利用基因工程改良植物脂肪酸和提高植物含油量的研究进展

植物脂肪酸和三酰甘油具有重要生理功能以及巨大的食用和工业价值,其生物合成途径较为复杂。近年来,植物脂肪酸和三酰甘油合成代谢途径的研究取得了很大进展,在多种植物中克隆了脂肪酸和三酰甘油合成代谢途径中的关键酶基因,同时发现一些转录因子在调控植物脂肪酸含量和组成中具有重要作用,并在此基础上开展了利用基因工程改良植物脂肪酸和提高植物含油量的研究,取得了一定的进展。以下系统介绍了植物脂肪酸和三酰甘油合成代谢途径中的关键酶基因以及在调控植物脂肪酸含量和组成中具有重要作用的转录因子,综述了基因工程研究所取得的成果,并对其应用前景进行了展望。     

植物含油量相关转录因子WRINKLED1的研究进展

植物油具有重要的食用和工业价值,对其需求越来越大。提高作物种子含油量育种一直在进行,与通过单一限速酶的遗传工程法相比,利用转相关转录因子基因改造植物脂肪代谢过程是更好选择之一。介绍植物含油量相关转录因子WRINKLED1的发现、结构、功能、调控、基因工程和聚类分析等,并对其应用前景进行展望。     

基于藜麦转录组的脂肪酸生物合成途径解析

藜麦营养丰富,油脂含量高,脂肪酸组成理想,是油脂提取物的潜在资源。植物油脂主要以三酰甘油的形式储存在作物种子和果实等器官中,其合成受到环境和基因水平的调控,涉及质体、内质网和油体等多个细胞器。该文基于藜麦转录组数据,对藜麦油脂合成相关的脂肪酸生物合成途径基因进行挖掘,并对基因表达模式进行分析。结果表明:在藜麦中,与脂肪酸生物合成相关的基因序列共87条,涉及乙酰CoA羧化酶和β-酮脂酰ACP合成酶等关键酶,其中编码长链酰基辅酶A合成酶基因和β-酮脂酰ACP还原酶数目最多。通过基因表达模式分析发现,与脂肪酸生物合成相关的基因在种子表达中呈现整体上调模式,可能与种子中油脂形成和积累密切相关。对藜麦乙酰CoA羧化酶亚基编码基因进行分析发现,accD基因在不同组织间无差异表达,表明在藜麦中accD编码的β-CT亚基可能不是影响乙酰CoA羧化酶发挥作用的限制因子。藜麦KASⅡ含有保守结构域,与其他组织相比,编码基因QcFb15、QcFb45和QcFb75在种子中均存在上调表达,参与藜麦脂肪酸碳链延伸及油脂形成。对藜麦脂肪酸生物合成途径相关基因的挖掘,为藜麦油脂合成和积累的研究提供了理论基础,对高油脂藜麦品种选育等后续研究也具有重要启示作用。     

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.     

不同海拔地区(南京和拉萨)种植的甘蓝型油菜的种子基因差异表达

分别在南京(海拔8.9 m)和拉萨(海拔3 658 m)2个不同海拔地区种植甘蓝型油菜(Brassica napus)高油品系H105, 该材料含油量在两地分别为(46.04±1.42)%和(53.09±1.35)%。利用拟南芥表达谱基因芯片检测两地种植的H105开花后30天种子基因的表达。以种植在南京的H105为对照, 差异表达分析结果显示有421个差异表达的基因, 其中229个基因表达下调, 192个基因表达上调。这些基因按功能可初步分为代谢相关、运输相关、结合相关、转录相关、结构相关、发育相关、信号转导相关、其它相关及功能未知基因等几大类别。一些与光合成、糖代谢以及脂肪酸合成相关的重要基因, 如叶绿素a-b结合蛋白基因家族、蔗糖合酶、丙酮酸激酶、6-磷酸葡萄糖酸脱氢酶、ATP-柠檬酸裂解酶、柠檬酸合酶、异柠檬酸脱氢酶、脂肪酸去饱和酶(FAD6和FAD7)基因等被鉴定为差异表达。研究结果初步揭示了相关基因的表达变化规律, 为探讨油菜在不同海拔地区含油量差异的分子遗传机理提供了重要信息。     

不同海拔地区(南京和拉萨)种植的甘蓝型油菜的种子基因差异表达

分别在南京(海拔8.9m)和拉萨(海拔3658m)2个不同海拔地区种植甘蓝型油菜(Brassica napus)高油品系H105,该材料含油量在两地分别为(46.04±1.42)%和(53.09±1.35)%。利用拟南芥表达谱基因芯片检测两地种植的H105开花后30天种子基因的表达。以种植在南京的H105为对照,差异表达分析结果显示有421个差异表达的基因,其中229个基因表达下调,192个基因表达上调。这些基因按功能可初步分为代谢相关、运输相关、结合相关、转录相关、结构相关、发育相关、信号转导相关、其它相关及功能未知基因等几大类别。一些与光合成、糖代谢以及脂肪酸合成相关的重要基因,如叶绿素a-b结合蛋白基因家族、蔗糖合酶、丙酮酸激酶、6-磷酸葡萄糖酸脱氢酶、ATP-柠檬酸裂解酶、柠檬酸合酶、异柠檬酸脱氢酶、脂肪酸去饱和酶(FAD6和FAD7)基因等被鉴定为差异表达。研究结果初步揭示了相关基因的表达变化规律,为探讨油菜在不同海拔地区含油量差异的分子遗传机理提供了重要信息。     

甘蓝型油菜中一类AP2/ERF转录因子的克隆和生物信息学分析

AP2/ERF转录因子家族是植物中广泛存在的一类转录因子,AP2/ERF这类转录因子主要参与植物的细胞周期、生长发育以及生物和非生物胁迫相关基因的表达调控。由于拟南芥和油菜同属于芸薹属,具有相似的基因信息,利用油菜UniGene数据库,以拟南芥ERF转录因子保守序列为探针,通过电子克隆从一个UniGene Cluster中分离得到三个同类的AP2/ERF转录因子,从cDNA序列、氨基酸序列的相似性、组成成分、理化性质、疏水性/亲水性分析、序列比对、进化树、功能域、二级结构、三级结构、无序化特性进行了预测和较为全面的分析。结果显示油菜来源的BnaERF1、BnaERF2和BnaERF3属于AP2/ERF转录因子的B-2亚族,是亲水性蛋白,在蛋白质的三级结构上与AtERF1相似。蛋白质无序化分析发现,油菜BnaERF1、BnaERF2和BnaERF3无序化程度小于拟南芥AtERF1。设计引物通过PCR和RT-PCR方法分别从甘蓝型双低油菜沪油15幼苗的DNA和cDNA中扩增了上述基因,初步分析BnaERF2没有内含子,BnaERF1和BnaERF3有内含子。另外,通过EST丰度分析显示,该类转录因子的表达最高峰在种子中,其次为花,在芽、茎和分生组织中没有检测出表达的存在。     

小桐子甘油-3-磷酸酰基转移酶（JcGPAT）cDNA的克隆与序列分析

甘油-3-磷酸酰基转移酶是植物生物合成储存油脂过程中的关键酶,对油料作物种子含油量具有重要的限制作用。本研究以植物甘油-3-磷酸酰基转移酶同源基因的保守区域序列为基础,设计简并引物,结合RACE技术,从能源植物小桐子种子中克隆获得JcGPAT基因的cDNA全长序列（GenBank登录号HQ395225）。JcGPAT cDNA核苷酸序列长度为1672bp,开放阅读框为1125bp,编码375个氨基酸。该基因具有明显的GPAT基因结构域,其编码的氨基酸序列与油桐、蓖麻等植物具有很高的同源性。RT-PCR表达分析表明,该基因在小桐子发育的种子、叶、根尖等多个组织表达。     

油菜含油量相关基因Gpat、Lpat在不同肥密条件下表达规律研究

油菜是我国第一大自产油料作物,具有重要的食用、饲用等价值。提高油菜籽含油量一直是育种家们不懈追求的目标之一,不同的栽培措施对油菜含油量有较大影响,为了研究不同施肥量、种植密度对油菜中控制含油量相关基因Gpat、Lpat表达量的影响,本研究采用qRT-PCR测定不同时期、不同肥密条件下Gpat、Lpat基因的表达量,并对其表达情况进行了分析。研究得知:Gpat、Lpat表达量变化规律是相同的,即:随着氮肥的增加而降低,随着密度的增加而升高,随着硼肥的增加而先升高后下降。此外,本研究在A1B3C1、A1B2C2及A2B3C2处理下可同时促进Gpat与Lpat的表达,是比较理想的肥密条件,可为提高油菜的含油量提供参考。     

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。     

Enzyme activities of intestinal triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.)

The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)) and DGAT activity (4 nmol [1-(14)C]palmitoyl-CoA incorporated min(-1) mg protein(-1)), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[(14)C]choline incorporated min(-1) mg protein(-1)). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet.     

植物脂肪酸代谢途径遗传调控的研究进展

目前,利用传统育种方法改良油料作物脂肪酸组分已取得巨大成功,通过有性杂交、X-射线或EMS处理等方法都可用来修饰存在于油菜中脂肪酸的性质。国外已培育出高棕榈酸、高或低亚油酸、高油酸和无芥酸的油菜品种。但由于油料作物基因池(Gene Pool)的局限性使得育种学家不得不寻找其他种质资源。随着基因克隆和遗传转化技术的进步,通过基因工程改良油料作物品质已成可能。本文主要介绍了植物脂肪酸的代谢途径以及通过操纵TAG的生物合成来改变油的成分等研究,其中主要包括脂肪酸链长度的改良、饱和度改良、增加脂肪酸含量以及新的不饱和脂肪酸的改良等方面。不久的将来,转基因油料作物中将会产生更有价值的脂肪酸造福于人类。     

Effect of rapeseed oil and dietary n-3 fatty acids on triacylglycerol synthesis and secretion in Atlantic salmon hepatocytes

Fish oil (FO) has traditionally been used as the dominating lipid component in fish feed. However, FO is a limited resource and the price varies considerably, which has led to an interest in using alternative oils, such as vegetable oils (VOs), in fish diets. It is far from clear how these VOs affect liver lipid secretion and fish health. The polyunsaturated fatty acids (PUFAs), eicosapentanoic acid (EPA) and docosahexanioc acid (DHA), reduce the secretion of lipoproteins rich in triacylglycerols (TAGs) in Atlantic salmon, as they do in humans. The mechanism by which n-3 fatty acids (FAs) in the diet reduce TAG secretion is not known. We have therefore investigated the effects of rapeseed oil (RO) and n-3 rich diets on the accumulation and secretion of (3)H-glycerolipids by salmon hepatocytes. Salmon, of approximately 90 g were fed for 17 weeks on one of four diets supplemented with either 13.5% FO, RO, EPA-enriched oil or DHA-enriched oil until a final average weight of 310 g. Our results show that the dietary FA composition markedly influences the endogenous FA composition and lipid content of the hepatocytes. The intracellular lipid level in hepatocytes from fish fed RO diet and DHA diet were higher, and the expressions of the genes for microsomal transfer protein (MTP) and apolipoprotein A1 (Apo A1) were lower, than those in fish fed the two other diets. Secretion of hepatocyte glycerolipids was lower in fish fed the EPA diet and DHA diet than it was in fish fed the RO diet. Our results indicate that EPA and DHA possess different hypolipidemic properties. Both EPA and DHA inhibit TAG synthesis and secretion, but only EPA induces mitochondrial proliferation and reduce intracellular lipid. Expression of the gene for peroxisome proliferator-activated receptor alpha (PPARalpha) was higher in the DHA dietary group than it was in the other groups.     

油菜油脂研究进展

油菜（Brassica napus）是世界范围内重要的油料作物,是植物油脂的第三大来源,其种植面积和总产量在油料作物中占有相当大的比例。我国油菜品种油脂含量普遍较国外低2—5个百分点,而油脂含量每增加1个百分点对产油量提高的贡献,相当于菜籽产量提高2．5个百分点。因此提高油菜油脂含量是解决油菜生产效益低的重要途径之一。本文综述了油菜油脂研究的状况,包括油脂积累的遗传学基础、油脂合成途径和调控、油脂含量的QTL定位及油脂含量与品质性状的遗传相关性,同时展望了油菜油脂研究前景,以期为油菜油脂含量的品种改良提供科学指导。     

油菜油脂研究进展

油菜(Brassica napus)是世界范围内重要的油料作物, 是植物油脂的第三大来源, 其种植面积和总产量在油料作物中占有相当大的比例。我国油菜品种油脂含量普遍较国外低2-5个百分点, 而油脂含量每增加1个百分点对产油量提高的贡献, 相当于菜籽产量提高2.5个百分点。因此提高油菜油脂含量是解决油菜生产效益低的重要途径之一。本文综述了油菜油脂研究的状况, 包括油脂积累的遗传学基础、油脂合成途径和调控、油脂含量的QTL定位及油脂含量与品质性状的遗传相关性, 同时展望了油菜油脂研究前景, 以期为油菜油脂含量的品种改良提供科学指导。     

Quantitative profiling and pattern analysis of triacylglycerol species in Arabidopsis seeds by electrospray ionization mass spectrometry

Plant triacylglycerols (TAGs), or vegetable oils, provide approximately 25% of dietary calories to humans and are becoming an increasingly important source of renewable bioenergy and industrial feedstocks. TAGs are assembled by multiple enzymes in the endoplasmic reticulum from building blocks that include an invariable glycerol backbone and variable fatty acyl chains. It remains a challenge to elucidate the mechanism of synthesis of hundreds of different TAG species in planta. One reason is the lack of an efficient analytical approach quantifying individual molecular species. Here we report a rapid and quantitative TAG profiling approach for Arabidopsis seeds based on electrospray ionization tandem mass spectrometry with direct infusion and multiple neutral loss scans. The levels of 93 TAG molecular species, identified by their acyl components, were determined in Arabidopsis seeds. Quantitative TAG pattern analyses revealed that the TAG assembly machinery preferentially produces TAGs with one elongated fatty acid. The importance of the selectivity in oil synthesis was consistent with an observation that an Arabidopsis mutant overexpressing a patatin‐like phospholipase had enhanced seed oil content with elongated fatty acids. This quantitative TAG profiling approach should facilitate investigations aimed at understanding the biochemical mechanisms of TAG metabolism in plants.     

Phosphorylation of the lipid droplet localized glycerol‑3‑phosphate acyltransferase Gpt2 prevents a futile triacylglycerol cycle in yeast

The proteome of lipid droplets, storage compartments of triacylglycerols (TAGs), comprises TAG synthesizing and TAG degrading enzymes. Thus, to prevent a futile cycle the activity of enzymes catalyzing key steps in TAG turnover has to be strictly coordinated. The first and committed reaction of TAG synthesis is catalyzed by a glycerol‑3‑phosphate acyltransferase (GPAT). Here we demonstrate that in the model organism yeast the lipid droplet associated GPAT Gpt2 requires phosphorylation at a conserved motif to prevent a futile TAG cycle. Phosphorylation deficiency at the conserved motif increases the enzyme activity of Gpt2 and consequently enhances TAG synthesis. In proliferating cells the phosphorylation deficient GPAT-form contributes to TAG metabolism similar to control. However, during lipolysis the increased activity of phosphorylation deficient Gpt2 causes a constant TAG level by using TAG-released fatty acids as substrate for TAG synthesis. These data strongly indicate that phosphorylation of Gpt2 at a conserved motif plays a critical role in coordinating the synthesis and degradation of TAGs.