嗜线虫致病杆菌产生抗生素的培养基及条件

本对嗜线虫致病杆菌（Xenorhadbus nematophilus）产生抗生素的发酵培养基和发酵条件进行了研究,同时对该菌代谢过程pH值、还原糖、总糖、氨基氮与抗生素产量的关系进行了分析,通过筛选该菌对碳源和氮源的要求,用正交试验初步确定了该菌产素的最佳发酵培养基和条件为：玉米粉1％,大豆粉3％,蔗糖1％,蛋白胨1.5％,KH2PO40.02％,MgSO40.2％,活化剂T0.1％;发酵培养基的起始pH值在6.0－8.0,种龄16h,接种量4％,500mL摇瓶装量15－150mL的条件下培养72h可获得较高的抗生素产量;产素量与菌代谢过程中pH、还原糖、总糖和氨基氮的变化有一定关系,通过培养基和培养条件的研究使该菌的产抗生素能力提高了56.3％。     

嗜线虫致病杆菌HB310菌株杀虫蛋白的纯化及活性鉴定

嗜线虫致病杆菌Xenorhabdus nematophila HB310是从河北省土壤中筛选出的一株昆虫病原线虫体内分离纯化获得的共生菌,该菌的发酵液对多种昆虫有较高的杀虫活性。利用85％饱和度的硫酸铵盐析分别获得胞内蛋白提取物和上清液中胞外蛋白提取物,生测结果表明这两种蛋白提取物中都含有胃毒素和血腔毒素。通过制备型非变性凝胶电泳对蛋白提取物进行分离和纯化,得到了3种有杀虫活性的毒素蛋白（毒素Ⅰ、毒素Ⅱ和毒素Ⅲ）,胞内的毒素蛋白与分泌到胞外上清液中的毒素蛋白是同种蛋白。毒素Ⅰ和毒素Ⅱ对棉铃虫初孵幼虫有明显的胃毒活性,但没有血腔毒性;毒素Ⅲ对大蜡螟幼虫有很强的血腔毒性,LD₅₀为0.18 μg/头。SDS-PAGE图谱显示毒素Ⅰ和毒素Ⅱ是由多个多肽组成的复合蛋白,而毒素Ⅲ只分离出一条多肽。毒素Ⅱ在50℃处理10 min,其杀虫活性没有显著变化;70℃处理10 min对毒素Ⅲ杀虫活性没有显著影响。     

嗜线虫致病杆菌CB6菌株培养特性的初步研究

研究了嗜线虫致病杆菌(Xenorhabdus nematophila)CB6菌株的菌体增殖规律及对主要营养成分的利用规律。结果表明,该菌延缓期、对数生长期、稳定期、衰亡期分别为0~6h、6~18h、18~48h和48h以后。培养18h时,糖和蛋白质的含量达到最低并保持稳定;氨基氮含量在6h时最低,以后逐渐升高,36h达到最高。药效实验表明,该菌培养42h时其菌悬液杀虫活性最高。     

嗜线虫致病杆菌产生抗生素的培养基及条件*

本文对嗜线虫致病杆菌 (Xenorhabdus nematophilus)产生抗生素的发酵培养基和发酵条件进行了研究 ,同时对该菌代谢过程 p H值、还原糖、总糖、氨基氮与抗生素产量的关系进行了分析。通过筛选该菌对碳源和氮源的要求 ,用正交试验初步确定了该菌产素的最佳发酵培养基和条件为 :玉米粉 1% ,大豆粉 3% ,蔗糖 1% ,蛋白胨 1.5% ,KH₂ PO₄ 0 .0 2 % ,Mg SO₄ 0 .2 % ,活化剂     

嗜线虫致病杆菌HB310培养基筛选和培养条件优化

嗜线虫致病杆菌Xenorhabdus nematophila是与小卷蛾斯氏线虫Steinernema carpocapsae互惠共生的一种革兰氏阴性菌,对多种农业害虫都具有很高的杀虫活性。本研究对多种影响嗜线虫致病杆菌HB310菌株发酵培养的因素进行了筛选优化。通过单因素试验确定最佳碳源、氮源和无机盐分别为葡萄糖、牛肉蛋白胨和KH2PO4;通过正交实验确定培养基的最优组合为：葡萄糖2 %、牛肉蛋白胨1 %、牛肉浸膏0.3% 和K2HPO4.1 %;最佳摇瓶培养条件为：接种量6%、培养基pH 7.5、摇床转速200 rpm、培养温度28℃和培养时间48 h,在此条件下,嗜线虫致病杆菌HB310菌株生长速率最快,菌液的杀虫活性最高。     

嗜线虫致病杆菌与七种杀虫剂混配对小菜蛾毒杀增效作用评价

采用浸叶法测定了嗜线虫致病杆菌Xenorhabdus nematophila HB310(Xn HB310)与7种常用杀虫剂混配后对小菜蛾3龄幼虫的毒杀增效作用,并对具有增效作用的混配组合进行了最佳配比的筛选和增效作用的评价.结果显示,Xn HB310仅与氯氰菊酯混配表现出明显的增效作用,协同毒力指数(cf)为30.9...     

昆虫病原线虫共生细菌致病机理的研究进展

昆虫病原线虫共生细菌是寄生于昆虫病原线虫肠道内的一种细菌,革兰氏染色呈阴性,属肠杆菌科(Ｅｎｔｅｒｏｂａｃｔｅｒｉａｃｅａｅ)细菌[1]。它包含两个属———嗜线虫致病杆菌属(Ｘｅｎｏｒｈａｂｄｕｓ)和发光杆菌属(Ｐｈｏｔｏｒｈａｂｄｕｓ),它们分别与斯氏线虫(Ｓｔｅｉｎｅｒｎｅｍａ)和异小杆线虫(Ｈｅｔｅｒｏｒｈａｂｄｉｔｉｓ)共生。这两种线虫由于杀虫能力强,是最有应用潜力的昆虫病原线虫。它们之间的共生关系可以概括为:共生菌存在于线虫的肠道内,线虫携带共生菌进入寄主昆虫体内,并将共生菌释放到昆虫的血腔中;共生菌在昆…     

嗜线虫致病杆菌Xna基因同源重组载体的构建及遗传转化体系的建立

构建嗜线虫致病杆菌Xna基因的同源重组载体和建立该细菌的遗传转化体系.扩增嗜线虫致病杆菌XNA基因的左右同源片段及质粒PEASY-T1上的卡那抗性基因Km;利用重叠延伸引物PCR法将这3个片段连接起来,然后将其克隆至自杀性载体PJQ200.采用电激法将同源重组载体DNA转化到供体细菌,再利用二亲本结合转移方法将供体细菌中的载体转化到受体细菌CB6菌株.结果成功地扩增了Xna基因的左右同源臂及卡那抗性基因的全长融合片段、构建了Xna基因的同源重组载体,并建立了嗜线虫致病杆菌北京变种的遗传转化体系.     

嗜线虫致病杆菌血腔毒素Tp40的纯化和特性分析

[目的]嗜线虫致病杆菌是一种昆虫病原线虫共生菌,它能够产生多种杀虫毒素.本研究旨在从嗜线虫致病杆菌Xenorhabdus nematophila HB310菌株的细胞内纯化新的杀虫蛋白毒素,并对其进行基因克隆和序列分析.[方法]应用盐析和制备型非变性凝胶电泳等方法纯化蛋白,再通过对5龄大蜡螟幼虫血腔注射进行活性筛选.对获得的目的蛋白与已知蛋白进行同源分析,克隆出该目的蛋白的基因序列,从而进行相应的基因和氨基酸序列分析.[结果]本研究纯化的Tp40蛋白对大蜡螟LD50为68.54 ng/头,其SDS-PAGE电泳图谱只显示出一条分子量约为42 kDa的多肽.Western印迹分析表明Tp40与已知的Txp40为同源蛋白,并且仅存在于细胞内.编码该蛋白的基因开放读码框全长1107bp(GenBank登录号:EU095326),编码368个氨基酸残基,预测分子量为41.5 kDa,等电点为8.66,与GenBank中的其余13株昆虫病原线虫共生菌所包含的相似基因核苷酸序列及推导的氨基酸序列比较,同源性分别为85%～99%和70%～99%.[结论]Tp40蛋白具有很高的血腔杀虫活性,其基因序列具有较强的保守性,是昆虫病原线虫共生菌复合体杀虫过程中的一种关键因子.     

微杆菌Sneb159杀线虫活性物质的分离与鉴定

【目的】有关Microbacterium maritypicum在植物线虫生物防治方面的研究较少,探究菌株M. maritypicum Sneb159的杀线虫活性,明确菌株发酵液中具有杀线虫活性的物质,为生物农药的开发提供理论依据。【方法】本研究检测了菌株Sneb159发酵液对大豆胞囊线虫二龄幼虫的触杀活性;并以触杀活性为追踪,采用有机溶剂萃取、硅胶柱层析及半制备高效液相色谱等技术对菌株Sneb159发酵滤液中的活性物质进行分离纯化;采用核磁共振波谱对纯化物进行结构鉴定。【结果】菌株Sneb159具有杀线虫活性,在24 h和48 h处理组中线虫的死亡率均显著高于对照组。从菌株Sneb159发酵滤液中分离得到杀线虫活性物质A6,经结构鉴定确定该物质为苯乙酰胺。【结论】首次发现M. maritypicum对大豆胞囊线虫的触杀作用,并且明确活性物质为苯乙酰胺。结果表明菌株M. maritypicum Sneb159和苯乙酰胺在大豆胞囊线虫的生物防治方面具有较好的应用潜力。     

Xenorhabdus nematophila发酵动力学研究

在分批发酵中,研究了Xenorhabdus nematophila YL001的生长、基质消耗及抗菌物质产生的特性.基于Logistic方程和Luedeking-Piret方程,得到了描述分批发酵过程的动力学模型及模型参数,同时对实验数据与模型进行了验证比较.模型计算值与实验数据拟合良好,模型基本反映了Xenorhabdus nematophila YL001分批发酵过程的动力学特征.分批发酵中细胞生长与产物合成属于偶联型.     

A toxin protein from Xenorhabdus nematophila var. pekingensis and insecticidal activity against larvae of Helicoverpa armigera

Xenorhabdus nematophila var. pekingensis, which is highly virulent for many insects, is a symbiotic bacterium of Steinernema carpocapsae isolated from Beijing soil in China. Previous studies demonstrated that the bacterium had high antifeedant activity against larvae of Helicoverpa armigera, Plutella xylostella and Spodoptera exigua. Herein, we report the purification, molecular cloning and antifeedant activity of an intracellular toxic protein from the bacterium. The purified protein displayed a single band and a relative molecular weight of over 212 kDa determined by SDS-PAGE. We designated the protein as XnAFP2. Peptide segments were obtained by MALDI-TOF and covered 40% of the amino acid sequence of a toxin protein from X. nematophilus PMFI1296. The full cDNA sequence encoding for XnAFP2 (Genbank accession number FJ222606) was amplified from X. nematophlia var. pekingensis and consists of 7575 bp. The gene showed homology with up to 99% identity to the A2 gene from X. nematophila strain BP (GenBank accession number AY282763) and 92% identity to the insecticidal toxin xptA2 gene from X. nematophila PMFI 1296 (GenBank accession number AJ308438). The protein caused a rapid cessation in feeding and reduction in larval weight of H. armigera. When fed to third instar larvae of H. armigera in an artificial diet at 6.0 µg/g (w/w) toxin protein, growth reduction reached 97.9%. The insecticidal protein greatly decreased fourth instar larval weight, lengthened larval stage, and reduced pupation and emergence rates. The antifeedant rate in choice and no-choice leaf disk tests against fifth instar larvae was 78.4 and 87.6% in 24 h, respectively.     

培养方式对Xenorhabdus nematophila生长与抗菌活性的影响

为了明确不同培养方式对Xenorhabdus nematophilaYL001生长和抗菌活性的影响,提高YL001菌株的抗菌活性。采用分批发酵方式研究了摇瓶与发酵罐培养对置nematophila YL001生长和抗菌活性的影响。实验结果表明：在通气量2．5L／min、搅拌转速300r／min条件下,发酵罐的体积氧传递系数KLa明显高于摇瓶,通气及供氧状况好于摇瓶,细胞生长及代谢旺盛,细胞生长量较摇瓶发酵增加了23．9％;但由于pH变化幅度大,抗菌物质的活性单位仅达到摇瓶的发酵水平,为229U／mL。发酵罐pH控制初步研究表明,发酵过程中控制pH为7．5时,细胞生长量和抗菌活性达到23．71g/L和290U／mL,较不控制pH分别增加了21％和25％。通风对X.nematophila YL001生长和抗菌物质的产生有较大的影响,发酵罐培养时通气及供氧状况好于摇瓶,有利于YL001菌株的生长和抗菌物质的产生。     

一株高毒力致病杆菌CB6的鉴定

从北京郊区果园采集的小卷蛾斯氏线虫（Steinernema carpocapsae）肠道内分离到一株具有较强杀虫和抑菌活性的致病杆菌菌株CB6。形态特征及生理生化特征测定结果表明,CB6菌株与致病杆菌属（Xenorhabdus）中的嗜线虫致病杆菌（X. nematophila）种的特征基本一致。测定了该菌株的16S rRNA序列并根据16S rRNA序列构建了系统发育树;在系统发育树中,CB6菌株与嗜线虫致病杆菌其他4个菌株形成一个类群,序列同源性大于99%。但CB6菌株的酪氨酸酶、脂酶（蛋黄）的产生、核糖产酸等生化特征与嗜线虫致病杆菌种内的其他菌株存在一定的差异,且具有更强的杀虫和抑菌活性。因此认为CB6菌株是嗜线虫致病杆菌的一个变种,命名为嗜线虫致病杆菌北京变种（X. nematophila var. pekingensis）。     

Optimization of fermentation condition for antibiotic production by Xenorhabdus nematophila with response surface methodology

Aims: To evaluate the influence of environmental parameters on the production of antibiotics (xenocoumacins and nematophin) by Xenorhabdus nematophila and enhance the antibiotic activity. Methods and Results: Response surface methodology (RSM) was employed to study the effects of five parameters (the initial pH, medium volume in flask, rotary speed, temperature and inoculation volume) on the production of antibiotics in flask cultures by X. nematophila YL001. A 2^5?1‐factorial central composite design was chosen to explain the combined effects of the five parameters and to design a minimum number of experiments. The experimental results and software‐predicted values of production of antibiotics were comparable. The statistical analysis of the results showed that, in the range studied, medium volume in flask, rotary speed, temperature and inoculation volume had a significant effect (P < 0·05) on the production of antibiotics at their individual level, medium volume in flask and rotary speed showed a significant influence at interactive level and were most significant at individual level. The maximum antibiotic activity was achieved at the initial pH 7·64, medium volume in 250 ml flask 25 ml, rotary speed of 220 rev min^?1, temperature 27·8°C and inoculation volume of 15·0%. Maximum antibiotic activity of 331·7 U ml^?1 was achieved under the optimized condition. Conclusions: As far as known, there are no reports of production of antibiotic from X. nematophila by engineering the condition of fermentation using RSM. The results strongly support the use of RSM for fermentation condition optimization. The optimization of the environmental parameters resulted not only in a 43·4% higher antibiotic activity than unoptimized conditions but also in a reduced amount of the experiments. The chosen method of optimization of fermentation condition was efficient, relatively simple and time and material saving. Significance and Impact of the Study: This study should contribute towards improving the antibiotics activity of X. nematophila. Integrated into a broader study of the impact of environmental factors on the production of antibiotic, this work should help to build more rational control strategy, possibly involving scale‐up of production of antibiotics by X. nematophila.     

Xenorhabdus nematophila BP杀虫毒素基因的序列分析及其杀虫活性

XnBP83是从Xenorhabdus nematophila BP基因组粘粒文库中筛选出的一个对棉铃虫有较强口服杀虫活性的克隆．采用亚克隆结合primer-walking DNA测序技术对粘粒XnBP83的插入片段进行序列测定．该插入片段全长38939bp,其中包括5个与杀虫活性相关的tc类基因xptA1、xptB1、xptC1、xptA2、xptD1．序列分析显示：a．插入片段中的xptD1不完整,与X．nematophila PMFI296 XptD1相应氨基酸序列有99％的相似性．b．BP xptA1读码框全长7569bp,编码2520个氨基酸,与PMFI296的XptA1氨基酸序列有98％的相似性,两者在第2200-2223氨基酸区域连续有23个氨基酸不同．c．BP xptB1读码框全长3051bp,编码1016个氨基酸,与PMFI296 XptB1氨基酸序列有98％的相似性,在第620-650氨基酸之间有28个氨基酸差异．d．BP xptC1读码框全长4225bp,编码1408个氨基酸,与PMFI296的XptC1氨基酸序列有96％的相似性．在BP的第232氨基酸后插入了一个TAQRYLAK的氨基酸序列,在第627-646氨基酸区域内,有18个氨基酸不同．e．BP xptA2读码框全长7574bp,编码2524个氨基酸,与PMFI296的XptA2氨基酸序列有90％的相似性,在BP品系XptA2的第788-855氨基酸和第1630～1784氨基酸有两个明显变异区．将XnBP83培养物上清和沉淀饲喂棉铃虫、甜菜夜蛾、斜纹夜蛾和粉纹夜蛾,结果表明XnBP83对所测昆虫有广谱杀虫活性．     

Toxic activity of a protein complex purified from Xenorhabdus nematophila HB310 to Plutella xylostella larvae

Abstract Xenorhabdus nematophila, a Gram‐negative proteobacterium belonging to the family Enterobacteriaceae and associated symbiotically with soil entomopathogenic nematodes, Steinernema carpocapsae, is pathogenic to a wide range of insects. A protein complex with insecticidal activity was isolated from the cells of X. nematophila HB310 strain using methods of salting out and native polyacrylamide gel electrophoresis (PAGE). Seven polypeptides ranging 50～250 kDa were well separated from the protein complex (named Xnpt) by sodium dodecyl sulfate (SDS)‐PAGE, five of which are identified as XptA2, xptC1, XptB1, GroEL and hypothetical protein by matrix‐assisted laser desorption‐time‐of‐flight mass spectrometry (MALDI‐TOFMS). Xnpt showed high oral virulence to larvae of diamondback moth (DBM), Plutella xylostella L. (Lepidoptera, Plutellidae) as its median lethal concentration (LC₅₀) against second and third instar larvae were 331.45 ng/mL and 553.59 ng/mL at 72 h, respectively. The histological analysis of Xnpt‐fed DBM larvae showed extensive histopathological effects on the midgut. Biochemical analysis indicated that Xnpt markedly inhibited the activities of three important enzymes in the midgut. Overall, our data showed that the protein complex isolated from X. nematophila HB310 induced the antifeedant and death of insects by destroying midgut tissues and inhibiting midgut proteases activities.