植物二酰甘油酰基转移酶基因(DGAT)研究进展

三酰甘油(TAG)是油料作物最主要的储藏脂类,二酰甘油酰基转移酶(DGAT,EC2.3.1.20)是TAG合成途径的限速酶,其主要作用是催化二酰甘油加上酰基脂肪酸形成三酰甘油.在植物中已发现了3种不同类型的DGAT基因,分别为DGAT1、DGAT2和DGAT3.该文对近年来国内外有关植物DGAT相关基因及其蛋白分类、定位、结构及其在脂肪酸合成、种子发育与萌发、幼苗发育、叶片新陈代谢等过程中的作用等研究进展进行综述.为提高油料作物种子油含量以及特定脂肪酸积累提供理论参考.     

植物中从二酰甘油到三酰甘油的两条合成新途径

过去认为植物中只有一条从二酰甘油到三酰甘油的合成途径。近年来,在一些植物体内又发现了从二酰甘油到三酰甘油合成的两条新途径。该文介绍这两条新途径及其意义。     

微藻三酰甘油合成途径及其最新研究进展

三酰甘油(triacylglycerols,TAGs)是动物、植物、微生物和微藻细胞主要的储藏性脂类,它可应用于食品、轻工业和生物燃料等方面,是一种新型可再生能源——生物柴油生产的重要原料。与高等油料作物相比,微藻具有光合作用效率高、生长速度快、油脂产量高、不占用农业耕地和适应多种生长环境等优势,是一种潜在的新型生物柴油生产原料。然而,目前人们对有机体,尤其是微藻细胞内TAG合成与积累的分子机制及细胞的代谢调控机制还知之甚少。对TAG合成的一系列重要过程,包括脂肪酸的合成,TAG生物合成的主要途径和旁路途径,以及与TAG合成相关的关键酶和重要基因等进行了综述,特别对微藻细胞中与TAG合成相关的关键基因的最新研究进展进行了总结,旨在更好地了解油脂代谢的调控途径,为最大限度地供应生物柴油的生产原料提供理论基础。     

磷脂酰甘油合成代谢研究

从磷脂酰甘油分子的结构特点和研究方法出发,重点介绍了磷脂酰甘油合成代谢近年来的研究进展,对磷脂酰甘油在光合作用中的作用作了综述,并对磷脂酰甘油的研究问题和前景作了展望。     

二脂酰甘油酰基转移酶2 (DGAT2)基因研究进展

二脂酰甘油酰基转移酶2 (Acyl CoA: Diacylgycerol Acyltransferase 2, DGAT2)是生物体内的一种非常重要的酶, 其主要机制是使二酰甘油加上脂肪酸酰基辅酶A以共价健结合形成三酰甘油。编码该酶的基因有DGAT2和DGAT1。文章综述了DGAT2基因的发现、定位、结构、生物学效应及其遗传多态性与生产性能的关系, 并对其应用前景进行了展望。     

大豆PDAT基因的鉴定、表达与进化分析

PDAT酶催化磷脂和二脂酰甘油反应,生成溶血磷脂和三脂酰甘油,在三脂酰甘油合成过程中扮演着重要作用。为探明大豆PDAT基因的起源与进化关系,本研究利用生物信息学手段剖析了大豆PDAT基因的序列、表达以及进化关系。系统进化关系分析显示,除低等植物外,大豆与其他被子植物PDAT基因被分为4种类群,每一个类群内基因结构与保守基序组成模式均高度保守。基于滑动窗口与位点模型检测选择压力,结果显示大豆PDAT基因均受到功能限制,受控于负选择;但位点-分枝模型发现大豆与其他植物类群Ⅰ和Ⅱ的祖先分支发生了适应性进化。表达分析表明,大豆PDAT基因具有多样化的转录模式,且同源基因对之间存在分化。这些结果为进一步功能分析大豆PDAT基因提供了帮助。     

二酰基甘油酰转移酶与肥胖研究新进展

甘油三酯(TG)是真核细胞中最重要的能量储存形式,尽管它是正常生理所必需,但过量堆积,就会导致肥胖.因此抑制TG的合成可能改善肥胖以及与之相关的症状.脂酰辅酶A:二酰基甘油酰转移酶(DGAT)是以甘油二酯和脂酰辅酶A为底物,催化甘油三酯合成途径的最后一步反应的关键酶.DGAT1基因敲除(Dgat1-/-)小鼠对肥胖有抵抗力,并且增加了对胰岛素和瘦素的敏感性,这种小鼠对饮食诱导的脂肪肝也有抵抗力.此外,DGAT1的缺乏影响脂肪源性因子的表达和分泌,从而调节能量和葡萄糖的代谢.这些研究提示DGAT1有望成为治疗肥胖和2-型糖尿病的新靶点.     

与磷脂酰甘油有关的植物抗冷机理研究进展

磷脂酰甘油是类囊体膜脂中较饱和、相变温度较高的类脂,冷敏感植物中含多较多饱和ＰＧ。捕光叶绿素蛋白复合体与之共阶连接,它的相变与光合冷敏性有关。３－磷磷甘油转酰酶的酰基选择性决定了ＰＧ的饱和度,该基因的克隆与转化的实验结果证实ＰＧ相变与植物光合冷害有关系。     

莱茵衣藻E2泛素结合酶CrUBC23调控油脂积累

【目的】为研究莱茵衣藻(Chlamydomonas reinhardtii)泛素结合酶(ubiquitin-conjugating enzymes,E2)CrUBC23在莱茵衣藻油脂代谢中的作用,为高产油微藻基因工程改良和揭示藻类油脂合成及代谢调控机理奠定基础。【方法】qRT-PCR分析莱茵衣藻在低氮、低磷胁迫下泛素结合酶CrUBC23表达情况;克隆CrUBC23同源基因干涉片段和全长基因,构建RNAi干涉载体和过量表达载体,转化莱茵衣藻并检测生物量和油脂含量;构建CrUBC23-GFP融合表达载体,用农杆菌浸染洋葱表皮细胞进行亚细胞定位。【结果】莱茵衣藻在低氮、低磷胁迫下CrUBC23基因表达量显著增加,增加幅度分别为正常培养的4.98–5.80倍和1.85–5.20倍。RNAi干扰结果显示,转基因藻细胞中性脂含量降低5.5%,总脂含量降低3.16%–17.6%。过量表达结果显示,转基因藻细胞中性脂含量增加8.8%,总脂含量增加4.51%–14.03%。【结论】CrUBC23正向调控莱茵衣藻油脂代谢,该基因定位于细胞核。     

烟草二脂酰甘油酰基转移酶基因(NtDGAT2)的克隆与功能分析

二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT2)是植物储存油脂生物合成过程中的关键酶,对种子储存油脂累积具有重要的生理作用。本文采用电子克隆与实验相结合的方法,从烟草种子cDNA中克隆到DGAT2基因的开放阅读框序列,命名为NtDGAT2(GenBank登录号JX843807),其序列长999bp,编码332个氨基酸。多序列比对和进化分析表明该基因编码蛋白与其他植物DGAT2具有较高相似性和典型的DGAT2结构域。利用Real-time PCR定量表达分析显示Nt-DGAT2在烟草种子、花、茎、叶和根里面都有表达,且在发育中的种子和花的发育过程大量表达。酵母互补实验证实该基因编码蛋白具有DGAT酶活性。     

A lecithin cholesterol acyltransferase-like gene mediates diacylglycerol esterification in yeast

The terminal step in triglyceride biosynthesis is the esterification of diacylglycerol. To study this reaction in the model eukaryote, Saccharomyces cerevisiae, we investigated five candidate genes with sequence conservation to mammalian acyltransferases. Four of these genes are similar to the recently identified acyl-CoA diacylglycerol acyltransferase and, when deleted, resulted in little or no decrease in triglyceride synthesis as measured by incorporation of radiolabeled oleate or glycerol. By contrast, deletion of LRO1, a homolog of human lecithin cholesterol acyltransferase, resulted in a dramatic reduction in triglyceride synthesis, whereas overexpression of LRO1 yielded a significant increase in triglyceride production. In vitro microsomal assays determined that Lro1 mediated the esterification of diacylglycerol using phosphatidylcholine as the acyl donor. The residual triglyceride biosynthesis that persists in the LRO1 deletion strain is mainly acyl-CoA-dependent and mediated by a gene that is structurally distinct from the previously identified mammalian diacylglycerol acyltransferase. These mechanisms may also exist in mammalian cells.     

Proteomic profiling of oil bodies isolated from the unicellular green microalga Chlamydomonas reinhardtii: with focus on proteins involved in lipid metabolism

Oil bodies are sites of energy and carbon storage in many organisms including microalgae. As a step toward deciphering oil accumulation mechanisms in algae, we used proteomics to analyze purified oil bodies from the model microalga Chlamydomonas reinhardtii grown under nitrogen deprivation. Among the 248 proteins (≥ 2 peptides) identified by LC-MS/MS, 33 were putatively involved in the metabolism of lipids (mostly acyl-lipids and sterols). Compared with a recently reported Chlamydomonas oil body proteome, 19 new proteins of lipid metabolism were identified, spanning the key steps of the triacylglycerol synthesis pathway and including a glycerol-3-phosphate acyltransferase (GPAT), a lysophosphatidic acid acyltransferase (LPAT) and a putative phospholipid:diacylglycerol acyltransferase (PDAT). In addition, proteins putatively involved in deacylation/reacylation, sterol synthesis, lipid signaling and lipid trafficking were found to be associated with the oil body fraction. This data set thus provides evidence that Chlamydomonas oil bodies are not only storage compartments but also are dynamic structures likely to be involved in processes such as oil synthesis, degradation and lipid homeostasis. The proteins identified here should provide useful targets for genetic studies aiming at increasing our understanding of triacyglycerol synthesis and the role of oil bodies in microalgal cell functions.     

The DGA1 gene determines a second triglyceride synthetic pathway in yeast.

Diacylglycerol esterification provides an excellent target for the pharmacological reduction of triglyceride accumulation in several human disease states. We have used Saccharomyces cerevisiae as a model system to study this critical component of triglyceride synthesis. Recent studies of an oleaginous fungus, Mortierella ramanniana, identified a new family of enzymes with in vitro acyl-CoA:diacylglycerol acyltransferase activity. We show here that DGA1, the sole member of this gene family in yeast, has a physiological role in triglyceride synthesis. Metabolic labeling of DGA1 deletion strains with triglyceride precursors detected significant reductions in triglyceride synthesis. Triglyceride synthesis was virtually abolished in four different growth conditions when DGA1 was deleted in concert with LRO1, an enzyme that esterifies diacylglycerol from a phospholipid acyl donor. The relative contributions of the two enzymes depended on growth conditions. The residual synthesis was lost when ARE2, encoding an acyl-CoA:sterol acyltransferase, was deleted. In vitro microsomal assays verified that DGA1 and ARE2 mediate acyl-CoA:diacylglycerol acyltransferase reactions. Three enzymes can thus account for diacylglycerol esterification in yeast. Yeast strains deficient in both diacylglycerol and sterol esterification showed only a slight growth defect indicating that neutral lipid synthesis is dispensable under common laboratory conditions.     

Extra virgin olive oil phenols down-regulate lipid synthesis in primary-cultured rat-hepatocytes

Hydroxytyrosol, tyrosol, and oleuropein, the main phenols present in extra virgin olive oil, have been reported to exert several biochemical and pharmacological effects.Here, we investigated the short-term effects of these compounds on lipid synthesis in primary-cultured rat-liver cells. Hydroxytyrosol, tyrosol and oleuropein inhibited both de novo fatty acid and cholesterol syntheses without an effect on cell viability. The inhibitory effect of individual compounds was already evident within 2 h of 25 μM phenol addition to the hepatocytes. The degree of cholesterogenesis reduction was similar for all phenol treatments (−25/30%), while fatty acid synthesis showed the following order of inhibition: hydroxytyrosol (−49%) = oleuropein (−48%) > tyrosol (−30%). A phenol-induced reduction of triglyceride synthesis was also detected.To clarify the lipid-lowering mechanism of these compounds, their influence on the activity of key enzymes of fatty acid biosynthesis (acetyl-CoA carboxylase and fatty acid synthase), triglyceride synthesis (diacylglycerol acyltransferase) and cholesterogenesis (3-hydroxy-3-methyl-glutaryl-CoA reductase) was investigated in situ by using digitonin-permeabilized hepatocytes. Acetyl-CoA carboxylase, diacylglycerol acyltransferase and 3-hydroxy-3-methyl-glutaryl-CoA reductase activities were reduced after 2 h of 25 μM phenol treatment. No change in fatty acid synthase activity was observed. Acetyl-CoA carboxylase inhibition (hydroxytyrosol, −41%, = oleuropein, −38%, > tyrosol, −17%) appears to be mediated by phosphorylation of AMP-activated protein kinase. These findings suggest that a decrease in hepatic lipid synthesis may represent a potential mechanism underlying the reported hypolipidemic effect of phenols of extra virgin olive oil.     

Phospholipid:diacylglycerol acyltransferase‐mediated triacylglycerol biosynthesis is crucial for protection against fatty acid‐induced cell death in growing tissues of Arabidopsis

Phospholipid:diacylglycerol acyltransferase (PDAT) and diacylglycerol:acyl CoA acyltransferase play overlapping roles in triacylglycerol (TAG) assembly in Arabidopsis, and are essential for seed and pollen development, but the functional importance of PDAT in vegetative tissues remains largely unknown. Taking advantage of the Arabidopsis tgd1–1 mutant that accumulates oil in vegetative tissues, we demonstrate here that PDAT1 is crucial for TAG biosynthesis in growing tissues. We show that disruption of PDAT1 in the tgd1–1 mutant background causes serious growth retardation, gametophytic defects and premature cell death in developing leaves. Lipid analysis data indicated that knockout of PDAT1 results in increases in the levels of free fatty acids (FFAs) and diacylglycerol. In vivo ¹⁴C‐acetate labeling experiments showed that, compared with wild‐type, tgd1–1 exhibits a 3.8‐fold higher rate of fatty acid synthesis (FAS), which is unaffected by disruption or over‐expression of PDAT1, indicating a lack of feedback regulation of FAS in tgd1–1. We also show that detached leaves of both pdat1–2 and tgd1–1 pdat1–2 display increased sensitivity to FFA but not to diacylglycerol. Taken together, our results reveal a critical role for PDAT1 in mediating TAG synthesis and thereby protecting against FFA‐induced cell death in fast‐growing tissues of plants.     

Hepatic sn-glycerol-3-phosphate acyltransferases: effect of monoacylglycerol analogs on mitochondrial and microsomal activities

The regulation of cellular diacylglycerol levels may have important consequences for protein kinase C activity. Because monoacylglycerols were said to inhibit the committed step of glycerolipid synthesis, the sn-glycerol-3-P acyltransferase (glycerol-P acyltransferase), we determined (1) whether both the mitochondrial and the microsomal glycerol-P acyltransferase isoenzymes were inhibited by 1- and 2-mono-18:1-glycerols, and their ether and amide analogs and (2) what the mechanism of inhibition was. 1- and 2-mono-18:1-glycerols, their ether and amide analogs, and 1-mono-18:1-glycerol 3-phosphate were all competitive inhibitors of the microsomal glycerol-P acyltransferase activity. The relative Ki values suggested that inhibition was strongest with the radyl group at the sn-1 position and that an oxygen bond is important at the sn-1 position. Although the monoacyl- and monoalkylglycerols were also competitive inhibitors of the mitochondrial glycerol-P acyltransferase, neither of the amide analogs was an inhibitor, suggesting that an oxygen bond is essential at both the sn-1 and sn-2 positions. Because monoradylglycerols inhibit several enzyme activities that contribute to the biosynthesis or the metabolism of diacylglycerol, these inhibitors may function within cells in part to regulate cellular diacylglycerol levels.     

Imaging of lipid biosynthesis: how a neutral lipid enters lipid droplets

The biosynthesis and storage of triglyceride (TG) is an important cellular process conserved from yeast to man. Most mammalian cells accumulate TG in lipid droplets, most prominent in adipocytes, which are specialized to store large amounts of the TG over long periods. In this study, we followed TG biosynthesis and targeting by fluorescence imaging in living 3T3-L1 adipocytes and COS7 fibroblasts. Key findings were (i) not only TG but also its direct metabolic precursor diacylglycerol, DG, accumulates on lipid droplets; (ii) the essential enzyme diacylglycerol acyltransferase 2 associates specifically with lipid droplets where it catalyzes the conversion of DG to TG and (iii) individual lipid droplets within one cell acquire TG at very different rates, suggesting unequal access to the biosynthetic machinery. We conclude that at least part of TG biosynthesis takes place in the immediate vicinity of lipid droplets. In vitro assays on purified lipid droplets show that this fraction of the biosynthetic TG is directly inserted into the growing droplet.     

Soybean phospholipid dependent reductions in triacylglycerol concentration and synthesis in the liver of fasted-refed rats.

The effect of dietary soybean phospholipid on the activities of hepatic triacylglycerol-synthesizing enzymes was compared with soybean oil in fasted-refed rats. Soybean oil at the dietary level corresponding to 20% but not at 5% fatty acid level (21.2 and 5.3% on weight bases, respectively) significantly decreased liver microsomal diacylglycerol acyltransferase activities measured with the endogenous diacylglycerol substrate. Dietary soybean phospholipid even at the dietary level corresponding to 2% fatty acids (3.4% on weight base) significantly decreased the acyltransferase activities measured with endogenous substrate. The dietary phospholipid further decreased the parameter as the dietary level increased, and at the 5% fatty acid level, it was lower than that obtained with soybean oil at 20% fatty acid level. Soybean oil and phospholipid decreased the diacylglycerol acyltransferase activities measured with the saturating concentration of exogenous dioleoylglycerol substrate only when the activities were expressed in terms of total activity (mumol/min per liver) but to much lesser extents. Dietary phospholipid compared to the oil profoundly decreased not only hepatic triacylglycerol but also microsomal diacylglycerol levels. It was indicated that the availability of microsomal diacylglycerol as the substrate for diacylglycerol transferase is the critical determinant in regulating hepatic triacylglycerol synthesis and concentration in this experimental situation. Alterations in the activities of microsomal glycerol 3-phosphate acyltransferase and of the enzymes in fatty acid synthesis could account for the phospholipid-dependent decrease in the microsomal concentration of this intermediate in triacylglycerol synthesis.     

Stability of diacylglycerol acyltransferase in dehydrated bovine muscle tissue

Meaningful estimates of diacylglycerol acyltransferase (EC 2.3.1.20) activity in different tissue samples require effective, unbiased methods of sample storage. Samples of the pars costalis diaphragmatis muscle (skirt muscle of the diaphragm) were obtained from 18- to 20-month-old cattle and assayed for microsomal protein content and diacylglycerol acyltransferase activity after having been stored under various conditions as dissected tissue or microsomes prepared from dissected tissue. There was relative enrichment of diacylglycerol acyltransferase specific activity (p<0.05) when samples prepared from the pars costalis diaphragmatis muscle were dehydrated and stored for 2 weeks, as compared to the control condition (in which the microsome fraction was prepared from fresh pars costalis diaphragmatis muscle and assayed immediately). The results suggested that dehydration was an effective method of storage for bovine muscle samples destined for estimation of the microsomal diacylglycerol acyltransferase activity. The dehydration approach for preparing samples for analysis of diacylglycerol acyltransferase activity might also prove useful to investigators who are interested in obtaining reliable estimates of the activity of other enzymes in tissue samples.