对甜菜夜蛾高杀虫毒力苏云金杆菌菌株的筛选及cry2Ah5的克隆表达
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摘要
利用生物活性测定方法从本实验室已分离保存的Bt菌株中筛选出了5株对甜菜夜蛾具有较高杀虫毒力的菌株。该5株Bt菌株对甜菜夜蛾初孵幼虫的LC50值为0.556~0.914 mg/mL胞晶混合物,其中GS3菌株杀虫活性最高,LC50值为0.556 mg/mL胞晶混合物;包含晶体形态主要是球形、大菱形、小菱形等;所含基因类型主要是cry1Aa、cry1Ab、cry1La、cry1Ia、cry1Ib、cry2Ab、cry2Ad、cry2Ah、cry7Ab、cry9Ba等;5株菌株均表达约60 kD和130~150 kD蛋白,其中2株还表达较弱的80 kD蛋白。从GS3菌株中克隆得到一种新的cry2A基因,Gen Bank登录号为KT692984,由Bt基因国际命名委员会命名为cry2Ah5。该基因开放阅读框为1899 bp,编码632个氨基酸,编码蛋白分子量为70.8 kD,等电点p I为8.18,与其他Cry2Ah蛋白序列相似性为97%~99%。该基因在大肠杆菌BL21(DE3)中表达约70 kD蛋白,与预测分子量相符。本研究筛选得到5株对甜菜夜蛾具有较高杀虫毒力的Bt菌株,克隆表达了一种新的cry2A基因cry2Ah5,为甜菜夜蛾的生物防治提供了新的菌株及基因资源。
According to the results of bioassay, five Bacillus thuringiensis strains with high toxicity against Spodoptera exigua were obtained. The median lethal concentrations(LC50) of these strains ranged from 0.556 to 0.914 mg/m L spores and crystals mixture, and the strain GS3 had the highest toxicity with a LC50 of 0.556 mg/m L. These strains contained cry1Aa, cry1Ab, cry1La, cry1Ia, cry1Ib, cry2Ab, cry2Ad, cry2Ah, cry7Ab and cry9Ba genes, forming globular or bipyramidal in different sizes crystals. All of them expressed 60 k D and 130—150 kD proteins, and two of them also lowly expressed 80 k D proteins. A new cry2A gene identified from the strain GS3 with an accession number of KT692984 was designated cry2Ah5 by the International Nomenclature Committee of Bt δ-endotoxin genes. Sequence analysis showed that cry2Ah5 contained a 1899 bp open reading frame, which encoded a 70.8 kD protein composing of 632 amino acids. The p I of this protein was 8.18. The Cry2Ah5 protein showed 97%—99% sequence identity with other Cry2Ah. The cry2Ah5 gene expressed a 70 k D protein in Escherichia coli BL21(DE3) corresponding to the predict molecular weight. Our research provided more strains and genetic resources for the biological control of S. exigua.
According to the results of bioassay, five Bacillus thuringiensis strains with high toxicity against Spodoptera exigua were obtained. The median lethal concentrations(LC50) of these strains ranged from 0.556 to 0.914 mg/m L spores and crystals mixture, and the strain GS3 had the highest toxicity with a LC50 of 0.556 mg/m L. These strains contained cry1Aa, cry1Ab, cry1La, cry1Ia, cry1Ib, cry2Ab, cry2Ad, cry2Ah, cry7Ab and cry9Ba genes, forming globular or bipyramidal in different sizes crystals. All of them expressed 60 k D and 130—150 kD proteins, and two of them also lowly expressed 80 k D proteins. A new cry2A gene identified from the strain GS3 with an accession number of KT692984 was designated cry2Ah5 by the International Nomenclature Committee of Bt δ-endotoxin genes. Sequence analysis showed that cry2Ah5 contained a 1899 bp open reading frame, which encoded a 70.8 kD protein composing of 632 amino acids. The p I of this protein was 8.18. The Cry2Ah5 protein showed 97%—99% sequence identity with other Cry2Ah. The cry2Ah5 gene expressed a 70 k D protein in Escherichia coli BL21(DE3) corresponding to the predict molecular weight. Our research provided more strains and genetic resources for the biological control of S. exigua.
引文
[1]Hofte H,Whiteley H R.Insecticidal crystal proteins of Bacillus thuringiensis[J].Fems Microbiology Reviews,1989,53(2):242-255.
[2]Raymond B,Johnston P R,Nielsen-Le Roux C,et al.Bacillus thuringiensis:an importent pathogen?[J].Trends in Microbiology,2010,18(5):189-194.
[3]Roh J Y,Choi J Y,Li M S,et al.Bacillus thuringiensis as a specific,safe,and effective tool for insect pest control[J].Journal of Microbiology and Biotechnology,2007,17(4):547-559.
[4]Palma L,Munoz D,Berry C,et al.Bacillus thuringiensis toxins:an overview of their biocidal activity[J].Toxins(Basel),2014,6(12):3296-3325.
[5]Ohba M,Mizuki E,Uemori A.Parasporin,a new anticancer protein group from Bacillus thuringiensis[J].Anticancer Research,2009,29(1):427-433.
[6]Schnepf H E,Whiteley H R.Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli[J].Proceedings of the National Academy of Sciences of the United States of America,1981,78(5):2893-2897.
[7]张彬,刘怀,王进军,等.甜菜夜蛾研究进展[J].中国农学通报,2008,24(10):427-433.
[8]江幸福,罗礼智.我国甜菜夜蛾发生为害特点及治理措施[J].长江蔬菜,2010,(18):93-95.
[9]刘向阳,朱福兴,张凯.甜菜夜蛾抗药性研究现状[J].昆虫知识,2007,44(5):632-636.
[10]郭巍.粉纹夜蛾对苏云金芽胞杆菌Cry1Ac的抗性及其防御体系的研究[D].广州:中山大学,2005.
[11]刘廷辉.苏云金芽胞杆菌菌株Bt11和GS8的研究[D].保定:河北农业大学,2009.
[12]贾春生.利用SPSS软件计算杀虫剂的LC50[J].昆虫知识,2006,43(3):414-417.
[13]刘晶晶,束长龙,张杰,等.苏云金芽胞杆菌内生质粒提取方法的改进[J].生物技术通报,2008,(6):120-123.
[14]Kuo W S,Chak K F.Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA[J].Applied and Environmental Microbiology,1996,62(4):1369-1377.
[15]宋福平,张杰,谢天健,等.苏云金芽孢杆菌cry基因PCR-RFLP鉴定体系的建立[J].中国农业科学,1998,31(3):11-14.
[16]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.
[17]Tabashnik B E,Sisterson M S,Ellsworth P C,et al.Suppressing resistance to Bt cotton with sterile insect releases[J].Nature Biotechnology,2010,28(12):1304-1307.
[18]Bravo A,Likitvivatanavong S,Gill S S,et al.Bacillus thuringiensis:A story of a successful bioinsecticide[J].Insect Biochemistry and Molecular Biology2011,41(7):423-431.
[19]Tabashnik B E,Gassmann A J,Crowder D W,et al.Insect resistance to Bt crops:evidence versus theory[J].Nature Biotechnology,2008,26(2):199-202.
[20]Storer N P,Babcock J M,Schlenz M,et al.Discovery and characterization of field resistance to Bt maize:Spodoptera frugiperda(Lepidoptera:Noctuidae)in Puerto Rico[J].Journal of Economic Entomology,2010,103(4):1031-1038.
[21]Bagla P.Hardy cotton-munching pests are latest blow to GM crops[J].Science,2010,327(5972):1439.
[22]Hernandez-Martinez P,Ferre J,Escriche B.Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis[J].Journal of Invertebrate Pathology,2008,97(3):245-250.
[23]Estruch J J,Warren G W,Mullins M A,et al.Vip3A,a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(11):5389-5394.
[24]Shu C,Zhang J,Chen G,et al.Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis[J].Journal of Invertebrate Pathology,2013,114(1):31-33.
[2]Raymond B,Johnston P R,Nielsen-Le Roux C,et al.Bacillus thuringiensis:an importent pathogen?[J].Trends in Microbiology,2010,18(5):189-194.
[3]Roh J Y,Choi J Y,Li M S,et al.Bacillus thuringiensis as a specific,safe,and effective tool for insect pest control[J].Journal of Microbiology and Biotechnology,2007,17(4):547-559.
[4]Palma L,Munoz D,Berry C,et al.Bacillus thuringiensis toxins:an overview of their biocidal activity[J].Toxins(Basel),2014,6(12):3296-3325.
[5]Ohba M,Mizuki E,Uemori A.Parasporin,a new anticancer protein group from Bacillus thuringiensis[J].Anticancer Research,2009,29(1):427-433.
[6]Schnepf H E,Whiteley H R.Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli[J].Proceedings of the National Academy of Sciences of the United States of America,1981,78(5):2893-2897.
[7]张彬,刘怀,王进军,等.甜菜夜蛾研究进展[J].中国农学通报,2008,24(10):427-433.
[8]江幸福,罗礼智.我国甜菜夜蛾发生为害特点及治理措施[J].长江蔬菜,2010,(18):93-95.
[9]刘向阳,朱福兴,张凯.甜菜夜蛾抗药性研究现状[J].昆虫知识,2007,44(5):632-636.
[10]郭巍.粉纹夜蛾对苏云金芽胞杆菌Cry1Ac的抗性及其防御体系的研究[D].广州:中山大学,2005.
[11]刘廷辉.苏云金芽胞杆菌菌株Bt11和GS8的研究[D].保定:河北农业大学,2009.
[12]贾春生.利用SPSS软件计算杀虫剂的LC50[J].昆虫知识,2006,43(3):414-417.
[13]刘晶晶,束长龙,张杰,等.苏云金芽胞杆菌内生质粒提取方法的改进[J].生物技术通报,2008,(6):120-123.
[14]Kuo W S,Chak K F.Identification of novel cry-type genes from Bacillus thuringiensis strains on the basis of restriction fragment length polymorphism of the PCR-amplified DNA[J].Applied and Environmental Microbiology,1996,62(4):1369-1377.
[15]宋福平,张杰,谢天健,等.苏云金芽孢杆菌cry基因PCR-RFLP鉴定体系的建立[J].中国农业科学,1998,31(3):11-14.
[16]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.
[17]Tabashnik B E,Sisterson M S,Ellsworth P C,et al.Suppressing resistance to Bt cotton with sterile insect releases[J].Nature Biotechnology,2010,28(12):1304-1307.
[18]Bravo A,Likitvivatanavong S,Gill S S,et al.Bacillus thuringiensis:A story of a successful bioinsecticide[J].Insect Biochemistry and Molecular Biology2011,41(7):423-431.
[19]Tabashnik B E,Gassmann A J,Crowder D W,et al.Insect resistance to Bt crops:evidence versus theory[J].Nature Biotechnology,2008,26(2):199-202.
[20]Storer N P,Babcock J M,Schlenz M,et al.Discovery and characterization of field resistance to Bt maize:Spodoptera frugiperda(Lepidoptera:Noctuidae)in Puerto Rico[J].Journal of Economic Entomology,2010,103(4):1031-1038.
[21]Bagla P.Hardy cotton-munching pests are latest blow to GM crops[J].Science,2010,327(5972):1439.
[22]Hernandez-Martinez P,Ferre J,Escriche B.Susceptibility of Spodoptera exigua to 9 toxins from Bacillus thuringiensis[J].Journal of Invertebrate Pathology,2008,97(3):245-250.
[23]Estruch J J,Warren G W,Mullins M A,et al.Vip3A,a novel Bacillus thuringiensis vegetative insecticidal protein with a wide spectrum of activities against lepidopteran insects[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(11):5389-5394.
[24]Shu C,Zhang J,Chen G,et al.Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis[J].Journal of Invertebrate Pathology,2013,114(1):31-33.