苏云金芽胞杆菌杀虫晶体蛋白Cry1D新基因的克隆与特性
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摘要
为扩充鳞翅目害虫杀虫基因资源,本研究从苏云金芽胞杆菌BN23-5中克隆得到一个新的cry基因,并对其进行鉴定和分析。该基因为一个完整的cry1D基因,全长3501 bp,编码1166个氨基酸残基。该氨基酸序列是一个新的Cry氨基酸序列,与Cry1Db1的同源性最高,为86%,命名为Cry1Dd1(登录号为KJ728844)。将该基因插入穿梭表达载体pSTK中,转入BT无晶体突变株-HD73中进行表达。结果表明,cry1Dd1基因能在BT无晶体突变株中表达,并形成菱形伴孢晶体。SDS-PAGE验证其分子量为132.2 kD,与预测的大小相符。生物活性测定表明,Cry1Dd1晶体蛋白对小菜蛾的幼虫具有杀虫活性,LC_(50)为13.1μg/mL;能明显抑制甜菜夜蛾幼虫的生长;但对棉铃虫幼虫没有杀虫活性。对cry1Dd1基因序列进行分析,cry1Dd1包含8个block保守区域,这和目前其他的cry基因相似;Cry1Dd1蛋白的活性区域为N端的37～593位氨基酸残基。
A novel holotype cry gene(cry1Dd1) was cloned from a Bt strain BN23-5, which enriches novel lepidopteran insecticidal Bt genes. In this study, the full length of the cry1Dd1 gene was cloned. The full-length cry1D gene was 3501 bp, encoding a polypeptide of 1166 amino acid residues. Its amino acid sequence was 86% identical to that of Cry1Db1, and the gene was named as cry1Dd1 by International Nomenclature Commitee of B. thuringiensis endotoxin with a Gen Bank number of KJ728844. The cry1Dd1 gene was inserted into a shuttle vector(p STK) and expressed in an acrystalliferous mutant B. thuringiensis HD73-. In this transformant, cry1Dd1 was expressed and diamond-shaped parasporal crystals were formed. SDS-PAGE analysis determined a molecular mass of approximately 130 kD of Cry1Dd1 protein. The Cry1Dd1 protein exhibited high larvicidal activity against the diamondback moth(Plutella xylostella), with an LC_(50) of 13.1 μg/mL, and obviously inhibited growth of Spodoptera exigua larvae, but showed no insecticidal activity against Helicoverpa armigera larvae. Sequence analysis of the gene indicated that the cry1Dd1 gene retained eight conserved regions commonly found in the existing cry genes, and the function domain of Cry1Dd1 protein was the 37 to 593 amino acid residues at the N-terminal.
A novel holotype cry gene(cry1Dd1) was cloned from a Bt strain BN23-5, which enriches novel lepidopteran insecticidal Bt genes. In this study, the full length of the cry1Dd1 gene was cloned. The full-length cry1D gene was 3501 bp, encoding a polypeptide of 1166 amino acid residues. Its amino acid sequence was 86% identical to that of Cry1Db1, and the gene was named as cry1Dd1 by International Nomenclature Commitee of B. thuringiensis endotoxin with a Gen Bank number of KJ728844. The cry1Dd1 gene was inserted into a shuttle vector(p STK) and expressed in an acrystalliferous mutant B. thuringiensis HD73-. In this transformant, cry1Dd1 was expressed and diamond-shaped parasporal crystals were formed. SDS-PAGE analysis determined a molecular mass of approximately 130 kD of Cry1Dd1 protein. The Cry1Dd1 protein exhibited high larvicidal activity against the diamondback moth(Plutella xylostella), with an LC_(50) of 13.1 μg/mL, and obviously inhibited growth of Spodoptera exigua larvae, but showed no insecticidal activity against Helicoverpa armigera larvae. Sequence analysis of the gene indicated that the cry1Dd1 gene retained eight conserved regions commonly found in the existing cry genes, and the function domain of Cry1Dd1 protein was the 37 to 593 amino acid residues at the N-terminal.
引文
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[2]Swiecicka I,Bideshi D K,Federici B A.Novel isolate of Bacillus thuringiensis subsp.thuringiensis that produces a quasicuboidal crystal of Cry1Ab21toxic to larvae of Trichoplusia ni[J].Applied and Environmental Microbiology,2008,74(4):923-930.
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[5]Schnepf E,Crickmore N V,Van Rie J,et al.Bacillus thuringiensis and its pesticidal crystal proteins[J].Microbiology and Molecular Biology Reviews,1998,62(3):775-806.
[6]Misztal L H,Mostowska A,Skibinska M,et al.Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants[J].Molecular Biotechnology,2004,26(1):17-26.
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[11]Sarfraz M,Keddie B A.Conserving the efficacy of insecticides against Plutella xylostella(L.)(Lep.,Plutellidae)[J].Journal of Applied Entomology,2005,129(3):149-157.
[12]Zhao J Z,Collins H L,Li Y X,et al.Monitoring of diamondback moth(Lepidoptera:Plutellidae)resistance to spinosad,indoxacarb,and emamectin benzoate[J].Journal of Economic Entomology,2006,99(1):176-181.
[13]Tan F,Zhu J,Tang J,et al.Cloning and characterization of two novel crystal protein genes,cry54Aa1 and cry30Fa1,from Bacillus thuringiensis strain Bt MC28[J].Current Microbiology,2009,58(6):654-659.
[14]Yu Z,Gong L,Li Q,et al.Diversity of insecticidal crystal protein genes of Bacillus thuringiensis isolated from soil and cloning of novel haplotypes of cry genes[J].Annals of Microbiology,2015,65(4):2179-2186.
[15]Yu Z,He L,Gong L,et al.Cloning and characterisation of a novel cry1H gene effective against Lepidopterous larvae from a Bacillus thuringiensis strain[J].Biocontrol Science and Technology,2015,25(12):1483-1492.
[16]Shu C,Liu R,Wang R,et al.Improving toxicity of Bacillus thuringiensis strain contains the cry8Ca gene specific to Anomala corpulenta larvae[J].Current Microbiology,2007,55(6):492.
[17]Reyes-Ramírez A,Ibarra J E.Plasmid patterns of Bacillus thuringiensis type strains[J].Applied and Environmental Microbiology,2008,74(1):125-129.
[18]Sambrook J E,Fritsch F,Maaiatis T.A Laboratory of Molecular Cloning,3rd edn[M].New York:Cold Spring Harbor Laboratory Press,2002.
[19]Mahillon J,Chungjatupornchai W,Decock J,et al.Transformation of Bacillus thuringiensis by electroporation[J].FEMS Microbiology Letters,1989,60(2):205-210.
[20]Park H W,Bideshi D K,Johnson J J,et al.Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB m RNA sequence[J].FEMS Microbiology Letters,1999,181(2):319-327.
[21]Liang H,Liu Y,Zhu J,et al.Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin,China[J].Brazilian Journal of Microbiology,2011,42(1):140-146.
[22]马君兰,束长龙,刘东明,等.大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析[J].生物技术通报,2011(2):80-84.
[23]Song F,Zhang J,Gu A,et al.Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene[J].Applied and Environmental Microbiology,2003,69(9):5207-5211.
[24]Crickmore N,Zeigler D R,Feitelson J,et al.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins[J].Microbiology and Molecular Biology Reviews,1998,62(3):807-813.
[25]Li J,Carroll J,Ellar D J.Crystal structure of insecticidalδ-endotoxin from Bacillus thuringiensis at 2.5?resolution[J].Nature,1991,353(6347):815.
[26]Wang L,Guo W,Tan J,et al.Construction of an engineering strain expressing cry7Ab7 gene cloned from Bacillus thuringiensis[J].Frontiers of Agriculture in China,2010,4(3):328-333.
[27]Guan P,Dai X,Zhu J,et al.Bacillus thuringiensis subsp.sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae[J].World Journal of Microbiology and Biotechnology,2014,30(4):1417-1421.
[28]Zhang Y,Zheng G,Tan J,et al.Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid(Coleoptera:Scarabaeidae)larvae[J].Microbiological Research,2013,168(8):512-517.
[2]Swiecicka I,Bideshi D K,Federici B A.Novel isolate of Bacillus thuringiensis subsp.thuringiensis that produces a quasicuboidal crystal of Cry1Ab21toxic to larvae of Trichoplusia ni[J].Applied and Environmental Microbiology,2008,74(4):923-930.
[3]Yi D,Cui L,Wang L,et al.Pyramiding of Bt cry1Ia8 and cry1Ba3 genes into cabbage(Brassica oleracea L.var.capitata)confers effective control against diamondback moth[J].Plant Cell,Tissue and Organ Culture(PCTOC),2013,115(3):419-428.
[4]Macharia I,L?hr B,De Groote H.Assessing the potential impact of biological control of Plutella xylostella(diamondback moth)in cabbage production in Kenya[J].Crop Protection,2005,24(11):981-989.
[5]Schnepf E,Crickmore N V,Van Rie J,et al.Bacillus thuringiensis and its pesticidal crystal proteins[J].Microbiology and Molecular Biology Reviews,1998,62(3):775-806.
[6]Misztal L H,Mostowska A,Skibinska M,et al.Expression of modified Cry1Ac gene of Bacillus thuringiensis in transgenic tobacco plants[J].Molecular Biotechnology,2004,26(1):17-26.
[7]Metz T D,Dixit R,Earle E D.Agrobacterium tumefaciens-mediated transformation of broccoli(Brassica oleracea var.italica)and cabbage(B.oleraceavar.capitata)[J].Plant Cell Reports,1995,15(3-4):287-292.
[8]Cao J,Tang J D,Strizhov N,et al.Transgenic broccoli with high levels of Bacillus thuringiensis Cry1C protein control diamondback moth larvae resistant to Cry1A or Cry1C[J].Molecular Breeding,1999,5(2):131-141.
[9]Cui L,Yang L M,Liu N,et al.Transformation and expressing of Bt gene cry1Ia8 in cabbage[J].Acta Horticulturae Sinica,2009,36(8):1161-1168.
[10]Yi D,Lei C U I,Liu Y,et al.Transformation of cabbage(Brassica oleracea L.var.capitata)with Bt cry1Ba3 gene for control of diamondback moth[J].Agricultural Sciences in China,2011,10(11):1693-1700.
[11]Sarfraz M,Keddie B A.Conserving the efficacy of insecticides against Plutella xylostella(L.)(Lep.,Plutellidae)[J].Journal of Applied Entomology,2005,129(3):149-157.
[12]Zhao J Z,Collins H L,Li Y X,et al.Monitoring of diamondback moth(Lepidoptera:Plutellidae)resistance to spinosad,indoxacarb,and emamectin benzoate[J].Journal of Economic Entomology,2006,99(1):176-181.
[13]Tan F,Zhu J,Tang J,et al.Cloning and characterization of two novel crystal protein genes,cry54Aa1 and cry30Fa1,from Bacillus thuringiensis strain Bt MC28[J].Current Microbiology,2009,58(6):654-659.
[14]Yu Z,Gong L,Li Q,et al.Diversity of insecticidal crystal protein genes of Bacillus thuringiensis isolated from soil and cloning of novel haplotypes of cry genes[J].Annals of Microbiology,2015,65(4):2179-2186.
[15]Yu Z,He L,Gong L,et al.Cloning and characterisation of a novel cry1H gene effective against Lepidopterous larvae from a Bacillus thuringiensis strain[J].Biocontrol Science and Technology,2015,25(12):1483-1492.
[16]Shu C,Liu R,Wang R,et al.Improving toxicity of Bacillus thuringiensis strain contains the cry8Ca gene specific to Anomala corpulenta larvae[J].Current Microbiology,2007,55(6):492.
[17]Reyes-Ramírez A,Ibarra J E.Plasmid patterns of Bacillus thuringiensis type strains[J].Applied and Environmental Microbiology,2008,74(1):125-129.
[18]Sambrook J E,Fritsch F,Maaiatis T.A Laboratory of Molecular Cloning,3rd edn[M].New York:Cold Spring Harbor Laboratory Press,2002.
[19]Mahillon J,Chungjatupornchai W,Decock J,et al.Transformation of Bacillus thuringiensis by electroporation[J].FEMS Microbiology Letters,1989,60(2):205-210.
[20]Park H W,Bideshi D K,Johnson J J,et al.Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB m RNA sequence[J].FEMS Microbiology Letters,1999,181(2):319-327.
[21]Liang H,Liu Y,Zhu J,et al.Characterization of cry2-type genes of Bacillus thuringiensis strains from soil-isolated of Sichuan basin,China[J].Brazilian Journal of Microbiology,2011,42(1):140-146.
[22]马君兰,束长龙,刘东明,等.大肠杆菌中利用苏云金芽胞杆菌强启动子p1Ac指导Cry1Ac蛋白表达的特性分析[J].生物技术通报,2011(2):80-84.
[23]Song F,Zhang J,Gu A,et al.Identification of cry1I-type genes from Bacillus thuringiensis strains and characterization of a novel cry1I-type gene[J].Applied and Environmental Microbiology,2003,69(9):5207-5211.
[24]Crickmore N,Zeigler D R,Feitelson J,et al.Revision of the nomenclature for the Bacillus thuringiensis pesticidal crystal proteins[J].Microbiology and Molecular Biology Reviews,1998,62(3):807-813.
[25]Li J,Carroll J,Ellar D J.Crystal structure of insecticidalδ-endotoxin from Bacillus thuringiensis at 2.5?resolution[J].Nature,1991,353(6347):815.
[26]Wang L,Guo W,Tan J,et al.Construction of an engineering strain expressing cry7Ab7 gene cloned from Bacillus thuringiensis[J].Frontiers of Agriculture in China,2010,4(3):328-333.
[27]Guan P,Dai X,Zhu J,et al.Bacillus thuringiensis subsp.sichuansis strain MC28 produces a novel crystal protein with activity against Culex quinquefasciatus larvae[J].World Journal of Microbiology and Biotechnology,2014,30(4):1417-1421.
[28]Zhang Y,Zheng G,Tan J,et al.Cloning and characterization of a novel cry8Ab1 gene from Bacillus thuringiensis strain B-JJX with specific toxicity to scarabaeid(Coleoptera:Scarabaeidae)larvae[J].Microbiological Research,2013,168(8):512-517.