苏云金芽胞杆菌Vip3AaAdAa嵌合蛋白的构建及其杀虫活性分析
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摘要
Vip3A类杀虫蛋白是一类新型杀虫蛋白,在氨基酸序列上与研究较为深入的Cry蛋白没有同源性,且昆虫受体结合位点与Cry蛋白也没有竞争关系。利用同源重组技术针对对甜菜夜蛾具有高活性的Vip3Aa39与无活性的Vip3Ad开展研究,成功构建了三个vip3AaAdAa型嵌合基因,将其转入大肠杆菌中表达嵌合蛋白Vip3AaAdAa1、Vip3AaAdAa2、Vip3AaAdAa3,并将这三种嵌合蛋白对甜菜夜蛾进行了杀虫活性测定。研究表明,三种嵌合蛋白对甜菜夜蛾具有杀虫活性,Vip3AaAdAa3蛋白的LC_(50)值比Vip3Aa39增加了1.4倍,Vip3AaAdAa1蛋白的LC_(50)值比Vip3Aa39增加了2.7倍,嵌合蛋白Vip3AaAdAa1和Vip3AaAdAa3的杀虫活性均低于Vip3Aa39;Vip3AaAdAa2的杀虫活性与Vip3Aa39差异不显著。
Vip3A-type insecticidal proteins are a new class of insecticidal proteins. There is no homology in amino acid sequences with well-studied protein Cry,and the insecticidal sites are not competitive with insect receptor binding sites of Cry proteins. Homologous recombinant technique was used to study the insecticidal activity of Vip3Aa39 and inactive Vip3Ad against Spodoptera exigua. Three vip3AaAdAa chimeric genes were successfully created and were transformed into Escherichia coli to express chimeric protein Vip3AaAda1,Vip3AaAda2,and Vip3AaAda3. The insecticidal activities of these 3 chimeric proteins against S. exigua were determined. The results showed that the 3 chimeric proteins presented high insecticidal activities against S. exigua,the LC_(50) of Vip3AaAda3 protein was 1.4 times higher than that of Vip3Aa39,and the LC_(50) of Vip3AaAda1 protein was 2.7 times higher than that of Vip3Aa39. The insecticidal activities of chimeric proteins Vip3AaAda1 and Vip3AaAda3 were lower than that of Vip3Aa39,and the insecticidal activity of Vip3AaAdAa2 was not significantly different from that of Vip3Aa39.
Vip3A-type insecticidal proteins are a new class of insecticidal proteins. There is no homology in amino acid sequences with well-studied protein Cry,and the insecticidal sites are not competitive with insect receptor binding sites of Cry proteins. Homologous recombinant technique was used to study the insecticidal activity of Vip3Aa39 and inactive Vip3Ad against Spodoptera exigua. Three vip3AaAdAa chimeric genes were successfully created and were transformed into Escherichia coli to express chimeric protein Vip3AaAda1,Vip3AaAda2,and Vip3AaAda3. The insecticidal activities of these 3 chimeric proteins against S. exigua were determined. The results showed that the 3 chimeric proteins presented high insecticidal activities against S. exigua,the LC_(50) of Vip3AaAda3 protein was 1.4 times higher than that of Vip3Aa39,and the LC_(50) of Vip3AaAda1 protein was 2.7 times higher than that of Vip3Aa39. The insecticidal activities of chimeric proteins Vip3AaAda1 and Vip3AaAda3 were lower than that of Vip3Aa39,and the insecticidal activity of Vip3AaAdAa2 was not significantly different from that of Vip3Aa39.
引文
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[22]Selvapandiyan A,Arora N,et al.Toxicity analysis of N-and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis[J].Appl Environ Microbiol,2001,67(12):5855-5858.
[23]Ahmad A,Javed MR,Rao AQ,et al.In-Silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against lepidopteran targets using molecular docking[J].Front Plant Sci,2015,6:1081.
[24]Lee MK,Walters FS,Hart H,et al.The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin[J].Appl Environ Microbiol,2003,69(8):4648-4657.
[2]张金波,李海涛,等.Bt菌株DQ89的sip基因的克隆、表达及杀虫活性分析[J].中国生物防治学报,2015,31(4):598-602.
[3]彭琦,周子珊,张杰.苏云金芽胞杆菌杀虫晶体蛋白研究进展[J].中国生物防治学报,2015,31(5):712-722.
[4]Tetreau G,Stalinski R,David JP,et al.Monitoring resistance to Bacillus thuringiensis subsp.israelensis in the field by performing bioassays with each Cry toxin separately[J].Mem Inst Oswaldo Cruz,2013,108(7):894-900.
[5]Tanaka S,Endo H,Adegawa S,et al.Functional characterization of Bacillus thuringiensis Cry toxin receptors explains resistance in insects[J].FEBS J,2016,283(24):4474-4490.
[6]Fang J,Xu X,Wang P,et al.Characterization of chimeric Bacillus thuringiensis Vip3 toxins[J].Appl Environ Microbiol,2007,73(3):956-961.
[7]Chakroun M,Banyuls N,Bel Y,et al.Bacterial vegetative insecticidal proteins(Vip)from entomopathogenic bacteria[J].Microbiol Mol Biol Rev,2016,80(2):329-350.
[8]Chen W,Liu C,Lu G,et al.Effects of Vip3AcAa+Cry1Ac cotton on midgut tissue in Helicoverpa armigera(Lepidoptera:Noctuidae)[J].J Insect Sci,2018,18(4).
[9]Chen WB,Lu GQ,Cheng HM,et al.Transgenic cotton co-expressing chimeric Vip3AcAa and Cry1Ac confers effective protection against Cry1Ac-resistant cotton bollworm[J].Transgenic Res,2017,26(6):763-774.
[10]Ferre J,Escriche B.Editorial for special issue:the insecticidal bacterial toxins in modern agriculture[J].Toxins(Basel),2017,9(12):396.
[11]Boukedi H,Tounsi S,Abdelkefi-Mesrati L.Insecticidal activity,putative binding proteins and histopathological effects of Bacillus thuringiensis Vip3(459)toxin on the lepidopteran pest Ectomyelois ceratoniae[J].Acta Trop,2018,182:60-63.
[12]Song F,Chen C,et al.Transcriptional profiling analysis of Spodoptera litura larvae challenged with Vip3Aa toxin and possible involvement of trypsin in the toxin activation[J].Sci Rep,2016,6:23861.
[13]Bel Y,Sheets JJ,et al.Toxicity and binding studies of Bacillus thuringiensis Cry1Ac,Cry1F,Cry1C,and Cry2A proteins in the soybean pests anticarsia gemmatalis and chrysodeixis(Pseudoplusia)includens[J].Appl Environ Microbiol,2017,83(11).
[14]徐曼,蒋健,束长龙,等.Cry1Ab_Cry1Ah杂合蛋白构建与功能研究[J].生物技术通报,31(9):91-96.
[15]De Maagd RA,Bakker PL,et al.Domain III of the Bacillus thuringiensis delta-endotoxin Cry1Ac is involved in binding to Manduca sexta brush border membranes and to its purified aminopeptidase N[J].Mol Microbiol,1999,31(2):463-471.
[16]Walters FS,Defontes CM,et al.Lepidopteran-active variableregion sequence imparts coleopteran activity in eCry3.1Ab,an engineered Bacillus thuringiensis hybrid insecticidal protein[J].Appl Environ Microbiol,2010,76(10):3082-3088.
[17]Wu ZL,Guo WY,Qiu JZ,et al.Cloning and localization of vip3Agene of Bacillus thuringiensis[J].Biotechnol Lett,2004,26(18):1425-1428.
[18]Boukedi H,Ben Khedher S,Abdelkefi-Mesrati L,et al.Comparative analysis of the susceptibility/tolerance of Spodoptera littoralis to Vip3Aa,Vip3Ae,Vip3Ad and Vip3Af toxins of Bacillus thuringiensis[J].J Invertebr Pathol,2018,152:30-34.
[19]Thornton JA.Splicing by overlap extension PCR to obtain hybrid DNA products[J].Methods Mol Biol,2016,1373:43-49.
[20]何晓明.苏云金芽胞杆菌新型vip3基因克隆、表达及活性分析[D].哈尔滨:东北农业大学,2011.
[21]Kunthic T,Surya W,Promdonkoy B,et al.Conditions for homogeneous preparation of stable monomeric and oligomeric forms of activated Vip3A toxin from Bacillus thuringiensis[J].Eur Biophys J,2017,46(3):257-264.
[22]Selvapandiyan A,Arora N,et al.Toxicity analysis of N-and C-terminus-deleted vegetative insecticidal protein from Bacillus thuringiensis[J].Appl Environ Microbiol,2001,67(12):5855-5858.
[23]Ahmad A,Javed MR,Rao AQ,et al.In-Silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against lepidopteran targets using molecular docking[J].Front Plant Sci,2015,6:1081.
[24]Lee MK,Walters FS,Hart H,et al.The mode of action of the Bacillus thuringiensis vegetative insecticidal protein Vip3A differs from that of Cry1Ab delta-endotoxin[J].Appl Environ Microbiol,2003,69(8):4648-4657.