紫苏PfLEC1基因的克隆及表达研究

紫苏是目前发现的α-亚麻酸含量最高的药食同源经济作物,其种子油脂中含60%以上的α-亚麻酸。该研究基于紫苏转录组测序结果,从紫苏种子中克隆获得植物种胚发生过程中的关键基因Leafy Cotyledon 1(Pf LEC1),并对其进行相关生物信息学分析、实时荧光定量PCR以及不同时期种子的脂肪酸含量测定,以探讨紫苏α-亚麻酸高效合成积累的分子调控机制。(1)序列分析结果表明,Pf LEC1基因编码区长度为621 bp,可编码206个氨基酸,属于NF-YB亚基家族,含有HAP3亚基的保守功能域B区域;在线预测该蛋白为不稳定亲水性蛋白,无信号肽和跨膜区域,定位于细胞质;进化分析表明,该蛋白序列与拟南芥、甘蓝型油菜、水稻和玉米关系较近。(2)实时荧光定量PCR分析表明,PfLEC1基因在紫苏根、茎、叶、花及种子中均有表达,且在种子中表达量最高,根中表达最低;PfLEC1在种子发育的前期表达较高,随着种子发育表达量显著下降。(3)不同阶段紫苏种子脂肪酸含量分析表明,油酸、α-亚麻酸含量随种子发育逐渐增加,而棕榈酸、硬脂酸、亚油酸含量变化则相反,其中变化最为明显的是α-亚麻酸,从种子发育5 d时的33.16%上升至20 d时的65.16%,表明紫苏种子中α-亚麻酸含量是随种子发育快速积累的。研究推测,PfLEC1可能与紫苏种子α-亚麻酸的高含量合成积累密切相关,该研究为今后深入探讨PfLEC1基因的功能奠定了分子基础。     

一株富含α-亚麻酸栅藻的异养培养条件优化

HSJ296是本实验室分离纯化的1株能够异养生长、富含α-亚麻酸的栅藻(Scenedesmus sp.)。研究比较了不同温度、氮源和葡萄糖浓度对其生长的影响, 结果显示, 其最适培养条件为30℃、4 g/L尿素和20—40 g/L葡萄糖。通过分析不同培养条件下HSJ296总脂中的脂肪酸组成, 发现主要含有十六碳脂肪酸(C16:0)、油酸(C18:1)、亚油酸(C18:2)和α-亚麻酸(α-C18:3), 并且α-亚麻酸的含量稳定在35%—45%。栅藻HSJ296发酵产品或可用作鱼类饲料添加剂以补充α-亚麻酸等营养。     

杜仲籽油与紫苏籽油脂肪酸组成的比较研究

利用气相色谱法,对杜仲籽油和紫苏籽油的脂肪酸组成、α-亚麻酸含量等进行了比较研究。结果发现,两者不仅脂肪酸GC指纹图谱较为相似(脂肪酸组成、含量基本相同),而且外观、气味、折光率等质量指标也非常相近。说明了杜仲籽油具有与紫苏籽油同样的开发价值。     

水酶法提取紫苏籽油脂工艺

采用中心组合设计(CCD)-响应面(RSM)优化紫苏籽油脂的水酶法提取工艺。在单因素试验的基础上采用中心组合设计方法,研究了酶的种类、酶解温度、pH、液(mL)固(g)比、加酶量、以及时间相互作用对紫苏油脂提取率的影响。结果显示,拟合得到方程显著,确定的紫苏油脂提取最优条件为:碱性蛋白酶在pH9.5条件下液(mL)固(g)比9.97∶1、加酶量2.75%、温度56.1℃、时间5.25h,该条件下紫苏油脂的提取率可达到37.65%,与理论值38.3%十分接近,建立的模型真实可靠,确定了紫苏油脂的最佳提取工艺。经气相色谱检测紫苏籽油中含有棕榈酸、硬脂酸、油酸、亚油酸、α-亚麻酸等脂肪酸,水酶法提取紫苏油脂的α-亚麻酸相对含量最高67.9%,且相对溶剂法及冷榨法理化指标最好。     

紫苏溶血磷脂酸酰基转移酶基因的克隆与功能分析

紫苏(Perilla frutescens)是一种重要食药同源油料作物,种子含油量高达46%−58%,其中α-亚麻酸(C18:3)含量占60%以上。溶血磷脂酸酰基转移酶(lysophosphatidic acid acyltransferase,LPAT)是植物种子三酰基甘油组装过程中的一类关键限速酶。本研究从紫苏发育种子中克隆了其编码基因(PfLPAT2),并利用qRT-PCR技术检测PfLPAT2基因在紫苏不同组织及不同发育时期种子的表达特性。构建PfLPAT2/GFP融合表达载体并通过农杆菌介导瞬时侵染本氏烟草叶片,检测PfLPAT2蛋白的亚细胞定位。构建大肠杆菌(Escherichia coli)表达载体、酵母表达载体和组成型植物过表达载体,分别转化大肠杆菌突变株SM2-1、酿酒酵母(Saccharomyces cerevisiae)野生型菌株INVSc1和普通烟草(Nicotiana tabacum),分析PfLPAT2蛋白的酶活性及生物学功能。结果表明,紫苏PfLPAT2基因ORF为1 155 bp,编码384个氨基酸。功能结构域预测显示PfLPAT2蛋白具有溶血磷脂酸酰基转移酶典型的保守区。PfLPAT2基因在紫苏根、茎、叶、花和开花后10、20、30、40 d的种子中均有表达,且在开花后20 d的种子中高表达。亚细胞定位结果显示PfLPAT2蛋白定位于细胞质。大肠杆菌功能互补测试表明,PfLPAT2可恢复SM2-1细胞膜脂生物合成,具有LPAT酶活性。与非转基因对照相比,转PfLPAT2基因酵母的总油脂含量显著提高,且脂肪酸各组分的含量发生改变,油酸(C18:1)含量增加明显,预示PfLPAT2对C18:1具有较高的底物偏好性。转基因烟草叶片总脂肪酸含量比对照组提高了约0.42倍,C18:1含量增加了约1倍。转基因株系总脂提高和脂肪酸组分的改变表明PfLPAT2异源表达可以促进宿主油脂合成和健康有益型脂肪酸(C18:1和C18:3)的积累。本研究为深入解析紫苏油脂特别是不饱和脂肪酸合成的分子调控机制和改良油料作物油脂品质提供理论依据和基因元件。     

发菜膜脂及其脂肪酸组成

对野生发菜(Nostocflagelliforme Bom.et Flab)的膜脂(主要成分为类囊体膜脂)及其脂肪酸组成进行了测定分析.发菜的膜脂由单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)、磷酯酰甘油(PG)和硫代异鼠李糖甘油二酯(SQDG)组成,其酯酰基连接有棕榈酸(16:0)、十六碳烯酸(16:1)、硬脂酸(18:0)、油酸(18:1)、亚油酸(18:2)和亚麻酸(18:3)6种脂肪酸.发菜的不饱和脂肪酸含量可达总脂的73%,特别是16:1和18:3分别高达29%和34%,远远高于已报道的其他蓝藻,说明了发菜类囊体膜具有较强的抗逆性特点.同时还对复水30 min和复水后生长24 h的发菜膜脂及其脂肪酸组成进行了分析.结果表明,复水对野生发菜的膜脂及其脂肪酸组成没有显著影响,说明发菜的膜脂和脂肪酸组成在干燥-吸水过程中能保持很高的稳定性.     

东方粘虫的飞翔活动和脂肪酸利用

应用GC和GC-MS分析了东方粘虫Mythimna separata(Walker)成虫脂肪体、血淋巴和飞翔肌内总脂类脂肪酸组成.它们的组成成分为肉豆蔻酸(C14:0),棕榈酸(C16:0),棕榈油酸(C16:1),硬脂酸(C18:0),油酸(C18:2),亚油酸(C18:2)和亚麻酸(C18:3);组成百分率为1-2:35:9-11:1:32:12-17:3-6.在吊飞1h后,脂肪体内的脂肪酸水平显著下降(20μg/mg组织·h~(-1),血淋巴内脂肪酸浓度明显升高,然而,飞翔肌内脂肪酸含量的变化不明显.从脂肪体、血淋巴和飞翔肌内脂肪酸组成成分的百分率变化可以发现东方粘虫飞翔肌在飞翔活动中主要选择性利用棕榈酸和油酸.     

侧柏叶子与种子脂肪酸的GC-MS比较分析研究

目的:研究柏子仁与侧柏叶的脂肪酸组成.方法:用GC-MS方法对侧柏叶子与种子油进行定性鉴定和定量分析.结果:鉴定了柏子仁油中的8种脂肪酸,占脂溶性成分的93.56%;侧柏叶子油中12种脂肪酸,占脂溶性成分的93.39%.柏子仁饱和脂肪酸主要是十六烷酸(8.11%)、硬脂酸(6.08%);不饱和脂肪酸主要为亚油酸(24.59%)、亚麻酸(59.77%),占脂肪酸的83.14%.侧柏叶子饱和脂肪酸饱和脂肪酸主要为十六烷酸(14.70%)、乙酸(3.20%)、十七烷酸(2.50%);不饱和脂肪酸主要为二十二碳四烯酸(40.48%)、亚油酸(10.69%)、亚麻酸(17.62%).结论:柏子仁和侧柏叶均含有合理的脂肪酸组成.     

13种微藻的脂肪酸组成分析

分析了13种微藻(包括7种绿藻,5种杂色藻和1种红藻)的总脂含量和脂肪酸组成,结果表明,不同门类微藻的脂肪酸组成差异较大:绿藻的脂肪酸组成以C16和C18为主;杂色藻类的脂肪酸组成相近,金藻门含有14:0、16:0、18:1、18:4等特征脂肪酸,三角褐指藻主要的脂肪酸为14:0、16:0、16:1、16:3和20:5,而粉核油球藻的脂肪酸以14:0、16:0、20:5为主;紫球藻的脂肪酸组成以16:0、20:4和20:5为主。在测试的13种微藻中,杜氏盐藻的亚麻酸含量最高,占总脂肪酸的60.9%;等鞭金藻的十八碳四烯酸含量最高,占总脂肪酸的19.6%;紫球藻和粉核油球藻中花生四烯酸与二十碳五烯酸(EPA)含量分别占总脂肪酸的17.1%和20.9%。     

菘蓝种子脂肪酸的GC-MS分析

目的:分析菘蓝种子中的脂肪酸。方法:分别利用索氏提取法,运用气相色谱-质谱(GC-MS)联用技术,计算机检索和人工解析对菘蓝种子中的脂肪酸进行分析和鉴定。结果:鉴定了11种脂肪酸成分。结论:菘蓝种子中脂肪酸成分主要是亚麻酸(24．72%)、芥酸(23．9%)、油酸(19．11%)和亚油酸(10．76%)。     

Fatty Acid Replacements in a Fatty Acid Auxotroph of Escherichia coli

Unsaturated fatty acids having structural features which are different from those of the monoenoic acids normally synthesized by Escherichia coli can serve as growth factors for an auxotroph requiring unsaturated fatty acids. These analogues were incorporated into the phospholipids, as shown by gas-liquid and thin-layer chromatographic analysis of the phospholipid fatty acid composition. Some of these fatty acids were cisDelta(5)- and cis-Delta(9)-tetradecenoic, cis-Delta(11)-eicosenoic, cis,cis-Delta(11,14)-eicosadienoic, cis,cis,cis-Delta(11,14,17)-eicosatrienoic, trans-Delta(9)- and trans-Delta(11)-octadecenoic acids. Although partial degradation of some of these analogues to shorter even-chain homologues occurred, chain elongation of the exogenous fatty acids was not detected. Trans-olefinic acids were utilized without stereochemical or positional isomerization. These studies provide a basis for exploring the properties of the fatty acids and phospholipids required for the formation, structure, and function of membranes.     

Saturated Fatty Acid Mutant of Saccharomyces cerevisiae with an Intact Fatty Acid Synthetase

To determine directly the effects of streptomycin on translational fidelity in intact cells, we studied the synthesis of beta-galactosidase and of the coat protein of bacteriophage R17 in an Escherichia coli mutant in which the bactericidal effects of streptomycin are delayed. After the addition of streptomycin to exponentially growing mutant cells, protein synthesis continues at an undiminished rate for approximately an hour; however, as measured by enzyme assays, little functional protein is produced. Serological assays designed to detect beta-galactosidase and bacteriophage R17 coat protein show that substantial amounts of the protein synthesized can react with antisera prepared against active beta-galactosidase and phage R17, indicating the aberrance of the protein produced in the presence of the antibiotic. The polypeptides synthesized in the presence of streptomycin are degraded in the cell to a much greater extent than protein synthesized in the absence of the antibiotic. The proteolytic attack on this protein is not affected by inhibitors of serine proteases, suggesting that enzymes other than those involved in "normal turnover" of cellular protein are responsible. In this strain, certain of the multiple effects of streptomycin are separated in time and the production of abnormal protein (enzymatically inactive and susceptible to proteolytic attack) could be studied in the absence of the lethal effect of the drug.     

Fatty Acid Metabolism in Microorganisms

The production of pimelic acid from azelaic acid by microorganisms was studied. About 100 strains of bacteria which were able to utilize azelaic acid as a sole carbon source were isolated from soil and other natural materials. Among these bacteria, several strains produced a large quantity of an organic acid (pimelic acid) from azelaic acid in their culture fluids during the cultivation. The acid was isolated from the culture fluid of strain A133 in crystalline form. The crystal was identified as pimelic acid by physicochemical and biological methods.

From the results of investigations on the morphological and physiological characters, the bacterial strain A133 was assumed to be Micrococcus sp.     

Thermodynamics of Fatty Acid Transfer

There is continuing controversy about the mechanism for transfer of fatty acids (FA) between plasma and the interior of cells and vice versa. One view is that this is a spontaneous process. The generally accepted view is that each step of the process is facilitated by a specialized protein. Whether uptake is spontaneous or facilitated, the components of the uptake system, e.g., albumin, water, FA, plasma membrane, and putative transport proteins of the plasma membrane, must behave according to the rules of the physical chemistry of the system. We review these features to illustrate the constraints they impose on the design of experiments to adduce the mechanism of uptake. Analysis of the literature in the context of the physical chemistry of the uptake system indicates that arguments for a facilitated mechanism of uptake for FA are not supported by any data extant. By contrast, comparison of the rates for individual steps of the pathway traversed by FA moving from albumin to the inside of a cell (or vesicles of a model system) with rates of uptake of FA of tissues in the steady state shows that the rates of the former are sufficient to account for the rate of the latter. Received: 18 January 2000/Revised: 17 April 2000     

Fatty Acid Metabolism in Microorganisms

During the investigation on the metabolism of azelaic acid by Micrococcus sp., it was found that the bacterium produced a large amount of keto acid (α-ketoglutaric acid) under the restricted condition for nitrogen source. The acid was identified as α-ketoglutaric acid by physico-chemical and biological methods. The mechanism of the production of α-ketoglutaric acid from azelaic acid was investigated. From the result, it was suggested that α-ketoglutaric acid production proceeded thrpugh the further oxidation of acetic acid produced from azelaic acid and that the production might be functioned by TCA cycle enzymes of the bacterium. Similarly, α-ketoglutaric acid was found to be produced remarkably from other various fatty acids.     

Fatty Acid Activation in Cyanobacteria Mediated by Acyl-Acyl Carrier Protein Synthetase Enables Fatty Acid Recycling

In cyanobacteria fatty acids destined for lipid synthesis can be synthesized de novo, but also exogenous free fatty acids from the culture medium can be directly incorporated into lipids. Activation of exogenous fatty acids is likely required prior to their utilization. To identify the enzymatic activity responsible for activation we cloned candidate genes from Synechocystis sp. PCC 6803 and Synechococcus elongatus PCC 7942 and identified the encoded proteins as acyl-acyl carrier protein synthetases (Aas). The enzymes catalyze the ATP-dependent esterification of fatty acids to the thiol of acyl carrier protein. The two protein sequences are only distantly related to known prokaryotic Aas proteins but they display strong similarity to sequences that can be found in almost all organisms that perform oxygenic photosynthesis. To investigate the biological role of Aas activity in cyanobacteria, aas knockout mutants were generated in the background of Synechocystis sp. PCC 6803 and S. elongatus PCC 7942. The mutant strains showed two phenotypes characterized by the inability to utilize exogenous fatty acids and by the secretion of endogenous fatty acids into the culture medium. The analyses of extracellular and intracellular fatty acid profiles of aas mutant strains as well as labeling experiments indicated that the detected free fatty acids are released from membrane lipids. The data suggest a considerable turnover of lipid molecules and a role for Aas activity in recycling the released fatty acids. In this model, lipid degradation represents a third supply of fatty acids for lipid synthesis in cyanobacteria.Cyanobacteria present a diverse group of Gram-negative bacteria capable of oxygenic photosynthesis (Margulis, 1975). Their two photosystems, as well as other genetic and morphological similarities, identified them as putative predecessors of chloroplasts of eukaryotic plants (Wallace, 1982; Pakrasi, 1995). The structural similarities of cyanobacteria and chloroplasts are reflected in part by equivalence of biochemical pathways and their components. For instance, cyanobacterial fatty acid and glycerolipid compositions closely resemble those of the inner envelope and thylakoid membranes of chloroplasts (Roughan et al., 1980; Heinz and Roughan, 1983). In cyanobacteria, as well as in chloroplasts, fatty acids are synthesized by a type II fatty acid synthase (FAS) complex utilizing a freely dissociable acyl carrier protein (ACP; Froehlich et al., 1990). The products of FAS are released as acyl ACPs and may serve directly as substrates for acyltransferases, incorporating the fatty acids into membrane lipids (Frentzen et al., 1983). The substrate specificity of the acyltransferases establishes in cyanobacteria as well as in plastids the typical prokaryotic fatty acid pattern characterized by C16 fatty acids esterified to the sn-2 position. The correspondence of metabolic pathways between cyanobacteria and chloroplasts is reflected by the shared presence of closely related enzymes that catalyze key reactions. Besides the many similarities, however, there are also clear discrepancies that in part account for the fact that cyanobacteria are unicellular organisms, whereas chloroplasts are embedded in the metabolism of a eukaryotic cell. In terms of lipid metabolism, such differences become obvious if one considers the fact that the plastidial FAS also supplies the extraplastidic compartment with fatty acids (Browse et al., 1986). Fatty acid export from the chloroplast necessitates the release of synthesized acyl chains from ACP to allow transport across both envelope membranes. The release is achieved by the action of acyl-ACP thioesterases that hydrolyze the acyl-ACP thioester to liberate the fatty acid (Voelker et al., 1997). In cyanobacteria such export would obviously result in an unfavorable loss of fatty acids, and consequently homologous proteins to acyl-ACP thioesterases cannot be found here. Whereas cyanobacteria seem to be unable to release fatty acids enzymatically from their activated state, all cyanobacterial genomes available to date encode an activity most likely responsible for the activation of free fatty acids. The respective sequences are annotated as acyl-CoA synthetases. Conserved motifs in the amino acid sequence identify these proteins as members of the well-established superfamily of AMP-binding proteins. This protein family comprises several hundred amino acid sequences spreading across all organisms analyzed so far. The family members are annotated in the PROSITE database under entry number PS00455. Although these predicted fatty acid-activating enzymes of cyanobacteria are annotated as acyl-CoA synthetases due to their sequence similarity to proteins with such enzymatic activity, there is a much higher degree of similarity to certain AMP-binding proteins of plant origin with less-well-established function. These plant proteins are predicted to reside in chloroplasts and one member of this subgroup from Arabidopsis (Arabidopsis thaliana) designated as AAE15 was recently described as acyl-ACP synthetase. The conclusions were based on the comparison of enzymatic activity between plant extracts of wild-type and knockout mutant lines (Koo et al., 2005). Whereas the biological role of this activity remained largely elusive, it was shown that the capacity of plant extracts to elongate supplied medium fatty acids depended on AAE15 activity. Since the elongation of medium chain fatty acids in the plastid depends on the FAS requiring acyl ACPs, it was concluded that the fatty acids must have been activated by ACP. The elongated fatty acids ultimately appeared in membrane lipids. Together these findings suggested that AAE15 is an acyl-ACP synthetase.Besides encoding a protein homologous to AAE15 from Arabidopsis, cyanobacteria are also able to utilize exogenous fatty acids like it was shown for isolated chloroplasts. It is well established that feeding different cyanobacteria with free fatty acids results in the incorporation of these fatty acids into membrane lipids. For this process the activation of the fatty acids is believed to be essential. This causal relationship was clearly shown at least for other unicellular organisms like Escherichia coli and yeast (Saccharomyces cerevisiae) where the deletion of acyl-CoA synthetase activity resulted in the inability to utilize exogenous fatty acids (Overath et al., 1969; Knoll et al., 1995). It is not easy to assess how regularly cyanobacterial cells are exposed to exogenous free fatty acids in nature but at least for marine strains this is most likely a rather artificial situation. Therefore, it can be speculated that the capacity to activate free fatty acids might be of different relevance in the lipid metabolism of cyanobacteria in vivo.In this article, we investigated the fatty acid metabolism of cyanobacteria. We isolated candidate genes potentially encoding enzymes involved in fatty acid activation from the strains Synechocystis sp. PCC 6803 (hereafter Synechocystis) and Synechococcus elongatus PCC 7942 (hereafter Synechococcus) and performed heterologous expression in E. coli. The recombinant proteins were shown to possess acyl-ACP synthetase activity with broad substrate specificity. Knockout mutant strains deficient in acyl-ACP synthetase activity were characterized by secretion of endogenous free fatty acids into the culture medium. Combined with labeling experiments, the results suggest an essential role for acyl-ACP synthetase in fatty acid recycling in cyanobacteria.     

Overproduction of a Functional Fatty Acid Biosynthetic Enzyme Blocks Fatty Acid Synthesis in Escherichia coli

β-Ketoacyl-acyl carrier protein (ACP) synthetase II (KAS II) is one of three Escherichia coli isozymes that catalyze the elongation of growing fatty acid chains by condensation of acyl-ACP with malonyl-ACP. Overexpression of this enzyme has been found to be extremely toxic to E. coli, much more so than overproduction of either of the other KAS isozymes, KAS I or KAS III. The immediate effect of KAS II overproduction is the cessation of phospholipid synthesis, and this inhibition is specifically due to the blockage of fatty acid synthesis. To determine the cause of this inhibition, we examined the intracellular pools of ACP, coenzyme A (CoA), and their acyl thioesters. Although no significant changes were detected in the acyl-ACP pools, the CoA pools were dramatically altered by KAS II overproduction. Malonyl-CoA increased to about 40% of the total cellular CoA pool upon KAS II overproduction from a steady-state level of around 0.5% in the absence of KAS II overproduction. This finding indicated that the conversion of malonyl-CoA to fatty acids had been blocked and could be explained if either the conversion of malonyl-CoA to malonyl-ACP and/or the elongation reactions of fatty acid synthesis had been blocked. Overproduction of malonyl-CoA:ACP transacylase, the enzyme catalyzing the conversion of malonyl-CoA to malonyl-ACP, partially relieved the toxicity of KAS II overproduction, consistent with a model in which high levels of KAS II blocks access of the other KAS isozymes to malonyl-CoA:ACP transacylase.     

海洋微藻脂肪酸去饱和酶

海洋微藻中富含多不饱和脂肪酸(polyunsaturated fatty acid,PUFA),在部分微藻中ω3 PUFA的量可达其总脂肪酸的30%～50%。而且微藻油具有鱼油所不可比拟的健康优势,也是唯一得到美国食品与药物管理局(FDA)认可的儿童DHA(二十二碳六烯酸)补充剂来源。由于用培养微藻来提取、纯化PUFA受到现有生产工艺的限制,使微藻油在国际食品(尤其是高质量食品)及保健品市场上供不应求。微藻脂肪酸去饱和酶(fatty aciddesaturase,FAD)是微藻PUFA合成的关键酶类,所以对微藻FAD的深入研究无疑将促进PUFA资源的合理开发和利用。