mel基因在苏云金芽孢杆菌中的转移和表达

苏云金芽孢杆菌(Bacillus thuringhensis)是研究较深入、使用较广泛的杀虫细菌,野外使用易受阳光中紫外线的影响而失活。黑色素具有很强的抗辐射作用。将构建的含有嗜麦芽假单胞菌mel基因的质粒pWSY通过原生质体转化导入苏云金芽孢杆菌体内,使后获得了稳定产生黑色素的能力。并在转化子细胞内检测到与供体菌相同的质粒。SDS-PAGE显示该转化子体内额外表达了一分子量约为18kD的蛋白,该蛋白很可能就是转化子表达的酪氨酸酶。经测定,转化子抗辐射作用明显增强,有效杀虫时间显延长。     

mel基因在苏云金芽孢杆菌中的转移和表达*

苏云金芽孢杆菌 (Bacillusthuringiensis)是研究较深入、使用较广泛的杀虫细菌 ,野外使用易受阳光中紫外线的影响而失活。黑色素具有很强的抗辐射作用。将构建的含有嗜麦芽假单胞菌mel基因的质粒pWSY通过原生质体转化导入苏云金芽孢杆菌体内 ,使后者获得了稳定产生黑色素的能力。并在转化子细胞内检测到与供体菌相同的质粒。SDS-PAGE显示该转化子体内额外表达了一分子量约为 18kD的蛋白 ,该蛋白很可能就是转化子表达的酪氨酸酶。经测定 ,转化子抗辐射作用明显增强 ,有效杀虫时间     

嗜碱芽孢杆菌Bacillus sp. N16-5表达载体的构建

【目的】构建可以在嗜碱芽孢杆菌Bacillus sp.N16-5中表达外源基因的表达载体。【方法】以枯草芽孢杆菌表达质粒pHCMC04为基本骨架,删除木糖诱导的启动子和木糖阻遏蛋白基因,分别插入枯草芽孢杆菌组成型强启动子P43和嗜碱芽孢杆菌N16-5的组成型启动子P EF,构建表达载体pABN165P43和pABN165P EF;将绿色荧光蛋白基因gfp作为报告基因连接至表达载体得到pABN165P43-gfp和pABN165P EF-gfp,利用原生质体转化法将其转化至Bacillus sp.N16-5,通过荧光显微镜和多功能荧光读板仪检测报告基因的表达情况。【结果】所构建的表达载体pABN165P43-gfp和pABN165P EF-gfp可以在Bacillus sp.N16-5中表达报告基因gfp,荧光定量数据显示,在7.5 h左右开始检测到绿色荧光蛋白的表达,从7.5 h到12 h荧光强度随时间增长迅速并在12 h左右达到最大,最大荧光值在7000左右。【结论】成功构建了2个嗜碱芽孢杆菌Bacillus sp.N16-5表达载体,实现了外源基因在嗜碱芽孢杆菌中的表达。     

绿色荧光蛋白基因原核表达载体的构建及其在昆虫肠道短短芽孢杆菌和苏云金芽孢杆菌中的表达

【目的】构建带有苏云金芽孢杆菌cry3a基因非芽孢依赖启动子和绿色荧光蛋白基因gfp(Green Fluorescent Protein)的原核表达载体,并转化从桑粒肩天牛幼虫肠道分离的两株常驻细菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,以检测cry3a启动子在昆虫肠道常驻菌中的启动子活性,获得GFP标记菌株,为常驻菌在昆虫幼虫肠道中的定殖情况和杀虫工程菌的构建奠定基础。【方法】采用重叠延伸PCR将cry3a基因启动子和gfp基因进行融合,并与pHT304载体连接构建重组质粒pHT3AG,获得的重组质粒以电脉冲转化肠道常驻菌短短芽孢杆菌CQUBb和苏云金芽孢杆菌CQUBt,于可见光和荧光显微镜下观察荧光并通过SDS-PAGE分析重组菌株的蛋白表达情况,然后对重组菌株进行生长动力学分析和稳定性测试。【结果】重组菌在营养期大量组成型表达GFP,经电泳分离在凝胶上出现约29kDa的特异蛋白条带;重组菌生长曲线与出发菌没有显著差异,说明外源质粒未对宿主菌的生长带来明显不利影响;抗性条件下传代30次后两菌株外源质粒稳定性仍可达95%、67%;两个菌株比较,CQUBb比CQUBt质粒转化率高、重组菌GFP表达时间长、表达量大,并且重组菌株稳定性好。【结论】成功地将cry3a基因核心启动子和gfp基因转入桑粒肩天牛幼虫肠道常驻菌,实现了该启动子在Bt之外的菌株中发挥作用,构建了两个GFP标记菌株;重组基因工程菌株表达量大,稳定性好,可以用作昆虫肠道内微生态研究和芽孢杆菌表达系统以及杀虫菌株的构建。     

苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白及其分子设计

cry1Ac编码的杀虫晶体蛋白是苏云金芽孢杆菌(Bt)产生的多种杀虫晶体蛋白中对鳞翅目昆虫有很高毒性的蛋白.第一个Cry1Ac杀虫晶体蛋白最早在库斯塔克亚种HD73中以伴胞晶体形式分离获得,其编码区为3 534 bp,编码蛋白分子量为133 kD,含1 178个氨基酸,等电点为4.84.自此以来,Cry1Ac杀虫晶体蛋白结构、功能以及应用研究一直是Bt杀虫晶体蛋白研究的重要方向.本文介绍了苏云金芽孢杆菌中应用最广泛的Cry1Ac杀虫晶体蛋白家族的结构、功能及其基因分类,并进一步就基于苏云金芽孢杆菌Cry1Ac杀虫晶体蛋白的基因工程研究做了分析,提出了持续利用BtCry1Ac杀虫晶体蛋白的一些见解.     

云南和广东部分热泉Alicyclobacillus分布及系统发育

从云南和广东热泉采集的样品中富集分离得到12株嗜热或微嗜热的嗜酸杆菌,革兰氏染色阳性或不定,营异养生长,最适pH为3．5～5．5,最适温度为43℃～52℃。测定其16S rDNA序列表明这些菌株与脂环酸芽孢杆菌属亲缘关系最近,结合其形态、生理等特性,鉴定这些菌株属于脂环酸芽孢杆菌。     

嗜碱性芽孢杆菌启动子的克隆及某些因子对表达的影响

从土壤中分离到一株嗜碱性芽孢杆菌(Bacillus sp. No．2),并初步分析了该菌的基本特性。以抗性基因启动子缺失质粒pG^_6为载体,从该菌的染色体DNA中,克隆了带有启动子的一些片段,并引入大肠杆菌得到表达。其中某些启动子的表达受到某些环境因子(如pH Nacl)的调控。     

嗜热脂肪芽孢杆菌pgiB基因上游452bp序列的功能分析

用化学诱变剂N甲基N′硝基N亚硝基胍进行随机诱变,获得了穿梭启动子探测质粒pPGV5的温度抗性突变型pPGV5(tr65),序列分析发现质粒上卡那霉素核苷转移酶基因kan的+238位碱基发生了G→T的单点突变。以来自嗜热脂肪芽孢杆菌FDTP3菌株的耐热邻苯二酚2,3双加氧酶基因pheB作为报道基因,构建了转录融合质粒pPGVPB452,用高压电穿孔法将其转化嗜热脂肪芽孢杆菌,通过报道蛋白活性的分析,证明了嗜热脂肪芽孢杆菌T521菌株的6磷酸葡萄糖异构酶同工酶基因pgiB上游含启动子样序列的425bp片段在嗜热脂肪芽孢杆菌中不具有启动子功能。     

利用PCR技术直接鉴定苏云金芽孢杆菌杀虫晶体蛋白基因型的快速方法

根据PCR程序中热变性温度可使菌体裂解,释放出DNA的原理,利用苏云金芽孢杆菌杀虫晶体蛋白不同基因型的特异混合引物,对苏云金芽孢杆菌营养体直接进行PCR分析。根据不同基因型的特异扩增产物片段的分子量大小便可直接确定该苏云金芽孢杆菌杀虫晶体蛋白的基因型。本文对本室筛选天然菌株和苏云金芽孢杆菌克隆菌株分别进行了cryl基因的PCR分析。表明,该法结果可靠快速易行,在方法学上,为苏云金芽孢杆菌菌株鉴定、新菌株的筛选和基因型分析提供了极大的方便。     

高温乳糖酶基因在大肠杆菌中的高效表达

来源于嗜热脂肪芽孢杆菌（Bacillus stearothermophilus)的β-半乳糖苷酶基因bgaB经克隆,测序后,转入大肠杆菌高效表达载体pET-20(b)中,重组菌在IPTG诱导下,表达出的重组蛋白比酶活量为6.66U/mg。比出发菌株高50倍。     

Revision of the Nomenclature for the Bacillus thuringiensis Pesticidal Crystal Proteins

The crystal proteins of Bacillus thuringiensis have been extensively studied because of their pesticidal properties and their high natural levels of production. The increasingly rapid characterization of new crystal protein genes, triggered by an effort to discover proteins with new pesticidal properties, has resulted in a variety of sequences and activities that no longer fit the original nomenclature system proposed in 1989. Bacillus thuringiensis pesticidal crystal protein (Cry and Cyt) nomenclature was initially based on insecticidal activity for the primary ranking criterion. Many exceptions to this systematic arrangement have become apparent, however, making the nomenclature system inconsistent. Additionally, the original nomenclature, with four activity-based primary ranks for 13 genes, did not anticipate the current 73 holotype sequences that form many more than the original four subgroups. A new nomenclature, based on hierarchical clustering using amino acid sequence identity, is proposed. Roman numerals have been exchanged for Arabic numerals in the primary rank (e.g., Cry1Aa) to better accommodate the large number of expected new sequences. In this proposal, 133 crystal proteins comprising 24 primary ranks are systematically arranged.     

核黄素基因工程研究进展

核黄素 (维生素B2 )为天然水溶性的B族维生素 ,是维持机体正常代谢所必须的物质 ,具有重要的生理功能。目前核黄素的生产方法主要有化学合成法和微生物发酵法。其中微生物发酵法是后来发展起来的一种十分经济有效的方法 ,并在核黄素主产中开始占据主导地位。为进一步获得核黄素高产菌株 ,人们对核黄素合成基因及其表达调控的机制做了深入细致的研究 ,并以此为依据 ,通过基因工程手段构建出了核黄素高产菌株 ,大大提高了核黄素的产量 ,其中尤以枯草芽孢杆菌最为成功。综述发酵法生产核黄素的现状、核黄素生物合成的分子生物学以及基因工程研究进展 ,讨论了其进一步的发展方向。     

芽胞杆菌高效分泌表达异源蛋白的研究进展

外泌蛋白易于分离纯化,有利于以较低的生产成本获得大量的目标蛋白,因此分泌表达是一种理想的外源基因的表达方式。芽胞杆菌由于其具有较好的分泌能力,被认为是一种理想的表达外源基因的宿主。到目前为止已有大量来源于不同生物的外源基因在芽胞杆菌中实现了高效分泌表达。但是芽胞杆菌的分泌表达系统仍然存在很多问题,如对某些目的蛋白的分泌量低等,限制了其作为“细胞工厂”生产目标蛋白的应用。综述了芽胞杆菌的外泌系统,分析了芽胞杆菌分泌蛋白过程中的限制因素,并总结了相关的解决方案。     

Phenotypic and genotypic comparisons of 23 strains from the Bacillus cereus complex for a selection of known and putative B. thuringiensis virulence factors

Sixteen Bacillus thuringiensis, four Bacillus cereus and three Bacillus anthracis isolates were screened for a selection of known and putative B. thuringiensis virulence factors. PCR primers were designed to detect genes for phosphatidylcholine specific phospholipase C, phosphatidylinositol specific phospholipase C, immune inhibitor A, vegetative insecticidal protein 3A, a protein proposed to be involved in capsule synthesis, a newly identified Ser/Thr kinase homologue and enterotoxin entS. Motility, the presence of flagella, haemolysis, chitinase and lecithinase production were also evaluated. The widely varying profiles of the 23 strains from the complex provide a pool of different genotypes that can help to identify factors involved in pathogenicity.     

多重PCR技术检测微生物肥料中巨大芽孢杆菌和蜡样群芽孢杆菌的研究与应用

巨大芽孢杆菌是微生物肥料生产中的常用菌种, 与之形态上相似的蜡样群芽孢杆菌(蜡样芽孢杆菌、苏云金芽孢杆菌、蕈状芽孢杆菌)则是产品中常见的污染菌, 传统方法区分两者费时费力, 有必要建立检测这两类芽孢杆菌的PCR方法。本文利用已登录的spoOA基因序列分别设计和筛选了上述两个种(群)的特异引物, 并建立了多重PCR检测技术。使用该方法对巨大芽孢杆菌、蜡样群芽孢杆菌和其他芽孢菌共3属13种24株标准菌株的基因组DNA进行扩增, 以检验其特异性。结果显示, 巨大芽孢杆菌、蜡样群芽孢杆菌基因组DNA分别产生大小不同的唯一产物, 其他芽孢杆菌均为阴性。该多重PCR检测方法的灵敏度经测定为105 CFU/mL。同时对10株待测菌株和8个微生物肥料产品进行检测, 其鉴定结果与常规鉴定结果一致。以上结果表明, 本文建立的多重PCR方法具有较高的特异性和灵敏度, 可快速、准确鉴定巨大芽孢杆菌和蜡样群芽孢杆菌, 在微生物肥料检测方面有良好的实用前景。     

苏云金杆菌杀虫蛋白的多样性

苏云金杆菌是生物防治中应用最为广泛的一种杀虫剂,其杀虫蛋白具有广泛的多样性。本文就苏云金杆菌杀虫蛋白的基因、基因分布、杀虫蛋白结构以及作用机制的多样性进行了概述。     

Quantification in soil of Bacillus thuringiensis var. kurstakiδ-endotoxin from transgenic plants

Transgenic plants that produce pesticidal proteins have the potential to release these products into the environment when the plants are incorporated into soil. This could result in novel exposure of soil organisms to these pesticidal proteins. There is a lack of knowledge about the fate and persistence of transgenic pesticidal products in the soil. A model system of transgenic cotton, which produces Bacillus thuringiensis kurstakiδ-endotoxin (Bt toxin), was used to address this issue. Methods were developed to quantify Btk toxin in soil and soil/plant litter by extraction of the Btk toxin with an aqueous buffer and quantification by ELISA. The highest recovery of Btk toxin from soil was obtained with a high salt, high pH buffer. In addition, for certain soil types, addition of a non-ionic detergent, Tween-20, was needed for optimal recovery. Recovery of Btk toxin from soil ranged from 60% for a low clay content, low organic matter soil to 27% for a high clay content, high organic matter soil. The limit of detection of this method is 0.5 ng of extractable toxin per g dry weight soil. The method was shown to be useful in tracking over time the persistence of both purified and transgenic Btk toxin in laboratory experiments.     

The use of bacterial spore formers as probiotics

The field of probiosis has emerged as a new science with applications in farming and aqaculture as alternatives to antibiotics as well as prophylactics in humans. Probiotics are being developed commercially for both human use, primarily as novel foods or dietary supplements, and in animal feeds for the prevention of gastrointestinal infections, with extensive use in the poultry and aquaculture industries. The impending ban of antibiotics in animal feed, the current concern over the spread of antibiotic resistance genes, the failure to identify new antibiotics and the inherent problems with developing new vaccines make a compelling case for developing alternative prophylactics. Among the large number of probiotic products in use today are bacterial spore formers, mostly of the genus Bacillus. Used primarily in their spore form, these products have been shown to prevent gastrointestinal disorders and the diversity of species used and their applications are astonishing. Understanding the nature of this probiotic effect is complicated, not only because of the complexities of understanding the microbial interactions that occur within the gastrointestinal tract (GIT), but also because Bacillus species are considered allochthonous microorganisms. This review summarizes the commercial applications of Bacillus probiotics. A case will be made that many Bacillus species should not be considered allochthonous microorganisms but, instead, ones that have a bimodal life cycle of growth and sporulation in the environment as well as within the GIT. Specific mechanisms for how Bacillus species can inhibit gastrointestinal infections will be covered, including immunomodulation and the synthesis of antimicrobials. Finally, the safety and licensing issues that affect the use of Bacillus species for commercial development will be summarized, together with evidence showing the growing need to evaluate the safety of individual Bacillus strains as well as species on a case by case by basis.     

Respective role of the 42- and 51-kDa components of the Bacillus sphaericus toxin overexpressed in Bacillus thuringiensis

Abstract The 42- and 51-kDa protein genes of Bacillus sphaericus 1593 have been subcloned independently downstream from the cytA gene promoter of Bacillus thuringiensis serovar israelensis and introduced into a non-mosquitocidal strain of Bacillus thuringiensis . Consequently, each protein was overproduced and accumulated as inclusion bodies which were purified. For the first time, the 42-kDa protein inclusions alone were found to be toxic to Culex pipiens larvae (LC₅₀ at 48 h 300 ng ml⁻¹); in contrast, the 51-kDa protein inclusions were not. Moreover, a synergistic effect between these two components was observed.     

炭疽芽胞杆菌疫苗研究进展

炭疽芽胞杆菌引起的炭疽病死亡率非常高 ,当前的疫苗具有效力不稳定、对吸入性炭疽的保护率低、免疫程序繁琐、存在副作用等缺点。近年来人们在改造传统疫苗的同时又有一些新的发现 ,如保护性抗原 (PA)的抗体在体内可杀死芽胞 ;通过粘膜免疫能够诱导机体分泌IgA抗体 ;抗多聚谷氨酸 (γ D PGA)抗体可以同炭疽杆菌的繁殖体作用 ,从而杀死繁殖体 ;寻找到新的免疫原。DNA疫苗、活载体疫苗的出现为新一代安全、免疫程序简单、具更高保护率的疫苗奠定了基础