消除内生质粒对苏云金芽胞杆菌YBT-1463特性的影响

研究了经完全消除苏云金芽胞杆菌野生菌株YBT-1463的内生质粒对该菌部分形态、遗传及生理生化特性的影响。结果表明,消除内生质粒后的无质粒突变株不形成伴胞晶体,但电转化4种供体质粒,即pBMBl21、pBMB304-1Ab、pBMBLC和pBMB9748的效率显著提高,转化频率最高比出发菌株提高6.8×10 倍,而无质粒突变株对红霉素等10种抗生素的敏感性,对葡萄糖等19种碳源和谷氨酸等12种氮源的利用能力及生长性能与出发菌株无明显差异。     

消除内生质粒对苏云金芽胞杆菌YBT-1463特性的影响

研究了经完全消除苏云金芽胞杆菌野生菌株ＹＢＴ－１４６３的内生质粒对该菌部分形态、遗传及生理生化特性的影响。结果表明,消除内生质粒后的无质粒突变株不形成伴胞晶体,但电转化４种供体质粒,即ｐＢＭＢ１２１、ｐＢＭＢ３０４－１Ａｂ、ｐＢＭＢＬＣ和ｐＢＭＢ９７４８的效率显著提高,转化频率最高比出发菌株提高６．８×１０^４倍,而无质粒突变株对红霉素等１０种抗生素的敏感性、对葡萄糖等１９种碳源和谷氨酸等１２种氮源的利用能力及生长性能与出发菌株无明显差异。     

苏云金芽胞杆菌质粒pBMB2062的克隆及遗传稳定载体的构建

从苏云金芽胞杆菌ＹＢＴ－１５２０菌株中克隆到１个小质粒ｐＢＭＢ２０６２,序列分析表明该质粒由２ ０６２个核苷酸组成。该质粒含２个编码框（ｏｒｆ１和ｏｒｆ２）,可分别编码由２８９个氨基酸和８０个氨基酸组成的蛋白质。这２个潜在的蛋白质分别与质粒的复制启始蛋白和复制蛋白同源。ｐＢＭＢ２０６２与２个已知同源质粒之间有２３个核苷酸的差异,这些差异引起了ｏｒｆ１编码框的变化。ｃＤＮＡ合成和ＰＣＲ检测显示与ｐＢ     

苏云金芽胞杆菌大质粒pBMB28的克隆

苏云金芽胞杆菌幕虫亚种YBT-020具有典型的晶胞粘连表型。在前期的研究中,通过质粒消除实验,推测晶胞粘连现象与YBT-020内生质粒pBMB28有关。为了定位质粒pBMB28上控制晶胞粘连表型的基因,首先对质粒pBMB28进行克隆。利用穿梭载体pEMB0557,成功构建了苏云金芽胞杆菌YBT-020的基因组人工染色体(BAC)文库。前期的研究表明晶体蛋白基因cry28Aa定位在质粒pBMB28上,根据cry28Aa基因序列设计引物,从文库中筛选到含有cry28Aa的重组质粒pBMB231。镜检和SDS-PAGE证明质粒pBMB231转化无晶体突变株BMB171形成的重组子BMB231可以产生Cry28Aa晶体蛋白,但不能恢复晶胞粘连表型。对重组质粒pBMB231的插入片段末端序列测定并设计引物筛选文库,通过染色体步移方式得到4个可以重叠覆盖质粒pBMB28不同区域的克隆子,从而克隆了该质粒。对这4个克隆子末端测序和酶切分析,测算出该质粒的大小约为140 kb。进一步确定应用基因组BAC文库以及重叠片段筛选的方法,可以快速有效的克隆苏云金芽胞杆菌大质粒。     

苏云金芽胞杆菌基因组研究

苏云金芽胞杆菌制剂是目前世界上产量最大、使用最广的微生物杀虫剂.近年来,国内外研究人员在苏云金芽胞杆菌的功能基因组学领域开展了深入的研究,取得了丰硕的研究成果.本文拟就苏云金芽胞杆菌的基因组、转录组、蛋白质组及代谢组研究进展进行简要论述.     

苏云金芽胞杆菌LM1212质粒缺失对细胞分化的影响

【目的】苏云金芽胞杆菌LM1212与传统Bt相比,芽胞和晶体形成产生了分化,研究目的是明确质粒缺失对LM1212菌株细胞分化的影响。【方法】采用高温法对含有cry35-like基因启动子与lac Z基因融合质粒的LM(p35'Z)菌株进行内源大质粒缺失。在含X-gal的HCO平板培养初步筛选缺失突变株,进一步提取野生型及突变株的质粒进行脉冲场凝胶电泳分析,并用cry基因引物进行鉴定、激光共聚焦扫描显微镜和光学显微镜观察、芽胞形成率分析及利用SDS-PAGE和LC-MS/MS(Q-TOF)质谱分析质粒缺失对LM1212细胞分化和Cry蛋白表达的影响。【结果】筛选得到两株质粒缺失突变株LM(p35'Z)-W菌株和LM(p35'Z)-DB菌株,在含X-gal的HCO平板上,LM(p35'Z)-W菌株菌落颜色为白色,LM(p35'Z)-DB菌株菌落颜色为深蓝色,说明cry35-like基因启动子活性在这两株菌中受到影响;细胞形态观察发现LM(p35'Z)-DB菌株形成更多晶体产生细胞,LM(p35'Z)-W菌株形成更少晶体产生细胞。SDS-PAGE结果表明LM(p35'Z)-DB菌株Cry蛋白表达量提高,LM(p35'Z)-W菌株Cry蛋白表达量减少。【结论】LM1212质粒缺失可以影响细胞分化和晶体蛋白产量,此发现为深入解析LM1212细胞分化的调控机制和Bt菌株的遗传改良奠定了基础。     

苏云金芽胞杆菌拟步行甲亚种质粒复制子oril65的克隆

以苏云金芽胞杆菌拟步行甲亚种菌株（Bacillus thuringiensis subsp.tenebrionis)YBT-1765作为出发菌株,克隆了一个包含复制子的EcoRI酶切片段,大小约为11kb,称为oril65。这是国内外从此亚种中克隆到的第一个复制子,缩小到8kb左右后仍然能够复制。杂交结果显示,此复制子来源于菌株YBT-1765可以检测到的分子量最大的质粒,以此复制子构建的穿梭载体pBMB6071在不同受体菌中的稳定性差异很大,其中在以色列亚种无晶体突变株4Q7中,传40后代,稳定性100%,质粒pBMB6071与含ori1030和ori2062在库斯塔克亚种无晶体突变株BMB171中是相容的。     

苏云金芽胞杆菌拟步行甲亚种质粒复制子ori165的克隆

以苏云金芽胞杆菌拟步行甲亚种菌株 (Bacillusthuringiensissubsp.tenebrionis)YBT 1 76 5作为出发菌株 ,克隆了一个包含复制子的EcoRI酶切片段 ,大小约为 1 1kb ,称为ori1 6 5。这是国内外从此亚种中克隆到的第一个复制子。缩小到 8kb左右后仍然能够复制。杂交结果显示 ,此复制子来源于菌株YBT 1 76 5可以检测到的分子量最大的质粒。以此复制子构建的穿梭载体pBMB6 0 71在不同受体菌中的稳定性差异很大 ,其中在以色列亚种无晶体突变株 4Q7中 ,传 40后代 ,稳定性 1 0 0 %。质粒pBMB6 0 71与含ori1 0 3 0和ori2 0 6 2在库斯塔克亚种无晶体突变株BMB1 71中是相容的     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

苏云金芽胞杆菌重组工程菌研究进展

苏云金芽胞杆菌(Bacillus thuringiensis,Bt)可以产生对多种农业害虫有毒性的晶体蛋白(Insecticidal crystal protein,ICP),是目前世界上使用范围最广、应用最成功的杀虫微生物.已经发现苏云金芽胞杆菌可以对包括鳞翅目、鞘翅目、直翅目等在内的9个目500多种昆虫具有杀虫活性①,同时对某些螨类、吸虫、鞭毛虫及动植物线虫也有一定的活性.     

Plasmid Patterns of Bacillus thuringiensis Type Strains

Practically all Bacillus thuringiensis strains contain a set of self-replicating, extrachromosomal DNA molecules or plasmids, which vary in number and size in the different strains. The plasmid patterns obtained from gel electrophoresis have previously been used as a tool to characterize strains, but comparison of the plasmid patterns has been limited in the number and diversity of strains analyzed. In this report, we were able to compare the plasmid patterns of 83 type strains (out of 84) and 47 additional strains from six serotypes. The information obtained from this comparison showed the importance of this tool as a strain characterization procedure and indicates the complexity and uniqueness of this feature. For example, with one exception, all type strains showed a unique plasmid pattern. All were unique in such a way that none showed even a single comigrating plasmid in the agarose gels, and therefore, cluster analysis was impossible, indicating that plasmid patterns are qualitative rather than quantitative features. Furthermore, comparison between strains belonging to the same serotype showed a great difference in variability. Some serotypes (e.g., israelensis) showed the same basic pattern among all its strains, while other serotypes (e.g., morrisoni) showed a great diversity of patterns. These results indicate that plasmid patterns are valuable tools to discriminate strains below the serotype level.     

苏云金芽孢杆菌大型质粒DNA的小量提取

以苏云金芽孢杆菌库斯塔克亚种、猝倒亚种及以色列亚种为材料,介绍了低拷贝大型质粒DNA的小量提取方法,该法采用PEG 6000进行质粒纯化,省略了苯酚和氯仿抽提过程。实验证明,该法结果稳定,提取的质粒DNA产量和质量均符合大多数分子生物学实验的要求。     

一种简单、快速的苏云金芽胞杆菌基因组DNA提取方法

从培养时间、裂解溶液、抽提和沉淀时间等方面对苏云金芽胞杆菌(Bacillus thuringiensis,Bt)基因组DNA提取技术进行改进.提取8株对蛴螬有杀虫活性的野生Bt菌株的基因组DNA,分析其纯度,并进行PCR分析与酶切分析.试验结果表明,新的提取方法耗时36 min,明显短于旧方法所用时间(97 min);两种方法提取的基因组DNA A260/A280均大于1.8,无明显区别;琼脂糖凝胶电泳结果显示,上样量相同时,新方法提取的基因组DNA浓度是旧方法的5倍以上;新方法提取的基因组DNA能作为模板灵敏地扩增出测试基因,所提取的DNA能被限制性内切酶完全酶切,证明DNA具有很高的纯度.本研究改进的基因组DNA提取方法耗时短、产量高,并能满足PCR扩增、酶切等分子生物学需要.     

番茄果实总RNA提取方法的优化

针对番茄等果实的特殊性,对商品化Trizol试剂盒提取总RNA的方法进行适当的改良:液氮研磨后加入预处理液除去果实中大部分糖类等次级代谢物质,裂解时加入β-巯基乙醇抑制酚类等物质的氧化,从富含多糖、多酚的番茄、枣果肉中成功提取总RNA。与其它方法相比,该改良方法具有操作步骤简单、快速等优点。利用RT-PCR技术,从所提取RNA中成功克隆出ζ-胡萝卜素脱氢酶基因片段,进一步表明其质量可以完全满足分子生物学研究的要求。     

苏云金杆菌对甜菜夜蛾毒性的筛选

测定了 10 0株野生型苏云金芽孢杆菌菌株对夜蛾科甜菜夜蛾幼虫的毒力活性 ,经回归分析 ,2 3株对甜菜夜蛾初孵幼虫的毒力与浓度有高相关性 (R >0 .90 ) ,按LC50 计算 ,CN73活性最强 ,LC50 达 2 .393微升培养液 /克饲料 ( μL/g) ;另有 2 3株进行了不同浓度下校正死亡率的比较 ,得到高毒株 13株 ,CN33在 5μL/g浓度下校正死亡率达到 10 0 %。该结果为进一步研究奠定了基础。     

New Strategy for Identification of Novel cry-Type Genes from Bacillus thuringiensis Strains

We designed five degenerate primers for detection of novel cry genes from Bacillus thuringiensis strains. An efficient strategy was developed based on a two-step PCR approach with these primers in five pair combinations. In the first step, only one of the primer pairs is used in the PCR, which allows amplification of DNA fragments encoding protein regions that include consensus domains of representative proteins belonging to different Cry groups. A second PCR is performed by using the first-step amplification products as DNA templates and the set of five primer combinations. Cloning and sequencing of the last-step amplicons allow both the identification of known cry genes encoding Cry proteins covering a wide phylogenetic distance and the detection and characterization of cry-related sequences from novel B. thuringiensis isolates.     

侧孢芽孢杆菌质粒提取方法的探究

以能够降解有机磷农药的两株侧孢芽孢杆菌BL-21和BL-22为研究对象,分别采用碱裂解法、试剂盒提取法和SDS法对侧孢芽孢杆菌BL-21和BL-22的质粒进行提取,并通过凝胶电泳和紫外分光光度法对提取结果进行分析,试验结果证明,适合侧孢芽孢杆菌BL-21和BL-22的质粒提取方法是SDS裂解法,该方法提取的质粒大小为10kb,且该方法提取的结果稳定,质粒的产量和质量均符合分子生物学实验的要求。     

Genotypic Diversity among Bacillus cereus and Bacillus thuringiensis Strains

Twenty-four strains of Bacillus cereus were analyzed by pulsed-field gel electrophoresis (PFGE) and compared with 12 Bacillus thuringiensis strains. In addition, the 36 strains were examined for variation in 15 chromosomal genes encoding enzymes (by multilocus enzyme electrophoresis [MEE]). The genome of each strain had a distinct NotI restriction enzyme digestion profile by PFGE, and the 36 strains could be assigned to 27 multilocus genotypes by MEE. However, neither PFGE nor MEE analysis could distinguish between the two species. Two of the B. cereus strains contained extrachromosomal DNA that hybridized to a cryIA insecticidal toxin probe, and seven strains contained DNA with homology to a Tn4430 transposon probe derived from B. thuringiensis. The results strongly indicate that B. cereus and B. thuringiensis should be regarded as one species.     

Strategy for Identification of Bacillus cereus and Bacillus thuringiensis Strains Closely Related to Bacillus anthracis

Bacillus cereus strains that are genetically closely related to B. anthracis can display anthrax-like virulence traits (A. R. Hoffmaster et al., Proc. Natl. Acad. Sci. USA 101:8449-8454, 2004). Hence, approaches that rapidly identify these “near neighbors” are of great interest for the study of B. anthracis virulence mechanisms, as well as to prevent the use of such strains for B. anthracis-based bioweapon development. Here, a strategy is proposed for the identification of near neighbors of B. anthracis based on single nucleotide polymorphisms (SNP) in the 16S-23S rRNA intergenic spacer (ITS) containing tRNA genes, characteristic of B. anthracis. By using restriction site insertion-PCR (RSI-PCR) the presence of two SNP typical of B. anthracis was screened in 126 B. cereus group strains of different origin. Two B. cereus strains and one B. thuringiensis strain showed RSI-PCR profiles identical to that of B. anthracis. The sequencing of the entire ITS containing tRNA genes revealed two of the strains to be identical to B. anthracis. The strict relationship with B. anthracis was confirmed by multilocus sequence typing (MLST) of four other independent loci: cerA, plcR, AC-390, and SG-749. The relationship to B. anthracis of the three strains described by MLST was comparable and even higher to that of four B. cereus strains associated with periodontitis in humans and previously reported as the closest known strains to B. anthracis. SNP in ITS containing tRNA genes combined with RSI-PCR provide a very efficient tool for the identification of strains closely related to B. anthracis.