莱茵衣藻磷脂二脂酰甘油酰基转移酶3在三酰甘油合成中的功能研究

为研究磷脂二脂酰甘油酰基转移酶（PDAT）在三酰甘油合成中的功能,克隆了莱茵衣藻（Chlamydomonas reinhardtii） PDAT同源基因CrPDAT3干涉片段,通过构建CrPDAT3 RNAi 干涉载体并转化莱茵衣藻,对CrPDAT3基因有效沉默,结果显示转基因藻株生长减缓,油脂含量下降14.65%-45.15%,说明CrPDAT3对油脂合成起到重要的作用。研究结果对于该基因应用于微藻油脂的遗传改良将起到重要作用。       

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。     

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。     

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。     

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。     

高山被孢霉中二酰甘油酰基转移酶2同源基因的克隆、表达和活性分析

[背景] 高山被孢霉（Mortierella alpina）是一种可积累大量花生四烯酸（Arachidonic Acid,AA）的产油丝状真菌,其所产脂肪酸主要被组装到甘油骨架上以三酰甘油（Triacylglycerol,TAG）形式存在。二酰甘油酰基转移酶（Diacylglycerol Acyltransferase,DGAT）是TAG生物合成途径的关键酶,对于高山被孢霉TAG的生产具有重要意义。[目的] 通过探究高山被孢霉DGAT2在TAG生物合成方面的功能特点,以期为提高产油真菌的TAG产量及改善TAG的脂肪酸组成提供参考。[方法] 利用序列比对在高山被孢霉ATCC32222基因组中筛选出2个编码DGAT2的候选基因MaDGAT2A/2B,在酿酒酵母（Saccharomyces cerevisiae）中异源表达后进行功能分析,并在外源添加AA条件下通过检测TAG产量进一步分析MaDGAT2A/2B的活性,最后在高山被孢霉中同源过表达MaDGAT2A/2B,通过检测重组菌总脂肪酸产量及组分以分析MaDGAT2A/2B的体内活性。[结果] MaDGAT2A在S. cerevisiae中异源表达时,重组酵母菌TAG的产量达到细胞干重的3.06%,为对照组的4.91倍;而MaDGAT2B未明显提高重组酵母菌TAG的产量。在外源添加AA时,MaDGAT2A/2B均可显著促进重组酵母菌中TAG合成,表达MaDGAT2A的重组酵母菌TAG含量为对照组的3.67倍,表达MaDGAT2B的重组酵母菌TAG含量为对照组的2.61倍。MaDGAT2A/2B在高山被孢霉中过表达对其总脂肪酸产量无显著影响,但可显著提高总脂肪酸中AA的含量,AA占总脂肪酸比例最高达到39.15%,相比对照组提高16.14%。[结论] MaDGAT2A/2B可以参与TAG的生物合成,表明2个候选基因编码的蛋白具有DGAT活性,并且可提高高山被孢霉脂肪酸中AA的含量,对于改善产油真菌的脂肪酸组成从而提高其应用价值具有重要意义。     

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。     

赖氨酸酰化酶的重组表达及其催化合成ε-月桂酰-L-赖氨酸

来源于链霉菌的赖氨酸酰化酶Sm-ELA能催化赖氨酸和月桂酸在水相中合成月桂酰赖氨酸,避免了采用化学合成法所必需的高温和有机溶剂条件,是一种节能、环境友好的替代方法。构建了过表达链霉菌赖氨酸酰化酶基因的重组质粒pET28a-SmELA和pTrcOmpXK₁₂₂SmELA,分别实现了该酶在大肠杆菌胞内和细胞表面的活性表达。比较两种不同表达方式的效果后,将重组酶应用于催化合成月桂酰赖氨酸的反应中,结果显示,在赖氨酸浓度为50 mmol/L,月桂酸浓度为10 mmol/L时,反应24 h,月桂酸转化率最高达到31.1%。     

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.     

Metabolic engineering of hydroxy fatty acid production in plants: RcDGAT2 drives dramatic increases in ricinoleate levels in seed oil

SUMMARY: A central goal of green chemistry is to produce industrially useful fatty acids in oilseed crops. Although genes encoding suitable fatty acid-modifying enzymes are available from many wild species, progress has been limited because the expression of these genes in transgenic plants produces low yields of the desired products. For example, Ricinus communis fatty acid hydroxylase 12 (FAH12) produces a maximum of only 17% hydroxy fatty acids (HFAs) when expressed in Arabidopsis. cDNA clones encoding R. communis enzymes for additional steps in the seed oil biosynthetic pathway were identified. Expression of these cDNAs in FAH12 transgenic plants revealed that the R. communis type-2 acyl-coenzyme A:diacylglycerol acyltransferase (RcDGAT2) could increase HFAs from 17% to nearly 30%. Detailed comparisons of seed neutral lipids from the single- and double-transgenic lines indicated that RcDGAT2 substantially modified the triacylglycerol (TAG) pool, with significant increases in most of the major TAG species observed in native castor bean oil. These data suggest that RcDGAT2 prefers acyl-coenzyme A and diacylglycerol substrates containing HFAs, and biochemical analyses of RcDGAT2 expressed in yeast cells confirmed a strong preference for HFA-containing diacylglycerol substrates. Our results demonstrate that pathway engineering approaches can be used successfully to increase the yields of industrial feedstocks in plants, and that members of the DGAT2 gene family probably play a key role in this process.     

GPAT3 and GPAT4 are regulated by insulin-stimulated phosphorylation and play distinct roles in adipogenesis

Acyl-CoA:glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first step during de novo synthesis of glycerolipids. Mammals have at least four GPAT isoforms. Here we report the further characterization of the two recently identified microsomal GPAT3 and GPAT4. Both enzymes are highly expressed in adipose tissues. However, while GPAT3 is highly (∼60-fold) induced during adipocyte differentiation, GPAT4 induction is only modest (∼5-fold), leading to a lower abundance of GPAT4 mRNA in adipocytes. While overexpression of GPAT3 and GPAT4 in either insect or mammalian cells results in a comparable increase of GPAT activity, shRNA-mediated knockdown of GPAT3, but not GPAT4, in 3T3-L1 adipocytes led to a significant decrease in GPAT activity, a profound inhibition of lipid accumulation, and a lack of expression of several adipogenic markers during adipocyte differentiation. These data suggest that GPAT3 may encode the major GPAT isoform in adipocytes and play an important role in adipogenesis. Furthermore, we have shown that both GPAT3 and GPAT4 are phosphorylated by insulin at Ser and Thr residues, leading to increased GPAT activity that is sensitive to wortmannin. Our results reveal a link between the lipogenic effects of insulin and microsomal GPAT3 and GPAT4, implying their importance in glycerolipid biosynthesis.     

Solid-state NMR characterization of triacylglycerol and polysaccharides in coffee beans

It is important to understand the structural characteristics of triacylglycerol (TAG), polysaccharides and trace elements in coffee beans, so that residues can be reutilized in applications including biodiesel oils. Here, we performed ¹H and ¹³C solid-state NMR measurements on Indonesian green beans, roasted beans, and spent coffee grounds (SCGs). In the NMR spectra, there were liquid-like TAG containing linoleic acids based on observed signals of -CH=CH-CH₂-CH=CH- group in an acyl chain, which play a role in decreasing TAG’s melting point. We found TAG was still abundant in the SCGs from NMR spectra. After lipids were removed from SCGs, the intensity of the TAG signal decreased considerably, with approximately 64% of the TAG was successfully extracted. We described the chemical structure of TAG in coffee beans and demonstrated that it is possible quantify the amount of extracted TAG using solid-state NMR.     

Some properties of diacylglycerol acyltransferase in a particulate fraction from maturing safflower seeds

The activity of diacylglycerol acyltransferase of a subcellular particulate fraction from maturing safflower seeds was remarkably stimulated by the addition of 1, 2-diacylglycerols which were previously emulsified in a gelatin solution by sonication. Metal ions were inhibitory to the reaction. Deoxycholate and diisopropyl fluorophosphate were the most effective inhibitors. Sulfhydryl groups seemed to be of limited significance in the enzyme. Both 1, 2-dioleoyl-sn-glycerol and 2, 3-dioleoyl-sn-glycerol were good substrates of diacylglycerol acyltransferase, but the 1, 3-isomer did not serve as an acyl acceptor. The enzyme showed broad specificity for synthetic rac-1, 2-diacylglycerols containing various fatty acids. However, rac-1, 2-diacetylglycerol and rac-1, 2-dibutyrylglycerol, which are soluble in water, were ineffective. The enzyme exhibited no significant specificity for saturated and unsaturated fatty acyl-CoA thioesters as acyl donors. This suggests that the fatty acid composition at the 3-position of the glycerol molecule of safflower triacylglycerols may depend on the composition of the endogenous acyl-CoA pool.     

Partial purification and specificity of triacylglycerol: Sterol acyltransferase from Sinapis alba

Triacylglycerol: sterol acyltransferase is present in roots of Sinapis alba seedlings. The enzyme is located predominantly in the cell membrane structures sedimenting at 300–16 000 g but can be solubilized by acetone treatment and buffer extraction. During gel filtration on Sephadex G-100 the acyltransferase activity was separated into two peaks corresponding to MW 1.8 × 10¹⁴ and MW ? 10⁵, respectively. A number of natural 3β-hydroxysterols can be esterified by the solubilized acyltransferase. The rate of esterification is much higher for sterols containing a planar ring system. The number and position of double bonds, as well as the structure of the side chain at C- 17 of the sterol molecule, are of secondary importance. Triacylglycerols containing fatty acids C, C₆-C₂₂ can be utilized as acyl donors. Among triacylglycerols containing saturated fatty acids, tripalmitoylglycerol (C_16:0) is the best acyl donor. For triacylglycerols containing C₁₈-fatty acids the following sequence was observed: trioleoylglycerol (C_18:1) > trilinoleoylglycerol (C_18:2) > trilinolenoylglycerol (C_18:3) > tristearoylglycerol (C_18:0).     

Enhancement of extraplastidic oil synthesis in Chlamydomonas reinhardtii using a type‐2 diacylglycerol acyltransferase with a phosphorus starvation–inducible promoter

When cultivated under stress conditions, many plants and algae accumulate oil. The unicellular green microalga Chlamydomonas reinhardtii accumulates neutral lipids (triacylglycerols; TAGs) during nutrient stress conditions. Temporal changes in TAG levels in nitrogen (N)‐ and phosphorus (P)‐starved cells were examined to compare the effects of nutrient depletion on TAG accumulation in C. reinhardtii. TAG accumulation and fatty acid composition were substantially changed depending on the cultivation stage before nutrient starvation. Profiles of TAG accumulation also differed between N and P starvation. Logarithmic‐growth‐phase cells diluted into fresh medium showed substantial TAG accumulation with both N and P deprivation. N deprivation induced formation of oil droplets concomitant with the breakdown of thylakoid membranes. In contrast, P deprivation substantially induced accumulation of oil droplets in the cytosol and maintaining thylakoid membranes. As a consequence, P limitation accumulated more TAG both per cell and per culture medium under these conditions. To enhance oil accumulation under P deprivation, we constructed a P deprivation‐dependent overexpressor of a Chlamydomonas type‐2 diacylglycerol acyl‐CoA acyltransferase (DGTT4) using a sulphoquinovosyldiacylglycerol 2 (SQD2) promoter, which was up‐regulated during P starvation. The transformant strongly enhanced TAG accumulation with a slight increase in 18 : 1 content, which is a preferred substrate of DGTT4. These results demonstrated enhanced TAG accumulation using a P starvation–inducible promoter.     

Functional characterization of two diacylglycerol acyltransferase 1 genes in Mortierella alpina

Diacylglycerol acyltransferase (DGAT) is a crucial enzyme in the triacylglycerol (TAG) biosynthesis pathway. The oleaginous fungus Mortierella alpina can accumulate large amounts of arachidonic acid (ARA, C20:4) in the form of TAG. Therefore, it is important to study the functional characteristics of its DGAT. Two putative genes MaDGAT1A/1B encoding DGAT1 were identified in M. alpina ATCC 32222 genome by sequence alignment. Sequence alignment with identified DGAT1 homologs showed that MaDGAT1A/1B contain seven conserved motifs that are characteristic of the DGAT1 subfamily. Conserved domain analysis showed that both MaDGAT1A and MaDGAT1B belong to the Membrane-bound O-acyltransferases superfamily. The transforming with MaDGAT1A/1B genes could increase the accumulation of TAG in Saccharomyces cerevisiae to 4·47 and 7·48% of dry cell weight, which was 7·3-fold and 12·3-fold of the control group, respectively, but has no effect on the proportion of fatty acids in TAG. This study showed that MaDGAT1A/1B could effectively promote the accumulation of TAG and therefore may be used in metabolic engineering aimed to increase TAG production of oleaginous fungi.     

蒺藜苜蓿DGAT1基因的克隆和功能鉴定

该研究采用RT-PCR与电子克隆的方法,从蒺藜苜蓿cDNA中克隆得到2个编码二脂酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)的基因MtDGAT1-1和MtDGAT1-2。MtDGAT1-1长1 620bp,编码539个氨基酸;MtDGAT1-2长1 524bp,编码507个氨基酸。多序列比对显示,MtDGAT1-1和MtDGAT1-2编码蛋白具有典型的植物DGAT1结构域。表达分析显示,MtDGAT1-1和MtDGAT1-2在根、茎、叶、花、种子中都有表达,在种子发育中高表达,且MtDGAT1-1于种子发育的中前期高表达,而MtDGAT1-2于种子发育的中后期高表达。酵母互补实验证实,MtDGAT1-2编码蛋白具有DGAT酶活性,能够恢复H1246的TAG合成和油体形成;而MtDGAT1-1编码蛋白不能恢复H1246的TAG合成和油体形成。