苏云金芽胞杆菌Vip3Aa11定点突变对棉铃虫杀虫活性的影响
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摘要
为了寻找苏云金芽胞杆菌Vip3Aa11中对杀虫活性有重要影响的氨基酸,为杀虫机理研究和改造杀虫蛋白提供理论依据,通过序列比对,利用PCR方法对Vip3Aa11中7个氨基酸位点进行定点突变,对各突变蛋白杀棉铃虫的生物活性进行测定。结果表明,获得的Vip3Aa11突变体I358V、I362M、K553I、I663T对棉铃虫的杀虫活性明显降低,而突变体I706N对棉铃虫的杀虫活性有所提高,各突变体对胰蛋白酶敏感性基本一致。说明第358位、362位、663位异亮氨酸及553位赖氨酸对Vip3Aa11杀棉铃虫活性有重要影响。
To find amino acids of Bt Vip3 Aa11 which had an important effect of insecticidal activity and to provide a theoretical basis for the research on pesticidal mechanism and recombination of insecticidal protein,7 amino acid sites in Vip3 Aa11 were conducted by PCR through sequences alignment to determine the biological activity of each mutation Helicoverpa Armigera.The results showed that the insecticidal activities of mutants I358 V,I362 M,K553 I,I663 T were significantly decreased while the mutant I706 N increased slightly.The sensitivity of the mutants on trypsin was basically consistent.The results indicated that isoleucine on site-358,362,663 and lysine on site-553 had important effects on the toxicity of Vip3 Aa11 against H.Armigera.
To find amino acids of Bt Vip3 Aa11 which had an important effect of insecticidal activity and to provide a theoretical basis for the research on pesticidal mechanism and recombination of insecticidal protein,7 amino acid sites in Vip3 Aa11 were conducted by PCR through sequences alignment to determine the biological activity of each mutation Helicoverpa Armigera.The results showed that the insecticidal activities of mutants I358 V,I362 M,K553 I,I663 T were significantly decreased while the mutant I706 N increased slightly.The sensitivity of the mutants on trypsin was basically consistent.The results indicated that isoleucine on site-358,362,663 and lysine on site-553 had important effects on the toxicity of Vip3 Aa11 against H.Armigera.
引文
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[20]Roh J Y,Nair M S,Liu X S,et al.Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin[J].Fems Microbiology Letters,2009,295(2):156-163.
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[22]Liu J,Song F,Zhang J,et al.Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene[J].Letters in Applied Microbiology,2007,45(4):432-438.
[23]Bhalla R,Dalal M,Panguluri S K,et al.Isolation,characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis[J].Fems Microbiology Letters,2005,243(2):467-472.
[24]何晓明,束长龙,刘晓垒,等.苏云金芽胞杆菌新型vip3Aa基因的克隆,表达与活性分析[J].农业生物技术学报,2011(5):932-937.
[25]Gayen S,Hossain M A,Sen S K.Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis[J].Journal of Plant Biochemistry&Biotechnology,2012,21(1):128-135.
[26]Li C,Xu N,Huang X,et al.Bacillus thuringiensis Vip3 mutant proteins:insecticidal activity and trypsin sensitivity[J].Biocontrol Science&Technology,2007,17(7):699-708.
[27]Dong F,Zhang S,Shi R,et al.Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis,Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity[J].Current Microbiology,2012,65(5):583-588.
[28]Fang J,Xu X,Wang P,et al.Characterization of chimeric Bacillus thuringiensis Vip3 toxins[J].Applied&Environmental Microbiology,2007,73(3):956-961.
[29]Chen J,Yu J,Tang L,et al.Comparison of the expression of Bacillus thuringiensis,full-length and N-terminally trun cated vip3A,gene in Escherichia coli[J].Journal of Applied Microbiology,2003,95(2):310-316.
[30]Ming L,Liu R,Luo G,et al.Effects of Site-Mutations Within the 22 kDa No-Core Fragment of the Vip3Aa11 Insecticidal Toxin of Bacillus thuringiensis[J].Current Microbiology,2017,74(5):655-659.
[31]雒国兴.Bt Vip3Aa11蛋白羧基端点突变对其杀虫活性和敏感性的影响[D].哈尔滨:东北农业大学,2017.
[32]Chakroun M,Banyuls N,Bel Y,et al.Bacterial Vegetative Insecticidal Proteins(Vip)from Entomopathogenic Bacteria[J].Microbiology&Molecular Biology Reviews Mmbr,2016,80(2):329-350.
[2]Donovan W P,Engleman J T,Donovan J C,et al.Discovery and characterization of Sip1A:a novel secreted protein from Bacillus thuringiensis,with activity against coleopteran larvae[J].Appl Microbiol Biotechnol,2006,72(4):713-719.
[3]Hofte H,Whiteley H R.Insecticidal crystal proteins of Bacillus thuringiensis.[J].Microbiol Rev,1989,53(2):242-255.
[4]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&Biotechnology,2007,17(4):547-559.
[5]Crickmore N,Baum J,Bravo A,et al Bacillus thuringiensis toxin nomenclature[DB/OL].(2017)[2017-12-07].http://www.btnomenclature.info/.
[6]Beard C E,Court L,Boets A,et al.Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection[J].Current Microbiology,2008,57(3):195-199.
[7]Ramasamy B,Nadarajah V D,Soong Z K,et al.A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thuringiensis isolates[J].Tropical Biomedicine,2008,25(1):64-74.
[8]Yu X,Tao L,Sun Z,et al.Co-expression and Synergism Analysis of Vip3Aa29 and Cyt2Aa3 Insecticidal Proteins from Bacillus thuringiensis[J].Current Microbiology,2012,64(4):326-331.
[9]Lee MK,Walters FS,Hart H,et al.The Mode of Action of the Bacillus thuringiensis Vegetative Insecticidal Protein Vip3A Differs from That of Cry1Abδ-Endotoxin[J].Applied&Environmental Microbiology,2003,69(8):4648-4657.
[10]Yu CG,Mullins MA,Warren GW,et al.The Bacillus thuringiensis vegetative insecticidal protein Vip3Aa lyses midgut epithelium cells of susceptible insects[J].Appl Environ Microbiol,1997,63(2):532-536.
[11]Yu J,Yi P,Tang M,et al.Highly Toxic and Broad-Spectrum Insecticidal Bacillus thuringiensis,Engineered by Using the Transposon Tn917 and Protoplast Fusion[J].Current Microbiology,2001,43(2):112-119.
[12]刘明,李海涛,刘荣梅,等.苏云金芽胞杆菌应用研究进展[J].黑龙江八一农垦大学学报,2014,26(4):9-13.
[13]Kono M,Miyazaki G,Nakamura H,et al.Site-directed mutagenesis in hemoglobin:attempts to control the oxygen affinity with cooperativity preserved[J].Protein Eng,1998,11(3):199-204.
[14]Bah A,Frankenhuyzen K V,Brousseau R,et al.The Bacillus thuringiensis,Cry1Aa toxin:effects of trypsin and chymotrypsin site mutations on toxicity and stability[J].Journal of Invertebrate Pathology,2004,85(2):120-127.
[15]Wu D,Aronson A I.Localized mutagenesis defines regions of the Bacillus thuringiensisδ-endotoxin involved in toxicity and specificity[J].Journal of Biological Chemistry,1992,267(4):2311-2317.
[16]Chandra A,Ghosh P,Mandaokar A D,et al.Amino acid substitution inα-helix 7 of Cry1Acδ-endotoxin of Bacillus thuringiensis,leads to enhanced toxicity to Helicoverpa armigera,Hubner[J].Febs Letters,1999,458(2):175-179.
[17]Wang Fa Xiang,Xia Li Qiu,Ding Xue Zhi,et al.N546 inβ18β19 loop is important for binding and toxicity of the Bacillus thuringiensis Cry1Ac toxin[J].Journal of Invertebrate Pathology,2009,101(2):119-123.
[18]任羽,刘华梅,宋福平,等.苏云金芽孢杆菌Cry1Ca7蛋白定点突变对甜菜夜蛾杀虫活性的影响[J].微生物学报,2008,48(6):733-738.
[19]Abdullah M A,Alzate O,Mohammad M,et al.Introduction of Culex Toxicity into Bacillus thuringiensis Cry4Ba by Protein Engineering[J].Applied&Environmental Microbiology,2003,69(9):5343-5353.
[20]Roh J Y,Nair M S,Liu X S,et al.Mutagenic analysis of putative domain II and surface residues in mosquitocidal Bacillus thuringiensis Cry19Aa toxin[J].Fems Microbiology Letters,2009,295(2):156-163.
[21]Mesrati L A,Tounsi S,Jaoua S.Characterization of a novel vip3-type gene from Bacillus thuringiensis,and evidence of its presence on a large plasmid[J].Fems Microbiology Letters,2005,244(2):353-358.
[22]Liu J,Song F,Zhang J,et al.Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene[J].Letters in Applied Microbiology,2007,45(4):432-438.
[23]Bhalla R,Dalal M,Panguluri S K,et al.Isolation,characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis[J].Fems Microbiology Letters,2005,243(2):467-472.
[24]何晓明,束长龙,刘晓垒,等.苏云金芽胞杆菌新型vip3Aa基因的克隆,表达与活性分析[J].农业生物技术学报,2011(5):932-937.
[25]Gayen S,Hossain M A,Sen S K.Identification of the bioactive core component of the insecticidal Vip3A toxin peptide of Bacillus thuringiensis[J].Journal of Plant Biochemistry&Biotechnology,2012,21(1):128-135.
[26]Li C,Xu N,Huang X,et al.Bacillus thuringiensis Vip3 mutant proteins:insecticidal activity and trypsin sensitivity[J].Biocontrol Science&Technology,2007,17(7):699-708.
[27]Dong F,Zhang S,Shi R,et al.Ser-Substituted Mutations of Cys Residues in Bacillus thuringiensis,Vip3Aa7 Exert a Negative Effect on its Insecticidal Activity[J].Current Microbiology,2012,65(5):583-588.
[28]Fang J,Xu X,Wang P,et al.Characterization of chimeric Bacillus thuringiensis Vip3 toxins[J].Applied&Environmental Microbiology,2007,73(3):956-961.
[29]Chen J,Yu J,Tang L,et al.Comparison of the expression of Bacillus thuringiensis,full-length and N-terminally trun cated vip3A,gene in Escherichia coli[J].Journal of Applied Microbiology,2003,95(2):310-316.
[30]Ming L,Liu R,Luo G,et al.Effects of Site-Mutations Within the 22 kDa No-Core Fragment of the Vip3Aa11 Insecticidal Toxin of Bacillus thuringiensis[J].Current Microbiology,2017,74(5):655-659.
[31]雒国兴.Bt Vip3Aa11蛋白羧基端点突变对其杀虫活性和敏感性的影响[D].哈尔滨:东北农业大学,2017.
[32]Chakroun M,Banyuls N,Bel Y,et al.Bacterial Vegetative Insecticidal Proteins(Vip)from Entomopathogenic Bacteria[J].Microbiology&Molecular Biology Reviews Mmbr,2016,80(2):329-350.