美国白蛾氨肽酶N的基因克隆表达及与苏云金杆菌3种毒素蛋白的结合特性分析
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摘要
美国白蛾(Hyphantria cunea)是桑树的重要害虫。为明确苏云金杆菌(Bacillus thuringiensis)杀虫晶体蛋白对该虫的作用机制,利用已知昆虫氨肽酶N基因序列设计引物,以美国白蛾中肠cDNA为模板,利用RACE-PCR技术获得美国白蛾中肠氨肽酶N基因hcapn1全长序列(GenBank登录号:KP053647)。该基因开放阅读框为3 000 bp,编码999个氨基酸,预测蛋白质的分子质量和等电点分别为113.14 kD和4.85。设计去信号肽引物PCR扩增获得hcapn1基因序列,构建原核重组表达载体pET30a-hcapn1,诱导表达的HcAPN1重组蛋白约110 kD。将苏云金杆菌Cry1Ac、Cry2Ab33和Cry9Ea6原毒素经胰蛋白酶消化后与HcAPN1重组蛋白分别进行体外配体印迹试验,发现HcAPN1重组蛋白与Cry9Ea6结合,而不与Cry2Ab33、Cry1Ac发生特异结合,推测HcAPN1可能为Cry9Ea6在美国白蛾中肠的受体蛋白。分别设计引物扩增HcAPN1蛋白2个结构域Asp_(34)～Asp_( 527)和Leu_(563)～Gln_(925)的编码序列,在大肠埃希菌中表达获得62 kD和48 kD 2种重组蛋白,体外结合试验显示Cry9Ea6与HcAPN1的结合发生在Leu_(563)～Gln_(925)之间。研究结果为深入理解苏云金杆菌Cry9Ea6蛋白对美国白蛾的杀虫机制提供了基础数据。
Hyphantria cunea Drury is an important pest of Morus alba L.. In order to classify the action mechanism of insecticidal toxins from Bacillus thuringiensis on H. cunea,the full length cDNA sequence of H. cunea midgut aminopeptidase N gene hcapn1( GenBank accession No.KP053647) was obtained through RACE-PCR using H.cunea midgut cDNA as template and specific primers,which was designed according to known insect aminopeptidase N gene sequence. The open reading frame of hcapn1 was 3 000 bp,encoding 999 amino acid residues. The predicted molecular weight and isoelectric point of HcAPN1 were 113. 14 k D and 4. 85,respectively.Signal peptide-removed hcapn1 gene was cloned by PCR and used to construct recombinant expression vector pET30 a-hcapn1 for prokaryotic expression. The molecular weight of induced HcAPN1 recombinant protein was about 110 k D. In vitro ligand blot assay was performed to ascertain the binding activity of HcAPN1 recombinant protein with B. thuringiensis bysepticus Cry1 Ac,Cry2 Ab33 and Cry9 Ea6 toxins after treated with trypsin. Results showed that HcAPN1 recombinant protein can bind specifically to Cry9 Ea6,while could not bind to Cry1 Ac and Cry2 Ab33. It was suggested that HcAPN1 might be the receptor of Cry9 Ea6 in midgut of H.cunea. Primers were designed to amplify the coding regions of two functional domains( Asp34-Asp527 and Leu563-Gln925)of HcAPN1 protein,respectively,and two recombinant proteins with molecular weight of 62 k D and 48 k D were expressed in Escherichia coli. In vitro binding test showed that the binding of Cry9 Ea6 with HcAPN1 occurred between Leu563 and Gln925. These results provide basic data for further understanding the mechanism of Cry9 Ea6 protein on H. cunea.
Hyphantria cunea Drury is an important pest of Morus alba L.. In order to classify the action mechanism of insecticidal toxins from Bacillus thuringiensis on H. cunea,the full length cDNA sequence of H. cunea midgut aminopeptidase N gene hcapn1( GenBank accession No.KP053647) was obtained through RACE-PCR using H.cunea midgut cDNA as template and specific primers,which was designed according to known insect aminopeptidase N gene sequence. The open reading frame of hcapn1 was 3 000 bp,encoding 999 amino acid residues. The predicted molecular weight and isoelectric point of HcAPN1 were 113. 14 k D and 4. 85,respectively.Signal peptide-removed hcapn1 gene was cloned by PCR and used to construct recombinant expression vector pET30 a-hcapn1 for prokaryotic expression. The molecular weight of induced HcAPN1 recombinant protein was about 110 k D. In vitro ligand blot assay was performed to ascertain the binding activity of HcAPN1 recombinant protein with B. thuringiensis bysepticus Cry1 Ac,Cry2 Ab33 and Cry9 Ea6 toxins after treated with trypsin. Results showed that HcAPN1 recombinant protein can bind specifically to Cry9 Ea6,while could not bind to Cry1 Ac and Cry2 Ab33. It was suggested that HcAPN1 might be the receptor of Cry9 Ea6 in midgut of H.cunea. Primers were designed to amplify the coding regions of two functional domains( Asp34-Asp527 and Leu563-Gln925)of HcAPN1 protein,respectively,and two recombinant proteins with molecular weight of 62 k D and 48 k D were expressed in Escherichia coli. In vitro binding test showed that the binding of Cry9 Ea6 with HcAPN1 occurred between Leu563 and Gln925. These results provide basic data for further understanding the mechanism of Cry9 Ea6 protein on H. cunea.
引文
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[8] 马文静,韩兰芝,尹新明,等.鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系[J].环境昆虫学报,2011,33(3):378-387
[9] PAN Z Z,XU L,LIU B,et al.PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis[J].Int J Biol Macromol,2017,105:516-521
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[11] PARK Y,HERRERO S,KIM Y.A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella[J].Insect Mol Biol,2015,24(6):624-633
[12] 王翠苹,赵丹,李少雅,等.美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析[J].植物保护,2016,42(2):62-67
[13] SIVAKUMAR S,RAJAGOPAL R,VENKATESH G R,et al.Knockdown of aminopeptidase N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac[J].J Biol Chem,2007,282(10):7312-7319
[14] HERRERO S,GONZáLEZ-CABRERA J,FERRé J,et al.Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains Ⅱ and Ⅲ in specificity towards Spodoptera exigua larvae[J].Biochem J,2004,384(Pt 3):507-513
[15] 杨明琪.不同气候情景下美国白蛾在我国的适生区预测[D].北京:中国林业科学研究院,2013
[16] GAHAN L J,PAUCHET Y,VOGEL H,et al.An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin[J/OL].PLoS Genet,2010,6(12):e1001248[2018-06-01].https://www.ncbi.nlm.nih.gov/pubmed/21187898.DOI:10.1371/journal.pgen.1001248
[17] YAOIA K,NAKANISHI K,KADOTANIB T,et al.Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N[J].FEBS Lett,1999,463(3):221-224
[18] XU L,WANG Z Y,ZHANG J,et al.Characterization of four midgut aminopeptidase N isozymes from Ostrinia furnacalis strains with different susceptibilities to Bacillus thuringiensis[J].J Invertebr Pathol,2014,115:95-98
[19] 齐佳.利用噬菌体展示技术筛选Cry2Ab毒素受体集结合表位的研究[D].北京:中国农业科学院研究生院,2011
[20] STEPHENS E,SUGARS J,MASLEN S L,et al.The N-linked oligosaccharides of aminopeptidase N from Manduca sexta:site localization and identification of novel N-glycan structures[J].Eur J Biochem,2004,271(21):4241-4258
[21] ZHANG S P,CHENG H M,GAO Y L,et al.Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac[J].Insect Biochem Mol Biol,2009,39(7):421-429
[22] 刘子欢.美国白蛾中肠HcAPN1的鉴定及其与Bt Cry蛋白的结合分析[D].保定:河北农业大学,2015
[2] 李路莎,袁郁斐,武磊,等.不同寄主植物对美国白蛾幼虫取食行为及解毒酶活性的影响[J].昆虫学报,2018,61(2):232-239
[3] 薛建杰,曲良建,王玉珠,等.美国白蛾核型多角体病毒多角体蛋白的SDS-PAGE分析[J].林业科学研究,2009,22(5):728-731
[4] 刘云飞,陆秀君,刘廷辉,等.苏云金杆菌与高效氯氰菊酯对美国白蛾的协同作用[J].农药学学报,2012,14(1):51-55
[5] BRAVO A,LIKITVIVATANAVONG S,GILL S S,et al.Bacillus thuringiensis: a story of a successful bioinsecticide[J].Insect Biochem Mol Biol,2011,41(7):423-431
[6] REN X L,CHEN R R,ZHANG Y,et al.A Spodoptera exigua cadherin serves as a putative receptor for Bacillus thuringiensis Cry1Ca toxin and shows differential enhancement of Cry1Ca and Cry1Ac toxicity[J].Appl Environ Microbiol,2013,79(18):5576-5583
[7] WANG J,WANG H D,LIU S Y,et al.CRISPR/Cas9 mediated genome editing of Helicoverpa armigera with mutations of an ABC transporter gene HaABCA2 confers resistance to Bacillus thuringiensis Cry2A toxins[J].Insect Biochem Mol Biol,2017,87:147-153
[8] 马文静,韩兰芝,尹新明,等.鳞翅目昆虫氨肽酶N与Bt毒素的结合及其与Bt抗性的关系[J].环境昆虫学报,2011,33(3):378-387
[9] PAN Z Z,XU L,LIU B,et al.PxAPN5 serves as a functional receptor of Cry2Ab in Plutella xylostella (L.) and its binding domain analysis[J].Int J Biol Macromol,2017,105:516-521
[10] GRANERO F,BALLESTER V,FERRé J,et al.Bacillus thuringiensis crystal proteins Cry1Ab and Cry1Fa share a high affinity binding site in Plutella xylostella (L.)[J].Biochem Biophys Res Commun,1996,224(3):779-783
[11] PARK Y,HERRERO S,KIM Y.A single type of cadherin is involved in Bacillus thuringiensis toxicity in Plutella xylostella[J].Insect Mol Biol,2015,24(6):624-633
[12] 王翠苹,赵丹,李少雅,等.美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析[J].植物保护,2016,42(2):62-67
[13] SIVAKUMAR S,RAJAGOPAL R,VENKATESH G R,et al.Knockdown of aminopeptidase N from Helicoverpa armigera larvae and in transfected Sf21 cells by RNA interference reveals its functional interaction with Bacillus thuringiensis insecticidal protein Cry1Ac[J].J Biol Chem,2007,282(10):7312-7319
[14] HERRERO S,GONZáLEZ-CABRERA J,FERRé J,et al.Mutations in the Bacillus thuringiensis Cry1Ca toxin demonstrate the role of domains Ⅱ and Ⅲ in specificity towards Spodoptera exigua larvae[J].Biochem J,2004,384(Pt 3):507-513
[15] 杨明琪.不同气候情景下美国白蛾在我国的适生区预测[D].北京:中国林业科学研究院,2013
[16] GAHAN L J,PAUCHET Y,VOGEL H,et al.An ABC transporter mutation is correlated with insect resistance to Bacillus thuringiensis Cry1Ac toxin[J/OL].PLoS Genet,2010,6(12):e1001248[2018-06-01].https://www.ncbi.nlm.nih.gov/pubmed/21187898.DOI:10.1371/journal.pgen.1001248
[17] YAOIA K,NAKANISHI K,KADOTANIB T,et al.Bacillus thuringiensis Cry1Aa toxin-binding region of Bombyx mori aminopeptidase N[J].FEBS Lett,1999,463(3):221-224
[18] XU L,WANG Z Y,ZHANG J,et al.Characterization of four midgut aminopeptidase N isozymes from Ostrinia furnacalis strains with different susceptibilities to Bacillus thuringiensis[J].J Invertebr Pathol,2014,115:95-98
[19] 齐佳.利用噬菌体展示技术筛选Cry2Ab毒素受体集结合表位的研究[D].北京:中国农业科学院研究生院,2011
[20] STEPHENS E,SUGARS J,MASLEN S L,et al.The N-linked oligosaccharides of aminopeptidase N from Manduca sexta:site localization and identification of novel N-glycan structures[J].Eur J Biochem,2004,271(21):4241-4258
[21] ZHANG S P,CHENG H M,GAO Y L,et al.Mutation of an aminopeptidase N gene is associated with Helicoverpa armigera resistance to Bacillus thuringiensis Cry1Ac[J].Insect Biochem Mol Biol,2009,39(7):421-429
[22] 刘子欢.美国白蛾中肠HcAPN1的鉴定及其与Bt Cry蛋白的结合分析[D].保定:河北农业大学,2015