野油菜黄单胞菌中烯脂酰ACP还原酶的功能鉴定

烯脂酰ACP还原酶是细菌脂肪酸合成的关键酶之一.本研究通过生物信息学分析发现,野油菜黄单胞菌Xanthomonas campestris(Xcc)8004基因组中XC_0119(Xccfab V)注释为反-2-烯脂酰Co A还原酶基因.但其编码产物与铜绿假单胞菌的烯脂酰ACP还原酶Fab V具有较高的同源性,并含有相同的催化活性中心Tyr-(Xaa)8-Lys序列.用携带Xccfab V的质粒载体互补大肠杆菌fab I温度敏感突变株JP1111,转化子能在42℃生长,表明Xccfab V能遗传互补大肠杆菌fab I突变.体外重建脂肪酸合成反应表明,Xcc Fab V能催化不同链长的烯脂酰ACP还原为脂酰ACP,且催化活性不受三氯森抑制.遗传学研究表明,Xccfab V是必需基因,不能获得Xccfab V基因敲除突变株.将携带大肠杆菌fab I的外源质粒导入野生菌后,可敲除染色体上的fab V基因,获得的替换突变株生长特性和脂肪酸组成未发生显著变化,但替换突变株对三氯森敏感.上述结果证实,野油菜黄单胞菌fab V是必需基因,编码烯脂酰ACP还原酶,参与脂肪酸从头合成反应,且Fab V是Xcc对三氯森耐受的根本原因.     

茄科雷尔氏菌脂酰-CoA合成酶的功能鉴定

【目的】茄科雷尔氏菌是一种常见的农作物致病菌,引起植物青枯病。研究其脂肪酸代谢途径将有助于寻找新的抗菌药物靶点,为防治青枯病害提供新的思路。【方法】利用大肠杆菌FadD序列,进行同源比对发现茄科雷尔氏菌GMI1000中RSc2857(RsFadD)具有较高的相似性,推测其具有脂酰-CoA合成酶活性。采用PCR扩增方法获得RsfadD基因,连入表达载体pBAD24M后互补大肠杆菌fadD突变株,并检测转化子的生长情况。RsfadD与pET-28b连接后,在大肠杆菌BL(DE3)中表达,并利用Ni-NTA纯化获得带有组氨酸标签的RsFadD,体外测定RsFadD的活性。利用同源重组方法,获得RsfadD敲除突变株,分析突变株的生长性状。【结果】RsfadD异体互补大肠杆菌fadD突变株,恢复突变株在以脂肪酸为碳源的基础培养基上生长。体外活性测定RsFadD具有脂酰-CoA合成酶活性,对不同链长的脂肪酸都具有活性,但活性低于大肠杆菌FadD。RsfadD突变株在添加不同链长脂肪酸的基础培养上仅能微弱生长,而在丰富培养基上生长无差异。【结论】茄科雷尔氏菌中RsfadD编码脂酰-CoA合成酶,在脂肪酸利用过程中发挥重要作用。但RsfadD突变株在基础培养基上微弱生长,说明茄科雷尔氏菌基因组中还有其他的脂酰-CoA合成酶基因。以上研究结果为进一步研究茄科雷尔氏菌中脂酰-CoA合成酶以及脂肪酸利用机制奠定了基础。     

大肠杆菌3-酮基脂酰ACP还原酶110位天冬酰胺突变后的结构与功能

3-酮基脂酰ACP还原酶催化3-酮基脂酰ACP还原为3-羟基脂酰ACP,是细菌脂肪酸合成反应的关键酶之一.为了明确该酶中110位的保守天冬酰胺残基在酶催化活性和酶结构中的作用,本研究采用基因定点突变和蛋白质表达纯化技术,获得了大肠杆菌3-酮基脂酰ACP还原酶FabG的两个突变蛋白：FabG N110Q和FabG N110L.圆二色谱结果显示,天冬酰胺残基的突变改变了FabG的空间结构,使突变蛋白的α螺旋结构明显增加.以3-酮脂酰ACP为底物的酶活性测定表明,突变蛋白的酶活性均有下降,但残存的酶活性达到了FabG的75%以上.突变蛋白FabG N110Q和FabG N110L具有3-酮基脂酰ACP还原酶的活性,能在体外重建细菌脂肪酸合成反应.对fabG温度敏感突变株的遗传互补分析表明,FabG蛋白110位天冬酰胺突变为谷氨酰胺或亮氨酸后,在一定的条件下仍能互补大肠杆菌的生长.本研究结果提示,FabG 110位的天冬酰胺残基不是参与3-酮基脂酰ACP还原酶催化反应的必需氨基酸,它只是作为结构氨基酸,在维持FabG的空间结构的稳定性方面起作用.     

Pseudomonas plecoglossicida NyZ12基因无痕敲除方法的建立

旨在建立一种适合环己胺降解菌NyZ12基因无痕敲除的可靠方法。通过overlapping PCR技术将目的基因上下游同源臂融合并克隆到自杀载体pEX18km上,将重组质粒转化到大肠杆菌S17pir中,再通过接合转移到假单胞菌NyZ12菌株内,经pEX18km质粒上sacB基因的反向筛选得到突变株并通过PCR方法和测序鉴定。结果显示,成功构建了假单胞菌NyZ12菌株orf4637的基因突变株(NyZ12Δ4637)。通过自杀载体同源重组可以成功获得敲除的无痕突变株,且突变株基因组上没有任何抗性筛选标记残留,为环己胺降解菌NyZ12基因功能研究提供了可靠的基因敲除技术。     

荧光假单胞菌M18的rpoS基因克隆及其功能分析

从荧光假单胞菌 (Pseudomonasfluorescentsp .)M1 8基因组中克隆了RNA聚合酶的稳定期σs 因子编码基因rpoS ,推测其氨基酸序列与铜绿假单胞菌、荧光假单胞菌和恶臭假单胞菌的同源性分别为 99 1 %、87 35 %和87 8%。利用体外定点插入突变和同源重组技术 ,构建了M1 8的rpoS突变株M1 8R- 。对突变株M1 8R- 合成抗生素吩嗪 1 羧酸 (PCA)和藤黄绿菌素 (Plt)的动力学分析结果表明 ,在KB或PPM培养基中 ,突变株合成PCA的能力比野生型分别提高了 2 5或 5 78倍 ,但Plt的积累量不受影响。与野生型相比 ,突变株对碳源饥饿的耐性下降。同时 ,在碳源饥饿条件下对过氧化氢、乙醇和和氯化钠等环境胁迫的交叉保护性减小 ,存活率显著降低     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

天蓝色链霉菌中长链3-酮脂酰ACP合成酶的功能

【背景】链霉菌属于放线菌科,在土壤环境中广泛分布。链霉菌具有复杂的形态分化和多样性的次生代谢网络,能产生大量具有生物活性的次级代谢产物,被广泛深入研究。【目的】天蓝色链霉菌是链霉菌的模式菌株,其脂肪酸合成代谢与次级代谢联系紧密,但目前脂肪酸合成代谢途径还不清楚,其长链3-酮脂酰ACP合成酶还未见报道。【方法】利用大肠杆菌FabF序列进行同源比对,发现天蓝色链霉菌A3(2)的基因组中,SCO2390(ScoFabF1)、SCO1266(ScoFabF2)、SCO0548(ScoFabF3)和SCO5886 (ScoRedR)具有较高的相似性,并具有保守的Cys-His-His催化活性中心,可能具有长链3-酮脂酰ACP合成酶活性。采用PCR扩增方法分别获得以上基因,连入表达载体pBAD24M后分别互补大肠杆菌fabB(ts)突变株和fabB(ts)fabF双突变株,并检测转化子的生长情况。以上基因与pET-28b连接后,在大肠杆菌BL21(DE3)中表达,并利用Ni-NTA纯化获得蛋白,体外测定其催化活性。将以上基因分别互补大肠杆菌fabF突变株后,GC-MS测定互补株的脂肪酸组成。【结果】4个同源基因中,只有ScofabF1能恢复fabB(ts)fabF双突变株42°C时在添加油酸条件下的生长,其他3个基因均不能恢复生长。而这4个基因都不能恢复fabB(ts)突变株42°C时生长。体外活性测定ScoFabF1具有长链3-酮脂酰ACP合成酶活性,其他3个蛋白都不具有该活性。仅ScofabF1能显著提高大肠杆菌fabF突变株的顺-11-十八碳烯酸(C18:1)比例,其他3个基因都不具有该功能。【结论】天蓝色链霉菌中ScofabF1编码长链3-酮脂酰ACP合成酶II,在脂肪酸利用过程中发挥重要作用。天蓝色链霉菌中没有发现编码长链3-酮脂酰ACP合成酶I的基因,其可能通过其他途径合成少量的不饱和脂肪酸。以上研究结果为进一步研究天蓝色链霉菌中脂肪酸合成机制奠定了基础。     

假单胞菌M18 pqsR突变株的构建及其对藤黄绿菌素合成的调控

假单胞菌M18是一株能同时合成藤黄绿脓菌素(Plt)和吩嗪-1-羧酸(PCA)两种抗生物质的植物根际促生细菌。运用PCR方法, 从M18基因组中扩增得到pqsR基因, 该基因编码LysR家族调控蛋白PqsR。通过同源重组技术, 构建假单胞菌M18的pqsR突变菌株M18PRG。比较野生型菌株M18和突变菌株M18PRG在KMB培养基的Plt产量, 发现M18PRG 菌株合成Plt的量约为野生型M18菌株的3~4倍。在pqsR突变株的反式互补实验中, Plt的产量回复到野生型水平。pltA′-′lacZ翻译融合的测定结果进一步证明PqsR对Plt生物合成基因簇具有负调控作用。分析M18野生型及其pqsR突变株的生长曲线, 发现PqsR对细菌的生长具有抑制作用。另外, 我们还发现pqsR基因调控红色色素的产生。上述结果表明, 在假单胞菌M18中, PqsR作为全局性调控因子参与了细胞内多种生理活动的调控。     

野油菜黄单胞菌中3-羟基脂酰ACP脱水酶功能研究

细菌采用II型脂肪酸系统合成脂肪酸,其中3-羟脂酰ACP脱水酶催化唯一的脱水反应,是细菌生长的关键酶之一.野油菜黄单胞菌(Xcc)引起几乎所有十字花科植物的黑腐病,在全球范围内造成广泛的经济损失.为研究Xcc中3-羟脂酰ACP脱水酶,本研究利用大肠杆菌3-羟脂酰ACP脱水酶FabZ序列同源比对时,发现其与XC₂876 (XcfabZ)编码蛋白具有同源性,序列一致性达到46.1%,同时还具有保守的α螺旋结构和活性位点.将XcfabZ异体遗传互补大肠杆菌fabZ(EcfabZ)条件突变株HW7,结果显示添加IPTG能恢复突变株的生长,初步表明XcFabZ具有3-羟脂酰ACP脱水酶活性.而体外活性分析显示,XcFabZ能在脂肪酸合成的起始反应和延伸反应中发挥3-羟脂酰ACP脱水酶活性作用.本研究不能直接获得XcfabZ基因敲除突变株,但将携带EcfabZ或XcfabZ的表达质粒导入后,获得基因替换突变株,证明XcfabZ是必需基因. EcfabZ替换突变株的脂肪酸组成与野生菌有差异,对逆境条件(高盐、低pH、H₂O₂和SDS)...     

假单胞菌M18调控基因ppbR的克隆及功能研究

假单胞菌(Pseudomonas sp.)M18是促进植物生长的根际细菌,能产生吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种不同的抗生素.根据生物信息学分析,铜绿假单胞菌PA2572基因编码蛋白可能是一个双元调控系统的应答调节子.本研究从假单胞菌M18基因组中扩增出PA2572同源基因片段ppbR,利用体外定点插入突变和同源重组技术构建了M18的ppbR突变株M18P.研究结果表明,突变株M18P在泳动能力和群集运动能力上有显著的下降.突变株合成PCA的能力比野生型有显著的下降,在发酵液中PCA积累量仅为野生型的50%.在KMB培养基中,突变株Plt的积累量和野生型没有显著的差异.     

Foreign exploration for Scirtothrips perseae Nakahara (Thysanoptera: Thripidae) and associated natural enemies on avocado (Persea americana Miller)

Scirtothrips perseae Nakahara was discovered attacking avocados in California, USA, in 1996. Host plant surveys in California indicated that S. perseae has a highly restricted host range with larvae being found only on avocados, while adults were collected from 11 different plant species. As part of a management program for this pest, a “classical” biological control program was initiated and foreign exploration was conducted to delineate the home range of S. perseae, to survey for associated natural enemies and inventory other species of phytophagous thrips on avocados grown in Mexico, Guatemala, Costa Rica, the Dominican Republic, Trinidad, and Brazil. Foreign exploration efforts indicate that S. perseae occurs on avocados grown at high altitudes (>1500 m) from Uruapan in Mexico south to areas around Guatemala City in Guatemala. In Costa Rica, S. perseae is replaced by an undescribed congener as the dominant phytophagous thrips on avocados grown at high altitudes (>1300 m). No species of Scirtothrips were found on avocados in the Dominican Republic, Trinidad, or Brazil. In total, 2136 phytophagous thrips were collected and identified, representing over 47 identified species from at least 19 genera. The significance of these species records is discussed. Of collected material 4% were potential thrips biological control agents. Natural enemies were dominated by six genera of predatory thrips (Aeolothrips, Aleurodothrips, Franklinothrips, Leptothrips, Scolothrips, and Karnyothrips). One genus each of parasitoid (Ceranisus) and predatory mite (Balaustium) were found. Based on the results of our sampling techniques, prospects for the importation of thrips natural enemies for use in a “classical” biological control program in California against S. perseae are not promising.     

Phylogeny of cardinalfishes (Teleostei: Gobiiformes: Apogonidae) and the evolution of visceral bioluminescence

The cardinalfishes (Apogonidae) are a diverse clade of small, mostly reef-dwelling fishes, for which a variety of morphological data have not yielded a consistent phylogeny. We use DNA sequence to hypothesize phylogenetic relationships within Apogonidae and among apogonids and other acanthomorph families, to examine patterns of evolution including the distribution of a visceral bioluminescence system. In conformance with previous studies, Apogonidae is placed in a clade with Pempheridae, Kurtidae, Leiognathidae, and Gobioidei. The apogonid genus Pseudamia is recovered outside the remainder of the family, not as sister to the superficially similar genus Gymnapogon. Species sampled from the Caribbean and Western Atlantic (Phaeoptyx, Astrapogon, and some Apogon species) form a clade, as do the larger-bodied Glossamia and Cheilodipterus. Incidence of visceral bioluminescence is found scattered throughout the phylogeny, independently for each group in which it is present. Examination of the fine structure of the visceral bioluminescence system through histology shows that light organs exhibit a range of morphologies, with some composed of complex masses of tubules (Siphamia, Pempheris, Parapriacanthus) and others lacking tubules but containing chambers formed by folds of the visceral epithelium (Acropoma, Archamia, Jaydia, and Rhabdamia). Light organs in Siphamia, Acropoma, Pempheris and Parapriacanthus are distinct from but connected to the gut; those in Archamia, Jaydia, and Rhabdamia are simply portions of the intestinal tract, and are little differentiated from the surrounding tissues. The presence or absence of symbiotic luminescent bacteria does not correlate with light organ structure; the tubular light organs of Siphamia and chambered tubes of Acropoma house bacteria, those in Pempheridae and the other Apogonidae do not.     

Biological control of three floating water weeds,<Emphasis Type="Italic">Eichhornia crassipes,Pistia stratiotes</Emphasis>, and <Emphasis Type="Italic">Salviniamolesta</Emphasis> in the Republic of Congo

Since 1999, four specific weevils (Coleoptera, Curculionidae) were released in the Republic of Congo against three exotic floating water weeds: Neochetina eichhorniae Warner and N. bruchi Hustache against water hyacinth, Neohydronomus affinis Hustache against water lettuce, and Cyrtobagous salviniae Calder and Sands against water fern. Recoveries of exotic weevils were made from all 24 release sites except one, and all four species have established and spread (up to 800 km for water hyacinth weevils). Within a few years of releases, control of water fern and water lettuce was such that fishing and navigation could be resumed, while reductions of water hyacinth populations were only beginning.     

A molecular phylogeny of Hebeloma species from Europe

In order to widen the scope of existing phylogenies of the ectomycorrhizal agaric genus Hebeloma a total of 53 new rDNA ITS sequences from that genus was generated, augmented by sequences retrieved from GenBank, and analysed using Bayesian, strict consensus and neighbour joining methods. The lignicolous Hebelomina neerlandica, Gymnopilus penetrans, and two species of Galerina served as outgroup taxa. Anamika indica, as well as representatives of the genera Hymenogaster and Naucoria, were included to test the monophyly of Hebeloma, which is confirmed by the results. Hebeloma, Naucoria, Hymenogaster and Anamika indica cluster in a strongly supported monophyletic hebelomatoid clade. All trees largely reflect the current infrageneric classification within Hebeloma, and divide the genus into mostly well-supported monophyletic groups surrounding H. crustuliniforme, H. velutipes, H. sacchariolens, H. sinapizans, and H. radicosum, with H. sarcophyllum being shown at an independent position; however this is not well supported. The section Indusiata divides with strong support into three groups, the position of the pleurocystidiate Hebeloma cistophilum suggests the possible existence of a third subsection within sect. Indusiata. Subsection Sacchariolentia is raised to the rank of section.     

Taxonomic status of the species of the green algal genusNeochloris

Komárek has recently reviewed the various species assigned to the green algal genusNeochloris Starr (Chlorococcales, Chlorococcaceae) and removed those with uninucleate vegetative cells to a new genus,Ettlia. Watanabe & Floyd, unaware ofKomárek's work, also reviewed the species ofNeochloris and distributed them among three genera—Neochloris, Chlorococcopsis gen. nov., andParietochloris gen. nov.—on the basis of details of the covering of the zoospore and the arrangement of the basal bodies of the flagellar apparatus. This paper reconciles these two treatments and makes additional recommendations at the ranks of genus, family, order, and class.     

Die GattungKarschia — Bindeglied zwischen bitunicaten Ascomyceten und lecanoralen Flechtenpilzen?

The genusKarschia, in the earlier sense, including saprophytes and parasites on lichens, has been thought to be a non-lichenized parallel genus of the lichen genusBuellia. Modern workers included it on the one hand inBuellia, on the other hand combined it with bitunicate ascomycetes. It is now proved thatKarschia is heterogeneous and contains but superficially similar members both of the genusBuellia of theLecanorales and of typical or masked bitunicateAscomycetes. Therefore, it can not be regarded as a link betweenLecanorales andDothideales. The type species ofKarschia belongs to theDothideales.

     

Demographic characteristics of an intertidal bay goby (Lepidogobius lepidus)

Synopsis In a fourteen month study (May 1976 – June 1977) I examined the following characteristics of an intertidal bay goby (Lepidogobius lepidus) in Morro Bay, California, U.S.A.: annual and seasonal patterns of abundance, age composition and growth rates, survivorship and mortality patterns, and the reproductive cycle for female gobies. Fishes were collected with the aid of quinaldine and otoliths and ovaries removed. Age and growth rates were estimated from otolith annuli using a back calculation formula and a Brody-Bertalanffy growth curve. Mortality rates were derived using the methods of Heincke (1913), Robson & Chapman (1960), mean age, and a catch curve (Ricker 1975). A gonad index was used to describe the annual reproductive cycle. Results indicated that abundance fluctuated seasonally and that these fluctuations appeared to be caused by reproductive emigrations. Bay gobies reached an age of 7+ and a standard length of 87 mm. Growth was relatively constant (6 mm yr⁻¹) until age 5, at which point it began to decline. The mean rates of survivorship, mortality, and instantaneous mortality were 0.75, 0.25, and 0.29 respectively. Mortality rates for individual age classes ranged from 0.13 to 0.51 and increased with age. This stock appears to reproduce mainly during the winter.     

Systematic significance of mature embryo of bamboos

The mature embryo of seven species belonging to five genera of Indian bamboos is described. In all these the basic pattern of embryo organisation is same: the scutellar and coleoptilar bundles are not separated by an internode, the epiblast is absent, the lower portion of the scutellum and the coleorhiza are separated by a cleft and the margins of embryonic leaves overlap. The features unique to fleshy fruited bamboos are: presence of a massive scutellum, the juxtaposition of plumule and radicle and the occurrence of a bud in the axil of the coleoptile. The fleshy fruit bearing bamboos should be classified into one group, the tribeMelocanneae. Evidence is provided to recognise additional groups in the subfamilyBambusoideae.