恒定磁场对苏云金芽孢杆菌的生长及毒力的影响

目的:研究恒定磁场对苏云金芽孢杆菌的生长和毒力的影响.方法:通过0-100 Gs的恒定磁场下对苏云金芽孢杆菌G-02、HD-1菌株的发酵,测定其生长和毒力的变化.结果:G-02、HD-1菌株分别在磁场强度43Gs和14Gs条件下发酵48h后的芽孢量和毒力达到最高,芽孢增长比分别为1.26和2.0,毒力增效比分别为1.27和1.25,苏云金芽孢杆菌G-02适宜的磁场强度为43-50Gs,HD-1菌株为7-29Gs.结论:低磁场强度对两种菌株的生长有一定的促进作用,而高强度磁场会对其生长产生抑制作用.     

苏云金芽孢杆菌的紫外线诱变及筛选实验初步设计

苏云金芽孢杆菌(Bacillus thuringiensis)由于其自身特点,由其制成的杀虫剂已成为目前微生物防治害虫的重要手段之一。虽然苏云金芽孢杆菌制剂目前应用效果良好,但也存在一些隐患:长期使用该制剂会使害虫抗性增加,因此寻找新的高毒力菌株势在必行。本文以苏云金芽孢杆菌的库尔斯塔克亚种为研究对象,设计实验以紫外线诱变的方法获得毒力更强的菌种。     

苏云金芽孢杆菌发酵液中晶体蛋白定量测定方法的探讨

探索了一种苏云金芽孢杆菌发酵液中晶体蛋白化学定量测定的方法。发酵液经离心技术处理后,其晶体蛋白由还原剂、变性剂组成的裂解液溶解,再用紫外光谱吸收法或考马斯亮蓝染色法定量。该方法测得的晶体蛋白量与经生物测定得到的毒力之间有较好的相关性,在苏云金芽孢杆菌的研究和生产上有一定的实用价值。     

苏云金芽孢杆菌转座因子研究进展

近年来的研究发现苏云金芽孢杆菌转座因子和许多毒力因子可能是紧密联系的。由于转座因子的特殊性质,使它们在现代农业生物技术中有着广泛的应用前景,科学家对苏云金芽孢杆菌转座因子的研究也在不断深入。本文主要针对苏云金芽孢杆菌转座因子的研究进展进行综述,并对发展前景进行展望。     

从湖北神农架原始森林土壤中分离的高毒力苏云金芽孢杆菌

从神农架原始森林土壤中分离出苏云金芽孢杆菌 9株。经过生理生化和血清学鉴定 ,此 9株苏云金芽孢杆菌分属于H7、H6和H14。生物测定结果表明 :两株H7型菌株对棉铃虫幼虫有较高的毒力 ;另两株对致倦库蚊幼虫和白纹伊蚊幼虫有很强的毒杀作用 ,此两株属苏云金芽孢杆菌H14。     

单宁对苏云金芽孢杆菌库斯塔克变种杀棉铃虫效力的作用(英文)

本文报道棉铃虫、苏云金芽孢杆菌库斯塔克变种和单宁三者间的相互作用。苏云金芽孢杆菌芽孢晶体混合粉以6个不同的浓度（0％,0．005％,0．01％,0．015％,0．02％,0．025％湿重）分别加入含0．025％单宁和不含单宁的人工饲料中。初孵幼虫分别在上述饲料中饲养48h后,转入相应的不含苏云金芽孢杆菌的人工饲料上饲养,直到留存的幼虫化蛹。结果表明,单宁与苏云金芽孢杆菌混用可提高苏云金芽孢杆菌对棉铃虫的毒力,LD50降低45％,并可显著抑制该虫的生长发育,但二者在抑制生长发育方面无交互作用。选择取食试验显示,苏云金芽孢杆菌对五龄幼虫有显著的抑食性,但单宁无此作用,单宁与苏云金芽孢杆菌混用不会增强抑食性,但单宁能抑制苏云金芽孢杆菌菌落的生长,浓度高于15mg／100ml还能抑制芽孢的萌发。     

农药助剂对苏云金杆菌毒力的影响及新液剂研制

研究了２０种化学物质对苏云金杆菌的毒力影响,分析了由乳化剂、湿润剂、防腐剂配制的几种不同农药助剂组合与该菌芽孢晶体混合物在不同贮存期的毒力变化及芽孢存活率。从供试的助剂组合中获得了一种最佳组合,其各项指标均优于中华人民共和国农业行业标准中所规定的各项要求,可发展成一种新的苏云金芽孢杆菌液剂。     

苏云金芽孢杆菌双组份信号转导系统的生物信息学分析

苏云金芽孢杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白等多种活性成分,是目前应用最广泛的微生物杀虫剂。本文采用生物信息学方法,系统分析了由本实验室完成全基因组测序的苏云金芽孢杆菌YBT-1520、CT-43和BMB171 3个菌株的双组分信号转导系统(Two-componentsignal transduction system,TCS)的分布、结构及功能,并初步构建了部分TCS的调控网络关系图。本研究旨在为深入研究苏云金芽孢杆菌的生长、代谢以及毒力因子的表达与调控,全面了解伴孢晶体的形成机制开辟新的研究方向。     

管蝶法测定苏云金杆菌上清液中增效物质含量的研究

以草生欧文氏杆菌LS005为指示菌,用管蝶法测定苏云金杆菌KN－11发酵液中增效物质的相对浓度。结果表明管蝶法和生物测定法在测定增效物质的代谢曲线有较好的一致性,均表明,菌株KN－11的增效物质城营养体时（约9h）开始产生,到芽孢初步形成时（约20h）已达到最高值的80％以上,之后是一个缓慢的积累过程。     

一种快速分离检测产脂肽类生物表面活性剂枯草芽孢杆菌的方法

脂肽(Lipopeptide)是由枯草芽孢杆菌(Bacillus subtilis)等微生物产生的一类具有较强表面活性的生物表面活性剂.枯革杆菌磷酸泛酰巯基转移酶基因(afp)是枯草芽孢杆菌中参与脂肽代谢的功能性基因.采用sfp基因PCR对从环境中得到的一组产生表面活性剂的微生物进行筛选,结合Tricine-SDS-PAGE电泳对PCR结果呈阳性的菌蛛的代谢粗初提物进行检测,初步鉴定得到两株枯草芽孢杆菌.进一步利用16S rDNA序列的系统发育学分析确定这两种菌株为枯草芽孢杆菌,并利用TLC、HPLC鉴定其产物为脂肽类表面活性剂,从而建立了一套快速分离检测产生脂肽类生物表面活性剂的枯草芽孢杆菌方法.     

Urea-mercaptoethanol-soluble protein from spores of Bacillus thuringiensis and other species

Treatment with urea-mercaptoethanol of purified spores of Bacillus thuringiensis, other Bacillus species, and Clostridium roseum solubilizes a protein fraction between 5 and 12% of the dry weight of the spores. This fraction behaves identically to the crystal protein of B. thuringiensis on acrylamide-gel electrophoresis. The protein from all of the Bacillus species shows partial homology with crystal protein, using the Ouchterlony immunodiffusion technique. A further fraction, similar in amount, can be removed from spores of B. thuringiensis by the addition of sodium lauryl sulfate to the urea-mercaptoethanol. Spores of B. thuringiensis extracted in these ways show no difference when compared to untreated spores with respect to viability or resistance to heat and ultraviolet-irradiation. The extracted spores do show differences in their germination requirements and their susceptibility to phase-darkening by lysozyme. It is concluded that an urea-mercaptoethanol-soluble protein or class of protein is a widespread component of bacterial spores, possibly located in the spore coat, and that this protein may be related to the crystal protein of B. thuringiensis.     

Arbitrary primer polymerase chain reaction, a powerful method to identify Bacillus thuringiensis serovars and strains.

Arbitrary primer polymerase chain reaction technology has been applied to the identification of commercial strains of Bacillus thuringiensis by using total DNAs extracted from single bacterial colonies as templates. Characteristic DNA banding patterns can be readily and reproducibly obtained by agarose gel electrophoresis. This method has been used to distinguish commercial products containing B. thuringiensis serovar kurstaki (3a3b). When a single primer was used this method was capable of producing discriminating DNA fingerprints for 33 known serovars. Differentiation from the closely related species Bacillus cereus is also readily achieved. This technique should prove to be a powerful tool for identification and discrimination of individual B. thuringiensis strains.     

Transcription of gene in an acrystalliferous strain of Bacillus thuringiensis XBU001 positively regulated by the metalloprotease camelysin gene at the onset of stationary phase

The calY gene, encoding metalloprotease camelysin in the Bacillus thuringiensis acrystalliferous strain XBU001, was amplified and sequenced. The camelysin from the calY sequence was 199 amino acids in size (c. 22?000?Da). The temperature-sensitive plasmid pKESX was used to construct a metalloprotease camelysin-deficient strain of B. thuringiensis. The calY gene was replaced by an erythromycin-resistant gene in KCTF. Sodium dodecyl sulfate polyacrylamide gel electrophoresis and MS analysis showed that the metalloprotease InhA was not expressed after knocking out the gene calY. The temperature-sensitive plasmid pKPC was used to construct a metalloprotease camelysin complementation strain KCTFC. The InhA protein was found in KCTFC. Analysis of the expression of InhA in the wild-type strain KCTF12, camelysin-deficient and complementation strains indicated that inhA expression depended on camelysin. Although camelysin did not directly regulate the expression of the InhA through binding to the promoter of the inhA, the results suggest that camelysin can positively regulate the expression of the InhA protein.     

抗水稻黄单胞菌的苏云金芽胞杆菌菌株筛选及其 活性物质

【目的】从400株苏云金芽胞杆菌菌株中筛选出拮抗水稻黄单胞菌活性最好的菌株YBT-2532,并对其抑菌活性物质进行分离。【方法】对苏云金芽胞杆菌YBT-2532产生的活性物质理化特性进行测定。【结果】该活性物质对温度、蛋白酶、pH均不敏感,70 °C处理1 h仍保留有75%的活性;活性物质在pH 2.0?12.0较稳定;该活性物质溶于甲醇、微溶于乙醇、不溶于丙酮、二氯甲烷和氯仿。利用凝胶过滤、离子交换层析、固相萃取、高效液相色谱技术,对抑菌组分进行分离,并通过HPLC-IT-MS方法确定其分子量。纯化的活性组分是一种分子量为797.8 Da的强极性水溶性小分子。【结论】该活性物质性质与已知的来源于苏云金芽胞杆菌的抗菌活性物质不同,可能为新型抗菌物质。     

Measurement of 25-hydroxyvitamin D in the clinical laboratory: Current procedures, performance characteristics and limitations

In this review we describe procedures, performance characteristics and limitations of methods available for the measurement of 25-hydroxyvitamin (25OHD) since the year 2000. The two main types of methods are competitive immunoassay and those based on chromatographic separation followed by non-immunological direct detection (HPLC, LC-MS/MS). Lack of a reference standard for 25OHD has, until recently, been a major issue resulting in poor between-method comparability. Fortunately this should soon improve due to the recent introduction of a standard reference material in human serum (SRM 972) from the National Institute of Standards and Technology (NIST). For immunoassay, specificity can be an issue especially in relation to the proportion of 25OHD2 that is quantified whereas HPLC and LC-MS/MS methods are able to measure the two major vitamin D metabolites 25OHD2 and 25OHD3 independently. HPLC and LC-MS/MS require more expensive equipment and expert staff but this can be offset against lower reagent costs. Increasingly procedures are being developed to semi-automate or automate HPLC and LC-MS/MS but run times remain considerably longer than for immunoassays especially if performed on automated platforms. For most HPLC and LC-MS/MS methods extraction and procedural losses are corrected for by the inclusion of an internal standard which, in part, may account for higher results compared to immunoassay. In general precision of immunoassay, HPLC and LC-MS/MS are comparable and all have the required sensitivity to identify severe vitamin D deficiency. Looking to the future it is hoped that the imminent introduction of a standard reference method (or methods) for 25OHD will further accelerate improvements in between method comparability.     

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis--one species on the basis of genetic evidence

Bacillus anthracis, Bacillus cereus, and Bacillus thuringiensis are members of the Bacillus cereus group of bacteria, demonstrating widely different phenotypes and pathological effects. B. anthracis causes the acute fatal disease anthrax and is a potential biological weapon due to its high toxicity. B. thuringiensis produces intracellular protein crystals toxic to a wide number of insect larvae and is the most commonly used biological pesticide worldwide. B. cereus is a probably ubiquitous soil bacterium and an opportunistic pathogen that is a common cause of food poisoning. In contrast to the differences in phenotypes, we show by multilocus enzyme electrophoresis and by sequence analysis of nine chromosomal genes that B. anthracis should be considered a lineage of B. cereus. This determination is not only a formal matter of taxonomy but may also have consequences with respect to virulence and the potential of horizontal gene transfer within the B. cereus group.     

Genetic differentiation between sympatric populations of Bacillus cereus and Bacillus thuringiensis

Little is known about genetic exchanges in natural populations of bacteria of the spore-forming Bacillus cereus group, because no population genetics studies have been performed with local sympatric populations. We isolated strains of Bacillus thuringiensis and B. cereus from small samples of soil collected at the same time from two separate geographical sites, one within the forest and the other at the edge of the forest. A total of 100 B. cereus and 98 B. thuringiensis strains were isolated and characterized by electrophoresis to determine allelic composition at nine enzymatic loci. We observed genetic differentiation between populations of B. cereus and B. thuringiensis. Populations of a given Bacillus species--B. thuringiensis or B. cereus--were genetically more similar to each other than to populations of the other Bacillus species. Hemolytic activity provided further evidence of this genetic divergence, which remained evident even if putative clones were removed from the data set. Our results suggest that the rate of gene flow was higher between strains of the same species, but that exchanges between B. cereus and B. thuringiensis were nonetheless possible. Linkage disequilibrium analysis revealed sufficient recombination for B. cereus populations to be considered panmictic units. In B. thuringiensis, the balance between clonal proliferation and recombination seemed to depend on location. Overall, our data indicate that it is not important for risk assessment purposes to determine whether B. cereus and B. thuringiensis belong to a single or two species. Assessment of the biosafety of pest control based on B. thuringiensis requires evaluation of the extent of genetic exchange between strains in realistic natural conditions.     

Detection of Bacillus thuringiensis kurstaki HD1 on cabbage for human consumption

The objectives of the study were to develop a specific procedure for quantification and identification of Bacillus thuringiensis kurstaki HD1, which is used as a biopesticide, and to quantify its presence in different kinds of cabbage for human consumption. We found that B. thuringiensis kurstaki HD1 can be distinguished from other B. thuringiensis strains by its unique random amplification of polymophic DNA-PCR pattern with the OPA9 primer and the presence of the flagellin genes, as detected by the primers FLAB1 and FLAB2. We detected from one to 100 Bacillus cereus-like bacteria in 10 batches of five different cabbage products for consumption. As many as 73 out of 134 isolates (53.7%) were identical with B. thuringiensis kurstaki HD1. The results show that B. thuringiensis kurstaki HD1 from biopesticides can be found in vegetables for human consumption.     

Correlation between serovars of Bacillus thuringiensis and type I beta-exotoxin production

beta-Exotoxin is a thermostable metabolite produced by some strains of Bacillus thuringiensis. Because of vertebrate toxicity, most commercial preparations of B. thuringiensis are prepared from isolates that do not produce beta-exotoxin. The aim of the present study was to find out the possible relationship between serovars of B. thuringiensis and beta-exotoxin production. A specific HPLC assay for type I beta-exotoxin has been used to detect this exotoxin in supernatants from final whole cultures of 100 strains belonging to four serovars of B. thuringiensis: thuringiensis, kurstaki, aizawai, and morrisoni. For each serovar, 25 strains randomly chosen from two Spanish collections were analyzed. Frequency of beta-exotoxin production was higher in B. thuringiensis serovar thuringiensis, whereas only two strains from serovar kurstaki showed beta-exotoxin production. None of the 25 strains belonging to serovars aizawai and morrisoni was found to produce this compound. Along with data from other studies, serovars can be classified as "common," "seldom," or "rare" beta-exotoxin producers. The serovar-dependent beta-exotoxin production is discussed in relation to the evolutionary process of serovar differentiation, the plasmid compatibility and limited plasmid exchange between serovars, and with the serovar-dependent regulation of plasmid-encoded genes.     

蛋白改造提高苏云金杆菌杀虫活力的研究进展

苏云金杆菌(Bt)已被广泛应用于农林害虫防治,但传统制剂仍然存在很大局限性,利用蛋白质工程对其改造能有效的克服这些缺点.主要概述了近年来利用结构域转换和定点突变技术改造Bt Cry蛋白并增强其杀虫活性的研究进展.