CN109628362A - Bti菌株Bt-59的sigK基因缺失突变体及其应用 - Google Patents
Bti菌株Bt-59的sigK基因缺失突变体及其应用 Download PDFInfo
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- C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
- C07K14/32—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Bacillus (G)
- C07K14/325—Bacillus thuringiensis crystal peptides, i.e. delta-endotoxins
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/75—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Bacillus
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
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- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
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Abstract
本发明涉及一种Bti菌株Bt‑59的sigK基因缺失突变体Bt59(ΔsigK)，保藏编号：CGMCC No.16822。本发明构建sigK基因缺失突变体Bt59(ΔsigK)，发现sigK基因的缺失导致Bt‑59完全丧失了形成芽胞的能力，延迟了母细胞的裂解，该结果可能有助于增强Bti的原毒素对环境因素的抗性并延长杀虫蛋白的持久性。另外，sigK缺失后，不再形成成熟的芽胞，释放晶体，并在T60母细胞裂解，在实际应用中，将来可通过高温灭菌后得到纯晶体产品，避免了微生物释放到环境中的风险。
Description
技术领域
本发明涉及生物技术领域，特别是涉及一种Bti菌株Bt-59的sigK基因缺失突变体及其 应用。
背景技术
苏云金芽胞杆菌(Bacillus thuringrensis，Bt)在形成芽胞的同时会产生一种或多种对 多种昆虫具有杀虫活性的杀虫晶体蛋白(ICPs)，是一种高效、特异且安全的微生物杀虫剂 [Sanahuja G,Banakar R,Twyman RM,Capell T,Christou P.2011.Bacillusthuringiensis: a century of research,development and commercialapplications.Plant Biotechnol J 9:283–300.]。但是野生型Bt菌株产生的ICPs自身毒力较低，并且对多种环境因素敏感， 例如紫外线(UV)[Manasherob R,Bendov E,XiaoqiangW,Boussiba S,Zaritsky A.2002. Protection from UV-B damage of mosquitolarvicidal toxins from Bacillus thuringiensis subsp.israelensis expressed inAnabaena PCC 7120.Curr Microbiol 45:217–220.]。 目前主要通过两种方式解决这些问题，一种是将编码强毒素的外源基因导入受体菌株以增加 其杀虫活性或扩大其杀虫谱[Yue C,Sun M,Yu Z.2010.Improved production of insecticidal proteins inBacillus thuringiensis strains carrying an additional cry1C gene in itschromosome.Biotechnol Bioeng 92:1–7.]，另一种是通过阻断母细胞裂解来 提高杀虫持久性[Sanchis V,Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C, DidierL.1999.Development and field performance of a broad-spectrum nonviableasporogenic recombinant strain of Bacillus thuringiensis with greater potencyand UV resistance.Appl Environ Microbiol 65:4032–4039.,Du L,Qiu L,Peng Q,Lereclus D,Zhang J,Song F,Huang D.2012.Identification of the promoter in theintergenic region between orf1and cry8Ea1controlled by sigma H factor.ApplEnviron Microbiol 78:4164–4168.]。苏云金芽胞杆菌以色列血清型变种(Bacillusthuringrensis var. israelesis，Bti)含有至少四种主要杀虫蛋白Cry4Aa，Cry4Ba，Cry11Aa和Cyt1Aa，对双 翅目昆虫表现出高毒性，例如蚊虫、黑蝇等[Bendov,E.2014.Bacillus thuringiensis subsp. israelensis and its Dipteran-specifictoxins.Toxins 6:1222–1243.]。其中Cry4Aa 对Culex具有高杀虫活性；Cry4Ba对Anopheles和Aedes具有高杀虫活性；Cry11Aa对Culex 和Aedes具有高杀虫活性；Cyt1Aa对所有蚊虫种类杀虫活性都很低，但可以协同其他杀虫蛋 白提高杀虫活性[Bendov,E.2014.Bacillus thuringiensis subsp.israelensis and its Dipteran-specifictoxins.Toxins 6:1222–1243.,Kang S,Odom OW,Malone CL, Thangamani S,HerrinDL.2018.Expression of a synthetic gene for the major Cytotoxin (Cyt1Aa)ofBacillus thuringiensis subsp.israelensis in the chloroplast of wild-typeChlamydomonas.Biology 7:29.]。前期研究中，在芽胞中期Bti所有主效杀虫基因都受σE因子的控制；在芽胞后期cry4Aa、cry11Aa和cyt1Aa受到σK因子的控制，但是具有低水平 转录活性[Yoshisue H,Fukada T,Yoshida K,Sen K,Kurosawa S,Sakai H,Komano T.1993.Transcriptional regulation of Bacillus thuringiensis subsp.israelensismosquito larvicidal crystal protein gene cryIVA.J Bacteriol 175:2750–2753.，Yoshisue H, Sakai H,Sen K,Yamagiwa M,Komano T.1997.Identification of a secondtranscriptional start site for the insecticidal protein gene cryIVA ofBacillus thuringiensis subsp. israelensis.Gene 185:251.，Yoshisue H,NishimotoT,Sakai H,Komano T.1993. Identification of a promoter for the crystalprotein-encoding gene cryIVB from Bacillus thuringiensissubsp.israelensis.Gene 137:247.，Dervyn E,Poncet S,Klier A,RapoportG.1995.Transcriptional regulation of the cryIVD gene operon from Bacillusthuringiensis subsp.israelensis.J Bacteriol 177:2283–2291.]。
在枯草芽孢杆菌(Bacillus subtilis，Bs)中，σK在孢子形成后期起着重要作用[Piggot J,Hilbert DW.2004.Sporulation of Bacillus subtilis.Curr Microbiol 7:579–586., Brehm SP,Staal SP,Hoch JA.1973.Phenotypes of pleiotropic-negativesporulation mutants of Bacillus subtilis.J Bacteriol 115:1063–1070.]。σK调节子中的基因编 码涉及σK自身催化，孢子外壳形成，母细胞裂解和孢子萌发[Zheng L,LosickR.1990. Cascade regulation of spore coat gene expression in Bacillussubtilis.J Mol Biol 212:645.，Nugroho FA,Yamamoto H,Kobayashi Y,SekiguchiJ.1999.Characterization of a new sigma-K-dependent peptidoglycan hydrolasegene that plays a role in Bacillus subtilis mother cell lysis.J Bacteriol181:6230–6237.，Steil L,Serrano M,Henriques AO, U.2005.Genome-wideanalysis of temporally regulated and compartment-specific gene expression insporulating cells of Bacillus subtilis. Microbiology 151:399.]。其中，与细胞壁水解酶相关的基因cwlC和cwlH参与母细胞的裂 解[Nugroho FA,Yamamoto H,KobayashiY,Sekiguchi J.1999.Characterization of a new sigma-K-dependent peptidoglycanhydrolase gene that plays a role in Bacillus subtilis mother cell lysis.JBacteriol 181:6230–6237.,Kuroda A,Asami Y,Sekiguchi J.1993. Molecular cloningof a sporulation-specific cell wall hydrolase gene of Bacillus subtilis.JBacteriol 175:6260–6268.]。cwlC和cwlH的单突变不影响Bs中的母细胞裂 解，而cwlC-cwlH双突变则阻断了母细胞的裂解[Nugroho FA,Yamamoto H,Kobayashi Y, SekiguchiJ.1999.Characterization of a new sigma-K-dependent peptidoglycan hydrolasegene that plays a role in Bacillus subtilis mother cell lysis.J Bacteriol181:6230–6237.,Kuroda A,Asami Y,Sekiguchi J.1993.Molecular cloning of asporulation-specific cell wall hydrolase gene of Bacillus subtilis.JBacteriol 175:6260–6268.]。另外，Bs的sigK突变体不产生成熟的孢子[Kunkel B,LosickR,Stragier. 1990.The Bacillus subtilis gene for the development transcriptionfactor sigma K is generated by excision of a dispensable DNA elementcontaining a sporulation recombinase gene.Gene Dev 4:525–535.]。而Bt与Bs之间sigK突变后存在一些差异。 Bravo等构建了B.thuringiensis 407菌株的sigK突变体，无芽胞形成并且母细胞不裂解， 但不影响Cry1Ac的蛋白产量[Bravo A,Agaisse H,SalamitouS,Lereclus D.1996.Analysis of cry1Aa expression in sigE and sigK mutants ofBacillus thuringiensis.Mol Genet Genomics 250:734–741.]；Sanchis等构建了B.thuringiensis subsp.kurstaki HD73(Kto) 菌株的sigK突变体，产生的晶体被包裹在细胞内以保护其免受紫外线的降解，提高了菌株对 环境的适应性，但减少了Cry蛋白的产量[Sanchis V,Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C,DidierL.1999.Development and field performance of a broad-spectrum nonviableasporogenic recombinant strain of Bacillus thuringiensis with greater potencyand UV resistance.Appl Environ Microbiol 65:4032–4039.]。 在B.thuringiensissubsp.kurstaki HD73中cwlC基因的表达受σK控制，与Bs中情况一 致，并且Chen等构建了cwlC突变体，证明cwlC单突变可以完全阻断母细胞裂解，而不影响 HD73中Cry1Ac的产量[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolaseCwlC from Bacillus thuringiensis is essential for mother cell lysis.ApplEnviron Microbiol 84:e02640-17.]。因此，Bti中σK的失活可能会改善杀虫 蛋白的持久性。然而，Bti中sigK的缺失对其芽胞形成、母细胞裂解和蛋白活性的影响尚不 清楚。
发明内容
本发明提供一种Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)，该突变体完全 丧失了形成芽胞的能力，并延迟了母细胞的裂解。
一种Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)，其保藏编号为CGMCCNo.16822。
一种Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)构建方法，采用同源重组构建 了Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)，所述Bti菌株Bt-59的保藏编号 为CGMCC No.16821。
所述构建方法步骤如下：
(1)以Bt-59全基因组为模板，sigK-1/sigK-2和sigK-3/sigK-4为引物分别扩增突变 盒sigKA即sigK基因上游和sigKB即sigK基因下游；
(2)以sigKA和sigKB为模板，sigK-1/sigK-4为引物扩增得到重叠片段1548 bp；再利用无缝克隆技术将重叠片段连接到用XmaI、EcoRI限制性内切酶双酶切成功的温敏载体pMAD上，构建敲除载体pMADΔsigK；
(3)将pMADΔsigK转化到Bt-59中后，构建sigK基因缺失突变体Bt59(ΔsigK)；
所述引物序列分别如下：
上述突变体Bt59(ΔsigK)在增强Bti的原毒素对环境因素的抗性并延长杀虫蛋白的持久 性中的应用。
本发明通过同源重组构建了Bti菌株Bt-59的sigK基因缺失突变体，并分析了突变体的 特征，包括芽胞和晶体的形成、母细胞的裂解和蛋白的活性，发现该突变体完全丧失了形成 芽胞的能力，并延迟了母细胞的裂解。
在B.subtilis中，sigK突变导致孢子的形成停止在孢子皮层发育的最早阶段[Cutting S,Driks A,Schmidt R,Kunkel B,Losick R.1991.Forespore-specifictranscription of a gene in the signal transduction pathway that governs Pro-sigma K processing in Bacillus subtilis.Gene Dev 5:456–466.]；Clostridium sp.、B.thuringiensis 407[Bravo A,Agaisse H,Salamitou S,Lereclus D.1996.Analysis ofcry1Aa expression in sigE and sigK mutants of Bacillus thuringiensis.MolGenet Genomics 250:734–741.]和 B.thuringiensis subsp.kurstaki HD73[Sanchis V,Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C,Didier L.1999.Developmentand field performance of a broad-spectrum nonviable asporogenic recombinantstrain of Bacillus thuringiensis with greater potency and UV resistance.ApplEnviron Microbiol 65:4032–4039.]的 sigK突变体都不再形成成熟的芽胞。但是，在Clostridium botulinum和Clostridium acetobutylicum的sigK突变体中，研究结果图片显示母细胞未裂解[Kirk DG,Dahlsten E, Zhang Z,Korkeala H,M.2012.Involvement of Clostridium botulinum ATCC 3502 sigma factor K inearly-stage sporulation.Appl Environ Microbiol 78:4590–4596., Alhinai MA,Jones SW,Papoutsakis ET.2014.σK of Clostridium acetobutylicum is the firstknown sporulation-specific sigma factor with two developmentally separatedroles,one early and one late in sporulation.J Bacteriol 196:287–299.]；B.thuringiensis 407和B.thuringiensis subsp.kurstaki HD73的sigK突变体都完全阻断了母细胞的裂解[Sanchis V,Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C,Didier L.1999.Development and field performance of a broad-spectrum nonviableasporogenic recombinant strain of Bacillus thuringiensis with greater potencyand UV resistance.Appl Environ Microbiol 65:4032–4039.,Bravo A,Agaisse H,Salamitou S,Lereclus D.1996.Analysis of cry1Aa expression in sigE and sigKmutants of Bacillus thuringiensis.Mol Genet Genomics 250:734–741.]，这些结果与本研究的发现不同，可 能是由于Bti中存在一些关键的水解酶。
母细胞的裂解与水解酶息息相关。在B.subtilis中，已经被证明存在三种主要的水解 酶CwlB、CwlC和CwlH，并且cwlC和cwlH基因的表达受σK控制[Nugroho FA,YamamotoH, Kobayashi Y,Sekiguchi J.1999.Characterization of a new sigma-K-dependentpeptidoglycan hydrolase gene that plays a role in Bacillus subtilis mothercell lysis. J Bacteriol 181:6230–6237.,Kuroda A,Asami Y,SekiguchiJ.1993.Molecular cloning of a sporulation-specific cell wall hydrolase geneof Bacillus subtilis.J Bacteriol 175:6260–6268.,Foster SJ.1992.Analysis ofthe autolysins of Bacillus subtilis 168during vegetative growth anddifferentiation by using renaturing polyacrylamide gel electrophoresis.JBacteriol 174:464–470.]。cwlB、cwlC或cwlH的单突变不影响 B.subtilis的母细胞裂解，而cwlB-cwlC或cwlC-cwlH双突变和cwlB-cwlC-cw1H三突变阻 断了母细胞的裂解[NugrohoFA,Yamamoto H,Kobayashi Y,Sekiguchi J.1999. Characterization of a new sigma-K-dependent peptidoglycan hydrolase gene that plays a role in Bacillussubtilis mother cell lysis.J Bacteriol 181:6230–6237.,Smith TJ,FosterSJ.1995.Characterization of the involvement of two compensatory autolysins inmother cell lysis during sporulation of Bacillus subtilis 168.J Bacteriol177:3855.]。在B.thuringiensis subsp.kurstaki HD73中，CwlB和CwlC参 与母细胞的裂解[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wallhydrolase CwlC from Bacillus thuringiensis is essential for mother celllysis. Appl Environ Microbiol 84:e02640-17.,Yang J,Peng Q,Chen Z,Deng C,ShuC,Zhang J,Huang D,Song F.2013.Transcriptional regulation and characteristicsof a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillusthuringiensis mother cell lysis.J Bacteriol 195:2887–2897.]，但CwlH尚未见报道。在HD73中，cwlB的突 变延迟了母细胞裂解，而cwlC的突变完全阻断了母细胞裂解[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolase CwlCfrom Bacillus thuringiensis is essential for mother cell lysis.Appl EnvironMicrobiol 84:e02640-17.,Yang J, Peng Q,Chen Z,Deng C,Shu C,Zhang J,Huang D,Song F.2013.Transcriptional regulation and characteristics of a novel N-acetylmuramoyl-L-alanine amidase gene involved in Bacillus thuringiensismother cell lysis.J Bacteriol 195:2887–2897.]。 因此，在Bt-59中，可能存在几种水解酶通过相互作用调节母细胞的裂解，如在B.subtilis 中；或者可能是某一种关键水解酶调节母细胞的裂解，如HD73中的CwlC。
由于cwlC的缺失完全阻断了HD73母细胞的裂解[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolase CwlC from Bacillusthuringiensis is essential for mother cell lysis.Appl Environ Microbiol 84:e02640-17.]，通过比较 Bt-59和HD73的cwlC启动子转录活性，证明了Bt-59与HD73 cwlC基因的转录受σK控制。 然而，Bt-59 CwlC无法恢复HD(ΔcwlC)的表型，表明Bt-59 CwlC的母细胞裂解功能可能丧 失。虽然Bt-59和HD73之间的CwlC保守结构域(MurNAc-LAA domain和amidase02 domain) 的氨基酸同源性非常高，CwlC(E24和E140)[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolase CwlC from Bacillusthuringiensis is essential for mother cell lysis.Appl Environ Microbiol 84:e02640-17.]的关键催化残基也相同， 然而，其保守结构域的一些差异可能就导致了Bt-59和HD73中CwlC的功能不同，以及Bt-59 CwlC的细胞壁水解酶活性可能丧失。
sigK基因的缺失降低了Bt-59对C.pipiens的毒性，但没有影响对A.albopictus的毒 性。可能是由于sigK缺失导致一些次要蛋白的变化影响了蛋白之间的协同作用，从而导致整 体杀虫活性的降低[Bideshi DK,Waldrop G,Fernandezluna MT,Diazmendoza M,Wirth MC, Johnson JJ,Park H,Federici BA.2013.Intermolecular interactionbetween Cry2Aa and Cyt1Aa and its effect on larvicidal activity against Culexquinquefasciatus.J Mol Biol 23:1107–1115.，Fernandezluna MT,Lanzmendoza H,GillSS,Bravo A,Soberon M, Mirandarios J.2010.Anα-amylase is a novel receptor forBacillus thuringiensis ssp.israelensis Cry4Ba and Cry11Aa toxins in themalaria vector mosquito Anopheles albimanus(Diptera:Culicidae).EnvironMicrobiol 12:746–757.，Elleuch J,Jaoua S,Darriet F,Chandre F,Tounsi S,ZghalRZ.2015.Cry4Ba and Cyt1Aa proteins from Bacillus thuringiensis israelensis:Interactions and toxicity mechanism against Aedes aegypti.Toxicon 104:83–90.]；另外也有研究显示野生型Bt的活芽胞存在低毒性 [Miyasono M,Inagaki S,Yamamoto M,Ohba K,Ishiguro T,Takeda R,Hayashi Y.1994. Enhancement of δ-endotoxin activity by toxin-free spore of Bacillus thuringiensis against thediamondback moth,Plutellaxylostella.J Invertebr Pathol 63:111–112., Tang JD,Shelton AM,VanRJ,De RS,Moar WJ,Roush RT,Peferoen M.1996.Toxicity of Bacillusthuringiensis spore and crystal protein to resistant diamondback moth(Plutella xylostella).Appl Environ Microbiol 62:564–569.]，这可能也是Bt59(ΔsigK) 杀虫活性降低的一个原因。尽管Bti Cry11Aa蛋白对Culex和Aedes都具有高毒性[Kang S, Odom OW,Malone CL,Thangamani S,Herrin DL.2018.Expression of asynthetic gene for the major Cytotoxin(Cyt1Aa)of Bacillus thuringiensissubsp.israelensis in the chloroplast of wild-type Chlamydomonas.Biology 7:29.]，但sigK的缺失不影响Bt-59 Cry11Aa蛋白的产量。因此，通过分析cry11Aa启动子的转录活性，表明cry11Aa基因表达 不依赖于σK。Dervyn等在B.subtilis 168中分析了Bti4Q2-81cry11Aa基因启动子的转 录活性，发现cry11Aa基因的转录可能在芽胞形成后期受σK控制，尽管其转录活性较弱 [Dervyn E,Poncet S,Klier A,RapoportG.1995.Transcriptional regulation of the cryIVD gene operon from Bacillusthuringiensis subsp.israelensis.J Bacteriol 177:2283–2291.]，这与本研究结果不一致。然而，在本研究中，cry11Aa启动子的转录活 性是在野生型Bt-59中进行了检测，结果可能更准确。
在B.thuringiensis中已经证明释放的晶体受到各种环境因素的影响，主要是 UV[Manasherob R,Bendov E,Xiaoqiang W,Boussiba S,Zaritsky A.2002.Protection fromUV-B damage of mosquito larvicidal toxins from Bacillus thuringiensis subsp.israelensis expressed in Anabaena PCC 7120.Curr Microbiol 45:217–220.,SanchisV, Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C,DidierL.1999.Development and field performance of a broad-spectrum nonviableasporogenic recombinant strain of Bacillus thuringiensis with greater potencyand UV resistance.Appl Environ Microbiol 65:4032–4039.]，而σK的失活母细胞裂解的阻断，从而保护晶体免于UV降解 [Sanchis V,Gohar M,Chaufaux J,Arante O,Meie A,Agaisse H,Jane C,Didier L.1999. Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensiswith greater potency and UV resistance. Appl Environ Microbiol 65:4032–4039.,Bravo A,Agaisse H,Salamitou S,Lereclus D. 1996.Analysis of cry1Aa expressionin sigE and sigK mutants of Bacillus thuringiensis. Mol Genet Genomics 250:734–741.]。在B.thuringiensis subsp.kurstaki HD73中，CwlC 的失活也阻断了母细胞的裂解，增加了对紫外线的抵抗力并延长了杀虫持久性[Chen X,Gao T, Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolase CwlC from Bacillusthuringiensis is essential for mother cell lysis.Appl Environ Microbiol 84:e02640-17.]。Bt-59 sigK的缺失延迟了母细胞的裂解，该结果可能有助于增强Bti的原 毒素对环境因素的抗性并延长杀虫蛋白的持久性。另外，sigK缺失后，不再形成成熟的芽胞，释放晶体，并在T60母细胞裂解，在实际应用中，将来可通过高温灭菌后得到纯晶体产品，避免了微生物释放到环境中的风险。
保藏信息：
Bt-59保藏编号：CGMCC No.16821；
分类命名苏云金芽孢杆菌以色列变种Bacillus thuringiensis var.israelesis
保藏日期：2018年11月26日
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心
保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所
Bt59(ΔsigK)保藏编号：CGMCC No.16822。
分类命名苏云金芽孢杆菌以色列变种Bacillus thuringiensis var.israelesis
保藏日期：2018年11月26日
保藏单位：中国微生物菌种保藏管理委员会普通微生物中心
保藏地址：北京市朝阳区北辰西路1号院3号，中国科学院微生物研究所
附图说明
图1 SDS-PAGE分析Bt-59和Bt59(ΔsigK)的晶体蛋白产量。
图2 Bt-59 cry11Aa的转录活性。(A)分析Bt-59 cry11Aa的上游区域。*表示转录起始位 点，蓝线区表示-35和-10区，P19和cry11Aa的翻译起始密码子用红线表示，核糖体结合位 点(RBS)用双下划线表示。(B)构建cry11Aa启动子与pHT304-18Z载体连接的重组载体示 意图。(C)测定Pcry11Aa在野生型Bt-59(●)和突变体Bt59(ΔsigK)(▲)中的β-半乳糖苷酶 活性。每个值代表至少三次独立重复的平均值。
图3 Bt59(ΔsigK)突变体的芽胞形成。(A)通过光学显微镜观察芽胞和晶体的形成。比 例尺，10μm。(B)Bt-59、Bt59(ΔsigK)和Bt59(HFsigK)的总细胞数和芽胞数。
图4野生型Bt-59、Bt59(ΔsigK)突变体和恢复株Bt59(HFsigK)的母细胞裂解。在T14、T22和T25观察野生型Bt-59和恢复株Bt59(HFsigK)母细胞的裂解。在T14、T22、T45和T60观察Bt59(ΔsigK)母细胞的裂解。比例尺，10μm。
图5 Bt-59 cwlC的转录由σK控制。(A)Bti Bt-59和B.thuringiensissubsp.kurstaki HD73的CwlC保守结构域。MurNAc-LAA(蓝色条)代表N-乙酰基胞壁酰基-L-丙氨酸酰胺酶结 构域。(B)Bt-59和HD73的cwlC启动子的序列比较。TSS表示转录起始位点，-35和-10区 用红色框标记，cwlC的翻译起始密码子用红线表示。(C)和(D)PBcwlC和PHcwlC在野生型Bt-59 (●)和突变体Bt59(ΔsigK)(△)中的β-半乳糖苷酶活性。每个值代表至少三次独立重复的平 均值。
图6用Bt-59 CwlC恢复HD(ΔcwlC)的母细胞裂解。通过光学显微镜观察15d后HD(ΔcwlC) 和HD(BHFcwlC)和在T24时的HD(HFcwlC)的母细胞裂解情况。比例尺，10μm。
图7比较Bt-59和HD73保守结构域。(A)MurNAc-LAA结构域氨基酸序列的比较。红框表 示两个保守的关键谷氨酸残基。(B)Amidase02-C结构域氨基酸序列的比较。
具体实施方式
下面结合实施例对本发明做进一步的详细说明。
实施例1
1材料和方法
1.1供试蚊虫、菌株、质粒和培养条件
白纹伊蚊(Aedes albopictus)，虫种引自江苏省血吸虫病防治研究所，淡色库蚊(Culex pipiens)，江苏里下河地区农业科学研究所饲养。所有菌株与质粒见表1，均可以对公众发 放。其中Bt-59菌株采集自邗江区小官桥6号，其保藏编号为CGMCC No.16821，江苏里下河 地区农业科学研究所有保存。E.coli ET用于对质粒进行去甲基化。所有E.coli菌株使用 LB培养基培养。Bt-59感受态制备培养基为BHI培养基(3.7％Brain HeartInfusion Broth)， 发酵培养基配方为4.5％豆饼粉、2％淀粉、2.0％玉米浆、0.1％MgSO4、0.1％CaCO3、0.1％KH2PO4。 SSM培养基(Schaeffer’s sporulation medium)：8g ofnutrient broth,，0.012％MgSO4， 0.1％KCl，1mM NaOH，用于胞子形成实验研究。
表1菌株和质粒
[25]Kirk DG,Dahlsten E,Zhang Z,Korkeala H,2012.Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation.Appl Environ Microbiol 78:4590–4596.
1.2 DNA操作
PrimeSTAR HS DNA聚合酶、限制性内切酶和T4DNA连接酶为TaKaRa公司产品(TaKaRa Biotechnology Corporation,Beijing,China)；TaqMix DNA聚合酶为BioMed公司产品 (BioMed,Beijing,China)；AxyPrep PCR纯化试剂盒、AxyPrep Plasmid Miniprep试剂盒 为Axygen公司产品(Axygen Biotechnology Corporation,Beijing,China)。引物合成由 Sangon Biotech(Beijing,China)，本研究所有引物序列见表2。
表2引物
菌液PCR按照以下方法操作，取1mL菌液10000×g离心1min；加入200μL ddH2O 悬浮沉淀，100℃下煮沸10min，然后10000×g离心1min，取上清液作为模板。PCR程 序如下：在98℃预变性5min，在98℃变性10s，在55℃退火15s，在72℃延伸1kb/min， 退火温度视情况而定。
1.3 Bt-59全基因组的提取及测序
利用150μL溶液SI(10.0mmol/L Tris-HCl，1.0mmol/L EDTA，1.0mo1/L蔗糖)、 适量溶菌酶和RNase A裂解100mg细菌菌体。再加入400μL异硫氰酸胍，离心10min。 取上清，加入与上清等体积的异丙醇沉淀DNA，最后加入适量ddH2O溶解沉淀，即得到Bt-59 的全基因组。利用Pacbio测序平台对Bt-59进行全基因组测序，使用HGAP(hierarchical genome-assembly process)对获得的测序数据进行组装，并使用RFam、Nr、KEGG、Swissprot 库对所有基因进行功能注释。
1.4质粒的构建与转化
通过同源重组的方法构建sigK基因缺失突变体Bt59(ΔsigK)。首先以Bt-59全基因组 为模板，sigK-1/sigK-2和sigK-3/sigK-4为引物分别扩增突变盒sigKA(sigK基因上游)和 sigKB(sigK基因下游)。以sigKA和sigKB为模板，sigK-1/sigK-4为引物扩增得到重叠片 段(1548bp)。再利用无缝克隆技术将重叠片段连接到用XmaI、EcoRI限制性内切酶双酶切成 功的温敏载体pMAD上，构建敲除载体pMADΔsigK。以Bt-59全基因组为模板，PBcw1C-F/PBcw1C-R为引物扩增617bp cwlC启动子区域(PBcwlC)，用PstI和BamHI双酶切 目的片段PBcwlC，并连接到含有无启动子lacZ基因的线性化载体pHT304-18Z中，构建重组载 体pHTPBcwlC。cry11Aa启动子(Pcry11Aa)连接到pHT304-18Z载体、sigK启动子和ORF(openreading frame)区域(PsigK-sigK)以及Bt-59 cwlC启动子和ORF区域(PBcwlC-BcwlC)连接到pHT315载体都是采用相同方法，将这些重组载体分别命名为pHTPcry11Aa，pHTHFsigK和pHTHFBcwlC。对所有重组载体进行PCR鉴定和酶切鉴定，并进行测序验证，然后将载体包括pHTPHcwlC(实验室库存，也称为pHTPcwlC)[20]去甲基化后转化到相应Bt菌株中获得所需菌株。通过化学转化[35]和电穿孔[36]将载体分别转化到E.coli和Bt中。根据需要在培养基中加入氨苄抗生素(终浓度为100μg/mL)或红霉抗生素(终浓度为5μg/mL)。将 pMADΔsigK转化到Bt-59中后，构建sigK基因缺失突变体Bt59(ΔsigK)，保藏编号：CGMCCNo.16822。将pHTPBcwlC和pHTPHcwlC分别转化到Bt-59和Bt59(ΔsigK)，获得菌株 Bt59(PBcwlC-lacZ)、Bt59(ΔsigK)(PBcwlC-lacZ)和Bt59(PHcwlC-lacZ)、Bt59(ΔsigK)(PHcwlC-lacZ)。 将pHTPcry11Aa转化到Bt-59和Bt59(ΔsigK)，构建菌株Bt59(Pcry11Aa-lacZ)和 Bt59(ΔsigK)(Pcry11Aa-lacZ)。将pHTHFsigK转化到Bt59(ΔsigK)中，构建恢复菌株 Bt59(HFsigK)。将pHTHFBcwlC转化到HD(ΔcwlC)，构建恢复菌株HD(BHFcwlC)。
1.5显微镜观察
将Bt-59和Bt59(ΔsigK)在100mL SSM培养基中，30℃220r/min条件下培养至指定时间。各取1mL菌液，离心后去上清，用去离子水清洗一遍，再加入适当蒸馏水稀释，从中 取1μL滴于载玻片上，盖上盖玻片，样品用BX61光学显微镜(Olympus,Japan)观察。
1.6芽胞和晶体蛋白产量测定
将Bt-59和Bt59(ΔsigK)在SSM培养基中培养，在细胞基本全部裂解时取1mL样品，65℃加热20min以杀死营养细胞，适当梯度稀释后涂布于LB固体平板上，统计样品中的活芽胞数。结果显示的数据为三次独立实验的平均值。
取样2mL，10000×g离心2min收集沉淀，加入500μL ddH2O重悬，利用BeBeBeater(Biospec Products,Inc.Bartlesville,OK)破碎。然后将上清液与5×蛋白上样缓冲液[Millet JH,Experiments in molecular genetics,1972.Cold Spring Harbor Press:Cold Spring Harbor,NY.]混合，煮沸10min后进行总蛋白质定量和SDS-PAGE(sodiumdodecyl sulfate polyacrylamide gel electrophoresis)[SambrookHC.1989.Molecular cloning: a laboratory manual.Cold Spring Harbor,NY.]。切下目的蛋白条带通过LC-MS/MS(liquid chromatograph-mass spectrometry)检测。
1.7 β-半乳糖苷酶活性分析
在指定的时间点(从T0到T12或T8到T20，间隔1h)收集在SSM中培养的2mL样品，将 样品10000×g离心2min收集菌体，测量β-半乳糖苷酶活性[Millet JH,Experiments inmolecular genetics,1972.Cold Spring Harbor Press:Cold Spring Harbor,NY.]。β-半乳糖苷酶活性以Miller units表示。结果显示数据是三次独立实验的平均值。
1.8生物活性测定
挑取Bt-59和Bt59(ΔsigK)单菌落，使用发酵培养基培养至晶体形成，以上述菌液为原 液，按0.156、0.312、0.625、1.250、2.500、5.000、10.000μl/L浓度梯度在罐子中配置上述菌株处理液，每处理分别接入4龄早期白色伊蚊或淡色库蚊的孑孓20头，每个蚊种每个浓度处理各三个重复，同时设蒸馏水为对照，置(28±0.5)℃的培养皿内培养，处理24h后统计死亡率，使用SPSS 22.0方差分析软件计算致死中浓度LC50和P值。
2结果
2.1 Bt-59全基因组和sigK基因序列分析
对Bt-59的全基因组进行全基因组测序，其全基因组由一个Genome(包括5,824个ORF) 和五个质粒(分别包括341、457、277、131和113个ORF)构成(表3)。质粒plasmid4包 含5个cry基因和3个cyt基因，即cry11Aa、cry4Ba、cry10Aa、cry4Aa和cyt1Aa、cyt2Ba、cyt1Ca，在其他质粒和基因组中未检测到cry/cyt基因。
表3 Bt-59全基因组的统计结果
a ORFs:开放阅读框；b CDS:蛋白质编码区。
根据B.thuringiensis subsp.kurstaki HD73的sigK基因序列，通过BLAST搜索找到 Bt-59全基因组中的sigK基因序列。Bt-59 sigK基因大小为714bp，编码237个氨基酸。Bt-59 sigK氨基酸序列与其他Bt亚种相似性为100％，例如B.thuringrensisvar.israelesis AM65-52、B.thuringiensis subsp.morrisoni HD12和B.thuringiensissubsp.kurstaki HD73；与Bacillus anthracis MCCC相似性为100％，与Bacillus cereusG9842相似性为100％， 但是与B.subtilis相似性为86％。
2.2 sigK突变对Cry产量和毒力的影响
构建sigK基因缺失突变体Bt59(ΔsigK)保藏编号：CGMCC No.16822。将Bt-59和Bt59(ΔsigK)在相同的培养条件下培养至大部分芽胞和晶体从母细胞中释放后，通过SDS-PAGE检测Cry蛋白的产量。与Bt-59相比，Bt59(ΔsigK)主要蛋白的产量有所变化，Cry4Aa/4Ba蛋白表达量略微升高，Cry11Aa和Cyt1Aa蛋白表达量基本不变(图1)。将切下 的主要蛋白条带(Bt-59和Bt59(ΔsigK))通过LC-MS/MS检测，证明四种蛋白的正确性(表 4)。
表4.Bt-59和Bt59(ΔsigK)晶体的质谱结果
通过生物活性测定(表5)，对于C.pipiens，Bt-59的LC50为1.20μl/L，95％置信区间为0.99–1.44μl/L；而Bt59(ΔsigK)的LC50为1.67μl/L，95％置信区间为1.46–1.99 μl/L，说明sigK基因的缺失显著降低了Bt-59对C.pipiens的杀虫活性(P＝0.001)。对 于A.albopictus，Bt-59的LC50为6.89μl/L，95％置信区间为4.76-13.38μl/L；而 Bt59(ΔsigK)的LC50为7.45μl/L，95％置信区间为5.00-17.48μl/L，说明sigK基因的缺 失不影响Bt-59对A.albopictus的杀虫活性(P＝0.360)。
表5 Bt-59和Bt59(ΔsigK)对Culex pipiens和Aedes albopictus的杀虫活性
2.3 cry11Aa的转录不依赖于σK
Bti Cry11Aa对Culex和Aedes都有很高的毒性[Kang S,Odom OW,Malone CL,Thangamani S,Herrin DL.2018.Expression of a synthetic gene for the majorCytotoxin(Cyt1Aa) of Bacillus thuringiensis subsp.israelensis in thechloroplast of wild-type Chlamydomonas.Biology 7:29.]，然而sigK的缺失不影响Bt-59 Cry11Aa蛋白的产量。因 此，分析了cry11Aa启动子的序列和其转录活性。由于P19、cry11Aa和P20构成一个操纵子 [11]，因此通过分析P19上游序列，推测在转录起始位点上游-10区和-35区的σE保守结合 序列分别为CATATATT和GCATCGT(图2中A)[Dervyn E,Poncet S,Klier A,Rapoport G. 1995.Transcriptional regulation of the cryIVDgene operon from Bacillus thuringiensis subsp.israelensis.J Bacteriol 177:2283–2291.]。通过检测cry11Aa 启动子在Bt-59和Bt59(ΔsigK)中的转录活性，发现sigK基因的缺失对早期cry11Aa'-lacZ 的表达无明显影响，但是从T8(T0表示指数生长期刚结束时；Tn表示T0之后n小时时期往后， 在Bt59(ΔsigK)中的转录活性要高于Bt-59(图2中B和图2中C)，说明cry11Aa基因的 表达应不受σK因子的控制。
2.4 sigK缺失对芽胞形成的影响
在SSM培养基中培养Bt-59、Bt59(ΔsigK)和Bt59(HFsigK)，并通过光学显微镜观察芽 胞的形成情况。Bt-59在晚期形成芽胞和晶体；而Bt59(ΔsigK)可产生晶体，但没有芽胞(图 3中A)。Bt-59的总细胞数和芽胞数分别为4.21×107CFU/ml和2.43×106CFU/ml(图3中 B)，而Bt59(ΔsigK)总细胞数为1.76×107CFU/mL(图3B)。通过恢复菌株Bt59(HFsigK)恢复了芽胞的形成，其总细胞数和芽胞数分别为4.64×107CFU/mL和3.33×106CFU/mL(图 3中B)。
2.5 sigK缺失对母细胞裂解的影响
在SSM培养基中将Bt-59、Bt59(ΔsigK)和Bt59(HFsigK)分别培养至指定时间，利用光 学显微镜观察菌株在不同生长期中的细胞表型。Bt-59、Bt59(ΔsigK)和Bt59(HFsigK)在T14时母细胞均未裂解(图4)。Bt-59在T22时约50％母细胞裂解，此时Bt59(ΔsigK)极少数母 细胞裂解，而Bt59(ΔsigK)在T45时达到约50％母细胞裂解(图4)。Bt-59和Bt59(ΔsigK) 分别在T25和T60时达到约100％的母细胞裂解(图4)。恢复菌株Bt59(HFsigK)在T25时达100％ 母细胞裂解，表明恢复了母细胞裂解的能力(图4)。这些结果表明sigK的缺失延迟了Bt-59 母细胞裂解。
2.6 Bt-59 cwlC的转录依赖于σK，且Bt-59 CwlC无法恢复HD(ΔcwlC)的表型
在B.thuringiensis subsp.kurstaki HD73中sigK缺失使母细胞的不再裂解，并且 HD73 cwlC的突变完全阻断了母细胞裂解[Chen X,Gao T,Peng Q,Zhang J,Chai Y,Song F.2018.The novel cell wall hydrolase CwlC from Bacillus thuringiensis isessential for mother cell lysis.Appl Environ Microbiol 84:e02640-17.]。比较Bt-59和HD73的 cwlC基因，CwlCs的氨基酸序列同源性为97％，并且都包含两个保守结构域N-端MurNAc-LAA domain和C-端amidase02 domain(图5中A)，两保守结构域的氨基酸同源性分别为99％和 97％，其关键催化位点(E24和E140)也相同(图7中B)[Chen X,Gao T,PengQ,Zhang J, Chai Y,Song F.2018.The novel cell wall hydrolase CwlC fromBacillus thuringiensis is essential for mother cell lysis.Appl EnvironMicrobiol 84:e02640-17.]，但是 MurNAc-LAA domain仍有2个氨基酸的差异，位于50和62位(图7中A)；amidase02 domain 存在1个氨基酸的差异，位于23位(图7中B)。推测Bt-59cwlC在转录起始位点上游-10 区和-35区的σK保守结合序列分别为AGCA和AATAAGATA(HDCA和CATANNNDD；H is A/C/T, D is A/G/T,and N is A/C/G/T)(图5中B)[Eichenberger P,Fujita M,Jensen ST,Conlon EM,Rudner DZ,Wang ST,Ferguson C,Haga K,Sato T,LiuJS,Losick R.2004.The program of gene transcription for a singledifferentiating cell type during sporulation in Bacillus subtilis.PLoS Biol2:e328.]。但是，与HD73 cwlC启动子区相比，有两段序列 在Bt-59 cwlC中丢失(图5中B)。通过比较Bt-59和HD73的cwlC启动子转录活性，两者 的cwlC启动子在Bt59(ΔsigK)中都没有转录活性(图5中C和图5中D)，说明Bt-59 cwlC 基因的表达受σK控制，这与先前对HD73cwlC基因表达的发现一致[Chen X,Gao T,Peng Q, Zhang J,Chai Y,Song F.2018.Thenovel cell wall hydrolase CwlC from Bacillus thuringiensis is essential formother cell lysis.Appl Environ Microbiol 84:e02640-17.]。然而，通过Bt-59 cwlC基因及其启动子区导入HD(ΔcwlC)构建的恢复菌 株HD(BHFcw1C)，母细胞未裂解，与HD(ΔcwlC)表型相同(图6)。结果表明，Bt-59 CwlC 无法恢复HD(ΔcwlC)的表型。
sigK基因的缺失降低了Bt-59对C.pipiens的毒性，但没有影响对A.albopictus的毒 性。尽管Bti Cry11Aa蛋白对Culex和Aedes都具有高毒性，但sigK的缺失不影响Bt-59Cry11Aa蛋白的产量。因此，通过分析cry11Aa启动子的转录活性，表明cry11Aa基因表达 不依赖于σK。
Bt-59 sigK的缺失延迟了母细胞的裂解，该结果可能有助于增强Bti的原毒素对环境因 素的抗性并延长杀虫蛋白的持久性。另外，sigK缺失后，不再形成成熟的芽胞，释放晶体， 并在T60母细胞裂解，在实际应用中，将来可通过高温灭菌后得到纯晶体产品，避免了微生物 释放到环境中的风险。
Claims (4)
1.一种Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)，保藏编号：CGMCCNo.16822。
2.权利要求1所述的Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)的构建方法，采用同源重组构建了Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)，所述Bti菌株Bt-59的保藏编号为CGMCC No.16821。
3.根据权利要求2所述构建方法，步骤如下：
(1)以Bt-59全基因组为模板，sigK-1/sigK-2和sigK-3/sigK-4为引物分别扩增突变盒sigKA即sigK基因上游和sigKB即sigK基因下游；
(2)以sigKA和sigKB为模板，sigK-1/sigK-4为引物扩增得到重叠片段1548bp；再利用无缝克隆技术将重叠片段连接到用XmaI、EcoRI限制性内切酶双酶切成功的温敏载体pMAD上，构建敲除载体pMADΔsigK；
(3)将pMADΔsigK转化到Bt-59中后，构建sigK基因缺失突变体Bt59(ΔsigK)；
所述引物序列分别如下：
4.根据权利要求1的Bti菌株Bt-59的sigK基因缺失突变体Bt59(ΔsigK)在增强Bti的原毒素对环境因素的抗性并延长杀虫蛋白的持久性中的应用。
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| US6096306A (en) * | 1995-10-27 | 2000-08-01 | Institut Pasteur | Strains of Bacillus thuringiensis and pesticide composition containing them |
| CN108411009A (zh) * | 2018-03-02 | 2018-08-17 | 蔡先全 | 检测食品中产气荚膜梭菌和sigK基因的引物、试剂盒和方法 |
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