Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

doi:10.1016/S2095-3119(14)60771-1

Journal of Integrative Agriculture ›› 2015, Vol. 14 ›› Issue (2): 337-346.DOI: 10.1016/S2095-3119(14)60771-1

Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

ZHANG Li-li, LIANG Ge-mei, GAO Xi-wu, CAO Guang-chun, GUO Yu-yuan

1、College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, P.R.China
2、State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

收稿日期:2013-12-31 出版日期:2015-02-01 发布日期:2015-02-11
通讯作者: LIANG Ge-mei, Tel: +86-10-62815929, E-mail: gmliang@ippcaas.cn; GUO Yu-yuan, E-mail: yyguo@ippcaas.cn
作者简介:ZHANG Li-li, E-mail: zhangliliwell@126.com
基金资助:
This research was supported by the Key Technologies R&D Program of China during the 12th Five-Year Plan period (2012BAD19B05) and the National Natural Science Foundation of China (30971921,31321004). We would like to thank Biotechnology Group in Institute of Plant Protection, Chinese Academy of Agricultural Sciences for provision of Cry1Ac strain.

Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

ZHANG Li-li, LIANG Ge-mei, GAO Xi-wu, CAO Guang-chun, GUO Yu-yuan

1、College of Agriculture and Biotechnology, China Agricultural University, Beijing 100193, P.R.China
2、State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, P.R.China

Received:2013-12-31 Online:2015-02-01 Published:2015-02-11
Contact: LIANG Ge-mei, Tel: +86-10-62815929, E-mail: gmliang@ippcaas.cn; GUO Yu-yuan, E-mail: yyguo@ippcaas.cn
About author:ZHANG Li-li, E-mail: zhangliliwell@126.com
Supported by:
This research was supported by the Key Technologies R&D Program of China during the 12th Five-Year Plan period (2012BAD19B05) and the National Natural Science Foundation of China (30971921,31321004). We would like to thank Biotechnology Group in Institute of Plant Protection, Chinese Academy of Agricultural Sciences for provision of Cry1Ac strain.

摘要/Abstract

摘要： Carbohydrate chains are the principal antigens by which Bacillus thuringiensis (Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length cDNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques (GAL-Harm, GenBank accession no.: GQ904195.1) with two potential N-glycosylation sites (157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1Ac enhanced the toxicity against the siRNA-treated larvae compared with non-siRNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1Ac in H. armigera.

关键词: &beta, -1 , 3-galactosyltransferase , Cry1Ac , resistance , Helicoverpa armigera , RNAi

Abstract: Carbohydrate chains are the principal antigens by which Bacillus thuringiensis (Bt) identify receptor proteins. The interaction between the antigen and Bt causes a pore in the membrane of midgut epithelial cells of insects. Receptor proteins, such as aminopeptidase N and alkaline phosphatase, are glycoproteins. Cadherin is another cell surface receptor protein which has potential glycosylation sites. Glycosyltransferase is very important for the synthesis and modification of receptor proteins. It can indirectly influence the function of Bt. The 1 950 bp full-length cDNA encoding β-1,3-galactosyltransferase was cloned from the the midgut of Helicoverpa armigera by degenerative PCR combined with RACE techniques (GAL-Harm, GenBank accession no.: GQ904195.1) with two potential N-glycosylation sites (157NNTI160 and 272NKTL275). Protein sequence alignments revealed that H. armigera β-1,3-galactosyltransferase shared high identity with β-1,3-galactosyltransferase in other insect species. The expression level of the β-1,3-galactosyltransferase gene in Cry1Ac-resistant H. armigera larvae was 9.2-fold higher than that in susceptible strain. The function of β-1,3-galactosyltransferase was investigated using RNAi technique. The result showed Cry1Ac enhanced the toxicity against the siRNA-treated larvae compared with non-siRNA-treated ones, which indicated β-1,3-galactosyltransferase played an important role for the insecticidal toxicity of Cry1Ac in H. armigera.

Key words: β-1 , 3-galactosyltransferase , Cry1Ac , resistance , Helicoverpa armigera , RNAi

ZHANG Li-li, LIANG Ge-mei, GAO Xi-wu, CAO Guang-chun, GUO Yu-yuan. Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)[J]. Journal of Integrative Agriculture, 2015, 14(2): 337-346.

参考文献

Agrawal N, Malhotra P, Bhatnagar R K. 2004. siRNA-directedsilencing of transgene expressed in cultured insect cells.Biochemical and Biophysical Research Communications,320, 428-434

Banks D J, Jurat-Fuentes J L, Dean D H, Adang M J 2001.Bacillus thuringiensis Cry1Ac and Cry1Fa δ-endotoxinbinding to a novel 110KDa aminopeptidase in Heliothisvirescens is not N-acetylgalactosamine mediated. InsectBiochemistry and Molecular Biology, 31, 909-918

Bradford M M. 1976. A rapid and sensitive method for thequantitation of microgram quantities of protein utilizing theprinciple of protein-dye binding. Analytical Biochemistry,72, 248-254

Bravo A, Gómez I, Conde J, Muñoz-Garay C, SánchezJ, Miranda R, Zhuang M, Gill S S, Soberón M. 2004.Oligomerization triggers binding of a Bacillus thuringiensisCry1Ab pore-forming toxin to aminopeptidase N receptorleading to insertion into membrane microdomains.Biochimica et Biophysica Acta-Biomembranes, 1667,38-46

Breton C, Šnajdrová L, Jeanneau C, Ko?a J, Imberty A. 2006.Structures and mechanisms of glycosyltransferases.Glycobiology, 16, 29-37

Burton S L, Ellar D J, Li J, Derbyshire D J. 1999.N-acetylgalactosamine on the putative insect receptoraminopeptidase N is recognized by a site on the domain IIIlectin-like fold of a Bacillus thuringiensis insecticidal toxin.Journal of Molecular Biology, 287, 1011-1022

Coates B S, Sumerford D V, Hellmicha R L, Lewis L C. 2007.A β-1,3-galactosyltransferase and brainiac/bre5 homologexpressed in the midgut did not contribute to a Cry1Abtoxin resistance trait in O. nubilalis. Insect Biochemistryand Molecular Biology, 37, 346-355

Finney D J. 1971. Probit Analysis. 3rd ed. Cambridge UniversityPress, Cambridge, UK.

Flores-Escobar B, Rodríguez-Magadan H, Bravo A, SoberónM, Gómez I. 2013. Differential role of Manduca sextaaminopeptidase-N and alkaline phosphatase have adifferential role in the mode of action of Cry1Aa, Cry1Aband Cry1Ac toxins from Bacillus thuringiensis. Applied and Environmental Microbiology, 79, 4543-4550

Gahan L J, Gould F, Heckel D G. 2001. Identification of agene associated with Bt resistance in Heliothis virescens.Science, 293, 857-860

Galili U, Mandrell R E, Hamadeh R M, Shohet S B, Griffiss J M.1988. Interaction between human natural anti-α-galactosylimmunoglobulin G and bacteria of the human flora. Infectionand Immunity, 56, 1730-1737

Gasteiger E, Gattiker A, Hoogland C, Ivanyi I, Appel R D,Bairoch A. 2003. ExPASy, the proteomics server for in-depthprotein knowledge and analysis. Nucleic Acids Research,31,3784-3788

Griffitts J S, Haslam S M, Yang T L, Garczynski S F, MulloyB, Morris H, Cremer P S, Dell A, Adang M J, Aroian R V.2005. Glycolipids as receptors for Bacillus thuringiensiscrystal toxin. Science, 307, 922-925

Griffitts J S, Huffman D L, Whitacre J L, Barrows B D, MarroquinL D, Müller R, Brown J R, Hennet T, Esko J D, Aroian RV. 2003. Resistance to a bacterial toxin is mediated byremoval of a conserved glycosylation pathway requiredfor toxin-host interactions. Journal of Biological Chemistry,278, 45594-45602

Griffitts S J, Whitacre L J, Stevens E D, Aroian R V. 2001.Bt toxin resistance from loss of a putative carbohydratemodifyingenzyme. Science, 293, 860-864

Heckel D G, Gahan L J, Baxter S W, Zhao J-Z, Shelton A M,Gould F, Tabashnik B E. 2007. The diversity of Bt resistancegenes in species of Lepidoptera. Journal of InvertebratePathology, 95, 192-197

Jurat-Fuentes J L, Adang M J. 2004. Characterization of aCry1Ac-receptor alkaline phosphatase in susceptible andresistant Heliothis virescens larvae. European Journal ofBiochemistry, 271,3127-3135

Jurat-Fuentes J L, Adang M J. 2006. The Heliothis virescenscadherin protein expressed in Drosophila S2 cells functionsas a receptor for Bacillus thuringiensis Cry1A but not Cry1Fatoxins. Biochemistry, 45, 9688-9695

Kumaraswami N S, Maruyama T, Kurabe S, Kishimoto T,Mitsui T, Hori H. 2001. Lipids of brush border membranevesicles (BBMV) from Plutella xylostella resistant andsusceptible to Cry1Ac δ-endotoxin of Bacillus thuringiensis.Comparative Biochemistry and Physiology (B-Biochemistryand Molecular Biology), 129, 173-183

Liang G M, Tan W J, Guo Y Y. 2000. Studies on the resistancescreening and cross-resistance of cotton bollworm toBacillus thuringiensis (Berliner). Scientia Agricultura Sinica,33, 46-53 (in Chinese)

Livak K J, Schmittgen T D. 2001. Analysis of relative geneexpression data using real-time quantitative PCR and the2-ΔΔCT method. Methods, 25, 402-408

Lu Q, Zhang Y J, Cao G C, Zhang L L, Liang G M, Lu Y H, WuK M, Gao X W, Guo Y Y. 2012. A fragment of cadherin-likeprotein enhances Bacillus thuringiensis Cry1B and Cry1Ctoxicity to Spodoptera exigua (Lepidoptera: Noctuidae).Journal of Integrative Agriculture, 11, 628-638

de Maagd R A, van der K, Bakker P L, Stiekema W J, Bosch,D. 1996. Different domains of Bacillus thuringiensisδ-endotoxins can bind to insect midgut membrane proteinson ligand blots. Applied and Environmental Microbiology,62, 2753-2757

Morin S, Biggs R W, Sisterson M S, Shriver L, Ellers-Kirk C,Higginson D, Holley D, Gahan L J, Heckel D G, CarrièreY, Dennehy T J, Brown J K, Tabashnik B E. 2003. Threecadherin alleles associated with resistance to Bacillusthuringiensis in pink bollworm. Proceedings of the NationalAcademy of Sciences of the United States of America, 100,5004-5009

Nielsen H, Engelbrecht J, Brunak S, von Heijine G. 1997.Identification of prokaryotic and eukaryotic signal peptidesand prediction of their cleavage sites. Protein Engineering,10, 1-6

Ning C M, Wu K M, Liu C X, Gao Y L, Jurat-Fuentes J L, Gao XW. 2010. Characterization of a Cry1Ac toxin-binding alkalinephosphatase in the midgut from Helicoverpa armigera(Hübner) larvae. Journal of Insect Physiology, 56, 666-672

Pardo-López L, Gómez I, Muñoz-Garay C, Jiménez-JuarezN, Soberón M, Bravo A. 2006. Structural and functionalanalysis of the pre-pore and membrane-inserted poreof Cry1Ab toxin. Journal of Invertebrate Pathology, 92,172-177

Perera O P, Willis J D, Adang M J, Jurat-Fuentes J L. 2009.Cloning and characterization of the Cry1Ac-binding alkalinephosphatase (HvALP) from Heliothis virescens. InsectBiochemistry and Molecular Biology, 39, 294-302

Pigott C R, Ellar D J. 2007. Role of receptors in Bacillusthuringiensis crystal toxin activity. Microbiology andMolecular Biology Reviews, 71, 255-281

Rajagopal S, Sivakumar N, Agrawal P, Malhotra R K, BhatnagarR. 2002. Silencing of midgut amiopeptidase N of Spodopteralitura by double-stranded RNA establishes its role asBacillus thuringiensis toxin receptor. Journal of BiologicalChemistry, 277, 46849-46851

Rodrigo-Simón A, Caccia S, Ferré J. 2008. Bacillus thuringiensisCry1Ac toxin-binding and pore-forming activity in brushborder membrane vesicles prepared from anterior andposterior midgut regions of Lepidopteran larvae. Appliedand Environmental Microbiology, 3, 1710-1716

Sarkar A, Hess D, Mondal H A, Banerjee S, Sharma H C, DasS. 2009. Homodimeric alkaline phosphatase located atHelicoverpa armigera midgut, a putative receptor of Cry1Accontains α-GalNAc in terminal glycan structure as interactiveepitope. Journal of Proteome Research, 8, 1838-1848

Schnepf E, Crickmore N, Van Rie J, Lereclus D, BaumJ, Feitelson J, Zeigler D R, Dean D H. 1998. Bacillusthuringiensis and its pesticidal crystal proteins. Microbiologyand Molecular Biology Reviews, 62, 775-806

Sivakumar S, Rajagopal R, Venkatesh G R, Srivastava A,Bhatnagar R K. 2007. Knockdown of aminopeptidase-Nfrom Helicoverpa armigera larvae and in transfected Sf21cells by RNA interference reveals its functional interactionwith Bacillus thuringiensis insecticidal protein Cry1Ac.Journal of Biological Chemistry, 282, 7312-7319

Soberón M, Pardo-López L, López I, Gómez I, Tabashnik B E,Bravo A. 2007. Engineering modified Bt toxins to counterinsect resistance. Science, 318, 1640-1642

Tabashnik B E, Brévault T, Carrière Y. 2013. Insect resistanceto Bt crops, lessons from the first billion acres. NatureBiotechnology, 31, 510-521

Tamura K, Peterson D, Peterson N, Stecher G, Nei M, KumarS. 2011. MEGA5, molecular evolutionary genetics analysisusing maximum likelihood, evolutionary distance, andmaximum parsimony methods. Molecular Biology andEvolution, 28, 2731-2739

Wu K M, Lu Y H, Feng H Q, Jiang Y Y, Zhao J Z. 2008.Suppression of cotton bollworm in multiple crops in Chinain areas with Bt toxin-containing cotton. Science, 321,1676-1678

Xie R, Zhuang M, Ross L S, Gomez I, Oltean D I, Bravo A,Soberon M, Gill S S. 2005. Single amino acid mutationsin the cadherin receptor from Heliothis virescens affect itstoxin binding ability to Cry1A toxins. Journal of BiologicalChemistry, 280, 8416-8425

Xu X J, Yu L Y, Wu Y D. 2005. Disruption of a cadherin geneassociated with resistance to Cry1Ac δ-endotoxin ofBacillus thuringiensis in Helicoverpa armigera. Applied andEnvironmental Microbiology, 71, 948-954

Yang Y J, Chen H Y, Wu Y D, Yang Y H, Wu S W. 2007. Mutatedcadherin alleles from a field population of Helicoverpaarmigera confer resistance to Bacillus thuringiensis toxinCry1Ac. Applied and Environmental Microbiology, 73,6939-6944

Zhang S P, Cheng H M, Gao Y L, Wang G R, Liang G M,Wu K M. 2009. Mutation of an aminopeptidase N geneis associated with Helicoverpa armigera resistance toBacillus thuringiensis Cry1Ac toxin. Insect Biochemistryand Molecular Biology, 39, 421-429

Zhang X B, Candas M, Griko N B, Rose-Young L, Bulla Jr LA. 2005. Cytotoxicity of Bacillus thuringiensis Cry1Ab toxindepends on specific binding of the toxin to the cadherinreceptor BT-R1 expressed in insect cells. Cell Death andDifferentiation, 12, 1407-1416

Zhang X B, Candas M, Griko N B, Taussig R, Bulla Jr LA.2006. A mechanism of cell death involving an adenylylcyclase/PKA signaling pathway is induced by the Cry1Abtoxin of Bacillus thuringiensis. Proceedings of the NationalAcademy of Sciences of the United States of America,103, 9897-9902

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Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

Characterization and functional analysis of β-1,3-galactosyltransferase involved in Cry1Ac resistance from Helicoverpa armigera (Hübner)

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