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.     

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。     

DGAT相关基因研究进展

DGAT是一种甘油酰基转移酶（Diacylgycerol Acyltransferase,DGAT）,该酶与脂肪代谢、脂类在组织中的沉积有很大关系,它的主要作用机制是使二酰甘油加上脂肪酸酰基形成三酰甘油。编码该酶的基因有DGAT1和GAAT2,前者属于ACAT基因家族,后者属于MGAT1基因家族。本文综述了动物DGAT相关基因定位、结构、生物学效应及其多态性与生产性能的关系。     

在基因家族背景下对四种植物中DGAT2的鉴定和序列分析

二酰甘油酰基转移酶2(Diacylglycerol O-acyltransferase 2,DGAT2)是植物中三羧酸甘油酯(TAG)合成途径的限速酶,其编码基因属于酰基转移酶基因超家族。本研究依托植物全基因组数据库Phytozome,通过BLAST搜索获得了蓖麻(Ricinus communis L.)、拟南芥(Arabidopsis thaliana Heynh.)、毛果杨(Populus tricho-carpa Torr.A.Gray.)和木薯(Manihot esculenta Crantz.)4种双子叶植物酰基转移酶基因超家族所编码的73条多肽序列,并从中鉴定出5条DGAT2序列。理化性质和跨膜结构域分析表明,5条DGAT2序列均为疏水性跨膜蛋白,其中木薯DGAT2为一次跨膜蛋白且在叶绿体膜中大量分布,这与其他植物的DGAT2序列存在差异;木薯DGAT2蛋白在进化过程中发生了功能分化且可能与木薯的抗逆作用有关。     

脑特异脂肪酸活化酶的克隆

东北大学基因实验室助教授山本德男小组克隆了特异存在于小鼠脑内、控制脂肪酸代谢和脂质生物合成的脂肪酸活化酶的cDNA.大脑干重的48%以上由脂质构成,是弄清脑脂质合成和代谢的最关键的物质. 脂肪酸活化酶(酰基CoA合成酶)是由脂肪酸和ATP、辅酶A(CoA)合成脂肪酸CoA的酶.脂肪酸被活化,转化成脂肪酸CoA之后,才能作为脂肪酸β氧化反应的能量以及乙酰基CoA的合成反应和中性脂肪、磷脂质、胆固醇酯等合成脂质的底物.由此看来,脂肪酸活化酶是一种重要的特异性酶.     

哺乳动物DGAT基因及其生物学功能研究进展

二酰基甘油酰基转移酶(DGAT, EC2.3.1.20)是一种微粒体酶, 与脂肪代谢、脂类在组织中的沉积有很大关系, 它的主要作用机制是使二酰甘油加上脂肪酸酰基形成三酰甘油。DGAT在细胞甘油代谢中起根本性的作用, 并在高等真核生物甘油三酯代谢途径如肠脂肪吸收、脂蛋白集合、脂肪形成和泌乳中发挥着重要的功能, 提示DGAT不仅是调控甘油三酯与脂肪酸之间的关键因子, 而且可能在动物脂肪沉积中起着关键的调控作用。     

食用植物油脂的代谢工程

植物种子油可提供人类营养所需的多种脂肪酸,也是工业用油的原料之一。文章结合我们对植物种子发育、脂肪酸生物合成途径和大豆油脂遗传改良的研究,重点论述参与脂肪酸合成及其调控的一些关键酶的基因、代谢工程改良植物油脂营养价值的技术策略及其研究进展,分析目前应用油料作物种子作为“生物反应器”规模化生产有重要营养价值和特殊用途的脂肪酸的问题及技术“瓶颈”,讨论未来植物脂肪酸代谢工程主攻方向以及在培育可再生资源和推动人类社会及经济可持续发展中的应用前景。     

花生DGAT3的生物信息学分析

三酰甘油(TAG)是真核细胞最重要的能量储存形式,而酰基-Co A:二酰甘油酰基转移酶(DGAT)是生物体内三酰甘油合成过程中的关键酶。DGAT分为DGAT1、DGAT2和DGAT3三个家族,其中DGAT1和DGAT2均为膜结合蛋白,而DGAT3为细胞质游离蛋白。花生DGAT3是第一种被发现的DGAT3家族成员,该酶的分子量为41±1.0 k D。通过生物信息学研究花生DGAT3的特性,对于进一步研究花生三酰甘油的合成过程以及通过生物工程提高花生产油量都有重要意义。     

同步抑制FAD2与FatB基因提高烟草种子油酸组分含量的研究

该研究以烟草品系NC89的无菌苗叶片为受体材料,采用前期构建的能同步抑制种子中FAD2(Δ12-油酸去饱和酶基因)与FatB(酰基转移酶基因)表达的RNAi载体,通过农杆菌介导转化获得了转基因烟草植株,分析转基因植株种子中的脂肪酸组分。结果显示:与对照相比,转基因植株种子中FAD2和FatB基因的表达水平分别降低了23%和11%;转基因植株种子的脂肪酸组分中,饱和脂肪酸棕榈酸和硬脂酸平均含量分别为8.02%和4.45%,多不饱和脂肪酸亚油酸平均含量为76.82%,较对照分别降低了2.91%、9.92%和3.47%;而转基因植株种子中单不饱和脂肪酸油酸含量高达7.48%,比对照提高46.38%。研究表明,同步抑制FAD2和FatB基因的表达能够显著提高烟草种子中油酸组分的含量,为进一步改良油料作物品质奠定了基础。     

沙棘种子高积累碳十八不饱和脂肪酸的多基因协同调控机制

为探讨沙棘种子油高积累碳十八不饱和脂肪酸的多基因协同作用机制,以近缘低油沙棘品系‘绥棘1号’和高油品系‘新俄3号’6个不同发育期的种子为材料,利用气相色谱飞行时间质谱法测定种子油脂肪酸组份,采用qRT-PCR方法分析不饱和脂肪酸合成积累相关基因KAR、FATB、Δ9 D、KASⅡ、SAD、FAD2、FAD3、FAD7和FAD8的表达模式,验证多基因表达对碳十八不饱和脂肪酸积累的影响。结果表明:(1)‘绥棘1号’和‘新俄3号’种子油均高积累碳十八不饱和脂肪酸,分别占总脂肪酸的87.71%和88.68%;种子发育期间,油酸相对含量一直呈上升趋势,亚油酸相对含量短时下降后上升趋稳,而亚麻酸相对含量则呈先上升后下降趋稳。(2)FATB基因下调表达协同Δ9 D基因低表达,使C16∶0-ACP转化为棕榈酸和棕榈油酸的代谢减弱,而KAR和KASⅡ基因的相对上调表达,促进了硬脂酸合成,为碳十八不饱和脂肪酸的合成积累了较多前体。(3)SAD基因的持续高表达催化硬脂酸去饱和为油酸,且持续上升的SAD/FATB基因表达比直接提高了脂肪酸的去饱和速率;FAD2、FAD3、FAD7和FAD8基因在亚油酸和亚麻酸快速合成期间同时出现明显的表达量峰值,进而促进油酸逐步去饱和为亚油酸和亚麻酸。研究认为,沙棘种子油高积累碳十八不饱和脂肪酸源于FATB和Δ9 D基因的低表达及KAR、KASⅡ、SAD、FAD2、FAD3、FAD7和FAD8基因的协同高表达,本研究结果为进一步理解种子油中碳十八不饱和脂肪酸的合成积累提供了理论依据,对改良植物油脂的不同脂肪酸比具有重要意义。     

Engineering the production of conjugated fatty acids in Arabidopsis thaliana leaves

The seeds of many nondomesticated plant species synthesize oils containing high amounts of a single unusual fatty acid, many of which have potential usage in industry. Despite the identification of enzymes for unusual oxidized fatty acid synthesis, the production of these fatty acids in engineered seeds remains low and is often hampered by their inefficient exclusion from phospholipids. Recent studies have established the feasibility of increasing triacylglycerol content in plant leaves, which provides a novel approach for increasing energy density of biomass crops. Here, we determined whether the fatty acid composition of leaf oil could be engineered to accumulate unusual fatty acids. Eleostearic acid (ESA) is a conjugated fatty acid produced in seeds of the tung tree (Vernicia fordii) and has both industrial and nutritional end‐uses. Arabidopsis thaliana lines with elevated leaf oil were first generated by transforming wild‐type, cgi‐58 or pxa1 mutants (the latter two of which contain mutations disrupting fatty acid breakdown) with the diacylglycerol acyltransferases (DGAT1 or DGAT2) and/or oleosin genes from tung. High‐leaf‐oil plant lines were then transformed with tung FADX, which encodes the fatty acid desaturase/conjugase responsible for ESA synthesis. Analysis of lipids in leaves revealed that ESA was efficiently excluded from phospholipids, and co‐expression of tung FADX and DGAT2 promoted a synergistic increase in leaf oil content and ESA accumulation. Taken together, these results provide a new approach for increasing leaf oil content that is coupled with accumulation of unusual fatty acids. Implications for production of biofuels, bioproducts, and plant–pest interactions are discussed.     

Industrial oils from transgenic plants

Unusual fatty acids that have useful industrial properties occur widely in the seed oils of many non-agronomic plant species. Researchers are attempting to use biotechnology to produce high levels of these fatty acids in the seeds of existing crop plants. cDNAs for a wide variety of unusual fatty acid biosynthetic enzymes have been identified, particularly through the use of expressed sequence tags. However, it has not yet been possible to use these cDNAs to produce large amounts of unusual fatty acids in seeds of transgenic plants. This difficulty points to the need for a greater understanding of fatty acid metabolism in oilseeds.     

Engineering industrial fatty acids in oilseeds

More than 300 types of modified fatty acids (mFA) are produced in triacylglycerols (TAG) by various plant species, with many of these unusual structures rendering unique physical and chemical properties that are desirable for a variety of bio-based industrial uses. Attempts to produce these mFA in crop species have thus far failed to reach the desired levels of production and highlighted the need to better understand how fatty acids are synthesized and accumulated in seed oils. In this review we discuss how some of the progress made in recent years, such as the improved TAG synthesis model to include acyl editing and new enzymes such as PDCT, may be utilized to achieve the goal of effectively modifying plant oils for industrial uses. Co-expressing several key enzymes may circumvent the bottlenecks for the accumulation of mFA in TAG through efficient removal of mFA from phosphatidylcholine. Other approaches include the prevention of feedback inhibition of fatty acid synthesis and improving primary enzyme activity in host transgenic plants. In addition, genomic approaches are providing unprecedented power to discover more factors that may facilitate engineering mFA in oilseeds. Based on the results of the last 20 years, creating a high mFA accumulating plant will not be done by simply inserting one or two genes; it is necessary to stack genes encoding enzymes with favorable kinetic activity or specificity along with additional complementary transgenes in optimized plant backgrounds to produce industrial fatty acids at desirable levels. Finally, we discuss the potential of Camelina as an industrial oilseed platform.     

Engineering oilseeds for sustainable production of industrial and nutritional feedstocks: solving bottlenecks in fatty acid flux

Oilseeds provide a unique platform for the production of high-value fatty acids that can replace non-sustainable petroleum and oceanic sources of specialty chemicals and aquaculture feed. However, recent efforts to engineer the seeds of crop and model plant species to produce new types of fatty acids, including hydroxy and conjugated fatty acids for industrial uses and long-chain omega-3 polyunsaturated fatty acids for farmed fish feed, have met with only modest success. The collective results from these studies point to metabolic 'bottlenecks' in the engineered plant seeds that substantially limit the efficient or selective flux of unusual fatty acids between different substrate pools and ultimately into storage triacylglycerol. Evidence is emerging that diacylglycerol acyltransferase 2, which catalyzes the final step in triacylglycerol assembly, is an important contributor to the synthesis of unusual fatty acid-containing oils, and is likely to be a key target for future oilseed metabolic engineering efforts.     

Tung tree DGAT1 and DGAT2 have nonredundant functions in triacylglycerol biosynthesis and are localized to different subdomains of the endoplasmic reticulum

Seeds of the tung tree (Vernicia fordii) produce large quantities of triacylglycerols (TAGs) containing approximately 80% eleostearic acid, an unusual conjugated fatty acid. We present a comparative analysis of the genetic, functional, and cellular properties of tung type 1 and type 2 diacylglycerol acyltransferases (DGAT1 and DGAT2), two unrelated enzymes that catalyze the committed step in TAG biosynthesis. We show that both enzymes are encoded by single genes and that DGAT1 is expressed at similar levels in various organs, whereas DGAT2 is strongly induced in developing seeds at the onset of oil biosynthesis. Expression of DGAT1 and DGAT2 in yeast produced different types and proportions of TAGs containing eleostearic acid, with DGAT2 possessing an enhanced propensity for the synthesis of trieleostearin, the main component of tung oil. Both DGAT1 and DGAT2 are located in distinct, dynamic regions of the endoplasmic reticulum (ER), and surprisingly, these regions do not overlap. Furthermore, although both DGAT1 and DGAT2 contain a similar C-terminal pentapeptide ER retrieval motif, this motif alone is not sufficient for their localization to specific regions of the ER. These data suggest that DGAT1 and DGAT2 have nonredundant functions in plants and that the production of storage oils, including those containing unusual fatty acids, occurs in distinct ER subdomains.     

Metabolic engineering of edible plant oils]

Plant seed oil is the major source of many fatty acids for human nutrition, and also one of industrial feedstocks. Recent advances in understanding of the basic biochemistry of seed oil biosynthesis, coupled with cloning of the genes encoding the enzymes involved in fatty acid modification and oil accumulation, have set the stage for the metabolic engineering of oilseed crops that produce "designer" plant seed oils with the improved nutritional values for human being. In this review we provide an overview of seed oil biosynthesis/regulation and highlight the key enzymatic steps that are targets for gene manipulation. The strategies of metabolic engineering of fatty acids in oilseeds, including overexpression or suppression of genes encoding single or multi-step biosynthetic pathways and assembling the complete pathway for the synthesis of long-chain polyunsaturated fatty acids (e.g. arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid) are described in detail. The current "bottlenecks" in using common oilseeds as "bioreactors" for commercial production of high-value fatty acids are analyzed. It is also discussed that the future research focuses of oilseed metabolic engineering and the prospects in creating renewable sources and promoting the sustainable development of human society and economy.     

Cloning and functional analysis of two type 1 diacylglycerol acyltransferases from Vernonia galamensis

Vernonia galamensis accumulates vernolic acid (cis-12-epoxyoctadeca-cis-9-enoic acid) as the major fatty acid in its seed oil. Such epoxy fatty acids are useful in a number of industrial applications. Successful genetic engineering of commercial oilseed crops to produce high levels of vernolic acid depends on a better understanding of the source plant enzymes for vernolic acid accumulation. Developing V. galamensis seed microsome assays demonstrate that diacylglycerol acyltransferase (DGAT), an enzyme for the final step of triacylglycerol synthesis, has a strong substrate preference for vernolic acid bearing substrates including acyl-CoA and diacylglycerol. There are two classes of DGATs known as DGAT1 and DGAT2. Here we report on the isolation, characterization, and functional analysis of two DGAT1 cDNAs from V. galamensis (VgDGAT1a and VgDGAT1b). VgDGAT1a and VgDGAT1b are expressed in all plant tissues examined with highest expression in developing seeds. Enzymatic assays using isolated microsomes from transformed yeast show that VgDGAT1a and VgDGAT1b have the same DGAT activity levels and substrate specificities. Oleoyl-CoA and sn-1,2-dioleoylglycerol are preferred substrates over vernoloyl-CoA and sn-1,2-divernoloylglycerol. This data indicates that the two VgDGAT1s are functional, but not likely to be responsible for the selective accumulation of vernolic acid in V. galamensis seed oil.     

FAD2类酶在合成工业用脂肪酸中的应用

许多工业用稀有脂肪酸存在于非食用植物种子油中,它们由不同脂肪酸Δ12-去饱和酶（FAD2）催化,在油酸Δ12位引入环氧基、羟基、形成三键或共扼双键。目前已从不同生物中克隆得到一系列FAD2酶基因,并在油料植物中获得成功表达。但总体上看,目标脂肪酸累积量还相对较低,稀有脂肪酸生物合成及其从磷脂酰胆碱（PC）到储存甘油三酯(IAG)的转化机制尚需要进一步阐明。     

Metabolic engineering of fatty acid biosynthesis in plants.

Fatty acids are the most abundant form of reduced carbon chains available from nature and have diverse uses ranging from food to industrial feedstocks. Plants represent a significant renewable source of fatty acids because many species accumulate them in the form of triacylglycerol as major storage components in seeds. With the advent of plant transformation technology, metabolic engineering of oilseed fatty acids has become possible and transgenic plant oils represent some of the first successes in design of modified plant products. Directed gene down-regulation strategies have enabled the specific tailoring of common fatty acids in several oilseed crops. In addition, transfer of novel fatty acid biosynthetic genes from noncommercial plants has allowed the production of novel oil compositions in oilseed crops. These and future endeavors aim to produce seeds higher in oil content as well as new oils that are more stable, are healthier for humans, and can serve as a renewable source of industrial commodities. Large-scale new industrial uses of engineered plant oils are on the horizon but will require a better understanding of factors that limit the accumulation of unusual fatty acid structures in seeds.