共查询到20条相似文献,搜索用时 93 毫秒
1.
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。 相似文献
2.
苏云金芽胞杆菌杀虫晶体蛋白基因的分类 总被引:9,自引:0,他引:9
1989年Hofte和Whiteley根据氨基到序列的同源性和杀虫谱双重标准将苏云金芽胸杆菌杀虫晶体蛋白基因划分为5类14亚类。根据双重标准出现的问题和冲突,给分类带来了困扰,为此1995年,只根据氨基酸序列的同源性进行分类。现已将基因分为21群,44亚群,64类,116个亚类。本文介绍了纳入新系统的条件、分类原则、命名方法、定名步骤和分类中值得注意的问题。 相似文献
3.
苏云金芽胞杆菌YBT1520杀虫晶体蛋白基因的属性 总被引:2,自引:1,他引:2
通过Southern杂交发现高毒力苏云金芽胞杆菌(Bacillus thuringiensis)TBT-1520菌株含有两个杀虫晶体蛋白基因片段,其5’=末端所在HindⅢ片段分别为6.8kb和4.6kb,它们对应的基因分别命名为cry218和cry4.6。经PCR鉴定,该菌含有cry1Aa、cry1Ab和cry1Ac基因,以及cry2基因,其中cry218属于cry1Ac。分析了cry1Ac基因 相似文献
4.
苏云金芽胞杆菌肠毒素基因的PCR检测 总被引:7,自引:0,他引:7
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 相似文献
5.
6.
1989年Hofte和Whiteley根据氨基酸序列的同源性和杀虫谱双重标准将苏云金芽胞杆菌杀虫晶体蛋白基因划分为5类14亚类。根据双重标准出现的问题和冲突,给分类带来了困扰,为此1995年,只根据氨基酸序列的同源性进行分类。现已将基因分为21群,44亚群,64类,116个亚类。本文介绍了纳入新系统的条件、分类原则、命名方法、定名步骤和分类中值得注意的问题。 相似文献
7.
8.
为了探索ERIC-PCR技术在苏云金芽胞杆菌和蜡状芽胞杆菌的鉴定及分型中的应用价值,本研究采用PCR方法初步检测苏云金芽胞杆菌杀虫晶体蛋白基因的组成,并对苏云金芽胞杆菌和蜡状芽胞杆菌的总DNA进行ERIC-PCR扩增,分析ERIC-PCR指纹图谱的特点并采用NTSYS2.10软件对其进行聚类。结果显示,各菌株的ERIC指纹图谱表现出不同程度的多态性,但图谱与菌株所含cry基因的类型存在一定的相关性。聚类分析结果显示,含有相同或相近cry基因类型的Bt菌株在进化树上趋向聚为一类,而不含cry基因的蜡状芽胞杆菌趋向于与不含cry基因的Bt菌株聚为一类或单独聚类。若在多种模式菌株的参考下,该方法可用于苏云金芽胞杆菌的初步鉴定和分型。 相似文献
9.
spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σE因子控制的cry1Aa基因表达。 相似文献
10.
转化cry1C基因对苏云金芽胞杆菌杀虫活性的影响 总被引:2,自引:0,他引:2
将含基因cry1C的质粒,通过电脉冲法转入含基因cry1C的质粒,通过电泳冲法转入含基因cry1Ab、cry1Ac和cry2的对小菜蛾具有高毒力的苏云金芽胞杆菌野生菌株YBT-803-1中,得到转化子MBMY-003。PCR和SDS-PAGE分析显示,cry1C可在其中正常复制、表达,但使受体菌部分内源质粒发生丢失。生物测定结果表明,转化子MBMY-003既对甜菜蛾有毒力,LC50值为1.178μL/mL,高于出发菌株YBT-803-1(LC501.879μL/mL),也对小菜蛾有毒力,LC50值为1.968μL/mL,低于YBKT-803-1(LC501.143μL/mL)。表明cry1C转入后,提高了野生菌株YBT-803-1对甜菜夜蛾的毒力,却降低了对小菜蛾的毒力。 相似文献
11.
摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。 相似文献
12.
13.
Takeshi Honda Atsuko Shiba Shigeko Seo Junko Yamamoto Junko Matsuyama Toshio Miwatani 《FEMS microbiology letters》1991,79(2-3):205-210
A hemolysin (Bt-hemolysin) produced by Bacillus thuringiensis var. kurstaki HD-1 producing crystalline toxin(s) was purified by successive treatments of ammonium sulfate (45-65%) and column chromatography using DEAE-cellulose, Sephadex G-75 and KB-002 (a hydroxyapatite column for fast protein liquid chromatography). A hemolysin (Bc-hemolysin) produced by B. cereus HG-6A was also purified by the same procedure. The purified Bt-hemolysin and Bc-hemolysin, both of which are thiol-activated hemolysins, were biologically, physicochemically and immunologically identical. These findings provide further evidence of the similarity of B. thuringiensis, which is being used as a biological insecticide, to B. cereus, a toxigenic organism of food poisoning. 相似文献
14.
添加剂对苏云金杆菌制剂杀虫效果的影响 总被引:2,自引:0,他引:2
供试13种化学物质中,12种物质对Bt制剂的杀虫效果有增效作用,各物质的不同质量浓度对杀虫效果影响差异显,在1g/L的剂量以下,多数添加剂的增效作用,随剂量的增加而增强,添加0.5g/L巯基乙醇增效作用最佳,增效倍数达3.61倍,0.5g/L的甲酸和尿素,及0.1g/L碳酸钾具有较好的增效作用,增效倍数分别为2.52,2.32和2.56和2.56倍,0.5g/L的马来酸,氯化钙,乙二胺四乙酸,氯化镁,硼酸,碳酸钠和1g/L的柠檬酸,磷酸氢二钾8种添加剂的增效倍数为0.53-1.88倍。 相似文献
15.
M. Bartoszewicz D.K. Bideshi A. Kraszewska E. Modzelewska I. Swiecicka 《Journal of applied microbiology》2009,106(6):1967-1975
Aim: To determine the potential of Bacillus thuringiensis , known primarily for its entomopathogenicity, to be a psychrotolerant contaminant of stored products.
Methods and Results: We determined the genetic properties and diversity of cold-adapted isolates of B. thuringiensis based on (i) the presence of cspA , a genetic determinant that confers psychrotolerance in Bacillus weihenstephanensis , (ii) 16S rRNA genes, and (iii) pulse-field gel electrophoretic (PFGE) genome profiles. We assessed the pathogenic potential of these isolates based on whether they harboured various combinations of known toxigenic-associated determinants ( nheA , hblA , cytK ). Of 36 nonclonal B. thuringiensis cultured from soil and milk, 21 harboured cspA , and of these, 16 (76%) were psychrotolerant and possessed genetic signatures typical of psychrotrophic Bacillus species. The majority of psychrotolerant isolates contained various combinations of nheA , hblA , and cytK .
Conclusion: Our results show that natural isolates of psychrotolerant B. thuringiensis occur in soil and milk, and suggest that psychrotolerance is determined by cspA .
Significance and Impact of the study: The presence of cspA in combination with nheA , hblA , and cytK could be of concern if commercial products are contaminated with strains that harbour these determinants. 相似文献
Methods and Results: We determined the genetic properties and diversity of cold-adapted isolates of B. thuringiensis based on (i) the presence of cspA , a genetic determinant that confers psychrotolerance in Bacillus weihenstephanensis , (ii) 16S rRNA genes, and (iii) pulse-field gel electrophoretic (PFGE) genome profiles. We assessed the pathogenic potential of these isolates based on whether they harboured various combinations of known toxigenic-associated determinants ( nheA , hblA , cytK ). Of 36 nonclonal B. thuringiensis cultured from soil and milk, 21 harboured cspA , and of these, 16 (76%) were psychrotolerant and possessed genetic signatures typical of psychrotrophic Bacillus species. The majority of psychrotolerant isolates contained various combinations of nheA , hblA , and cytK .
Conclusion: Our results show that natural isolates of psychrotolerant B. thuringiensis occur in soil and milk, and suggest that psychrotolerance is determined by cspA .
Significance and Impact of the study: The presence of cspA in combination with nheA , hblA , and cytK could be of concern if commercial products are contaminated with strains that harbour these determinants. 相似文献
16.
17.
本研究检测了在不同遗传背景的苏云金芽胞杆菌菌株中不同碳源对杀虫晶体蛋白cry1D-lacZ融合基因表达的影响。研究表明,cry1D-lacZ在同一菌株中的表达因碳源不同而异,在碳源相同的条件下不同菌株的表达也不相同,而衍生菌株HD-133-5的代谢途径非常特殊。琥珀酸钠作碳源时导致芽胞形成提前,cry1D-lacZ融合基因表达也随之提前。而以乙酸钠作碳源时,在不同亚种的菌株中都能高水平、持续稳定地表达。
Abstract:In order to investigate the influence of the carbon source on the expression of cry1D-lacZ in Bacillus thuringiensis with different genetic backgrounds,cry1D-lacZ was introduced into different strains and cultured in liquid medium containing different carbon sources.Analysis of activity of β-galactosidase showed that cry1D-lacZ expressed differenfly in the same host because of different carbon sources and differenly in different hosts utlizing same carbon sources,suggesting that metabolism pathway of carbon sourcein HD-133-5 was special.Sodium succinate as carbon source could lead to earlier.sporulation and earlier expression of cry1D-lacZ.When sodium acetate acted as carbonsource,cry1D-lacZ could keep high,stable expression in different hosts.These results arehelpful for future fermentation of Bacillus thuringiensis. 相似文献
18.
Fang Y Li Z Liu J Shu C Wang X Zhang X Yu X Zhao D Liu G Hu S Zhang J Al-Mssallem I Yu J 《遗传学报》2011,38(12):567-576
Bacillus thuringiensis (B.thuringiensis) is a soil-dwelling Gram-positive bacterium and its plasmid-encoded toxins (Cry) are commonly used as biological alternatives to pesticides.In a pangenomic study,we sequenced seven B.thuringiensis isolates in both high coverage and base quality using the next-generation sequencing platform.The B.thuringiensis pangenome was extrapolated to have 4196 core genes and an asymptotic value of 558 unique genes when a new genome is added.Compared to the pangenomes of its closely related species of the same genus,B.thuringiensis pangenome shows an open characteristic,similar to B.cereus but not to B.anthracis; the latter has a closed pangenome.We also found extensive divergence among the seven B.thuringiensis genome assemblies,which harbor ample repeats and single nucleotide polymorphisms (SNPs).The identities among orthologous genes are greater than 84.5% and the hotspots for the genome variations were discovered in genomic regions of 2.3-2.8 Mb and 5.0-5.6 Mb.We concluded that high-coverage sequence assemblies from multiple strains,before all the gaps are closed,are very useful for pangenomic studies. 相似文献
19.
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. 相似文献
20.
根据蜡状芽胞杆菌plcR基因和papR基因序列设计特异引物,对6个Bt菌株(WB1、WB7、WB9、HD98、8010、8311)及5个Bc菌株(6A1、6A2、6A3、6A4、6S1)进行了PCR检测.结果显示,3个Bt菌株及4个Bc菌株含有plcR-papR基因.克隆了Bt8010、Bc6A2和6A3的plcR、papR基因,核苷酸序列分析表明,三个菌株的plcR、papR基因与NCBI数据库中的Bt、Bc及Ba相应序列都有很高的相似性.Bt8010的plcR基因编码框由846个核苷酸组成,可编码282个氨基酸;papR基因的编码框由144个核苷酸组成,可编码48个氨基酸.推导的氨基酸序列分析表明,Bt8010 的PapR有21个氨基酸的信号肽序列,PlcR没有信号肽序列.与Bc6A2、6A3和Bc 569相比,Bt8010 的PlcR和PapR在氨基酸序列上与Bc 相应序列存在相对较大的差异.将plcR-papR基因连接到表达载体pHT304中,并转化至大肠杆菌JM109中成功进行了表达,为研究Bt plcR基因的功能奠定了基础. 相似文献