Clinic of the University of Crete. Depression was assessed through the use of the Beck Depression Inventory (BDI) and the Center for Epidemiologic Studies Depression Scale (CES-D). Unlike other studies, there were no significant relations between adipose tissue n-3 or n-6 polyunsaturated fatty acids and depression. BDI correlated positively with adipose tissue C20:3n-6/C18:3n-6 ratio, while CES-D correlated positively with adipose tissue (C20:3n-6+C22:5n-3)/(C18:3n-6+C20:5n-3) ratio. Depressed subjects (BDI>16, CES-D>16) had significantly elevated adipose tissue C20:3n-6/C18:3n-6 and (C20:3n-6+C22:5n-3)/(C18:3n-6+C20:5n-3) ratios, than non-depressed subjects. The observed positive relation between depression and the particular fatty acid ratios, in the present study, appears to indicate increasing activity of elongases, the enzymes responsible for elongating polyunsaturated fatty acids into their longer-chain derivatives, with increasing depression. This is the first literature report of a possible relation between elongases and depression. The observed relation may stem from a possible over-expression of the HELO1 (ELOVL5) gene, the gene encoding a protein responsible for elongating long-chain polyunsaturated fatty acids, in the adipose tissue of depressed adolescents.     

ELOVL2 controls the level of n-6 28:5 and 30:5 fatty acids in testis, a prerequisite for male fertility and sperm maturation in mice

ELOVL2 is a member of the mammalian microsomal ELOVL fatty acid enzyme family, involved in the elongation of very long-chain fatty acids including PUFAs required for various cellular functions in mammals. Here, we used ELOVL2-ablated (Elovl2(-/-)) mice to show that the PUFAs with 24-30 carbon atoms of the ω-6 family in testis are indispensable for normal sperm formation and fertility in male mice. The lack of Elovl2 was associated with a complete arrest of spermatogenesis, with seminiferous tubules displaying only spermatogonia and primary spermatocytes without further germinal cells. Furthermore, based on acyl-CoA profiling, heterozygous Elovl2(+/-) male mice exhibited haploinsufficiency, with reduced levels of C28:5 and C30:5n-6 PUFAs, which gave rise to impaired formation and function of haploid spermatides. These new insights reveal a novel mechanism involving ELOVL2-derived PUFAs in mammals and previously unrecognized roles for C28 and C30 n-6 PUFAs in male fertility. In accordance with the function suggested for ELOVL2, the Elovl2(-/-) mice show distorted levels of serum C20 and C22 PUFAs from both the n-3 and the n-6 series. However, dietary supplementation with C22:6n-3 could not restore male fertility to Elovl2(+/-) mice, suggesting that the changes in n-6 fatty acid composition seen in the testis of the Elovl2(+/-) mice, cannot be compensated by increased C22:6n-3 content.     

Biochemical characterization of the very long-chain fatty acid elongase ELOVL7

Very long-chain fatty acids (VLCFAs) have a variety of physiological functions and are related to numerous disorders. The key step of VLCFA elongation is catalyzed by members of the elongase family, ELOVLs. Mammals have seven ELOVLs (ELOVL1-7), yet none of them has been purified and analyzed. In the presented study we purified ELOVL7 and measured its activity by reconstituting it into proteoliposomes. Purified ELOVL7 exhibited high activity toward acyl-CoAs with C18 carbon chain length. The calculated K(m) values toward C18:3(n-3)-CoA and malonyl-CoA were both in the μM range. We also found that progression of the VLCFA cycle enhances ELOVL7 activity.     

Effects of ambient temperature on lipid and fatty acid composition in the oviparous lizards, Phrynocephalus przewalskii

This study was designed to assess the effect of ambient temperature on lipid content, lipid classes and fatty acid compositions of heart, liver, muscle and brain in oviparous lizards, Phrynocephalus przewalskii, caught in the desert area of China. Significant differences could be observed in the contents of the total lipid and fatty acid compositions among different temperatures (4, 25 and 38 degrees C). The study showed that liver and muscle were principal sites of lipid storage. Triacylglycerol (TAG) mainly deposited in the liver, while phospholipids (PL) was identified as the predominant lipid class in the muscle and brain. Palmitic and stearic acid generally occupied the higher proportion in saturated fatty acids (SFA), while monounsaturated fatty acids (MUFAs) and polyunsaturated fatty acids (PUFAs) consisted mainly of 16:1n-7, 18:1n-9, 18:2n-6, 18:3n-3, 20:4n-6 and 22:6n-3 regardless of tissue and temperature. These predominant fatty acids proportion fluctuations caused by temperature affected directly the ratio of unsaturated to saturated fatty acids. There was a tendency to increase the degree of unsaturation in the fatty acids of TAG and PL as environmental temperature dropped from 38 to 4 degrees C, although the different extent in different tissues. These results suggested that lipid characteristics of P. przewalskii tissues examined were influenced by ambient temperature.     

(n-7) and (n-9) cis-Monounsaturated fatty acid contents of 12 Brassica species

cis-Vaccenic acid or cis-11-octadecenoic acid, a C18:1 (n-7) isomer of oleic acid (C18:1 (n-9)) has been found in several oilseeds. It is synthesized from palmitic acid (C16:0) via production of C16:1 (n-7) by a Delta9 desaturase and elongation by an elongase giving C18:1 (n-7). In this study, the fatty acid composition of 12 Brassica species was analyzed by GC-FID and confirmed by GC-MS. All species contained C18:1 (n-7), C20:1 (n-7) and C22:1 (n-7) fatty acid isomers, suggesting that C18:1 (n-7) was elongated. The levels of these fatty acids varied according to the species. C18:1(n-7)) represented from 0.4% to 3.3% of the total relative fatty acid contents of the seeds. The contents of C20:1(n-7) and C22:1(n-7) levels were lower than C18:1(n-7) contents; the relative fatty acid composition varied from 0.02% to 1.3% and from below the limit of detection to 1.3% for C20:1 (n-7) and C22:1 (n-7), respectively. The ratios of (n-7)/(n-9) ranged from 2.8% to 16.7%, 0.6% to 29.5% and 0% to 2.6% for C18:1, C20:1 and C22:2, respectively. Using statistical similarities or differences of the C18:1 (n-7)/(n-9) ratios for chemotaxonomy, the surveyed species could be arranged into three groups. The first group would include Brassica napus, B. rapa, and B. tournefortii with Eruca sativa branching only related to B. napus. The second group would include B. tournefortii, Raphanus sativus and Sinapis alba. The last group would include B. juncea, B. carinata and B. nigra with no similarity/relationship between them and between the other species. Results suggested that the level of C20:1 (n-7) influenced the levels of all monounsaturated fatty acids with chain length higher than 20 carbons. On the other hand, palmitoleic acid (C16:1) levels, C16:1 being the parent of all (n-7) fatty acids, had no statistically significant correlation with the content of any of the fatty acids of the (n-7) or (n-9) family.     

An alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi (Lates calcarifer)

Desaturase and elongase are two key enzyme categories in the long-chain polyunsaturated fatty acid (LCPUFA) pathway that convert dietary α-linolenic acid (18:3n-3) to docosahexaenoic acid (22:6n-3). The Δ6 desaturase is considered as rate limiting in the conversion. In a previous study in barramundi we demonstrated that the desaturase had a low Δ6 activity but noted that the enzyme also possessed Δ8 ability that utilised 20-carbon fatty acids. This observation suggests that an alternative pathway may exist in the barramundi via elongases to form 20-carbon metabolites from 18:3n-3 to 20:3n-3 and then Δ6/8 desaturase to 20:4n-3. Cloning of the barramundi elongation of very long-chain fatty acid gene (ELOVL) and heterologous expression of the corresponding elongase were performed to examine activity with regard to time course, substrate concentration and substrate preference. Results revealed that the barramundi elongase showed a broad range of substrate specificity including 18-carbon PUFA (including 18:3n-3 and 18:2n-6), 20- and 22-carbon LCPUFA, with greater activity towards omega-3 (n-3) than n-6 fatty acids. The findings from this study provide molecular evidence for an alternative n-3 fatty acid elongation pathway utilising 18:3n-3 in barramundi.     

苏太猪ELOVL7基因的生物信息学分析

不同于人、鼠等物种ELOVL7基因的高相似度,不同品种的猪ELOVL7基因相似度较低。为了探究该基因的特性,本研究运用生物信息学的方法对苏太猪ELOVL7基因及其氨基酸序列的同源性、理化性质、保守结构域、亚细胞定位、信号肽、跨膜结构域、亲水性/疏水性、二级结构、功能预测以及磷酸化位点等进行预测分析。结果表明：在苏太猪中,ELOVL7全长2 324 bp,编码区为846 bp,共编码281个氨基酸。其结构稳定,分子量为33 387.4 Da,带正电荷,偏碱性。该基因所编码的蛋白质最可能位于细胞膜上,主要的功能是运输和结合,为跨膜、非分泌型疏水蛋白质,含有1个GNS1/SUR4家族的保守结构域,并有15个丝氨酸激酶、15个苏氨酸激酶和20个酪氨酸激酶潜在磷酸化位点。α螺旋是ELOVL7二级结构和三级结构中最主要的结构元件。另外ELOVL7与大部分物种的氨基酸序列相似性达90%以上,且亲缘关系较近。分析ELOVL7基因及其氨基酸序列的特征,能够为进一步挖掘该基因内的突变对长链脂肪酸表型的影响以及合成、代谢机理提供分子依据。     

Effect of ELOVL6 on the lipid metabolism of bovine adipocytes

This study investigated the effect of ELOVL6 (elongation of very long chain fatty acids protein 6) and its underlying mechanism on lipid metabolism in bovine adipocytes. The ELOVL6 gene was overexpressed in bovine adipocytes by adenoviruses, and RNA sequencing was performed. Overexpression of ELOVL6 showed reduced proportions of C14:0 (Myristic) and C16:0 (palmitate) fatty acids and increased proportions of C18.0 (stearate) and C20:4n6 (arachidonic) fatty acids in adipocytes. In addition, a total of 2170 differentially expressed genes (DEGs) were found, containing 1802 up-regulated and 368 down-regulated genes. KEGG pathway analysis revealed that the down-regulated genes were linked with the regulation of lipolysis and the Wnt signaling pathway. The up-regulated genes were mainly involved in the FoxO signaling pathway; the PI3K-Akt signaling pathway; and the cAMP signaling pathway. In conclusion, our results suggest that ELOVL6 could affect the fatty acid composition in bovine adipocytes. We identified numerous related DEGs and pathways, which may provide a basis for studying the function and molecular mechanism of the ELOVL6 gene in regulating lipid metabolism.