苏云金芽胞杆菌spoIIID基因缺失突变株的特点

摘要：【目的】构建苏云金芽胞杆菌spoIIID基因缺失突变株,并研究其与出发菌株的表型及性质差异。【方法】采用基因同源重组技术敲除了苏云金芽胞杆菌HD-73菌株中的spoIIID基因,构建了spoIIID缺失突变株,测定生长曲线,并通过扫描电子显微镜观察,芽胞计数分析及SDS-PAGE 蛋白电泳比较突变株与出发菌株的差异。构建遗传互补菌株,观察菌株性状的回复情况。【结果】通过温敏载体同源重组敲除技术获得了苏云金芽胞杆菌HD-73菌株spoIIID基因缺失突变株,生长曲线测定表明,突变株较出发菌株在平稳期后期生长较缓和;扫描电子显微镜观察和芽胞计数分析显示,突变株基本丧失了形成芽胞的能力,但依然形成晶体。SDS-PAGE结果显示,在 SSM培养基中,突变株对伴胞晶体蛋白的形成量影响并不显著;在营养较富集的Luria-Bertani培养基中,突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低。利用载体pHT315携带spoIIID操纵子互补突变株,互补株恢复了产生晶体和芽胞的能力。【结论】本研究证明spoIIID基因是苏云金芽胞杆菌芽胞形成所必需,同时与晶体蛋白的表达相关。     

苏云金芽胞杆菌产苏云金素菌株CT-43及其 缺失突变株的差异蛋白

[目的]苏云金素(Thuringiensin)的合成和代谢途径的相关研究在国内外一直进展缓慢,本文拟从蛋白质组水平揭示与苏云金素合成或代谢相关的蛋白.[方法]利用双向电泳技术研究了高产苏云金素的苏云金芽胞杆菌野生菌株CT-43、其高产突变菌株CT-43-1C及不产突变菌株BMB0806在蛋白表达水平的差异,然后对差异蛋白点进行质谱鉴定,最后对鉴定出的蛋白进行生物信息学分析.[结果]与野生型和高产菌株相比,在BMB0806中发现了13个差异显著的蛋白点,鉴定出了其中的9个,生物信息学结果显示有6个蛋白可能与苏云金素合成或代谢相关.[结论]通过蛋白质组研究找到了6个可能与苏云金素合成或代谢相关的蛋白,为苏云金素合成基因簇的克隆和合成途径的验证提供了有力的证据.     

多粘类芽胞杆菌SC2基因敲除体系的建立

摘要:【目的】建立多粘类芽胞杆菌SC2 的基因敲除体系。【方法】利用电转化把温敏型自杀质粒pRN5101导入到多粘类芽胞杆菌SC2中。采用基因重组技术敲除SC2 中的多粘菌素基因E(pmxE),得到突变株SC2-E。利用抗细菌性能检测和高效液相色谱分析合成多粘菌素的能力,来证实pmxE基因是否被敲除。【结果】成功构建了多粘类芽胞杆菌SC2 的基因敲除体系。pRN5101转入SC2后能够在28℃复制,39℃自杀。突变株失去了合成多粘菌素的能力,成功敲除pmxE基因,验证了此体系的可用性。【结论】首次构建了多粘类芽胞杆菌的基因敲除体系,拓展了pRN5101的使用范围,为研究多粘类芽胞杆菌的基因功能提供了高效的遗传操作工具。     

含质粒复制起始区ori44的苏云金芽胞杆菌解离载体的构建

将苏云金芽胞杆菌转座子Tn4430的解离酶识别位点res分别插入克隆载体pRSET B和pUC19得到质粒pBMB1201和pBMB1202。这两个质粒分别经BamHI/Hin dⅢ和EcoRI/HindⅢ双酶切回收含res位点的小DNA片段,与穿梭载体pHT3101经EcoRI/HindⅢ双酶切后加收的含大肠杆菌复制起始区、氨苄青霉素抗性基因和红霉素抗性基因的3．3kb片段连接,获得重组质粒pBMB1203。封闭pBMB1203两res位点外的BamHI和EcoRI位点后,得到解离载体pBMB1204。将来源于苏云金芽胞杆菌库斯塔克亚种YBT－1520的质粒复制起始区ori44片段插入pBMB1204的两res位点之间,得到解离穿梭载体pBMB1205。该解离载体插入壮观霉素抗性基因后电转化无晶体突变株,在辅助质粒所提供的解离酶作用下可发生解离消除抗性基因,解离频率为100％,解离后的质粒稳定性为93％。利用解离穿梭载体pBMB1205可在用抗性筛选到转化子后特定消除抗性标记基因和其它非苏云金芽胞杆菌DNA片段。     

炭疽芽胞杆菌mntA基因缺失突变株的构建及鉴定

目的:通过同源重组的方法敲除炭疽芽胞杆菌减毒AP422株的mntA基因,使菌株进一步减毒,用于构建新的疫苗候选株。方法:利用PCR方法扩增mntA基因上下游同源臂后与温敏质粒连接,构建打靶载体,并转化炭疽芽胞杆菌减毒AP422株;利用抗生素和温度2种选择压力实现同源重组,敲除目标基因mntA,然后利用Cre-LoxP系统去除抗性筛选标记,得到无抗性标记的缺失突变株,并利用PCR和Western印迹等方法对重组菌进行系统鉴定,最后分析突变株的生物学性状。结果:敲除了AP422株的mntA基因,获得了无抗性标记的缺失突变株,突变株的生存竞争能力比原始菌株明显减弱。结论:突变株获得了进一步减毒,可用于构建新的疫苗候选株。     

ste7与ste15双基因敲除对依博素生物合成的影响

摘要：【目的】研究ste7和ste15基因双敲除对依博素生物合成的影响。【方法】通过基因同源重组双交换,对ste15基因缺失突变株Streptomyces sp. 139 (ste15 -)再进行ste7基因的敲除,经Southern杂交验证,获得了ste7和ste15双基因缺失变株Streptomyces sp. 139 (ste7 - ste15 -)。对该突变株进行了基因互补。气相色谱分析ste7和ste15双基因缺失突变株及互补株产生的胞外多糖单糖组分,排阻色谱测定衍生物的重均分子量,ELISA法     

苏云金芽胞杆菌HD-73菌株sigE基因敲除突变株的构建与特点

SigE因子是芽胞杆菌芽胞形成过程中起重要作用的sigma因子,它控制着众多芽胞形成相关基因的表达.在苏云金芽胞杆菌中,Cryl等杀虫晶体蛋白的表达也受SigE因子的控制.本研究利用同源重组技术构建了苏云金芽胞杆菌库斯塔克亚种标准菌株HD-73 sigE缺失突变株.研究表明:突变株衰亡期提前,丧失了形成芽胞和晶体的能力:cryIAa指导的β-半乳糖苷酶活性分析表明sigE基因对crylAa基因的转录有较大影响.利用载体pHT315携带sigE所在操纵子spoIIG及其启动子序列在突变株中表达,使突变株恢复了产生芽胞和形成杀虫晶体的能力,生长基本恢复正常,这些结果表明SigE因子是Bt库斯塔克亚种菌株产生芽胞和形成晶体所必需的.     

苏云金芽胞杆菌spoⅢD基因缺失突变株的特点

微镜观察和芽胞计数分析显示,突变株基本丧失了形成芽胞的能力,但依然形成晶体.SDS-PAGE结果显示,在SSM培养基中,突变株对伴胞晶体蛋白的形成量影响并不显著;在营养较富集的Luria-Bertani培养基中,突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低.利用载体pHT315携带spoⅢD操纵子互补突变株,互补株恢复了产生晶体和芽胞的能力.[结论]本研究证明spoⅢD基因是苏云金芽胞杆菌芽胞形成所必需,同时与晶体蛋白的表达相关.     

炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体的构建

目的：构建炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体,以进行后续SLP的功能研究,为炭疽芽孢杆菌重要基因功能的研究建立技术平台。方法：利用PCR技术,分别扩增得到目的基因的上游同源臂（slp-F）和下游同源臂（slp-R）,将抗性基因（S）和上、下游同源臂先后连入穿梭质粒pKSV7,构建打靶载体pKSV7-FSR,经去甲基化后,电转化入炭疽芽胞杆菌A16R,通过同源重组敲除slp基因,并通过DNA测序和Western blot实验验证;对野生株和突变株37℃时的生长曲线及生化反应进行比较研究。结果：分别从DNA水平和蛋白质水平证实slp基因被成功敲除;突变株对数期生长较快,衰退较慢,与野生株的生化反应差异不明显。结论：获得了炭疽芽胞杆菌假想S-层蛋白SLP缺失突变体。     

苏云金杆菌毒性质粒在蜡状芽胞杆菌群间的水平转移

以苏云金芽胞杆菌KT0为质粒供体菌,在液体环境中与蜡状芽胞杆菌组成员菌(Bacillus cereus sensu lato group strains)发生接合转移。对所筛选到的接合转移子进行了质粒稳定性检测、PCR验证以及供、受体菌染色体背景RAPD图谱分析。不同培养基中质粒转移率数据表明:在人工LB培养基及灭菌牛乳中,质粒pHT73均能以不同频率转移至蜡状芽胞杆菌组受体菌,并且在牛乳中转移活性更高,最高可这4.8×10~(-4)cfu/ donor。获得质粒的受体菌同时具备了质粒所编码基因功能,产生其编码的杀虫晶体蛋白。SDS-PAGE电泳和光镜结果显示转化菌在得到外源质粒pHT73后,能产生由cry1Ac基因编码的130 ku蛋白Cry1Ac,在芽胞期能形成稳定的菱形晶体。     

Genome-wide Screening Reveals the Genetic Determinants of an Antibiotic Insecticide in Bacillus thuringiensis

Thuringiensin is a thermostable secondary metabolite in Bacillus thuringiensis and has insecticidal activity against a wide range of insects. Until now, the regulatory mechanisms and genetic determinants involved in thuringiensin production have remained unclear. Here, we successfully used heterologous expression-guided screening in an Escherichia coli–Bacillus thuringiensis shuttle bacterial artificial chromosome library, to clone the intact thuringiensin synthesis (thu) cluster. Then the thu cluster was located on a 110-kb endogenous plasmid bearing insecticide crystal protein gene cry1Ba in strain CT-43. Furthermore, the plasmid, named pBMB0558, was indirectly cloned and sequenced. The gene functions on pBMB0558 were annotated by BLAST based on the GenBank^TM and KEGG databases. The genes on pBMB0558 could be classified into three functional modules: a thuringiensin synthesis cluster, a type IV secretion system-like module, and mobile genetic elements. By HPLC coupling mass spectrometer, atmospheric pressure ionization with ion trap, and TOF technologies, biosynthetic intermediates of thuringiensin were detected. The thuE gene is proved to be responsible for the phosphorylation of thuringiensin at the last step by vivo and vitro activity assays. The thuringiensin biosynthesis pathway was deduced and clarified. We propose that thuringiensin is an adenine nucleoside oligosaccharide rather than an adenine nucleotide analog, as is traditionally believed, based on the predicted functions of the key enzymes, glycosyltransferase (ThuF) and exopolysaccharide polymerization protein (Thu1).     

黑暗链霉菌中tbmA基因的功能研究

PCR获得tbmA基因内部863 bp片段,构建基因阻断穿梭载体pSPU112-1,经接合转移导入Strepto-myces tenebrariusH6,筛选单交换阻断变株,并用Southern blot验证阻断变株的tbmA已经被破坏。经发酵产物分析,阻断变株不再合成氨甲酰妥布霉素,只合成安普霉素。首次从分子水平证明了tbmA只参与氨甲酰妥布霉素生物合成,而不参与安普霉素的生物合成。     

Construction of an <Emphasis Type="Italic">Escherichia coli</Emphasis> to <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> shuttle vector for large DNA fragments

Shuttle vectors for Bacillus thuringiensis or Bacillus cereus usually cannot hold fragments larger than 20 kb. With the development of genome research, shuttle vectors with higher loading capacity are necessary. We constructed an Escherichia coli to B. thuringiensis shuttle vector, pEMB0557, with a large loading capacity. This vector incorporated the ori60 replicon from B. thuringiensis subsp. kurstaki YBT-1520, erythromycin resistance (B. thuringiensis), and chloromycetin resistance (E. coli) genes. A bacterial artificial chromosome library of B. thuringiensis strain CT-43 was constructed and pEMB0557 was able to accommodate at least a 70-kb DNA fragment. Simultaneously, the cry1B gene on a 40-kb fragment could express a 140-kDa protein in plasmid-cured B. thuringiensis BMB171. Due to its high capacity and utility in expressing exogenous genes, pEMB0557 will be useful in cloning (especially silencing genes) and expressing large DNA fragments (e.g., gene clusters) in B. thuringiensis. Plasmid pEMB0557 provides a new tool for B. thuringiensis genome or B. cereus group research.     

Genetic analysis of the Serratia marcescens N28b O4 antigen gene cluster

The Serratia marcescens N28b wbbL gene has been shown to complement the rfb-50 mutation of Escherichia coli K-12 derivatives, and a wbbL mutant has been shown to be impaired in O4-antigen biosynthesis (X. Rubirés, F. Saigí, N. Piqué, N. Climent, S. Merino, S. Albertí, J. M. Tomás, and M. Regué, J. Bacteriol. 179:7581-7586, 1997). We analyzed a recombinant cosmid containing the wbbL gene by subcloning and determination of O-antigen production phenotype in E. coli DH5alpha by sodium dodecyl sulfate-polyacrylamide electrophoresis and Western blot experiments with S. marcescens O4 antiserum. The results obtained showed that a recombinant plasmid (pSUB6) containing about 10 kb of DNA insert was enough to induce O4-antigen biosynthesis. The same results were obtained when an E. coli K-12 strain with a deletion of the wb cluster was used, suggesting that the O4 wb cluster is located in pSUB6. No O4 antigen was produced when plasmid pSUB6 was introduced in a wecA mutant E. coli strain, suggesting that O4-antigen production is wecA dependent. Nucleotide sequence determination of the whole insert in plasmid pSUB6 showed seven open reading frames (ORFs). On the basis of protein similarity analysis of the ORF-encoded proteins and analysis of the S. marcescens N28b wbbA insertion mutant and wzm-wzt deletion mutant, we suggest that the O4 wb cluster codes for two dTDP-rhamnose biosynthetic enzymes (RmlDC), a rhamnosyltransferase (WbbL), a two-component ATP-binding-cassette-type export system (Wzm Wzt), and a putative glycosyltransferase (WbbA). A sequence showing DNA homology to insertion element IS4 was found downstream from the last gene in the cluster (wbbA), suggesting that an IS4-like element could have been involved in the acquisition of the O4 wb cluster.     

苏云金杆菌δ-内毒素、芽孢及苏云金素A对棉铃虫毒性及拒食性的比较

苏云金杆菌库斯塔克变种HD-1的晶体蛋白与芽孢、HD-1无晶体突变株(Cry-)的芽孢以及苏云金素A对棉铃虫Helicoverpa armigera毒性和拒食性的比较研究显示,HD-1晶体蛋白对棉铃虫的杀虫毒力高, 拒食作用强; HD-1芽孢对棉铃虫具有一定的杀虫活性和生长抑制作用, 并有很强的拒食作用; HD-1无晶体突变株(Cry-)芽孢对棉铃虫无毒也无拒食作用;苏云金素A对棉铃虫的生长发育有极显著的抑制作用, 但对棉铃虫无拒食作用,由此证明晶体蛋白是苏云金杆菌杀虫活性和拒食作用的主要来源。苏云金素A与苏云金杆菌芽孢晶体混合物一起使用, 可使棉铃虫的死亡率显著提高。     

Complete genome sequence of Bacillus thuringiensis subsp. chinensis strain CT-43

Bacillus thuringiensis has been widely used as an agricultural biopesticide for a long time. As a producing strain, B. thuringiensis subsp. chinensis strain CT-43 is highly toxic to lepidopterous and dipterous insects. It can form various parasporal crystals consisting of Cry1Aa3, Cry1Ba1, Cry1Ia14, Cry2Aa9, and Cry2Ab1. During fermentation, it simultaneously generates vegetative insecticidal protein Vip3Aa10 and the insecticidal nucleotide analogue thuringiensin. Here, we report the finished, annotated genome sequence of B. thuringiensis strain CT-43.     

人类免疫缺陷病毒1型Tat蛋白N末端缺失突变体融合蛋白的表达及其免疫原性分析

本研究采用PCR方法从人类免疫缺陷病毒1型（Human immunodeficiency virus 1,HIV-1）HXB2株tat基因中扩增编码Tat蛋白N末端1-21位氨基酸缺失的突变体Tat22-101基因片段,构建其原核表达质粒pET32a-Tat22-101,经双酶切及测序验证后,转化大肠埃希菌BL21（DE3）,进行IPTG诱导表达及Ni²⁺-NTA柱亲和层析纯化。纯化后的突变体融合蛋白PET32a-Tat22-101经SDS-PAGE及Western blotting鉴定,其相对分子质量约为26.9kD。该融合蛋白免疫BALB/c小鼠,经ELISA检测结果表明,pET32a-Tat22-101融合蛋白不仅较好地保留其免疫原性,而且能诱导产生高滴度的针对Tat N末端区之外的Tat其他功能区表位的抗体,为进一步研究Tat生物学功能和研制新型HIV Tat疫苗奠定试验基础。     

以CotX为分子载体在枯草芽胞杆菌芽胞表面展示绿色荧光蛋白

芽胞衣壳蛋白CotB、CotC、CotG等可作为芽胞表面展示外源蛋白的分子载体,制备口服重组疫苗或具有催化活性的重组酶。CotX为枯草芽胞杆菌Bacillussubtilis芽胞衣壳中的另一种结构蛋白。为证明CotX能否作为分子载体将外源蛋白展示在芽胞表面,本研究将cotX基因与绿色荧光蛋白基因gfp的编码序列进行基因重组,构建融合表达CotX-GFP的整合型重组质粒,将该质粒转化枯草芽胞杆菌,筛选重组菌株并诱导产生芽胞,观察到重组芽胞表面具有GFP绿色荧光。结果表明枯草芽胞杆菌的芽胞衣壳蛋白CotX位于芽胞衣壳外层,可作为芽胞表面展示外源蛋白的载体分子。