四个脂肪酸合成酶基因在哺乳动物细胞中的超表达提高长链多不饱和脂肪酸生物合成效率

DHA(22:6n-3)、EPA(20:5n-3)和ARA(20:4n-6)三种长链多不饱和脂肪酸在生物体内活性最强,它们在促进大脑发育和功能维持以及在预防和治疗心血管疾病、炎症、癌症等多种疾病方面有着重要作用。然而,尽管哺乳动物体内有完整的长链多不饱和脂肪酸合成酶系,但哺乳动物合成这些长链多不饱和脂肪酸的效率很低而主要依赖于食物获取。本研究应用转基因方法,将哺乳动物来源的Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶这4种酶的编码基因构建成为一个多基因表达载体,然后转染哺乳动物细胞HEK293T,实现了4个目的基因的超表达,再通过气质联用(GC-MS)分析证实了DHA、EPA和ARA等长链多不饱和脂肪酸的合成效率及水平显著增加,DHA的水平更是提高了2.5倍。由此可见,哺乳动物具有某种抑制长链多不饱和脂肪酸高水平合成的机制,但通过Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶的超表达,能够打破哺乳动物这种抑制机制,从而显著提高DHA、EPA、ARA等的合成水平。同时,本研究的思路也为在转基因动物中生产长链多不饱和脂肪酸提供了重要的启示。     

“自我剪切”2A肽介导的Δ-12和ω-3脂肪酸脱氢酶以及过氧化氢酶在转基因小鼠肌肉表达研究

哺乳动物因为缺乏Δ-12和ω-3脂肪酸脱氢酶,不能自身合成必需的多不饱和脂肪酸．目前,通过转基因技术在哺乳动物体内表达ω-3脂肪酸脱氢酶,能将长链的n-6多不饱和脂肪酸转化成n-3多不饱和脂肪酸,造成体内长链的n-6多不饱和脂肪酸含量显著减低．本研究通过自我剪切2A肽介导Δ-12和ω-3脂肪酸脱氢酶(FAT-2和FAT-1)以及人过氧化氢酶(human catalase,hCAT)在小鼠的肌肉同时表达．结果表明,转基因小鼠肌肉中长链n-3多不饱和脂肪酸含量提高2.6倍,长链n-6多不饱和脂肪酸含量没有显著变化,而n-6/n-3比例显著降低(P < 0.01)．同时蛋白质印迹检测到人过氧化氢酶hCAT在小鼠的肌肉组织中表达,且过氧化氢酶活性比野生型小鼠显著提高(P < 0.01)．     

基于Cre/loxP系统的睾丸组织特异性敲除Elovl4基因小鼠模型的构建及敲除效率检测

极长链多不饱和脂肪酸(very long chain polyunsaturated fatty acids,VLC-PUFAs)是哺乳动物视网膜、睾丸等极少数组织中特有的脂肪酸,其生物合成的关键酶为极长链脂肪酸延长酶4(very long chain fatty acid elongase 4,Elovl4)。建立组织特异性敲除Elovl4基因的动物模型有利于深入研究VLC-PUFAs的生物学功能,因此,本研究基于Cre/loxP系统,先分别构建了Stra8-Cre小鼠和Elovl4 floxed小鼠,通过杂交获得(Elovl4[flox/+],Stra8-Cre)杂合子基因敲除小鼠,再选择雌鼠与Elovl4 floxed纯合子雄鼠即Elovl4 [flox/flox]雄鼠杂交,通过基因型鉴定筛选获得(Elovl4[flox/flox], Stra8-Cre)纯合子小鼠。利用RT-PCR、qRT-PCR、Western blotting、免疫组化和免疫荧光检测Elovl4在睾丸组织中的敲除效率,结果表明,无论是杂合子还是纯合子基因敲除小鼠,其睾丸组织中Elovl4的表达在mRNA及蛋白水平显著下调,但其他组织未受影响。本研究成功构建了睾丸组织特异性敲除Elovl4基因小鼠,为后续研究VLC-PUFAs对雄性小鼠生殖功能的影响及相关分子机制提供可靠的动物模型。     

达氏鲟Elovl4、ELovl5和Elovl7克隆、组织分布及饥饿对其表达的影响

为研究长链脂肪酸延长酶(Elovls)作用机制, 实验根据前期达氏鲟转录组测序获得的unigene序列为基础, 得到Elovl4、Elovl5和Elovl7 CDS区序列, 并分析了3个基因在各组织及饥饿胁迫下的表达情况。结果显示, 达氏鲟Elovl4、Elovl5和Elovl7编码区为753、885和846 bp, 分别编码250、294和281个氨基酸。达氏鲟Elovl4、Elovl5和Elovl7氨基酸序列与斑点雀鳝Elovls具有较高的同源性; 组织分布结果显示, 达氏鲟Elovl4在卵巢和眼中表达最高; Elovl5在肌肉中的表达显著高于其他各个组织; Elovl7在卵巢中表达最高(P<0.05), 鳃、精巢和肌肉中表达较多。在饥饿条件下, Elovl4、Elovl5和Elovl7在肝脏中的表达量均显著下调(P<0.05), 肠道中的趋势各有不同; 饥饿3d时, Elovl4、Elovl5和Elovl7在胃中的表达量显著降低, 随后上调(P<0.05); 饥饿7d时, 脑和肌肉中Elovl4、Elovl5和Elovl7的表达量显著高于其他各组, 随后显著下降(P<0.05)。这说明Elovl4、Elovl5和Elovl7在达氏鲟营养调控过程中发挥的作用可能存在差异, 其具体作用机制需要进一步研究。     

长链多不饱和脂肪酸的替代来源——转基因油料作物

长链多不饱和脂肪酸(LCPUFA)对人体健康和发育具有极为重要的作用.由于海洋鱼类资源过度捕捞和环境污染的加剧,长链多不饱和脂肪酸的来源日益枯竭.利用转基因油料作物生产LCPUFA,特别是DHA和EPA成为目前研究的热点.归纳了LCPUFA合成代谢途径相关基因的最新研究进展,描述了利用植物基因工程合成长链多不饱和脂肪酸(特别是EPA和DHA)取得的主要成果,讨论了影响转基因油料作物合成DHA和EPA的关键因素,最后对利用转基因油料作物合成DHA和EPA的研究前景进行了展望.     

水生动物脂肪酸延长酶基因研究进展

多不饱和脂肪酸(polyunsaturated fatty acids,PUFAs)对人类具有重要的生理作用,但由于人类缺乏从头合成这些脂肪酸的能力,而必须从食物中获得,鱼类等水生动物中二十碳五烯酸(20:5n-3,EPA)和二十二碳六烯酸(22:6n-3,DHA)等多不饱和脂肪酸含量丰富,从而使得鱼类等水生动物成为人们获得PUFAs的主要途径。鱼类等水生动物体内PUFAs的合成是在一系列去饱和酶和延长酶的作用下实现的,其中长链脂肪酸延长酶是合成PUFAs必不可少的一类关键酶。本研究总结了水生动物长链多不饱和脂肪酸合成的过程,综述了水生动物中脂肪酸延长酶基因的研究进展和未来的发展方向。     

哺乳动物细胞多不饱和脂肪酸合成酶系的重建将亚油酸代谢转变为二十二碳六烯酸

二十二碳六烯酸(DHA,22:6n-3)是一种长度为22个碳原子且含有6个双键的ω-3系多不饱和脂肪酸,在人体中具有重要生物学功能。人体及其他哺乳动物体内只能合成少量的DHA,更多的需求必须从食物中获取。然而,DHA的天然资源(主要是深海鱼类等海洋产品)日趋枯竭,开发新型资源以满足不断扩大的市场需求势在必行。本研究利用转基因技术,在哺乳动物细胞中使Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶超表达,同时表达来源于秀丽隐杆线虫Caenorhabditis elegans的Δ15去饱和酶和小眼虫Euglena gracilis的Δ4去饱和酶,结果表明,这6种酶的表达或超表达能将ω-6系的亚油酸(LA,18:2n-6)有效地转化为DHA(22:6n-3),后者的含量从对照组的16.74%提高到实验组的25.3%。本研究的策略及技术路线为将来利用遗传改造的哺乳动物生产珍稀的DHA(22:6n-3)等长链多不饱和脂肪酸产品提供了重要的启示。     

灵活操控果蝇翅芽中wingless基因表达水平的策略

【目的】灵活操控靶基因的表达水平对于研究基因的功能十分重要。Gal4/UAS系统已被广泛应用于调控基因表达,可研究果蝇Drosophila等模式生物复杂的生物学问题。受采用载体的特性及插入位点的影响,Gal4或UAS转基因品系在构建好之后,其调控靶基因的能力基本是确定的。本研究旨在在现有Gal4/UAS系统的基础上,开发一种新的策略,实现在果蝇翅芽中灵活操控wingless(wg)基因的表达水平。【方法】用遗传学手段将黑腹果蝇Drosophila melanogaster品系的UAS-wg和UAS-wg-RNAi转基因重组到同一黑腹果蝇品系中。将该重组黑腹果蝇品系与dpp-Gal4黑腹果蝇品系杂交,同时驱动UAS-wg和UAS-wg-RNAi在果蝇幼虫翅芽中共表达。杂交子代幼虫分别放置在不同的温度(18, 25和30℃)下培养。将幼虫翅芽解剖并进行免疫组化染色,测量染色的荧光强度,分析翅芽中wg的表达水平。【结果】在低温(18℃)下,UAS-wg在基因表达调控中起主要作用,wg表现为超表达,但其超表达的效率可被UAS-wg-RNAi有效地削弱。相反,在高温(30℃)下,UAS-wg-RNAi起主导作用,wg的表达受到抑制。并且通过转换温度,可实现wg在翅芽发育的不同阶段在超表达和抑制之间相互转化,从而灵活地操控wg基因在翅芽中的表达水平。【结论】该方法可以灵活操控果蝇翅芽中wg基因的表达水平,对于调控转基因的表达有重要的意义。     

转gfp基因大麦中gfp基因表达的半定量RT-PCR方法分析

本研究以5种转绿色荧光蛋白基因(gfp)大麦的不同株系(A,B,C,D,F)及野生型Igri为材料,对供试材料的叶片中gfp基因的荧光表达量利用荧光显微镜测定,并对各株系进行半定量RT-PCR分析。研究结果表明:5种转基因大麦叶片的gfp基因荧光表达量不同,检测结果为FA≈B≈CD;同一转基因材料(C、F)的gfp基因表达存在组织差异,其花粉、叶片及根尖等不同器官中的gfp表达强度不同;转gfp基因D系的gfp基因沉默是gfp基因在转录水平失活。     

Production of dicarboxylic acids from novel unsaturated fatty acids by laccase-catalyzed oxidative cleavage

The establishment of renewable biofuel and chemical production is desirable because of global warming and the exhaustion of petroleum reserves. Sebacic acid (decanedioic acid), the material of 6,10-nylon, is produced from ricinoleic acid, a carbon-neutral material, but the process is not eco-friendly because of its energy requirements. Laccase-catalyzing oxidative cleavage of fatty acid was applied to the production of dicarboxylic acids using hydroxy and oxo fatty acids involved in the saturation metabolism of unsaturated fatty acids in Lactobacillus plantarum as substrates. Hydroxy or oxo fatty acids with a functional group near the carbon–carbon double bond were cleaved at the carbon–carbon double bond, hydroxy group, or carbonyl group by laccase and transformed into dicarboxylic acids. After 8 h, 0.58 mM of sebacic acid was produced from 1.6 mM of 10-oxo-cis-12,cis-15-octadecadienoic acid (αKetoA) with a conversion rate of 35% (mol/mol). This laccase-catalyzed enzymatic process is a promising method to produce dicarboxylic acids from biomass-derived fatty acids.     

Discovery and fine-mapping of loci associated with MUFAs through trans-ethnic meta-analysis in Chinese and European populations

MUFAs are unsaturated FAs with one double bond and are derived from endogenous synthesis and dietary intake. Accumulating evidence has suggested that plasma and erythrocyte MUFA levels are associated with cardiometabolic disorders, including CVD, T2D, and metabolic syndrome (MS). Previous genome-wide association studies (GWASs) have identified seven loci for plasma and erythrocyte palmitoleic and oleic acid levels in populations of European origin. To identify additional MUFA-associated loci and the potential functional variant at each locus, we performed ethnic-specific GWAS meta-analyses and trans-ethnic meta-analyses in more than 15,000 participants of Chinese and European ancestry. We identified novel genome-wide significant associations for vaccenic acid at FADS1/2 and PKD2L1 [log₁₀(Bayes factor) ≥ 8.07] and for gondoic acid at FADS1/2 and GCKR [log₁₀(Bayes factor) ≥ 6.22], and also observed improved fine-mapping resolutions at FADS1/2 and GCKR loci. The greatest improvement was observed at GCKR, where the number of variants in the 99% credible set was reduced from 16 (covering 94.8 kb) to 5 (covering 19.6 kb, including a missense variant rs1260326) after trans-ethnic meta-analysis. We also confirmed the previously reported associations of PKD2L1, FADS1/2, GCKR, and HIF1AN with palmitoleic acid and of FADS1/2 and LPCAT3 with oleic acid in the Chinese-specific GWAS and the trans-ethnic meta-analyses. Pathway-based analyses suggested that the identified loci were in unsaturated FA metabolism and signaling pathways. Our findings provide novel insight into the genetic basis relevant to MUFA metabolism and biology.     

Effects of Different Dietary Fat Types on Postprandial Appetite and Energy Expenditure

Objective: Observational studies suggest that monounsaturated (MUFA) and trans fatty acids (TRANS) are more fattening than polyunsaturated fatty acids (PUFA). Therefore, the aim of this study was to investigate the acute effect of intake of PUFA, MUFA, or TRANS on appetite and energy expenditure (EE). Research Methods and Procedures: Three test meals were randomly given in a cross‐over design to 19 overweight (BMI: 26.8 ± 0.4 kg/m²), young (25.2 ± 0.7 years) men. The fat‐rich breakfasts (0.8 g fat/kg body weight, 60% energy from fat) varied only in the source of C:18‐fat. EE was measured continuously in a respiration chamber, and appetite sensations were rated by visual analog scales before and every 30 minutes, for 5 hours, after the meal. After 5 hours, an ad libitum meal was served, and energy intake was registered. Sensory evaluations of all meals were given using visual analog scales. Data were analyzed by two‐way ANOVA. Results: There were no differences in basal or postprandial values of appetite ratings and EE, in subsequent ad libitum energy intake, or in the sensory evaluation of the test meals among the 3 test days. Discussion: Giving acutely large amounts of MUFA, PUFA, or TRANS did not impose any differences in appetite and EE in overweight humans. However, studies with extended protocols and other subject groups are warranted to investigate the long‐term effect of dietary fat quality on the regulation of energy balance and body weight.     

Gestational and hormonal regulation of human placental lipoprotein lipase

The fetal demand for FFA increases as gestation proceeds, and LPL represents one potential mechanism for increasing placental lipid transport. We examined LPL activity and protein expression in first trimester and term human placenta. The LPL activity was 3-fold higher in term (n = 7; P < 0.05) compared with first trimester (n = 6) placentas. The LPL expression appeared lower in microvillous membrane from first trimester (n = 2) compared with term (n = 2) placentas. We incubated isolated placental villous fragments with a variety of effectors [GW 1929, estradiol, insulin, cortisol, epinephrine, insulin-like growth factor-1 (IGF-1), and tumor necrosis factor-alpha] for 1, 3, and 24 h to investigate potential regulatory mechanisms. Decreased LPL activity was observed after 24 h of incubation with estradiol (1 micro g/ml), insulin, cortisol, and IGF-1 (n = 12; P < 0.05). We observed an increase in LPL activity after 3 h of incubation with estradiol (20 ng/ml) or hyperglycemic medium plus insulin (n = 7; P < 0.05). To conclude, we suggest that the gestational increase in placental LPL activity represents an important mechanism to enhance placental FFA transport in late pregnancy. Hormonal regulation of placental LPL activity by insulin, cortisol, IGF-1, and estradiol may be involved in gestational changes and in alterations in LPL activity in pregnancies complicated by altered fetal growth.     

Syntheses and reactions of methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate

The syntheses and reactions of two epoxyketoacids (methyl (Z)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (IV) and methyl (E)-9,10-epoxy-13-oxo-(E)-11-octadecenoate (V)) are described. The synthetic method is based on the stereoselective oxidation of linoleic acid by soybean lipoxygenase to produce the corresponding 13-hydroperoxide. Reduction of the hydroperoxide with sodium borohydride followed by oxidation, esterification and epoxidation yielded the compounds IV and V with a global yield of 14% and 3%, respectively, referred to the diasteromerically pure isolated compounds. Confirmation of the structures was carried out by reduction of the ketone group with sodium borohydride and by the opening of the oxirane ring with methanolic boron trifluoride. The reduction of compounds IV and V with hydrogen mainly yielded the tetrahydrofuranoid fatty acid, methyl 10,13-epoxyoctadecanoate. This reaction may be considered a new procedure to obtain tetrahydrofuranoid fatty acids.     

Effects of flaxseed,raw soybeans and calcium salts of fatty acids on apparent total tract digestibility,energy balance and milk fatty acid profile of transition cows

Oilseeds offer some protection to the access of ruminal microorganisms and may be an alternative to calcium salts of fatty acids (FA), which are not fully inert in the ruminal environment. This study aimed to evaluate the effects of different sources of FA supplementation on apparent total tract nutrient digestibility, milk yield and composition, and energy balance (EB) of cows during the transition period and early lactation. We compared diets rich in C18:2 and C18:3 FA. Multiparous Holstein cows were randomly assigned to receive one of the four diets: control (n=11); whole flaxseed (WF, n=10), 60 and 80 g/kg (diet dry matter (DM) basis) of WF during the prepartum and postpartum periods, respectively; whole raw soybeans (WS, n=10), 120 and 160 g/kg (diet DM basis) of WS during the prepartum and postpartum periods, respectively; and calcium salts of unsaturated fatty acids (CSFA, n=11), 24 and 32 g/kg (diet DM basis) of CSFA during the prepartum and postpartum periods, respectively. Dry cows fed WF had higher DM and net energy of lactation (NE_L) intake than those fed WS or CSFA. The FA supplementation did not alter DM and NDF apparent total tract digestibility, dry cows fed WF exhibited greater NDF total tract digestion than cows fed WS or CSFA. Feeding WS instead of CSFA did not alter NE_L intake and total tract digestion of nutrients, but increased milk fat yield and concentration. Calculated efficiency of milk yield was not altered by diets. FA supplementation increased EB during the postpartum period. Experimental diets increased long-chain FA (saturated and unsaturated FA) in milk. In addition, cows fed WS and CSFA had higher C18:1 trans-11 FA and C18:2 cis, and lower C18:3 FA in milk than those fed WF. Furthermore, cows fed CSFA had higher C18:1 trans-11 and cis-9, trans-11 FA than cows fed WS. Although supplemental C18:2 and C18:3 FA did not influence the milk yield of cows, they positively affected EB and increased unsaturated long-chain FA in milk fat.     

Effect of Randomly Interesterified Triacylglycerol Containing Medium- and Long-Chain Fatty Acids on Hepatic Fatty Acid Oxidation after a Single Administration to Rats

MLCTs, which are randomly interesterified triacylglycerol containing medium- and long-chain fatty acids in the same glycerol molecule, showed significantly higher acyl-CoA dehydrogenase activity when measured by using butyryl-CoA, octanoyl-CoA, and palmitoyl-CoA as substrates than long-chain triacylglycerol one hour after a single administration to rats. These results suggest that not only medium-chain fatty acid oxidation, but also long-chain fatty acid oxidation were increased in the liver of rats administered with MLCT.     

Fatty acid interactions with native and mutant fatty acid binding proteins

The interactions of long chain fatty acids (FA) with wild type (WT) fatty acid binding proteins (FABP) and engineered FABP mutants have been monitored to determine the equilibrium binding constants as well as the rate constants for binding and dissociation. These measurements have been done using the fluorescent probes, ADIFAB and ADIFAB2, that allow the determination of the free fatty acid (FFA) concentration in the reaction of FA with proteins and membranes. The results of these studies indicate that for WT proteins from adipocyte, heart, intestine, and liver, Kd values are in the nM range and affinities decrease with increasing aqueous solubility of the FA. Binding affinities for heart and liver are generally greater than those for adipocyte and intestine. Moreover, measurements of the rate constants indicate that binding equilibrium at 37øC is achieved within seconds for all FA and FABPs. These results, together with the level of serum (unbound) FFA, suggests a buffering action of FABPs that helps to maintain the intracellular concentration of FFA so that the flux of FFA between serum and cells occurs down a concentration gradient. Measurements of the temperature dependence of binding reveal that the free energy is predominately enthalpic and that the enthalpy of the reaction results from FA-FABP interactions within the binding cavity. The nature of these interactions were investigated by determining the thermodynamics of binding to engineered point mutants of the intestinal FABP. These measurements showed that binding affinities did not report accurately the changes in protein-FA interactions because changes in the binding entropy and enthalpy tend to compensate. For example, an alanine substitution for arginine 106 yields a 30 fold increase in binding affinity, because the loss in enthalpy due to the elimination of the favorable interaction between the FA carboxylate and Arg106, is more than compensated for by an increase in entropy. Thus understanding the effects of amino acid replacements on FA-FABP interactions requires measurements of enthalpy and entropy, in addition to affinity.