黄色粘球菌发育阶段外膜蛋白表达的变化

【目的】黄色粘球菌是研究原核发育的一种模式生物,对其膜蛋白的研究仍然十分缺乏。【方法】利用6种预测软件,在黄色粘球菌的基因组中筛选编码外膜蛋白(OMP)的基因。根据报告基因lacZ,检测这些基因在营养性生长和发育阶段的表达。【结果】基于生物信息学分析,筛选出11个编码外膜蛋白的基因。其中2个基因(MXAN3106和MXAN3883)在发育阶段表达量上升,它们分别编码Secretin家族和Fimbrial usher protein (FUP)家族转运蛋白。其余9个基因在发育起始阶段表达量降低或保持较低水平,它们均编码TonB依赖型受体或外排蛋白。【结论】这些数据提示,黄色粘球菌由生长到发育的转换过程,伴随着膜蛋白表达的显著变化。     

Natural variation of gliding motility in a centimetre-scale population of Myxococcus xanthus

A major challenge in microbial evolutionary ecology is to understand how fitness-related traits vary in natural populations of microorganisms at defined spatial scales and subsequently to identify the forces that maintain such variation. The Gram-negative soil bacterium Myxococcus xanthus is a model system for the study of gliding motility, which is driven by two complementary motility systems in this species and is central to its social lifestyle. We tested whether the ecological context of a centimetre-scale M. xanthus population allows the coexistence of diverse motility-related phenotypes. Swarming rates among 26 clones isolated at the centimetre scale were found to vary greatly in multiple laboratory environments. This variation appears to be motility-specific, as it is not explained by a correlated variation in intrinsic growth rate. In contrast to the common reference strain DK1622, most isolates swarmed faster on hard agar than on soft agar, highlighting the difficulty of inferring species characteristics from laboratory reference strains. These isolates also varied greatly in swarm morphology and in the effect of nutrient limitation on swarming rate. Our results show that diverse swarming phenotypes can coexist in a small-scale bacterial population.     

胸膜肺炎放线杆菌萘啶酸抗性菌株的选育和信号标签突变株的构建

胸膜肺炎放线杆菌(APP)是重要的猪呼吸道病原菌,给世界养猪业造成严重的经济损失.信号标签突变(STM)技术是在宿主动物体内鉴定病原菌毒力因子的高通量方法.通过体外传代选育出APP血清1型和3型萘啶酸抗性菌株,再以萘啶酸抗性菌株为受体菌,以携带mini-Tn10的标签质粒(pLOF/TAG1-48)的E.coli CC118 λ pir或S17-1λpir为供体菌,在或不在E.coli DH5α(pRK2073)的辅助下,进行三亲本或两亲本接合,通过抗性筛选、PCR和Southern杂交鉴定转座突变株.结果表明:体外萘啶酸加压传代很容易选育出萘啶酸抗性APP菌株,该抗性的产生与DNA促旋酶A亚基基因gyrA的突变有关.在APP与E. coli接合实验中,两亲本接合比三亲本接合操作更简单,效率也较高;APP不同菌株在接合和转座效率上存在很大差异,血清1型菌株高于血清3型菌株,3型标准菌株高于地方分离株JL03-R.本研究为APP STM突变体库的构建与毒力基因的鉴定奠定了基础.     

Increases in the intracellular concentration of glycerol during development in Myxococcus xanthus S. Courtney Frasch

Abstract The role of glycerol as a natural morphogen of myxospore formation in Myxococcus xanthus was examined. Glycerol was extracted from cells undergoing development and analyzed by gas chromatography. Glycerol is present in cells, and the intracellular level undergoes a series of transient increases during development. The data suggest a role for glycerol in myxosporulation and fruiting body morphogenesis supporting the notion that this chemical induction of sporulation may represent a physiological pathway in development.     

四个脂肪酸合成酶基因在哺乳动物细胞中的超表达提高长链多不饱和脂肪酸生物合成效率

DHA(22:6n-3)、EPA(20:5n-3)和ARA(20:4n-6)三种长链多不饱和脂肪酸在生物体内活性最强,它们在促进大脑发育和功能维持以及在预防和治疗心血管疾病、炎症、癌症等多种疾病方面有着重要作用。然而,尽管哺乳动物体内有完整的长链多不饱和脂肪酸合成酶系,但哺乳动物合成这些长链多不饱和脂肪酸的效率很低而主要依赖于食物获取。本研究应用转基因方法,将哺乳动物来源的Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶这4种酶的编码基因构建成为一个多基因表达载体,然后转染哺乳动物细胞HEK293T,实现了4个目的基因的超表达,再通过气质联用(GC-MS)分析证实了DHA、EPA和ARA等长链多不饱和脂肪酸的合成效率及水平显著增加,DHA的水平更是提高了2.5倍。由此可见,哺乳动物具有某种抑制长链多不饱和脂肪酸高水平合成的机制,但通过Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶的超表达,能够打破哺乳动物这种抑制机制,从而显著提高DHA、EPA、ARA等的合成水平。同时,本研究的思路也为在转基因动物中生产长链多不饱和脂肪酸提供了重要的启示。     

Improved ethanol tolerance of Saccharomyces cerevisiae strains by increases in fatty acid unsaturation via metabolic engineering

To enhance the ethanol tolerance of Saccharomyces cerevisiae, the Arabidopsis thaliana FAD2 gene and/or the S. cerevisiae OLE1 gene were over-expressed in this yeast. The transformant over-expressing both these genes could not only synthesize dienoic fatty acids but also increased the unsaturated fatty acid content of membrane lipid and then showed the highest viability in the presence of 15% (v/v) ethanol.     

哺乳动物细胞多不饱和脂肪酸合成酶系的重建将亚油酸代谢转变为二十二碳六烯酸

二十二碳六烯酸(DHA,22:6n-3)是一种长度为22个碳原子且含有6个双键的ω-3系多不饱和脂肪酸,在人体中具有重要生物学功能。人体及其他哺乳动物体内只能合成少量的DHA,更多的需求必须从食物中获取。然而,DHA的天然资源(主要是深海鱼类等海洋产品)日趋枯竭,开发新型资源以满足不断扩大的市场需求势在必行。本研究利用转基因技术,在哺乳动物细胞中使Δ6和Δ5脂肪酸去饱和酶以及Δ6和Δ5脂肪酸延长酶超表达,同时表达来源于秀丽隐杆线虫Caenorhabditis elegans的Δ15去饱和酶和小眼虫Euglena gracilis的Δ4去饱和酶,结果表明,这6种酶的表达或超表达能将ω-6系的亚油酸(LA,18:2n-6)有效地转化为DHA(22:6n-3),后者的含量从对照组的16.74%提高到实验组的25.3%。本研究的策略及技术路线为将来利用遗传改造的哺乳动物生产珍稀的DHA(22:6n-3)等长链多不饱和脂肪酸产品提供了重要的启示。     

Transcriptional regulation of genes required for antibiotic TA synthesis in Myxococcus xanthus

Abstract Regulatory mutants were isolated for six genes required for the production of antibiotic TA in Myxococcus xanthus , using Tn5lac as a promoter probe. Two of the regulatory mutants were closely linked to their corresponding Tn5lac insertions and three were separated by at least 40 kb from their original insertions. All of the β-galactosidase-overproducing mutants showed higher specific activities than their parent strains in three different media. The specific activity was higher throughout the growth cycle, starting earlier and finishing higher. In addition to helping to understand the mechanism of repression of TA genes, the mutants are potentially useful in the construction of antibiotic-overproducing strains.     

DHA合成途径中关键酶基因在毕赤酵母中的表达研究

DHA（二十二碳六烯酸）是人体所需的一种非常重要的多不饱和脂肪酸,主要存在于深海鱼类和海洋微藻类生物中。由于高等植物自身不能合成DHA,因此通过基因工程方法在作物(如玉米)中表达DHA,将会成为最健康的DHA来源,同时也能够减轻人类对海洋资源的破坏。从深海藻类中挑选了5个DHA合成途径中的关键酶基因,分别是Δ4脱饱和酶基因（D4）、Δ5脱饱和酶基因（D5）、Δ6脱饱和酶基因（D6）、C18延长酶基因（E18）和C20延长酶基因（E20）。密码子优化并合成这5个基因,为确定优化后的核苷酸序列是否能在真核生物中正确表达,以pPIC9K为基础载体连入目的基因转化到毕赤酵母GS115 (His4+MUts)中进行表达分析。结果表明,这些基因在甲醇诱导96 h后蛋白表达量最大,Western Blot结果表明表达产物为目的蛋白。该结果为进一步培育能够自身合成DHA的作物奠定了基础。     

粘球菌基因功能和表达情况分析质粒pZCY11的构建及其应用

【目的】旨在构建一个用于粘球菌基因插入失活、同时可通过报告基因分析插入位点基因表达情况的质粒载体,并应用该质粒对黄色粘球菌MXAN1334基因功能和表达情况进行分析。【方法】通过PCR、酶切和连接等方法构建质粒载体pZCY11。从黄色粘球菌DK1622基因组上PCR扩增MXAN1334基因内部部分片段,插入载体pZCY11上lacZ基因的上游,构建重组质粒pZCY13,将其转入DK1622菌株,获得MXAN1334基因插入失活突变株ZC16-18。【结果】基因功能和表达情况分析质粒载体pZCY11含有抗性标记基因aph、自杀性质粒复制子OriR6K和无启动子的报告基因lacZ。突变株ZC16-18在CTT软硬琼脂平板上菌落扩展结果显示,MXAN1334基因插入失活会造成黄色粘球菌S运动能力缺陷。通过X-gal检测突变株ZC16-18中β-半乳糖苷酶酶活,实验结果显示,含有X-gal的平板上培养的ZC16-18菌落呈现蓝色,表明MXAN1334基因能够表达;颜色呈现的时间分析结果显示,MXAN1334基因表达时间较早。【结论】构建的质粒载体pZCY11不仅能够对目的基因进行功能的分析,而且能够同时通过报告基因分析基因的表达情况,可简化粘球菌中基因功能及表达情况的研究。     

Effect of different contents of extruded linseed in the sow diet on piglet fatty acid composition and hepatic desaturase expression during the post-natal period

n-3 polyunsaturated fatty acids (n-3 PUFA) contribute to the normal growth and development of numerous organs in the piglet. The fatty acid composition of piglet tissues is linked to the fatty acid composition of sow milk and, consequently, to the composition of sow diet during the gestation and lactation period. In this study, we investigated the impact of different contents of extruded linseed in the sow diet on the fatty acid composition and desaturase gene expression of piglets. Sows received a diet containing either sunflower oil (low 18:3n-3 with 18:3n-3 representing 3% of total fatty acids) or a mixture of extruded linseed and sunflower oil (medium 18:3n-3 with 9% of 18:3n-3) or extruded linseed (high 18:3n-3 with 27% of 18:3n-3) during gestation and lactation. Fatty acid composition was evaluated on sow milk and on different piglet tissues at days 0, 7, 14, 21 and 28. The postnatal evolution of delta5 (D5D) and delta6 (D6D) desaturase mRNA expression was also measured in the liver of low 18:3n-3 and high 18:3n-3 piglets. The milk of high 18:3n-3 sows had higher proportions of n-3PUFA than that of low 18:3n-3 and medium 18:3n-3 sows. Piglets suckling the high 18:3n-3 sows had greater proportions of 18:3n-3, 20:5n-3, 22:5n-3 and 22:6n-3 in the liver, and of 22:5n-3 and 22:6n-3 in the brain than low 18:3n-3 and medium 18:3n-3 piglets. D5D and D6D mRNA expressions in piglet liver were not affected by the maternal diet at any age. In conclusion, extruded linseed in the sow diet modifies the n-3PUFA status of piglets during the postnatal period. However, a minimal content of 18:3n-3 in the sow diet is necessary to increase the n-3PUFA level in piglet liver and brain. Moreover, modifications in the n-3PUFA fatty acid composition of piglet tissue seem linked to the availability of 18:3n-3 in maternal milk and not to desaturase enzyme expression.     

Isolation and Characterization of Δ6-Desaturase,an ELO-Like Enzyme and Δ5-Desaturase from the Liverwort Marchantia Polymorpha and Production of Arachidonic and Eicosapentaenoic Acids in the Methylotrophic Yeast Pichia Pastoris

The liverwort Marchantia polymorpha contains high proportions of arachidonic and eicosapentaenoic acids. In general, these C20 polyunsaturated fatty acids (PUFA) are synthesized from linoleic and alpha -linolenic acids, respectively, by a series of reactions catalyzed by Delta(6)-desaturase, an ELO-like enzyme involved in Delta(6) elongation and Delta(5)-desaturase. Here we report the isolation and characterization of the cDNAs, MpDES6, MpELO1 and MpDES5, coding for the respective enzymes from M. polymorpha. Co-expression of the MpDES6, MpELO1 and MpDES5 cDNAs resulted in the accumulation of arachidonic and eicosapentaenoic acids in the methylotrophic yeast Pichia pastoris. Interestingly, Delta(6) desaturation by the expression of the MpDES6 cDNA appears to occur both in glycerolipids and the acyl-CoA pool, although other lower-plant Delta(6)-desaturases are known to have a strong preference for glycerolipids.     

Propionyl-CoA carboxylase of Myxococcus xanthus: catalytic properties and function in developing cells

An acyl-coenzyme A carboxylase that carboxylates acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA was purified from Myxococcus xanthus. Since the enzyme showed maximal rates of carboxylation with propionyl-CoA, the enzyme is thought to be propionyl-CoA carboxylase. The apparent K _m values for acetyl-CoA, butyryl-CoA, propionyl-CoA, and succinyl-CoA were found to be 0.2, 0.2, 0.03, and 1.0 mM, respectively. The native enzyme has a molecular mass of 605–615 kDa and is composed of nonidentical subunits (α and β) with molecular masses of 53 and 56 kDa, respectively. The enzyme showed maximal activity at pH 7.0–7.5 and at 25–30°C, and was affected by variation in concentrations of ATP and Mg²⁺. During development of M. xanthus, the propionyl-CoA carboxylase activity increased gradually, with maximum activity observed during the sporulation stage. Previous work has shown that a propionyl-CoA-carboxylase-deficient mutant of M. xanthus reduces levels of long-chain fatty acids. These results suggest that the propionyl-CoA carboxylase is also responsible for the carboxylation of acetyl-CoA to malonyl-CoA used for the synthesis of long-chain fatty acids during development. Received: 24 February 1998 / Accepted: 25 May 1998     

脂肪酸脱饱和酶的研究进展

脂肪酸脱饱和酶催化与载体结合的饱和脂肪酸或不饱和脂肪酸在脂酰链上形成双键。脂肪酸脱饱和酶分为脂酰ＣｏＡ脱饱和酶、脂酰ＡＣＰ脱饱和酶和脂酰脂脱饱和酶三类。它在控制生物膜的形成与物理性质,保护光合机构和决定贮脂与膜脂的脂肪酸组成与不饱和度等方面起着关键作用。     

花生Δ~(12)脂肪酸脱氢酶基因高效表达载体构建与原核表达(英文)

花生是世界范围内广泛种植的重要油料作物之一,其种子中富含油酸和亚油酸。△12脂肪酸脱氢酶(FAD2)是亚油酸合成的关键酶,催化油酸(18:1)在△12位上脱氢生成亚油酸(18:2),但由于△12脂肪酸脱氢酶本身的特性,目前还没有有效的方法将其纯化并在蛋白水平作进一步的研究,尚需对其结构和功能之间以及表达调控进行更深入全面的研究。本文利用从花生中克隆的△12脂肪酸脱氢酶基因(GenBank接受号为AY1006)构建高效表达载体,把花生?12脂肪酸脱氢酶基因全长序列插入到大肠杆菌高效表达载体pRSETB中,构建了pRSET/HO-A融合表达载体,并转化到大肠杆菌表达菌BL21(DE3)pLysS中,在IPTG诱导下,pRSET/HO-A融合表达载体在BL21(DE3)pLysS菌株中高效表达了?12脂肪酸脱氢酶。利用Clon-Tech蛋白纯化Kit进一步分离了目的蛋白,同时加入外源性底物油酸在20℃温育6h后,进行脂肪酸甲酯化处理,通过气相色谱(GC)和气相色谱/质谱(GC-MS)分析表明,所编码的酶具有?12脂肪酸脱氢酶的活性,能将外源性的底物油酸转化为亚油酸,转化率为11.8%。花生△12脂肪酸脱氢酶基因的原核表达目前国内外还未见报导,本实验为其进一步的大量纯化和结构功能分析奠定了基础。     

The expression of three desaturase genes of Spirulina platensis in Escherichia coli DH5α – Heterologous expression of Spirulina-desaturase genes

The genes from a cyanobacterium--Spirulina platensis strain C1--that encode the acyl-lipid desaturases (desC, desA and desD) involved in gamma-linolenic (GLA) synthesis have been successfully expressed for the first time in Escherichia coli by employing a pTrcHisA expression system. In this report, the authors describe the expression of the three Spirulina N-terminal 6xHis-desaturases as well as the functional analysis of these recombinant proteins. The gene products of desC, desA and desD have approximate molecular masses of 37, 45, and 47 kDa, respectively. Enzymatic activity measurement of these products was carried out in vivo to demonstrate that (i) the expressed proteins are in functional form, and (ii) the cofactors of the host system can complement the system of Spirulina platensis. The study demonstrated that the gene products of desC and desA catalyzed the reactions in vivo where the enzyme substrates were provided in appropriate concentration. This indicates that the delta9 and delta12 desaturases were expressed in the heterologous host in their active form, and that these two reactions can be carried out in an E. coli host cell using its cofactors system. In contrast, delta6 desaturase activity can be detected only in vitro where electron carriers are provided. This suggests that while this enzyme is expressed in the heterologous host in its active form, its function in vivo is suppressed, as the electron carriers of the host system cannot complement the system of Spirulina platensis.