晶胞粘连苏云金芽胞杆菌C15杀线虫毒力因子发掘与功能鉴定
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摘要
苏云金芽胞杆菌(Bacillus thuringiensis,Bt)制剂作为一种高效的微生物杀虫剂,在植物病虫害防控领域有着广泛的应用。Bt制剂的主效成分为杀虫晶体和芽胞,其中,杀虫晶体的环境低持久性是Bt农药应用的重要限制因素之一。自然界中存在着一些Bt菌株,其产生的杀虫晶体位于芽胞外壁和芽胞衣之间,这种特殊的表型被称为晶胞粘连(spore-crystal association,SCA)表型。由于芽胞外壁对晶体的保护作用,SCA表型可以提升晶体抵抗不良环境因素的能力,是开发新型Bt生物囊杀虫剂的有效育种策略。本文选取对线虫具有强毒杀能力的SCA菌株C15作为研究对象。获得了C15菌株的完整基因组序列,包括一个5 637 049 bp的环状染色体和8个不同大小的环形质粒(240 314 bp到3 188 bp)。C15基因编码了5个杀虫蛋白(Cry蛋白)基因:cry21-99、cry21-67、cry21-66、cry21-46和cry-N。在Bt无晶体突变株BMB171中异源表达cry21-99基因,发现其表达产物形成菱形晶体,且对秀丽隐杆线虫(Caenorhabditis elegans)和南方根结线虫(Meloidogyne incognita)均有毒杀活性。同时,还在全基因组范围内预测了Cry毒素以外的杀线虫毒力因子和次生代谢产物。此外,在C15基因组中预测了本团队已报道的苏云金芽胞杆菌幕虫亚种(B. thuringiensis serovar finitimus)菌株YBT-020 SCA表型决定因子的同源基因,缺失后突变体仍然保留稳定的SCA表型,说明C15菌株的SCA表型形成机制与YBT-020不同,该菌株代表了一种新的SCA表型形成机制。本研究为转基因作物防控线虫提供了新的遗传资源,也为研究SCA表型形成机制,开发新型高效Bt制剂提供了新线索。
As one of the most successful microbial pesticide,Bacillus thuringiensis(Bt)products are widely used in the field of plant disease and pest control. The main components of Bt products are insecticidal crystals and spores. One of the limiting factors in Bt based products usage in agriculture is that the sprayed crystals are sensitive to the sun radiation and degraded in few days. In some Bt strains,the mature crystals are located between the exosporium and the spore coat.This special phenotype is called as spore-crystal association(SCA). In SCA phenotype strains,exosporium provides a natural defense,improving the crystal resistance to adverse environmental conditions. Artificial simulated SCA is an ef-fective breeding strategy to develop new Bt capsule pesticide. Bt C15 is highly toxic to nematode and has a SCA pheno-type. Here we presented the complete genome sequence of C15,which consists of a 5,637,049 bp circular chromosome,and 8 plasmids(from 240 314 bp to 3 188 bp). C15 genome encodes five novel insecticidal crystal potein(Cry)genes,in-cluding cry21-99,cry21-67,cry21-66,cry21-46 and cry-N. In this work,the cry21-99 gene was cloned and expressed in the acrystalliferous Bt mutant BMB171. The purified Cry21-99 toxin showed high toxicity against Caenorhabditis elegans and Meloidogyne incognita. In addition,genes of other nematocidal factors and secondary metabolites were predicted.This work provides novel genetic resources for transgenic crops to control nematodes. Previously,we have identified the SCA controller in another B. thuringiensis SCA stain YBT-020(serovar finitimus). The homolog genes of these SCA controller in C15 genome were predicted. However,the gene interrupted mutant maintained stable SCA phenotype. It in-dicates that the SCA formation in C15 is different from that of YBT-020,which represents a new SCA phenotype forma-tion mechanism. This work provides clues for understanding the mechanism of SCA phenotype and development of novel high efficiency Bt capsule pesticides.
As one of the most successful microbial pesticide,Bacillus thuringiensis(Bt)products are widely used in the field of plant disease and pest control. The main components of Bt products are insecticidal crystals and spores. One of the limiting factors in Bt based products usage in agriculture is that the sprayed crystals are sensitive to the sun radiation and degraded in few days. In some Bt strains,the mature crystals are located between the exosporium and the spore coat.This special phenotype is called as spore-crystal association(SCA). In SCA phenotype strains,exosporium provides a natural defense,improving the crystal resistance to adverse environmental conditions. Artificial simulated SCA is an ef-fective breeding strategy to develop new Bt capsule pesticide. Bt C15 is highly toxic to nematode and has a SCA pheno-type. Here we presented the complete genome sequence of C15,which consists of a 5,637,049 bp circular chromosome,and 8 plasmids(from 240 314 bp to 3 188 bp). C15 genome encodes five novel insecticidal crystal potein(Cry)genes,in-cluding cry21-99,cry21-67,cry21-66,cry21-46 and cry-N. In this work,the cry21-99 gene was cloned and expressed in the acrystalliferous Bt mutant BMB171. The purified Cry21-99 toxin showed high toxicity against Caenorhabditis elegans and Meloidogyne incognita. In addition,genes of other nematocidal factors and secondary metabolites were predicted.This work provides novel genetic resources for transgenic crops to control nematodes. Previously,we have identified the SCA controller in another B. thuringiensis SCA stain YBT-020(serovar finitimus). The homolog genes of these SCA controller in C15 genome were predicted. However,the gene interrupted mutant maintained stable SCA phenotype. It in-dicates that the SCA formation in C15 is different from that of YBT-020,which represents a new SCA phenotype forma-tion mechanism. This work provides clues for understanding the mechanism of SCA phenotype and development of novel high efficiency Bt capsule pesticides.
引文
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[7]Bravo A,Likitvivatanavong,S,Gill,S S,et al.Bacillus thuringiensis:a story of a successful bioinsecticide[J].Insect Biochem Mol Biol,2011,41(7):423-431.
[8]Sanahuja G,Banakar R,Twyman R M,et al.Bacillus thuringiensis:a century of research,development and commercial applications[J].Plant Biotechnol J,2011,9(3):283-300.
[9]Bechtel D B,Bulla L A Jr.Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis[J].J Bacteriol,1976,127(3):1472-1481.
[10]Myasnik M,Manasherob R,Ben-Dov E,et al.Com-parative sensitivity to UV-B radiation of two Bacillus thuringiensis subspecies and other Bacillus sp.[J].Curr Microbiol,2001,43(2):140-143.
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[13]Wojciechowska J A,Lewitin E,Revina L P,et al.Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp.finitimus[J].FEBS Lett,1999,453(1-2):46-48.
[14]Ji F,Zhu Y,Ju S,et al.Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp.finitimus strain YBT-020[J].FEMS Microbiol Lett,2009,300(1):11-17.
[15]Zhu Y,Shang H,Zhu Q,et al.Complete genome se-quence of Bacillus thuringiensis serovar finitimus strain YBT-020[J].J Bacteriol,2011,193(9):2379-2380.
[16]Iatsenko I,Corton C,Pickard D J,et al.Draft genome sequence of highly nematicidal Bacillus thuringiensis DB27[J].Genome Announc,2014,2(1):e00101-14.
[17]Ammons D R,Reyna A,Granados J C,et al.A novel Bacillus thuringiensis Cry-like protein from a rare fila-mentous strain promotes crystal localization within the exosporium[J].Appl Environ Microbiol,2013,79(18):5774-5776.
[18]Ammons D,Toal G,Roman A,et al.Cry-like genes,in an uncommon gene configuration,produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis[J].FEMS Microbiol Lett,2016,363(4):fnw010.
[19]Ruan L F,Crickmore N,Peng D H,et al.Are Nema-todes a missing link in the confounded ecology of the en-tomopathogen Bacillus thuringiensis?[J].Trends Mi-crobiol,2015,23(6):341-346.
[20]Jones J T,Haegeman A,Danchin E G J,et al.Top 10plant-parasitic Nematodes in molecular plant pathology[J].Mol Plant Pathol,2013,14(9):946-961.
[21]Tian B Y,Yang J K,Zhang K Q.Bacteria used in the biological control of plant-parasitic nematodes:popula-tions,mechanisms of action,and future prospects[J].FEMS Microbiol Ecol,2007,61(2):197-213.
[22]Andrup L,Damgaard J,Wassermann K.Mobilization of small plasmids in Bacillus thuringiensis subsp.israelensis is accompanied by specific aggregation[J].J Bacte-riol,1993,175(20):6530-6536.
[23]Quail M A,Kozarewa I,Smith F,et al.A large ge-nome center's improvements to the Illumina sequencing system[J].Nat Methods,2008,5(12):1005-1010.
[24]Rhoads A,Au K F.PacBio sequencing and its applications[J].Genom Proteom Bioinform,2015,13(5):278-289.
[25]Jackman S D,Vandervalk B P,Mohamadi H,et al.ABySS 2.0:resource-efficient assembly of large ge-nomes using a Bloom filter[J].Genome Res,2017,27(5):768-777.
[26]Nadalin F,Vezzi F,Policriti A.GapFiller:a de novo assembly approach to fill the gap within paired reads[J].BMC Bioinform,2012,13(S14):S8.
[27]Ye W X,Zhu L,Liu Y Y,et al.Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a com-putational pipeline[J].Appl Environ Microbiol,2012,78(14):4795-4801.
[28]Li L,Yang C,Liu Z D,et al.Screening of acrystallif-erous mutants from Bacillus thuringiensis and their transformation properties[J].Acta Microbiol Sin,2000,40(1):85-90.李林,杨超,刘子铎,等.苏云金芽胞杆菌无晶体突变株的逐级升温筛选及其转化性能[J].微生物学报,2000.40(1):85-90.
[29]Arantes O,Lereclus D.Construction of cloning vectors for Bacillus thuringiensis[J].Gene,1991,108(1):115-119.
[30]Sambrook J,Russell D W.Molecular Cloning:A Labo-ratory Manual[M].New York:Cold Spring Harbor Laboratory Press:2001.
[31]Bischof L J,Huffman D L,Aroian R V.Assays for toxicity studies in C.elegans with Bt crystal proteins[M]//Strange K.C.elegans.New Jersey:Humana Press:139-154.
[32]Luo X X,Chen L,Huang Q,et al.Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor[J].Appl Environ Microbiol,2013,79(2):460-468.
[33]Peng D H,Lin J,Huang Q,et al.A novel metallopro-teinase virulence factor is involved in Bacillus thuringiensis pathogenesis in Nematodes and insects[J].Envi-ron Microbiol,2016,18(3):846-862.
[34]Du Y,Wang Y,Huang T,et al.Identification and characterization of the biosynthetic gene cluster of poly-oxypeptin A,a potent apoptosis inducer[J].BMC Mi-crobiol,2014,14:30.
[35]Trudgill D L,Blok V C.Apomictic,polyphagous root-knot nematodes:exceptionally successful and damaging biotrophic root pathogens[J].Annu Rev Phytopathol,2001,39:53-77.
[2]van Frankenhuyzen K.Insecticidal activity of Bacillus thuringiensis crystal proteins[J].J Invertebr Pathol,2009,101(1):1-16.
[3]Wei J Z,Hale K,Carta L,et al.Bacillus thuringiensis crystal proteins that target nematodes[J].Proc Natl Acad Sci U S A,2003,100(5):2760-2765.
[4]Mizuki E,Ohba M,Akao T,et al.Unique activity as-sociated with non-insecticidal Bacillus thuringiensis para-sporal inclusions:in vitro cell-killing action on human cancer cells[J].J Appl Microbiol,1999,86(3):477-486.
[5]Yamashita S,Akao T,Mizuki E,et al.Characteriza-tion of the anti-cancer-cell parasporal proteins of a Bacillus thuringiensis isolate[J].Can J Microbiol,2000,46(10):913-919.
[6]Roh J Y,Choi J Y,Li M S,et al.Bacillus thuringiensis as a specific,safe,and effective tool for insect pest con-trol[J].J Microbiol Biotechnol,2007,17(4):547-559.
[7]Bravo A,Likitvivatanavong,S,Gill,S S,et al.Bacillus thuringiensis:a story of a successful bioinsecticide[J].Insect Biochem Mol Biol,2011,41(7):423-431.
[8]Sanahuja G,Banakar R,Twyman R M,et al.Bacillus thuringiensis:a century of research,development and commercial applications[J].Plant Biotechnol J,2011,9(3):283-300.
[9]Bechtel D B,Bulla L A Jr.Electron microscope study of sporulation and parasporal crystal formation in Bacillus thuringiensis[J].J Bacteriol,1976,127(3):1472-1481.
[10]Myasnik M,Manasherob R,Ben-Dov E,et al.Com-parative sensitivity to UV-B radiation of two Bacillus thuringiensis subspecies and other Bacillus sp.[J].Curr Microbiol,2001,43(2):140-143.
[11]Debro L,Fitz-James P C,Aronson A.Two different para-sporal inclusions are produced by Bacillus thuringiensis subsp.finitimus[J].J Bacteriol,1986,165(1):258-268.
[12]Zhu Y,Ji F,Shang H,et al.Gene clusters located on two large plasmids determine spore crystal association(SCA)in Bacillus thuringiensis subsp.finitimus strain YBT-020[J].PLoS One,2011,6(11):e27164.
[13]Wojciechowska J A,Lewitin E,Revina L P,et al.Two novel delta-endotoxin gene families cry26 and cry28 from Bacillus thuringiensis ssp.finitimus[J].FEBS Lett,1999,453(1-2):46-48.
[14]Ji F,Zhu Y,Ju S,et al.Promoters of crystal protein genes do not control crystal formation inside exosporium of Bacillus thuringiensis ssp.finitimus strain YBT-020[J].FEMS Microbiol Lett,2009,300(1):11-17.
[15]Zhu Y,Shang H,Zhu Q,et al.Complete genome se-quence of Bacillus thuringiensis serovar finitimus strain YBT-020[J].J Bacteriol,2011,193(9):2379-2380.
[16]Iatsenko I,Corton C,Pickard D J,et al.Draft genome sequence of highly nematicidal Bacillus thuringiensis DB27[J].Genome Announc,2014,2(1):e00101-14.
[17]Ammons D R,Reyna A,Granados J C,et al.A novel Bacillus thuringiensis Cry-like protein from a rare fila-mentous strain promotes crystal localization within the exosporium[J].Appl Environ Microbiol,2013,79(18):5774-5776.
[18]Ammons D,Toal G,Roman A,et al.Cry-like genes,in an uncommon gene configuration,produce a crystal that localizes within the exosporium when expressed in an acrystalliferous strain of Bacillus thuringiensis[J].FEMS Microbiol Lett,2016,363(4):fnw010.
[19]Ruan L F,Crickmore N,Peng D H,et al.Are Nema-todes a missing link in the confounded ecology of the en-tomopathogen Bacillus thuringiensis?[J].Trends Mi-crobiol,2015,23(6):341-346.
[20]Jones J T,Haegeman A,Danchin E G J,et al.Top 10plant-parasitic Nematodes in molecular plant pathology[J].Mol Plant Pathol,2013,14(9):946-961.
[21]Tian B Y,Yang J K,Zhang K Q.Bacteria used in the biological control of plant-parasitic nematodes:popula-tions,mechanisms of action,and future prospects[J].FEMS Microbiol Ecol,2007,61(2):197-213.
[22]Andrup L,Damgaard J,Wassermann K.Mobilization of small plasmids in Bacillus thuringiensis subsp.israelensis is accompanied by specific aggregation[J].J Bacte-riol,1993,175(20):6530-6536.
[23]Quail M A,Kozarewa I,Smith F,et al.A large ge-nome center's improvements to the Illumina sequencing system[J].Nat Methods,2008,5(12):1005-1010.
[24]Rhoads A,Au K F.PacBio sequencing and its applications[J].Genom Proteom Bioinform,2015,13(5):278-289.
[25]Jackman S D,Vandervalk B P,Mohamadi H,et al.ABySS 2.0:resource-efficient assembly of large ge-nomes using a Bloom filter[J].Genome Res,2017,27(5):768-777.
[26]Nadalin F,Vezzi F,Policriti A.GapFiller:a de novo assembly approach to fill the gap within paired reads[J].BMC Bioinform,2012,13(S14):S8.
[27]Ye W X,Zhu L,Liu Y Y,et al.Mining new crystal protein genes from Bacillus thuringiensis on the basis of mixed plasmid-enriched genome sequencing and a com-putational pipeline[J].Appl Environ Microbiol,2012,78(14):4795-4801.
[28]Li L,Yang C,Liu Z D,et al.Screening of acrystallif-erous mutants from Bacillus thuringiensis and their transformation properties[J].Acta Microbiol Sin,2000,40(1):85-90.李林,杨超,刘子铎,等.苏云金芽胞杆菌无晶体突变株的逐级升温筛选及其转化性能[J].微生物学报,2000.40(1):85-90.
[29]Arantes O,Lereclus D.Construction of cloning vectors for Bacillus thuringiensis[J].Gene,1991,108(1):115-119.
[30]Sambrook J,Russell D W.Molecular Cloning:A Labo-ratory Manual[M].New York:Cold Spring Harbor Laboratory Press:2001.
[31]Bischof L J,Huffman D L,Aroian R V.Assays for toxicity studies in C.elegans with Bt crystal proteins[M]//Strange K.C.elegans.New Jersey:Humana Press:139-154.
[32]Luo X X,Chen L,Huang Q,et al.Bacillus thuringiensis metalloproteinase Bmp1 functions as a nematicidal virulence factor[J].Appl Environ Microbiol,2013,79(2):460-468.
[33]Peng D H,Lin J,Huang Q,et al.A novel metallopro-teinase virulence factor is involved in Bacillus thuringiensis pathogenesis in Nematodes and insects[J].Envi-ron Microbiol,2016,18(3):846-862.
[34]Du Y,Wang Y,Huang T,et al.Identification and characterization of the biosynthetic gene cluster of poly-oxypeptin A,a potent apoptosis inducer[J].BMC Mi-crobiol,2014,14:30.
[35]Trudgill D L,Blok V C.Apomictic,polyphagous root-knot nematodes:exceptionally successful and damaging biotrophic root pathogens[J].Annu Rev Phytopathol,2001,39:53-77.