苏云金芽胞杆菌Vip3Aa11蛋白定点突变对甜菜夜蛾和棉铃虫杀虫活性的影响
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摘要
营养期杀虫蛋白(vegetative insecticidal proteins, Vips)与杀虫晶体蛋白相比,在氨基酸序列进化上没有同源性,杀虫作用位点上也无竞争关系。由于Vips蛋白结构尚未解析,其杀虫作用机制的研究相对滞后。为明确影响苏云金芽胞杆菌(Bacillus thuringiensis, Bt) Vip3Aa11蛋白杀虫活性的关键氨基酸,本研究对Vip3Aa11中3个氨基酸位点进行定点突变,构建3个突变体蛋白G200S、F442S、S726T,并对其进行甜菜夜蛾(Spodoptera exigua)及棉铃虫(Heliothis armigera)的杀虫活性测定。结果显示,突变体蛋白S726T对甜菜夜蛾的杀虫活性是野生型Vip3Aa11的4倍,对棉铃虫的杀虫活性没有显著变化;其他突变体蛋白对甜菜夜蛾及棉铃虫的杀虫活性均无显著变化。Vip3Aa11与各突变体蛋白对胰蛋白酶的敏感性一致。二级结构预测表明突变体蛋白S726T的构象发生了α-螺旋后移,说明S726T突变体引起的杀虫活性提高可能与蛋白结构的空间细微变化有关。本研究比较了Vip3Aa11与各突变体蛋白之间的杀虫活性差异,并初步分析了差异产生的原因,为研究Vip3Aa类蛋白的结构和杀虫机理提供了参考依据。
Compared with the insecticidal crystal proteins, vegetative insecticidal proteins(Vips) have no homology of amino acid sequences and have no competitive relationship with the sites of insecticidal action.As the protein structure of Vips has not yet been elucidated, the mechanistic study on insecticidal action has lagged behind. In order to identify the key amino acids affecting insecticidal activity of Bacillus thuringiensis(Bt) Vip3 Aa11 protein, site-directed mutagenesis was performed on 3 amino acid sites in Vip3 Aa11, and 3 mutant proteins G200 S, F442 S and S726 T were successfully constructed, and their insecticidal activities against Spodoptera exigua and Helicoverpa armigera were determined. The results showed that the insecticidal activity of mutant protein S726 T against S. exigua was 4 times of that of Vip3 Aa11, while the mutant proteins G200 S and F442 S showed no significant change in insecticidal activity against S. exigua and H. Armigera. To find out the reason of activity change in mutants, 3 mutants and the wild type Vip3 Aa11 were treated with trypsin. The results indicated that both variants and wild type Vip3 Aa11 could yield a 62～66 k D fragment by trypsin digestion in vitro and the effects of the 3 mutants on trypsin sensitivity were basically consistent.Through secondary structure prediction, it was found that the conformation of mutant protein S726 T was shifted backward on α-helix which indicated that the increase of insecticidal activity caused by S726 T mutant might be related to the slight change of protein structure space. In this study, the difference in insecticidal activity between Vip3 Aa11 protein and each mutant protein for different insects was compared, and the reason for the difference was preliminarily analyzed, the results may provide a guideline for further study on the structure and function of Vip3 Aa proteins.
Compared with the insecticidal crystal proteins, vegetative insecticidal proteins(Vips) have no homology of amino acid sequences and have no competitive relationship with the sites of insecticidal action.As the protein structure of Vips has not yet been elucidated, the mechanistic study on insecticidal action has lagged behind. In order to identify the key amino acids affecting insecticidal activity of Bacillus thuringiensis(Bt) Vip3 Aa11 protein, site-directed mutagenesis was performed on 3 amino acid sites in Vip3 Aa11, and 3 mutant proteins G200 S, F442 S and S726 T were successfully constructed, and their insecticidal activities against Spodoptera exigua and Helicoverpa armigera were determined. The results showed that the insecticidal activity of mutant protein S726 T against S. exigua was 4 times of that of Vip3 Aa11, while the mutant proteins G200 S and F442 S showed no significant change in insecticidal activity against S. exigua and H. Armigera. To find out the reason of activity change in mutants, 3 mutants and the wild type Vip3 Aa11 were treated with trypsin. The results indicated that both variants and wild type Vip3 Aa11 could yield a 62～66 k D fragment by trypsin digestion in vitro and the effects of the 3 mutants on trypsin sensitivity were basically consistent.Through secondary structure prediction, it was found that the conformation of mutant protein S726 T was shifted backward on α-helix which indicated that the increase of insecticidal activity caused by S726 T mutant might be related to the slight change of protein structure space. In this study, the difference in insecticidal activity between Vip3 Aa11 protein and each mutant protein for different insects was compared, and the reason for the difference was preliminarily analyzed, the results may provide a guideline for further study on the structure and function of Vip3 Aa proteins.
引文
何晓明,束长龙,刘晓垒,等.2011.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,19(5):932-937.(He X M,Shu C L,Liu X L,et al.2011.Cloning,expression and analysis of insecticidal activity of a novel vip3Aa-type gene from Bacillus thuringiensis[J].Journal of Agricultural Biotechnology,19(5):932-937.)
林承喜,束长龙,翟元娜,等.2011.苏云金芽胞杆菌Cry1Ba3蛋白定点突变对杀小菜蛾活性的影响[J].植物保护,37(6):75-81.(Lin C X,Shu C L,Zhai Y N,et al.2011.The influences of site-directed mutation on the toxicity of Bacillus thuringiensis Cry1Ba3 polypeptide against Plutella xylostella[J].Plant Protection,37(6):75-81.)
徐沙,夏立秋,丁学知.2013.Bt营养期杀虫蛋白Vip3及其作用受体的研究进展[J].中国生物防治学报,29(4):620-625.(Xu S,Xia L Q,Ding X Z.2013.Research advances on the Bacillus thuringiensis vegetative insecticidal protein Vip3 and their receptors[J].Chinese Journal of Biological Control,29(4):620-625.)
Beard C E,Court L,Boets A,et al.2008.Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection[J].Current Microbiology,57(3):195-199.
Bhalla R,Dalal M,Panguluri S K,et al.2005.Isolation,characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis[J].Fems Microbiology Letters,243(2):467-472.
Chakroun M,Banyuls N,Bel Y,et al.2016.Bacterial vegetative insecticidal proteins(Vip)from entomopathogenic bacteria[J].Microbiology and Molecular Biology Reviews,80(2):329-350.
Chi B Y,Luo G X,Zhang J B,et al.2017.Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests[J].Biocontrol Science and Technology,27(12):1-10.
Dong F,Zhang S,Shi R,et al.2012.Ser-substituted mutations of cys residues in Bacillus thuringiensis,Vip3Aa7 exert a negative effect on its insecticidal activity[J].Current Microbiology,65(5):583-588.
Estruch J J,Warren G W,Mullins M A,et al.1996.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 USA,93(11):5389-5394.
Liu J,Song F,Zhang J,et al.2007.Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene[J].Letters in Applied Microbiology,45(4):432-438.
Liu M,Liu R M,Luo G X,et al.2017.Effects of site-mutations within the 22 kD no-core fragment of the Vip3Aa11 insecticidal toxin of Bacillus thuringiensis[J].Current Microbiology,74(5):655-659.
Mesrati L A,Tounsi S,Jaoua S.2005.Characterization of a novel vip3-type gene from Bacillus thuringiensis,and evidence of its presence on a large plasmid[J].Fems Microbiology Letters,244(2):353-358.
Ramasamy B,Nadarajah V D,Soong Z K,et al.2008.A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thuringiensis isolates[J].Tropical Biomedicine,25(1):64-74.
Song F,Chen C,Wu S,et al.2016.Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation[J].Scientific Reports,6:23861.
Yu C G,Mullins M A,Warren G W,et al.1997.The Bacillus thuringiensis vegetative insecticidal protein Vip3Aa lyses midgut epithelium cells of susceptible insects[J].Applied and Environmental Microbiology,63(2):532-536.
Yu J,Yi P,Tang M,et al.2001.Highly toxic and broad-spectrum insecticidal Bacillus thuringiensis,engineered by using the transposon tn917 and protoplast fusion[J].Current Microbiology,43(2):112-119.
Yu X,Tao L,Sun Z,et al.2012.Co-expression and synergism analysis of Vip3Aa29 and Cyt2Aa3 insecticidal proteins from Bacillus thuringiensis[J].Current Microbiology,64(4):326-331.
林承喜,束长龙,翟元娜,等.2011.苏云金芽胞杆菌Cry1Ba3蛋白定点突变对杀小菜蛾活性的影响[J].植物保护,37(6):75-81.(Lin C X,Shu C L,Zhai Y N,et al.2011.The influences of site-directed mutation on the toxicity of Bacillus thuringiensis Cry1Ba3 polypeptide against Plutella xylostella[J].Plant Protection,37(6):75-81.)
徐沙,夏立秋,丁学知.2013.Bt营养期杀虫蛋白Vip3及其作用受体的研究进展[J].中国生物防治学报,29(4):620-625.(Xu S,Xia L Q,Ding X Z.2013.Research advances on the Bacillus thuringiensis vegetative insecticidal protein Vip3 and their receptors[J].Chinese Journal of Biological Control,29(4):620-625.)
Beard C E,Court L,Boets A,et al.2008.Unusually high frequency of genes encoding vegetative insecticidal proteins in an Australian Bacillus thuringiensis collection[J].Current Microbiology,57(3):195-199.
Bhalla R,Dalal M,Panguluri S K,et al.2005.Isolation,characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis[J].Fems Microbiology Letters,243(2):467-472.
Chakroun M,Banyuls N,Bel Y,et al.2016.Bacterial vegetative insecticidal proteins(Vip)from entomopathogenic bacteria[J].Microbiology and Molecular Biology Reviews,80(2):329-350.
Chi B Y,Luo G X,Zhang J B,et al.2017.Effect of C-terminus site-directed mutations on the toxicity and sensitivity of Bacillus thuringiensis Vip3Aa11 protein against three lepidopteran pests[J].Biocontrol Science and Technology,27(12):1-10.
Dong F,Zhang S,Shi R,et al.2012.Ser-substituted mutations of cys residues in Bacillus thuringiensis,Vip3Aa7 exert a negative effect on its insecticidal activity[J].Current Microbiology,65(5):583-588.
Estruch J J,Warren G W,Mullins M A,et al.1996.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 USA,93(11):5389-5394.
Liu J,Song F,Zhang J,et al.2007.Identification of vip3A-type genes from Bacillus thuringiensis strains and characterization of a novel vip3A-type gene[J].Letters in Applied Microbiology,45(4):432-438.
Liu M,Liu R M,Luo G X,et al.2017.Effects of site-mutations within the 22 kD no-core fragment of the Vip3Aa11 insecticidal toxin of Bacillus thuringiensis[J].Current Microbiology,74(5):655-659.
Mesrati L A,Tounsi S,Jaoua S.2005.Characterization of a novel vip3-type gene from Bacillus thuringiensis,and evidence of its presence on a large plasmid[J].Fems Microbiology Letters,244(2):353-358.
Ramasamy B,Nadarajah V D,Soong Z K,et al.2008.A preliminary study of the bioactivity of vegetative proteins extracted from Malaysian Bacillus thuringiensis isolates[J].Tropical Biomedicine,25(1):64-74.
Song F,Chen C,Wu S,et al.2016.Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation[J].Scientific Reports,6:23861.
Yu C G,Mullins M A,Warren G W,et al.1997.The Bacillus thuringiensis vegetative insecticidal protein Vip3Aa lyses midgut epithelium cells of susceptible insects[J].Applied and Environmental Microbiology,63(2):532-536.
Yu J,Yi P,Tang M,et al.2001.Highly toxic and broad-spectrum insecticidal Bacillus thuringiensis,engineered by using the transposon tn917 and protoplast fusion[J].Current Microbiology,43(2):112-119.
Yu X,Tao L,Sun Z,et al.2012.Co-expression and synergism analysis of Vip3Aa29 and Cyt2Aa3 insecticidal proteins from Bacillus thuringiensis[J].Current Microbiology,64(4):326-331.
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