苏云金芽胞杆菌Cry8Ea1与Cry8Ca2蛋白纯化
摘要
金龟子属于鞘翅目金龟总科(Scarabaeoidae)，其幼虫(俗称蛴螬)是一类重要的世界性分布的地下害虫，可危害多种农林植物。20年来全国各地的大量调查表明，蛴螬在地下害虫中危害居首位。本文围绕对鞘翅目暗黑鳃金龟(Holotrichia parallela)铜绿丽金龟(Anomala carpulenta)的幼虫有活性的苏云金芽孢杆菌Cry8Ea1和Cry8Ca2蛋白的表达和纯化进行了研究。
HD8E和HBF-1菌株是苏云金芽孢杆菌的菌株，分别含有cry8Ea1和cry8Ca2的基因，编码Cry8Ea1和Cry8Ca2的原毒素(伴孢晶体)。研究表明，两个菌株表达的伴孢晶体均为球形晶体，相对分子质量为130kU。用胰蛋白酶和胰凝乳蛋白酶消化得到60kU的活性毒素，利用分子筛层析(Superdex200)收集60kU的毒素，然后使用阴离子交换层析进一步纯化得到纯的60kU的毒素。对纯蛋白进行生物活性测定，结果表明Cry8Ea1的原毒素与毒素对暗黑鳃金龟幼虫均具有较高活性，Cry8Ea1原毒素的LC50值为9.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为35.39μg／克土；Cry8Ca2的原毒素和毒素对铜绿丽金龟幼虫也具有很高的活性，Cry8Ca2原毒素的LC50值为0.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为0.08μg／克土，胰蛋白酶消化后纯化的毒素的LC50值为0.003μg／克土。本研究建立了Cry8Ea1与Cry8Ca2活性毒素蛋白的纯化方法，通过该方法可大量获得该毒素的纯品，为进一步研究该蛋白的结构与功能，揭示毒素的杀虫机理奠定了良好的基础。
研究中还发现Cry8Ea1和Cry8Ca2蛋白是与DNA相结合的。在本实验中对DNA的作用进行了研究，用透析的方法分别对两种毒素的DNA复合体和去除了DNA的毒素在不同pH条件及不同缓冲液条件下的稳定性进行了比较，结果表明，Cry8Ea1和Cry8Ca2毒素与DNA的复合体的稳定性要比不含DNA的毒素稳定，不易发生沉淀，DNA在维持毒素稳定性中起到重要作用。
The Cockchafer larvae are important underground pest that make damage in agriculture.Cry8Ea1and Cry8Ca2 protein expressed in Bacillus thuringiensis are toxic to Cockchafer larvae.In the studs; we investigated the expression and purification of these two proteins and the lethalconcentration 50 percent of both protein was also evaluated.
HD8E and HBF-1 are Bt strains, with genes that encoding Cry8Ea1and Cry8Ca2 protoxinseperatly. The protoxin expressed by these two Bt strains were analysed by SDS-PAGE. One majorband with an estimated molecular mass of 130 kU was detected. Cry8Ea1 and Cry8Ca2 protoxinwere activated by trpsin or chymotrypsin and the toxin with molecular mass of 60kU were obtained.Both toxin was purified by size exclusion chromatography first and further by anion exchangechromatography. The purification method of Cry8Ea1 and Cry8Ca2 toxin were established in thisstudy. Great amount of pure toxin of Cry8Ea1 and Cry8Ca2 can be obtained by using this method.It was a great foundation for futher investigation on structure and function of these two protein.
The insecidical activity of both pure toxin was tested by bioassays against scarab. Cry8Ea1protoxin and toxin are both toxic to Holotrichia parallela (scarab). The lethal concentration 50percent of of Cry8Ea1 protoxin was 9.46μg per gram soil, while that of the Cry8Ea1 toxin activatedby chymotrypsin was 35.39μg per gram soil. Cry8Ca2 protoxin and toxin are both toxic toAnomala carpulenta (scarab). The lethal concentration 50 percent of Cry8Ca2 protoxin was 0.47μgper gram soil, while that of Cry8Ca2 toxin activated by trypsin was 0.003μg per gram soil,Cry8Ca2 toxin activated by chymotrypsin was 0.08μg per gram soil.
HD8E和HBF-1菌株是苏云金芽孢杆菌的菌株，分别含有cry8Ea1和cry8Ca2的基因，编码Cry8Ea1和Cry8Ca2的原毒素(伴孢晶体)。研究表明，两个菌株表达的伴孢晶体均为球形晶体，相对分子质量为130kU。用胰蛋白酶和胰凝乳蛋白酶消化得到60kU的活性毒素，利用分子筛层析(Superdex200)收集60kU的毒素，然后使用阴离子交换层析进一步纯化得到纯的60kU的毒素。对纯蛋白进行生物活性测定，结果表明Cry8Ea1的原毒素与毒素对暗黑鳃金龟幼虫均具有较高活性，Cry8Ea1原毒素的LC50值为9.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为35.39μg／克土；Cry8Ca2的原毒素和毒素对铜绿丽金龟幼虫也具有很高的活性，Cry8Ca2原毒素的LC50值为0.47μg／克土，胰凝乳蛋白酶消化后纯化的毒素的LC50值为0.08μg／克土，胰蛋白酶消化后纯化的毒素的LC50值为0.003μg／克土。本研究建立了Cry8Ea1与Cry8Ca2活性毒素蛋白的纯化方法，通过该方法可大量获得该毒素的纯品，为进一步研究该蛋白的结构与功能，揭示毒素的杀虫机理奠定了良好的基础。
研究中还发现Cry8Ea1和Cry8Ca2蛋白是与DNA相结合的。在本实验中对DNA的作用进行了研究，用透析的方法分别对两种毒素的DNA复合体和去除了DNA的毒素在不同pH条件及不同缓冲液条件下的稳定性进行了比较，结果表明，Cry8Ea1和Cry8Ca2毒素与DNA的复合体的稳定性要比不含DNA的毒素稳定，不易发生沉淀，DNA在维持毒素稳定性中起到重要作用。
The Cockchafer larvae are important underground pest that make damage in agriculture.Cry8Ea1and Cry8Ca2 protein expressed in Bacillus thuringiensis are toxic to Cockchafer larvae.In the studs; we investigated the expression and purification of these two proteins and the lethalconcentration 50 percent of both protein was also evaluated.
HD8E and HBF-1 are Bt strains, with genes that encoding Cry8Ea1and Cry8Ca2 protoxinseperatly. The protoxin expressed by these two Bt strains were analysed by SDS-PAGE. One majorband with an estimated molecular mass of 130 kU was detected. Cry8Ea1 and Cry8Ca2 protoxinwere activated by trpsin or chymotrypsin and the toxin with molecular mass of 60kU were obtained.Both toxin was purified by size exclusion chromatography first and further by anion exchangechromatography. The purification method of Cry8Ea1 and Cry8Ca2 toxin were established in thisstudy. Great amount of pure toxin of Cry8Ea1 and Cry8Ca2 can be obtained by using this method.It was a great foundation for futher investigation on structure and function of these two protein.
The insecidical activity of both pure toxin was tested by bioassays against scarab. Cry8Ea1protoxin and toxin are both toxic to Holotrichia parallela (scarab). The lethal concentration 50percent of of Cry8Ea1 protoxin was 9.46μg per gram soil, while that of the Cry8Ea1 toxin activatedby chymotrypsin was 35.39μg per gram soil. Cry8Ca2 protoxin and toxin are both toxic toAnomala carpulenta (scarab). The lethal concentration 50 percent of Cry8Ca2 protoxin was 0.47μgper gram soil, while that of Cry8Ca2 toxin activated by trypsin was 0.003μg per gram soil,Cry8Ca2 toxin activated by chymotrypsin was 0.08μg per gram soil.
引文
蔡峻．2002．苏云金芽孢杆菌Cyt蛋白研究进展．微生物学报．42(4)：514-519
张杰．2000．31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究．微生物学报．40(4)：372-378
冯书亮等．2000．一株对金龟子类幼虫具有杀虫活性的苏云金杆菌新分离株．中国生物防治．16(2)：74～78．
黄大昉，林敏等．2001．农业微生物基因工程．科学出版社．235～236
胡宏源．2004．苏云金芽孢杆菌伴孢晶体20Kb DNA中cry1Ac基因的克隆、高效表达和生物活性研究．生物工程学报．20(5)：656-661
胡琼波等．2004．我国地下害虫蛴螬的发生与防治研究进展．湖北农业科学．6：87～92
刘凯于，姚汉超，杨红，洪华珠．2004．昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展．昆虫知识．41(3)203～207
孙明，喻子牛等．1996．苏云金芽孢杆菌中华亚种CT-43菌株伴孢晶体蛋白的特性．微生物学报．36(4)：303～306
邵宗泽．2000．苏云金芽胞杆菌杀虫晶体蛋白的结构与功能研究进展．生物化学与生物物理进展．27(5)
徐建国，张明考等．2002．暗黑鳃金龟生活习性观察及防治技术研究．植保技术与推广．22(11)：9～10
魏鸿钧等．1989．中国地下害虫．上海：上海科学技术出版社．1～41
王永祥等．1998．冀中平原区蛴螬种类及综合防治技术．河北师范大学学报(自然科学版)．22(2)：268～270
Abaci, Andre, R., Duck Nicholas, B., Feng, Xiang. et. al 2002. Genes encoding novel proteins with pesticidal activity against Coleopterans. WO 02/34774 A2
Abdullah. 2003. Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering. Appl. Environ. Microbiol. 69:5343～5353.
Angsuthanasombat, C. 1993. Effects on toxicity of eliminating a cleavage site in a predicted interhelieal loop in Bacillus thuringiensis CryIVB δ-endotoxin. FEMS Mierobiol. Letters. 111, 255～262.
Baroy F, Lecadet M M, Deleluse A. 1998. Cloning and Sequencing of Three New Putative Toxin Genes From Clostridium Bifermentans. Gene. 211:293～295
Bietlot H P, Vishnubhatla I, Carey P R, et al. 1990. Characterization of the Insecticidal Protein from Bacillus. Biochem J, 267: 309～315.
Bietlot H P, Schernthaner J P, Miine R E, et al. 1993 Evidence that Cry Ⅰ A protein from Bacillus thuringiensis is associated with DNA. J Biol Chem. 268(11): 8240～8245
Boonhiang Promdonkoy. 2004 Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis.Biochemical and Biophysical Research Communications.317:744-748
Bravo,A. 1992. Immunocy to chemical localization of Bacillus thuringiensis insecticidal crystal proteins in intoxicated insects.J.Invertebr.Pathol. 60:237—246
Carroll.J Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var.tenebrionis. Biochem.J.261: 99—105.
Chen X J, Lee M K, Dean D H. 1993. Site-Directed Mutations in a Highly Conserved Region of Bacillus thuringiensis 5-Endotoxin Affect Inhibition of Short Circuit Current Across Bombyx mori midguts. Proc. Natl. Acad. Sci. USA, 90:9041—9045
Chilcott C N, Ellar D J. 1988. Comparative Study of Bacillus thuringiensis var. israelensis Crystal Proteins in vivo and in vitro. J. Gen. Micro-biol. 134: 2551—2558
Clairmont F R, Milne R E, Pham V T, et al .1998. Role of DNA in the activation of the CryM Insecticidai Crystal Protein fiom Bacillus thrinensis. JBiol Chem, 273(15): 9292—9296.
Clairmont F R, Milne R E, Pham V T, et al. 1998. Role of DNA in the Activation of the CryM Insecticidai Crystal Protein fiom Bacillus thrinensis. JBiol Chem. 273(15): 9292—9296.
Claudia perez. 2005. Bacillus thuringiensis subsp.israelensis CytlAa synergizes CryllAa toxin by functioning as a membrane-bound receptor.PNAS. 102(51) 18303—18308
Coux,F. 2001. Role of interdomain salt bridges in the pore-forming ability of the Bacillus thutingiensis toxins CrylAa and Cry1Ac. J.Biol.Chem.276,35546—35551
Crickmore N, Zeigler D R, Schnepf E et al 2001. Bacillus thuringiensis Toxin Nomenclature 5
Denolf,P. 1993. Tow different Bacillus thuringiensis 8-endotoxin receptors in the midgut brush border membrane of the European corn borer,Ostrinia nubilalis.Appl.Environ.Microbiol. 59:1828— 1837
E.Schnepe and J.Feitelson. 1998. Bacillus thuringiensis and its pesticidal crystal proteins.Microbiology and Molecular biology reviews.Rev 62:775—806
Edwards D L, Payne J, Soares G 1988. Novel isolates of Bacillus thuringiensis having Activity against Nematodes. European Patent Application, 303 —426
Faust G M, Abe K, Held G A, Lizuka T, Bulla L A, Meyers C L. 1983. Evidence for Plasmid-Associated Crystal Toxin Production in Bacillus thuringiensis subsp. Israelensis, Plasmid, (9):98—103
Fernandez,L.E. 2005. CryllAa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loopα8 of domain II. FEBS Lett. 579,3508—3514.
Gazit E, Shai Y. 1995. The Assembly and Organization of the α5 and α7 Helices from the Pore-Forming Domain of Bacillus thuringiensis 5-Endotoxin. J. Biol. Chem. 270:2571—2578
Gazit E. 1998. The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis 5-endotoxin are consistent with an "umbrella-like" structure of the pore. Proc. Natl Acad. Sci. USA, 95,12289—12294.
Gill S S, Cowles E A, Pietrantonio P V. 1992. The Mode of Action of Bacillus thuringiensis Endotoxins. Annu. Rev. Entomol. 37:615-636
Grochulski,p. 1995. Bacillus thuringiensis CryIAa insecticidal toxin crystal structure and channel formation. J.Mol.Biol. 254,447-464
Hodgman C. 1990. Models for the structure and function of the Bacillus thuringiensis δ-endotoxin determined by compilational analysis.J.DNA Seque. Map. 1,97-106
Hodgman T C, Ellar D J. 1990. Models for the structure and function of the Bacillus thuringiensis δ-Endotoxins Determined by Compicational Analysis. DNA Sequence. 1:97—106
Hofmann C, Luthy P, Hutter R, Pliska V. 1988. Binding of the Deltaendotoxin from Bacillus thuringiensis to Brushborder Membran-Evesicles of the Cabbage Butterfly(Pieris brassicae). Eur J Biochem. 173:85—91
Hofte,H.,H.R.Whiteley.l989.Insecticidal crystal proteins of Bacillus thutingiensis Microbiol. Rev. 53:242-255
Isabel Gomez. 2006. Specific epitopes of Domains II and III of Bacillus thuringiensis CrylAb toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta. JBC Papers in Press. Published on September 12, as Manuscript M604721200
Jade Li. 1991. Crystal structure of insecticidal 8-endotoxin from Bacillus thuringiensis at 2.5A resolution. Nature.Vol353.815-821
Jade Li. 1996. Structure of the Mosquitocidalδ-endotoxin CytB from Bacillus thuringiensis sp.kyushuensis and implications for membrane pore formationJ.Mol.Biol. 257:129-152
Juan L. 2004. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. Eur.J.Biochem. 271,3127—3135.
Judith L. 1988. Crystallization and preliminary X-ray diffraction studies of a toxic crystal protein from a subspecies of Bacillus thuringiensis.The Journal of Bioloyical Chemistry.Vol 263,No.24,pp. 11800—11801
Kanintronkul 1998. Specific mutations within thea4-α5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170. Mol.Biotechnol.24,11—20.
Knowles B H, 1984. Thomas W E, Ellar D J. Lectin-like Binding of Bacillus thuringiensis var. kurstaki lepidopteran-Specific Toxin is an Initial Step in Insecticidal Action. FEBS Lett. 168:197— 202
Knowles B H, Ellar D J. 1987. Colloid-Osmotic Lysis is a General Feature of the Mechanism of Action of Bacillus thiringiensis δ-Endotoxins with Different Insect Specificity. Biochim. Biophys. Acta. 924:509—518
Knowles B H. 1994. Mechanism of Action of Bacillus thuringiensis Insecticidal δ-Endotoxins. Adv. Insect Physiol. 24:275—307
Kusol Pootanakit. 2003. Identification of tow isoforms of aminopeptidase N in Aedes aegypti larval midgut. Journal of Biochemistry and Molecular Biology. Vol.36, No.5, pp. 508—51
Luisa E.FERNANDEZ. 2006. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry 11 Aa toxin inAedes aegypti larvae.Biochem.J. 394,77—84
Luthy P, Ebersold H R. 1981. Bacillus thuringiensis Delta-Endotoxins: Histopathology and Molecular Mode of Action. In:Davidson E W ed. Pathogenesis of Invertebrate Microbial Diseases. New Jersey: Allanheld, 235—267
M.-E. Nunez-Valdez. 2001. Structural and functional studies of α-helix 5 region from Bacillus thuringiensis Cry1Abδ-endotoxin.Biochimica et Biophysica Acta 1546,122-131
M.Schiffer,C.H.Chang. 1992. The functions of tryptophan residues in membrane proteins. Protein Eng. 5.213—214
Masashi Yamagiwa. 2004. Functional Analysis of two processed fragments of Bacillus thuringiensis Cry11A toxin.Biosci Biotechem.Biochem, 68(3),523—528
McNall,R.J. 2003. Dentication of novel Bacillus thuringiensis Cry1Ac toxin binding proteins in Manduca seta midgut through proteomic analysis.Insect Biochem.Mol.Biol. 33, 999—1010.
Mi Kyong Lee. 2000. Role of two arginine residues in domain II,loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis δ-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.Molecular Microbiology. 38(2), 289—298
Mi Kyong Lee, 2001. Mutations at the arginine residues ina8 loop of Bacillus thuringiensis δ-endotoxin CrylAc affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.FEBS Letters 497(2001 )108-112
Natalie J.Tigue. 2001. The a-helix 4 residue,Asnl35,is involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins. Applied and environmental Microbiology,p. 5715-5720
Nicholls 1989. Evidence for two different types of insecticidal P2 toxins with dual specificity in bacillus thuringiensis subspecies. J.Bacteriol.171,5141—5147
Nikolai Galitsky. 2001. Structure of the insecticidal bacterial 5-endotoxin Cry3Bb1 of Bacillus thuringiensis. ISSN0909—4449
Novillo C, Castan-era P, Ortego F. 1997.Charaterization and Dis-Tribution of Chymotrypsin-Like and Other Digestive Proteases in Colorado Potato Beetle Larvae. Arch. Insect. Biochem. Physiol., 36:181-201. Serotype. Curr. Microbiol. 32:48—54
Ohba M et al. 1992. A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology. 14: 54—57
Panadda Boonserm. 2004. Crystallization and preliminary crystallographic study of the functional form of the Bacillus thuringiensis mosquito-larvicidal Cry4Aa mutant toxin.ISSN 0907-4449
Panadda Boonserm. 2005. Crystal structure of the mosquito-larvicidal toxin cry4Ba and its biological implications. J. Mol. Biol. 348, 363—382
Panadda Boonserm. 2006. Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-Angstrom resolution.JB. 188.9.3391—3401
Parker 1993 Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxin. Trends Biochem.Sci. 18,391 —395.
Puntheeranurak, T., Stroh, C., Zhu, R., Angsuthanasombat, C., Hinterdorfer, P. 2005. Structure and domain lstribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes. Ultramicroscopy, 105:115～124
R.J.Morse. 2001. Structure of Cry2Aa suggests an unexpec ted receptor binding epitope.Rev 9:409～417
R.Rajagopalt. 2002. Silencing of midgut aminopetidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiemis toxin receptor. The journal of Biological Chemistry. Vol.277.No.49.pp. 46849～46851
Rausell, C., Pardo-Lopez, L., Sanchez, J., Munoz-Garay, C., Morera, C., Soberon, M., Bravo A. 2004. Unfol domain lng events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.J. Biol. Chem. 279(53):55168～55175
Ruiyu Xie. 2005. Single Amino Acid Mutations in the Cadherin Receptor from Heliothis virescens Affect Its Toxin Binding Ability to CrylA Toxins. The Journal of Biological Chemistry. Vol.280,No.9,pp.8416～8425
Salvador HERRERO. 2004. Mutations in the Bacillus thuringiemis Cry1Ca toxin demonstrate the role of domains Ⅱ and Ⅲ in specificity towards Spodoptera exigua larvae.Biochem.J. 384,507～5
Satia Tapaneeyakom. 2005. Structural requirements of the unique disulphide bond and the praline-rich motif within the α4-α5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa δ-endotoxin. Biochemical and Biophysical Research Communications330(2005)519～525
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH. 1998. Bacillus thuringiensis and Its Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews. 62:775～806
Schwartz. J.L 2000. Membrane protein insertion and pore formation. In Entomopathogenic Bacteria: from Laboratory to Field Application pp.1～19.
Schwartz,J.L Restriction of intramolecutar movements within the Cry1Aa toxin molecule of Bacillus thudngiensis through disulfide bond engineering.FEBS Letters,410,397～402.
Schwartz,J-L Membrane permeabilization by Bacillus thutingiensis toxins:protein insertion and pore formation.In Entomopathogenic Bacteria: from Laboratory to Field Application Charles.A.pp. 1～19
Sheng-Jiun wu. 2000. Enhanced toxicity of Bacillus thuringiensis Cry3A δ-endoxin in coleopterans by mutagenesis in a receptor binding loop.FEBS Letters 473.227～232
Shin-ichiro Asano, Yamashita C, Iizuka T, Takeuchi K, Yamanaka S, Cerf D, Yamamoto T 2003. A strain of Bacillus thuringiensis subsp, galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biol Control 28: 191～196
Shogo Atsumi. 2005. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.Applied and Environmental.Microbiology, 3966～3977
Sick; August J., Schwab; George E., Payne; Jewel M., 1994. Genes encoding nematode-active toxins cloned from Bacillus thuringiensis isolate PS17, USP5281530
Somphob Leetachewa. 2006. Novel preparation and characterization of the α4-loop-α5 membrane-perturbing peptide from the Bacillus thuringiensis Cry4BaS-endotoxin.Journal of Biochemistry and Molecular Biology. Vol.39,No.3,pp. 270—277
Supaporn Likitvivatanavong. 2006. Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.Archives of Biochemistry and Biophysics 445(2006)46-55
Svetlana Borisova. 1994. Crystalization and preliminary X-ray diffraction studies of the lepidopteran-specifif insecticidal crystal protein Cry2Aa.J.Mol.Biol. 243:530—532
Tojo A, Aizawa K. 1983. Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori. Appl. Environ. Microbiol. 45:576-580
Tracy E. Michaels, Kenneth E. Narva, Foncerrada, 1994. Bacillus thuringiensis toxins active against scarab pests. USP5554534.
Uawithya 1998 Effects on larvicidal activity of single praline substitutions inα.3 orα4 of the Bacillus thuringiensis Cry4Ba toxin.Biochem.Mol.Biol.Int.44,825—832.
Vachon,V. 2002. Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1A.Biochemistry, 41,6178—6184
Vincent Vachon. 2004. Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities.Applied and environmental Microbiology,p. 6123—6130
Walairat Pornwiroon. 2004. Aromaticity of Tyr-202 in the a4-a5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin.Journal of Biochemistry and Molecular Biology. Vol.37,No.3,pp.292—297
Wu S J, Dean D H. 1996. Functional Significance of Loops in the Receptor Binding Domain of Bacillus thuringiensis CryIIIA 8-Endotoxin. J. Mol. Biol. 255:628—640
XINTIAN Wei, XuDAN Xu, C.Jack Deloach 1995. Biological control of white grubs (Coleopera: Scarabaeidae) by larvae of Promachus yesonicus (Diptera:AsiIidae) in China Bio Control 5 :290—296
Yoko TSUDA. 2003. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori(silkworm).Biochem.J. 369,697-703
张杰．2000．31株苏云金芽孢杆菌杀虫晶体蛋白基因型鉴定及表达产物研究．微生物学报．40(4)：372-378
冯书亮等．2000．一株对金龟子类幼虫具有杀虫活性的苏云金杆菌新分离株．中国生物防治．16(2)：74～78．
黄大昉，林敏等．2001．农业微生物基因工程．科学出版社．235～236
胡宏源．2004．苏云金芽孢杆菌伴孢晶体20Kb DNA中cry1Ac基因的克隆、高效表达和生物活性研究．生物工程学报．20(5)：656-661
胡琼波等．2004．我国地下害虫蛴螬的发生与防治研究进展．湖北农业科学．6：87～92
刘凯于，姚汉超，杨红，洪华珠．2004．昆虫中肠Bt杀虫晶体蛋白毒素受体氨肽酶N的研究进展．昆虫知识．41(3)203～207
孙明，喻子牛等．1996．苏云金芽孢杆菌中华亚种CT-43菌株伴孢晶体蛋白的特性．微生物学报．36(4)：303～306
邵宗泽．2000．苏云金芽胞杆菌杀虫晶体蛋白的结构与功能研究进展．生物化学与生物物理进展．27(5)
徐建国，张明考等．2002．暗黑鳃金龟生活习性观察及防治技术研究．植保技术与推广．22(11)：9～10
魏鸿钧等．1989．中国地下害虫．上海：上海科学技术出版社．1～41
王永祥等．1998．冀中平原区蛴螬种类及综合防治技术．河北师范大学学报(自然科学版)．22(2)：268～270
Abaci, Andre, R., Duck Nicholas, B., Feng, Xiang. et. al 2002. Genes encoding novel proteins with pesticidal activity against Coleopterans. WO 02/34774 A2
Abdullah. 2003. Introduction of Culex toxicity into Bacillus thuringiensis Cry4Ba by protein engineering. Appl. Environ. Microbiol. 69:5343～5353.
Angsuthanasombat, C. 1993. Effects on toxicity of eliminating a cleavage site in a predicted interhelieal loop in Bacillus thuringiensis CryIVB δ-endotoxin. FEMS Mierobiol. Letters. 111, 255～262.
Baroy F, Lecadet M M, Deleluse A. 1998. Cloning and Sequencing of Three New Putative Toxin Genes From Clostridium Bifermentans. Gene. 211:293～295
Bietlot H P, Vishnubhatla I, Carey P R, et al. 1990. Characterization of the Insecticidal Protein from Bacillus. Biochem J, 267: 309～315.
Bietlot H P, Schernthaner J P, Miine R E, et al. 1993 Evidence that Cry Ⅰ A protein from Bacillus thuringiensis is associated with DNA. J Biol Chem. 268(11): 8240～8245
Boonhiang Promdonkoy. 2004 Trp132, Trp154, and Trp157 are essential for folding and activity of a Cyt toxin from Bacillus thuringiensis.Biochemical and Biophysical Research Communications.317:744-748
Bravo,A. 1992. Immunocy to chemical localization of Bacillus thuringiensis insecticidal crystal proteins in intoxicated insects.J.Invertebr.Pathol. 60:237—246
Carroll.J Proteolytic processing of a coleopteran-specific delta-endotoxin produced by Bacillus thuringiensis var.tenebrionis. Biochem.J.261: 99—105.
Chen X J, Lee M K, Dean D H. 1993. Site-Directed Mutations in a Highly Conserved Region of Bacillus thuringiensis 5-Endotoxin Affect Inhibition of Short Circuit Current Across Bombyx mori midguts. Proc. Natl. Acad. Sci. USA, 90:9041—9045
Chilcott C N, Ellar D J. 1988. Comparative Study of Bacillus thuringiensis var. israelensis Crystal Proteins in vivo and in vitro. J. Gen. Micro-biol. 134: 2551—2558
Clairmont F R, Milne R E, Pham V T, et al .1998. Role of DNA in the activation of the CryM Insecticidai Crystal Protein fiom Bacillus thrinensis. JBiol Chem, 273(15): 9292—9296.
Clairmont F R, Milne R E, Pham V T, et al. 1998. Role of DNA in the Activation of the CryM Insecticidai Crystal Protein fiom Bacillus thrinensis. JBiol Chem. 273(15): 9292—9296.
Claudia perez. 2005. Bacillus thuringiensis subsp.israelensis CytlAa synergizes CryllAa toxin by functioning as a membrane-bound receptor.PNAS. 102(51) 18303—18308
Coux,F. 2001. Role of interdomain salt bridges in the pore-forming ability of the Bacillus thutingiensis toxins CrylAa and Cry1Ac. J.Biol.Chem.276,35546—35551
Crickmore N, Zeigler D R, Schnepf E et al 2001. Bacillus thuringiensis Toxin Nomenclature 5
Denolf,P. 1993. Tow different Bacillus thuringiensis 8-endotoxin receptors in the midgut brush border membrane of the European corn borer,Ostrinia nubilalis.Appl.Environ.Microbiol. 59:1828— 1837
E.Schnepe and J.Feitelson. 1998. Bacillus thuringiensis and its pesticidal crystal proteins.Microbiology and Molecular biology reviews.Rev 62:775—806
Edwards D L, Payne J, Soares G 1988. Novel isolates of Bacillus thuringiensis having Activity against Nematodes. European Patent Application, 303 —426
Faust G M, Abe K, Held G A, Lizuka T, Bulla L A, Meyers C L. 1983. Evidence for Plasmid-Associated Crystal Toxin Production in Bacillus thuringiensis subsp. Israelensis, Plasmid, (9):98—103
Fernandez,L.E. 2005. CryllAa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loopα8 of domain II. FEBS Lett. 579,3508—3514.
Gazit E, Shai Y. 1995. The Assembly and Organization of the α5 and α7 Helices from the Pore-Forming Domain of Bacillus thuringiensis 5-Endotoxin. J. Biol. Chem. 270:2571—2578
Gazit E. 1998. The structure and organization within the membrane of the helices composing the pore-forming domain of Bacillus thuringiensis 5-endotoxin are consistent with an "umbrella-like" structure of the pore. Proc. Natl Acad. Sci. USA, 95,12289—12294.
Gill S S, Cowles E A, Pietrantonio P V. 1992. The Mode of Action of Bacillus thuringiensis Endotoxins. Annu. Rev. Entomol. 37:615-636
Grochulski,p. 1995. Bacillus thuringiensis CryIAa insecticidal toxin crystal structure and channel formation. J.Mol.Biol. 254,447-464
Hodgman C. 1990. Models for the structure and function of the Bacillus thuringiensis δ-endotoxin determined by compilational analysis.J.DNA Seque. Map. 1,97-106
Hodgman T C, Ellar D J. 1990. Models for the structure and function of the Bacillus thuringiensis δ-Endotoxins Determined by Compicational Analysis. DNA Sequence. 1:97—106
Hofmann C, Luthy P, Hutter R, Pliska V. 1988. Binding of the Deltaendotoxin from Bacillus thuringiensis to Brushborder Membran-Evesicles of the Cabbage Butterfly(Pieris brassicae). Eur J Biochem. 173:85—91
Hofte,H.,H.R.Whiteley.l989.Insecticidal crystal proteins of Bacillus thutingiensis Microbiol. Rev. 53:242-255
Isabel Gomez. 2006. Specific epitopes of Domains II and III of Bacillus thuringiensis CrylAb toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta. JBC Papers in Press. Published on September 12, as Manuscript M604721200
Jade Li. 1991. Crystal structure of insecticidal 8-endotoxin from Bacillus thuringiensis at 2.5A resolution. Nature.Vol353.815-821
Jade Li. 1996. Structure of the Mosquitocidalδ-endotoxin CytB from Bacillus thuringiensis sp.kyushuensis and implications for membrane pore formationJ.Mol.Biol. 257:129-152
Juan L. 2004. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. Eur.J.Biochem. 271,3127—3135.
Judith L. 1988. Crystallization and preliminary X-ray diffraction studies of a toxic crystal protein from a subspecies of Bacillus thuringiensis.The Journal of Bioloyical Chemistry.Vol 263,No.24,pp. 11800—11801
Kanintronkul 1998. Specific mutations within thea4-α5 loop of the Bacillus thuringiensis Cry4B toxin reveal a crucial role for Asn-166 and Tyr-170. Mol.Biotechnol.24,11—20.
Knowles B H, 1984. Thomas W E, Ellar D J. Lectin-like Binding of Bacillus thuringiensis var. kurstaki lepidopteran-Specific Toxin is an Initial Step in Insecticidal Action. FEBS Lett. 168:197— 202
Knowles B H, Ellar D J. 1987. Colloid-Osmotic Lysis is a General Feature of the Mechanism of Action of Bacillus thiringiensis δ-Endotoxins with Different Insect Specificity. Biochim. Biophys. Acta. 924:509—518
Knowles B H. 1994. Mechanism of Action of Bacillus thuringiensis Insecticidal δ-Endotoxins. Adv. Insect Physiol. 24:275—307
Kusol Pootanakit. 2003. Identification of tow isoforms of aminopeptidase N in Aedes aegypti larval midgut. Journal of Biochemistry and Molecular Biology. Vol.36, No.5, pp. 508—51
Luisa E.FERNANDEZ. 2006. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry 11 Aa toxin inAedes aegypti larvae.Biochem.J. 394,77—84
Luthy P, Ebersold H R. 1981. Bacillus thuringiensis Delta-Endotoxins: Histopathology and Molecular Mode of Action. In:Davidson E W ed. Pathogenesis of Invertebrate Microbial Diseases. New Jersey: Allanheld, 235—267
M.-E. Nunez-Valdez. 2001. Structural and functional studies of α-helix 5 region from Bacillus thuringiensis Cry1Abδ-endotoxin.Biochimica et Biophysica Acta 1546,122-131
M.Schiffer,C.H.Chang. 1992. The functions of tryptophan residues in membrane proteins. Protein Eng. 5.213—214
Masashi Yamagiwa. 2004. Functional Analysis of two processed fragments of Bacillus thuringiensis Cry11A toxin.Biosci Biotechem.Biochem, 68(3),523—528
McNall,R.J. 2003. Dentication of novel Bacillus thuringiensis Cry1Ac toxin binding proteins in Manduca seta midgut through proteomic analysis.Insect Biochem.Mol.Biol. 33, 999—1010.
Mi Kyong Lee. 2000. Role of two arginine residues in domain II,loop 2 of Cry1Ab and Cry1Ac Bacillus thuringiensis δ-endotoxin in toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.Molecular Microbiology. 38(2), 289—298
Mi Kyong Lee, 2001. Mutations at the arginine residues ina8 loop of Bacillus thuringiensis δ-endotoxin CrylAc affect toxicity and binding to Manduca sexta and Lymantria dispar aminopeptidase N.FEBS Letters 497(2001 )108-112
Natalie J.Tigue. 2001. The a-helix 4 residue,Asnl35,is involved in the Oligomerization of Cry1Ac1 and Cry1Ab5 Bacillus thuringiensis toxins. Applied and environmental Microbiology,p. 5715-5720
Nicholls 1989. Evidence for two different types of insecticidal P2 toxins with dual specificity in bacillus thuringiensis subspecies. J.Bacteriol.171,5141—5147
Nikolai Galitsky. 2001. Structure of the insecticidal bacterial 5-endotoxin Cry3Bb1 of Bacillus thuringiensis. ISSN0909—4449
Novillo C, Castan-era P, Ortego F. 1997.Charaterization and Dis-Tribution of Chymotrypsin-Like and Other Digestive Proteases in Colorado Potato Beetle Larvae. Arch. Insect. Biochem. Physiol., 36:181-201. Serotype. Curr. Microbiol. 32:48—54
Ohba M et al. 1992. A unique isolate of Bacillus thuringiensis serovar japonensis with a high larvicidal activity specific for scarabaeid beetles, Letters in Applied Microbiology. 14: 54—57
Panadda Boonserm. 2004. Crystallization and preliminary crystallographic study of the functional form of the Bacillus thuringiensis mosquito-larvicidal Cry4Aa mutant toxin.ISSN 0907-4449
Panadda Boonserm. 2005. Crystal structure of the mosquito-larvicidal toxin cry4Ba and its biological implications. J. Mol. Biol. 348, 363—382
Panadda Boonserm. 2006. Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-Angstrom resolution.JB. 188.9.3391—3401
Parker 1993 Rendering a membrane protein soluble in water: a common packing motif in bacterial protein toxin. Trends Biochem.Sci. 18,391 —395.
Puntheeranurak, T., Stroh, C., Zhu, R., Angsuthanasombat, C., Hinterdorfer, P. 2005. Structure and domain lstribution of the Bacillus thuringiensis Cry4Ba toxin in lipid membranes. Ultramicroscopy, 105:115～124
R.J.Morse. 2001. Structure of Cry2Aa suggests an unexpec ted receptor binding epitope.Rev 9:409～417
R.Rajagopalt. 2002. Silencing of midgut aminopetidase N of Spodoptera litura by double-stranded RNA establishes its role as Bacillus thuringiemis toxin receptor. The journal of Biological Chemistry. Vol.277.No.49.pp. 46849～46851
Rausell, C., Pardo-Lopez, L., Sanchez, J., Munoz-Garay, C., Morera, C., Soberon, M., Bravo A. 2004. Unfol domain lng events in the water-soluble monomeric Cry1Ab toxin during transition to oligomeric pre-pore and membrane-inserted pore channel.J. Biol. Chem. 279(53):55168～55175
Ruiyu Xie. 2005. Single Amino Acid Mutations in the Cadherin Receptor from Heliothis virescens Affect Its Toxin Binding Ability to CrylA Toxins. The Journal of Biological Chemistry. Vol.280,No.9,pp.8416～8425
Salvador HERRERO. 2004. Mutations in the Bacillus thuringiemis Cry1Ca toxin demonstrate the role of domains Ⅱ and Ⅲ in specificity towards Spodoptera exigua larvae.Biochem.J. 384,507～5
Satia Tapaneeyakom. 2005. Structural requirements of the unique disulphide bond and the praline-rich motif within the α4-α5 loop for larvicidal activity of the Bacillus thuringiensis Cry4Aa δ-endotoxin. Biochemical and Biophysical Research Communications330(2005)519～525
Schnepf E, Crickmore N, Van Rie J, Lereclus D, Baum J, Feitelson J, Zeigler DR, Dean DH. 1998. Bacillus thuringiensis and Its Pesticidal Crystal Proteins, Microbiology and Molecular Biology Reviews. 62:775～806
Schwartz. J.L 2000. Membrane protein insertion and pore formation. In Entomopathogenic Bacteria: from Laboratory to Field Application pp.1～19.
Schwartz,J.L Restriction of intramolecutar movements within the Cry1Aa toxin molecule of Bacillus thudngiensis through disulfide bond engineering.FEBS Letters,410,397～402.
Schwartz,J-L Membrane permeabilization by Bacillus thutingiensis toxins:protein insertion and pore formation.In Entomopathogenic Bacteria: from Laboratory to Field Application Charles.A.pp. 1～19
Sheng-Jiun wu. 2000. Enhanced toxicity of Bacillus thuringiensis Cry3A δ-endoxin in coleopterans by mutagenesis in a receptor binding loop.FEBS Letters 473.227～232
Shin-ichiro Asano, Yamashita C, Iizuka T, Takeuchi K, Yamanaka S, Cerf D, Yamamoto T 2003. A strain of Bacillus thuringiensis subsp, galleriae containing a novel cry8 gene highly toxic to Anomala cuprea (Coleoptera: Scarabaeidae). Biol Control 28: 191～196
Shogo Atsumi. 2005. Location of the Bombyx mori aminopeptidase N type 1 binding site on Bacillus thuringiensis Cry1Aa toxin.Applied and Environmental.Microbiology, 3966～3977
Sick; August J., Schwab; George E., Payne; Jewel M., 1994. Genes encoding nematode-active toxins cloned from Bacillus thuringiensis isolate PS17, USP5281530
Somphob Leetachewa. 2006. Novel preparation and characterization of the α4-loop-α5 membrane-perturbing peptide from the Bacillus thuringiensis Cry4BaS-endotoxin.Journal of Biochemistry and Molecular Biology. Vol.39,No.3,pp. 270—277
Supaporn Likitvivatanavong. 2006. Asn183 in α5 is essential for oligomerisation and toxicity of the Bacillus thuringiensis Cry4Ba toxin.Archives of Biochemistry and Biophysics 445(2006)46-55
Svetlana Borisova. 1994. Crystalization and preliminary X-ray diffraction studies of the lepidopteran-specifif insecticidal crystal protein Cry2Aa.J.Mol.Biol. 243:530—532
Tojo A, Aizawa K. 1983. Dissolution and Degradation of Bacillus thuringiensis δ-Endotoxin by Gut Juice Protease of the Silkworm Bombyx mori. Appl. Environ. Microbiol. 45:576-580
Tracy E. Michaels, Kenneth E. Narva, Foncerrada, 1994. Bacillus thuringiensis toxins active against scarab pests. USP5554534.
Uawithya 1998 Effects on larvicidal activity of single praline substitutions inα.3 orα4 of the Bacillus thuringiensis Cry4Ba toxin.Biochem.Mol.Biol.Int.44,825—832.
Vachon,V. 2002. Role of helix 3 in pore formation by the Bacillus thuringiensis insecticidal toxin Cry1A.Biochemistry, 41,6178—6184
Vincent Vachon. 2004. Helix 4 mutants of the Bacillus thuringiensis insecticidal toxin Cry1Aa display altered pore-forming abilities.Applied and environmental Microbiology,p. 6123—6130
Walairat Pornwiroon. 2004. Aromaticity of Tyr-202 in the a4-a5 loop is essential for toxicity of the Bacillus thuringiensis Cry4A toxin.Journal of Biochemistry and Molecular Biology. Vol.37,No.3,pp.292—297
Wu S J, Dean D H. 1996. Functional Significance of Loops in the Receptor Binding Domain of Bacillus thuringiensis CryIIIA 8-Endotoxin. J. Mol. Biol. 255:628—640
XINTIAN Wei, XuDAN Xu, C.Jack Deloach 1995. Biological control of white grubs (Coleopera: Scarabaeidae) by larvae of Promachus yesonicus (Diptera:AsiIidae) in China Bio Control 5 :290—296
Yoko TSUDA. 2003. Cytotoxic activity of Bacillus thuringiensis Cry proteins on mammalian cells transfected with cadherin-like Cry receptor gene of Bombyx mori(silkworm).Biochem.J. 369,697-703