苏云金芽胞杆菌杀虫晶体蛋白研究进展

doi:10.16409/j.cnki.2095-039x.2015.05.011

摘要/Abstract

摘要： 苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt),属于蜡样芽胞杆菌族,芽胞杆菌属的革兰氏阳性细菌,与其它芽胞杆菌的区别是在形成芽胞的同时,伴随有杀虫晶体蛋白(Cry蛋白)的产生。Cry蛋白对鳞翅目、鞘翅目、膜翅目和双翅目等多种害虫具有特异的杀虫作用,因此,Cry蛋白以微生物杀虫剂或转Bt基因植物的形式而被广泛应用于害虫生物防治中。本文从Bt菌株的发掘、cry基因的转录调控机制、Cry蛋白的结构功能及作用机制等方面进行综述,为Bt杀虫蛋白的推广应用、Bt蛋白的定向改造和构建高效广谱的工程菌提供参考。

关键词: 苏云金芽胞杆菌, 杀虫晶体蛋白, 转录调控, 作用机制

Abstract: Bacillus thuringiensis is Gram-positive, spore-forming bacterium belonging to the Bacillus cereus group, and differs from other Bacillus species by its ability to produce crystalline inclusions. These parasporal crystal proteins possess highly specialized insecticidal activity against a large number of insect species. Because of the insecticidal properties of the crystal protein, B. thuringiensis has been used for many years as pesticide in biocontrol applications or transgenic plant. Isolation of Bt strains, transcriptional regulation mechanism of cry gene, and structure, function and mechanism of Cry protein, were summarized in this paper. All the summarization will provide reference on popularization, application, and directional modification of Cry protein, and construction of Bt engineered strains.

Key words: Bacillus thuringiensis, insecticidal crystal proteins, transcriptional regulation, action mechanism

中图分类号:

S476.11

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.

PENG Qi, ZHOU Zishan, ZHANG Jie. Research Prospects in Insecticidal Crystal Proteins of Bacillus thuringiensis[J]. journal1, 2015, 31(5): 712-722.

参考文献

[1] Estruch J J, Warren G W, Mullins M A, et al. 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 United States of America, 1996, 93(11): 5389-5394.
[2] Donovan W P, Engleman J T, Donovan J C, et al. Discovery and characterization of Sip1A: A novel secreted protein from Bacillus thuringiensis with activity against coleopteran larvae[J]. Applied Microbiology and Biotechnology, 2006, 72(4): 713-719.
[3] Aronson A I, Beckman W, Dunn P. Bacillus thuringiensis and related insect pathogens[J]. Microbiological Reviews, 1986, 50(1): 1-24.
[4] Schnepf H E, Whiteley H R. Cloning and expression of the Bacillus thuringiensis crystal protein gene in Escherichia coli[J]. Proceedings of the National Academy of Sciences of the United States of America, 1981, 78(5): 2893-2897.
[5] Ben-Dov E, Zaritsky A, Dahan E, et al. Extended screening by PCR for seven cry-group genes from field-collected strains of Bacillus thuringiensis[J]. Applied and Environmental Microbiology, 1997, 63(12): 4883-4890.
[6] Ben-Dov E, Wang Q, Zaritsky A, et al. Multiplex PCR screening to detect cry9 genes in Bacillus thuringiensis strains[J]. Applied and Environmental Microbiology, 1999, 65(8): 3714-3716.
[7] Noguera P A, Ibarra J E. Detection of new cry genes of Bacillus thuringiensis by use of a novel PCR primer system[J]. Applied and Environmental Microbiology, 2010, 76(18): 6150-6155.
[8] Fang J, Xu X, Wang P, et al. Characterization of chimeric Bacillus thuringiensis Vip3 toxins[J]. Applied and Environmental Microbiology, 2007, 73(3): 956-961.
[9] Shu C, Zhang J, Chen G, et al. Use of a pooled clone method to isolate a novel Bacillus thuringiensis Cry2A toxin with activity against Ostrinia furnacalis[J]. Journal of Invertebrate Pathology, 2013, 114(1): 31-33.
[10] Agaisse H, Lereclus D. How does Bacillus thuringiensis produce so much insecticidal crystal protein?[J]. Journal of Bacteriology, 1995, 177(21): 6027-6032.
[11] Zhou Z, Yang S, Shu C, et al. Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 stain[J]. Journal of Integrative Agriculture, 2015, 14(8): 1598-1603.
[12] Piggot P J, Hilbert D W. Sporulation of Bacillus subtilis[J]. Current Opinion in Microbiology, 2004, 7(6): 579-586.
[13] Aronson A. Sporulation and delta-endotoxin synthesis by Bacillus thuringiensis[J]. Cellular and Molecular Life Sciences: CMLS, 2002, 59(3): 417-425.
[14] Bravo A, Agaisse H, Salamitou S, et al. Analysis of cryIAa expression in sigE and sigK mutants of Bacillus thuringiensis[J]. Molecular and General Genetics, 1996, 250(6): 734-741.
[15] Yang H, Wang P, Peng Q, et al. Weak transcription of the cry1Ac gene in nonsporulating Bacillus thuringiensis cells[J]. Applied and Environmental Microbiology, 2012, 78(18): 6466-6474.
[16] Poncet S, Dervyn E, Klier A, et al. Spo0A represses transcription of the cry toxin genes in Bacillus thuringiensis[J]. Microbiology, 1997, 143 (8): 2743-2751.
[17] Du L, Qiu L, Peng Q, et al. Identification of the promoter in the intergenic region between orf1 and cry8Ea1 controlled by sigma H factor[J]. Applied and Environmental Microbiology, 2012, 78(12): 4164-4168.
[18] Zhang J, Schairer H U, Schnetter W, et al. Bacillus popilliae cry18Aa operon is transcribed by sigmaE and sigmaK forms of RNA polymerase from a single initiation site[J]. Nucleic Acids Research, 1998, 26(5): 1288-1293.
[19] Perez-Garcia G, Basurto-Rios R, Ibarra J E. Potential effect of a putative sigma (H)-driven promoter on the over expression of the Cry1Ac toxin of Bacillus thuringiensis[J]. Journal of Invertebrate Pathology, 2010, 104(2): 140-146.
[20] Brown K L. Transcriptional regulation of the Bacillus thuringiensis subsp. thompsoni crystal protein gene operon[J]. Journal of Bacteriology, 1993, 175(24): 7951-7957.
[21] Widner W R, Whiteley H R. Two highly related insecticidal crystal proteins of Bacillus thuringiensis subsp. kurstaki possess different host range specificities[J]. Journal of Bacteriology, 1989, 171(2): 965-974.
[22] Baum J A, Malvar T. Regulation of insecticidal crystal protein production in Bacillus thuringiensis[J]. Molecular Microbiology, 1995, 18(1): 1-12.
[23] Agaisse H, Lereclus D. Expression in Bacillus subtilis of the Bacillus thuringiensis cryIIIA toxin gene is not dependent on a sporulation-specific sigma factor and is increased in a spo0A mutant[J]. Journal of Bacteriology, 1994, 176(15): 4734-4741.
[24] Salamitou S, Agaisse H, Bravo A, et al. Genetic analysis of cryIIIA gene expression in Bacillus thuringiensis[J]. Microbiology, 1996, 142(8): 2049-2055.
[25] Walter T, Aronson A. Specific binding of the E2 subunit of pyruvate dehydrogenase to the upstream region of Bacillus thuringiensis protoxin genes[J]. The Journal of Biological Chemistry, 1999, 274(12): 7901-7906.
[26] Doruk T, Avican U, Camci I Y, et al. Overexpression of polyphosphate kinase gene (ppk) increases bioinsecticide production by Bacillus thuringiensis[J]. Microbiology Research, 2013, 168(4): 199-203.
[27] Banerjee-Bhatnagar N. Modulation of Cry IV A toxin protein expression by glucose in Bacillus thuringiensis israelensis[J]. Biochemical and Biophysical Research Communications, 1998, 252(2): 402-406.
[28] Kant S, Kapoor R, Banerjee N. Identification of a catabolite-responsive element necessary for regulation of the cry4A gene of Bacillus thuringiensis subsp. israelensis[J]. Journal of Bacteriology, 2009, 191(14): 4687-4692.
[29] Khan S R, Banerjee-Bhatnagar N. Loss of catabolite repression function of HPr, the phosphocarrier protein of the bacterial phosphotransferase system, affects expression of the cry4A toxin gene in Bacillus thuringiensis subsp. israelensis[J]. Journal of Bacteriology, 2002, 184(19): 5410-5417.
[30] Glatron M F, Rapoport G. Biosynthesis of the parasporal inclusion of Bacillus thuringiensis: half-life of its corresponding messenger RNA[J]. Biochimie, 1972, 54(10): 1291-1301.
[31] Wong H C, Chang S. Identification of a positive retroregulator that stabilizes mRNAs in bacteria[J]. Proceedings of the National Academy of Sciences of the United States of America, 1986, 83: 3233-3237.
[32] Yu Z, Bai P, Ye W, et al. A novel negative regulatory factor for nematicidal Cry protein gene expression in Bacillus thuringiensis[J]. Journal of Microbiology and Biotechnology, 2008, 18(6): 1033-1039.
[33] Agaisse H, Lereclus D. Structural and functional analysis of the promoter region involved in full expression of the cryIIIA toxin gene of Bacillus thuringiensis[J]. Molecular Microbiology, 1994, 13(1): 97-107.
[34] Agaisse H, Lereclus D. STAB-SD: a Shine-Dalgarno sequence in the 5' untranslated region is a determinant of mRNA stability[J]. Molecular Microbiology, 1996, 20(3): 633-643.
[35] Mathy N, Benard L, Pellegrini O, et al. 5'-to-3' exoribonuclease activity in bacteria: role of RNase J1 in rRNA maturation and 5' stability of mRNA[J]. Cell, 2007, 129(4): 681-692.
[36] Hambraeus G, Karhumaa K, Rutberg B. A 5' stem-loop and ribosome binding but not translation are important for the stability of Bacillus subtilisapre leader mRNA[J]. Microbiology, 2002, 148(6): 1795-1803.
[37] Aronson J N, Borris D P, Doerner J F, et al. Gamma-aminobutyric acid pathway and modified tricarboxylic acid cycle activity during growth and sporulation of Bacillus thuringiensis[J]. Applied Microbiology, 1975, 30(3): 489-492.
[38] Nickerson K W, de Pinto J, Bulla L A, Jr. Sporulation of Bacillus thuringiensis without concurrent derepression of the tricarboxylic acid cycle[J]. Journal of Bacteriology, 1974, 117(1): 321-323.
[39] Zhu L, Peng Q, Song F, et al. Structure and regulation of the gab gene cluster, involved in the gamma-aminobutyric acid shunt, are controlled by a sigma54 factor in Bacillus thuringiensis[J]. Journal of Bacteriology, 2010, 192(1): 346-355.
[40] Peng Q, Yang M, Wang W, et al. Activation of gab cluster transcription in Bacillus thuringiensis by gamma-aminobutyric acid or succinic semialdehyde is mediated by the Sigma 54-dependent transcriptional activator GabR[J]. BioMed Central Microbiology, 2014, 14: 306.
[41] Zhang Z, Yang M, Peng Q, et al. Transcription of the Lysine-2,3-Aminomutase gene in the kam locus of Bacillus thuringiensis subsp. kurstaki HD73 is controlled by both sigma54 and sigmaK factors[J]. Journal of Bacteriology, 2014, 196: 2934-2943.
[42] Peng Q, Wang G, Liu G, et al. Identification of metabolism pathways directly regulated by sigma (54) factor in Bacillus thuringiensis[J]. Frontiers in Microbiology, 2015, 6: 407.
[43] Gong Y, Li M, Xu D, et al. Comparative proteomic analysis revealed metabolic changes and the translational regulation of Cry protein synthesis in Bacillus thuringiensis[J]. Journal of Proteomics, 2012, 75(4): 1235-1246.
[44] Wu D, He J, Gong Y, et al. Proteomic analysis reveals the strategies of Bacillus thuringiensis YBT-1520 for survival under long-term heat stress[J]. Proteomics, 2011, 11(13): 2580-2591.
[45] Ruan L, Yu Z, Fang B, et al. Melanin pigment formation and increased UV resistance in Bacillus thuringiensis following high temperature induction[J]. Systematic and Applied Microbiology, 2004, 27(3): 286-289.
[46] Zhang J T, Yan J P, Zheng D S, et al. Expression of mel gene improves the UV resistance of Bacillus thuringiensis[J]. Journal of Applied Microbiology, 2008, 105(1): 151-157.
[47] Sanchis V, Gohar M, Chaufaux J, et al. Development and field performance of a broad-spectrum nonviable asporogenic recombinant strain of Bacillus thuringiensis with greater potency and UV resistance[J]. Applied and Enviromental Microbology, 1999, 65(9): 4032-4039.
[48] Zhou C, Zheng Q, Peng Q, et al. Screening of cry-type promoters with strong activity and application in Cry protein encapsulation in a sigK mutant[J]. Applied Microbiology and Biotechnology, 2014, 98(18): 7901-7909.
[49] Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a) insecticidal toxin: crystal structure and channel formation[J]. Journal of Molecular Biology, 1995, 254(3): 447-464.
[50] Derbyshire D J, Ellar D J, Li J. Crystallization of the Bacillus thuringiensis toxin Cry1Ac and its complex with the receptor ligand N-acetyl-D-galactosamine[J]. Acta Crystallographica. Section D, Biological Crystallography, 2001, 57: 1938-1944.
[51] Li J D, Carroll JEllar D J. Crystal structure of insecticidal delta-endotoxin from Bacillus thuringiensis at 2.5 A resolution[J]. Nature, 1991, 353(6347): 815-821.
[52] Galitsky N, Cody V, Wojtczak A, et al. Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis[J]. Acta Crystallographica. Section D, Biological Crystallography, 2001, 57(8): 1101-1109.
[53] Guo S, Ye S, Liu Y, et al. Crystal structure of Bacillus thuringiensis Cry8Ea1: An insecticidal toxin toxic to underground pests, the larvae of Holotrichia parallela[J]. Journal of Structural Biology, 2009, 168(2): 259-266.
[54] Boonserm P, Mo M, Angsuthanasombat C, et al. Structure of the functional form of the mosquito larvicidal Cry4Aa toxin from Bacillus thuringiensis at a 2.8-angstrom resolution[J]. Journal of Bacteriology, 2006, 188(9): 3391-3401.
[55] Boonserm P, Davis P, Ellar D J, et al. Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications[J]. Journal of Molecular Biology, 2005, 348(2): 363-382.
[56] Morse R J, Yamamoto T, Stroud R M. Structure of Cry2Aa suggests an unexpected receptor binding epitope[J]. Structure, 2001, 9(5): 409-417.
[57] Han S, Craig J A, Putnam C D, et al. Evolution and mechanism from structures of an ADP-ribosylating toxin and NAD complex[J]. Nature Structural Biology, 1999, 6: 932-936.
[58] Pardo-Lopez L, Soberon M, Bravo A. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection[J]. FEMS Microbiology Reviews, 2013, 37(1): 3-22.
[59] Bravo A, Gomez I, Porta H, et al. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity[J]. Microbial Biotechnology, 2013, 6(1): 17-26.
[60] Lin X, Parthasarathy K, Surya W, et al. A conserved tetrameric interaction of Cry toxin helix α3 suggests a functional role for toxin oligomerization[J]. Biochimica et Biophysica Acta, 2014, 1838(7): 1777-1784.
[61] Bravo A, Gomez I, Conde J, et al. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains[J]. Biochimica et Biophysica Acta, 2004, 1667(1): 38-46.
[62] Zhang X, Candas M, Griko N B, et al. A mechanism of cell death involving an adenylyl cyclase/PKA signaling pathway is induced by the Cry1Ab toxin of Bacillus thuringiensis[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(26): 9897-9902.
[63] Gomez I, Arenas I, Benitez I, et al. Specific epitopes of domains II and III of Bacillus thuringiensis Cry1Ab toxin involved in the sequential interaction with cadherin and aminopeptidase-N receptors in Manduca sexta[J]. The Journal of Biological Chemistry, 2006, 281(45): 34032-34039.
[64] Pacheco S, Gomez I, Arenas I, et al. Domain II loop 3 of Bacillus thuringiensis Cry1Ab toxin is involved in a ""ping pong"" binding mechanism with Manduca sexta aminopeptidase-N and cadherin receptors[J]. The Journal of Biological Chemistry, 2009, 284(47): 32750-32757.
[65] Arenas I, Bravo A, Soberon M, et al. Role of alkaline phosphatase from Manduca sexta in the mechanism of action of Bacillus thuringiensis Cry1Ab toxin[J]. Journal of Biological Chemistry, 2010, 285(17): 12497-12503.
[66] Vadlamudi R K, Ji T H, Bulla L A Jr. A specific binding protein from Manduca sexta for the insecticidal toxin of Bacillus thuringiensis subsp. berliner[J]. The Journal of Biological Chemistry, 1993, 268(17): 12334-12340.
[67] Endo H, Kobayashi Y, Hoshino Y, et al. Affinity maturation of Cry1Aa toxin to the Bombyx mori cadherin-like receptor by directed evolution based on phage display and biopanning selections of domain II loop 2 mutant toxins[J]. Microbiology Open, 2014, 54(3): 568-577.
[68] Hua G, Park Y, Adang M J. Cadherin AdCad1 in Alphitobius diaperinus larvae is a receptor of Cry3Bb toxin from Bacillus thuringiensis[J]. Insect Biochemistry and Molecular Biology, 2014, 45(1): 11-17.
[69] Tanaka S, Miyamoto K, Noda H, et al. The ATP-binding cassette transporter subfamily C member 2 in Bombyx mori larvae is a functional receptor for Cry toxins from Bacillus thuringiensis[J]. The FEBS Journal, 2013, 280(8): 1782-1794.
[70] Griffitts J S, Haslam S M, Yang T, et al. Glycolipids as receptors for Bacillus thuringiensis crystal toxin[J]. Science, 2005, 307(5711): 922-925.
[71] Valaitis A P, Jenkins J L, Lee M K, et al. Isolation and partial characterization of gypsy moth BTR-270, an anionic brush border membrane glycoconjugate that binds Bacillus thuringiensis Cry1A toxins with high affinity[J]. Archives of Insect Biochemistry and Physiology, 2001, 46(4): 186-200.
[72] Hossain D M, Shitomi Y, Moriyama K, et al. Characterization of a novel plasma membrane protein, expressed in the midgut epithelia of Bombyx mori, that binds to Cry1A toxins[J]. Applied and Environmental Microbiology, 2004, 70(8): 4604-4612.
[73] Khajuria C, Buschman L L, Chen M S, et al. Identification of a novel aminopeptidase P-like gene (OnAPP) possibly involved in Bt toxicity and resistance in a major corn pest (Ostrinia nubilalis)[J]. PLoS ONE, 2011, 6: e23983.
[74] Zhang H, Du B, Yang Y, et al. Cadherin mutation linked to resistance to Cry1Ac affects male paternity and sperm competition in Helicoverpa armigera[J]. Journal of Insect Physiology, 2014, 70: 67-72.
[75] Lee Su-Bum, Aimanova K G. Gill S S. Alkaline phosphatases and aminopeptidases are altered in a Cry11Aa resistant strain of Aedes aegypti[J]. Insect Biochemistry and Molecular Biology, 2014, 54: 112-121.
[76] Bosch D, Schipper B, van der Kleij H, et al. Recombinant Bacillus thuringiensis crystal proteins with new properties: possibilities for resistance management[J]. Biotechnology (N Y), 1994, 12(9): 915-918.
[77] Karlova R, Weemen-Hendriks M, Naimov S, et al. Bacillus thuringiensis delta-endotoxin Cry1Ac domain III enhances activity against Heliothis virescens in some, but not all Cry1-Cry1Ac hybrids[J]. Journal of Invertebrate Pathology, 2005, 88(2): 169-172.
[78] Soberon M, Pardo-Lopez L, Lopez I, et al. Engineering modified Bt toxins to counter insect resistance[J]. Science, 2007, 318(5856): 1640-1642.
[79] Mandal C C, Gayen S, Basu A, et al. Prediction-based protein engineering of domain I of Cry2A entomocidal toxin of Bacillus thuringiensis for the enhancement of toxicity against lepidopteran insects[J]. Protein Engineering, Design &Selection : PEDS, 2007, 20: 599-606.
[80] Leeuwen E, O’Neill S, Matthews A, et al. Making pathogens sociable: The emergence of high relatedness through limited host invisibility[J]. The ISME Journal, 2015, 9(1): 1-9.
[81] Lereclus D, Agaisse H, Grandvalet C, et al. Regulation of toxin and virulence gene transcription in Bacillus thuringiensis[J]. International Journal of Medical Microbiology, 2000, 290(4-5): 295-299.
[82] Lee S B, Chen J, Aimanova K G, et al. Aedes cadherin mediates the in vivo toxicity of the Cry11Aa toxin to Aedes aegypti[J]. Peptides, 2015, 68: 140-147.
[83] Aroonkesorn A, Pootanakit K, Katzenmeier G, et al. Two specific membrane-bound aminopeptidase N isoforms from Aedes aegypti larvae serve as functional receptors for the Bacillus thuringiensis Cry4Ba toxin implicating counterpart specificity[J]. Biochemical and Biophysical Research Communications, 2015, 461: 300-306.
[84] Han G, Li X, Zhang T, et al. Cloning and tissue-specific expression of a chitin deacetylase gene from Helicoverpa armigera (Lepidotera: Noctuidae) and its response to Bacillus thuringensis[J]. Journal of Insect Science, 2015, 15(1): 95-101.
[85] Feng D, Chen Z, Wang Z, et al. Domain III of Bacillus thuringiensis Cry1Ie toxin plays an important role in binding to peritrophic membrane of asian corn borer[J]. PLoS ONE, 2015, 10: e0136430.
[86] Vellichirammal1 N N, Wang H, Eyun S, et al. Transcriptional analysis of susceptible and resistant European corn borer strains and their response to Cry1F protoxin[J]. BioMed Central Genomics, 2015, 16: 558-568.
[87] Song X, Kain W, Cassidy D, et al. Resistance to Bacillus thuringiensis toxin Cry2Ab in Trichoplusia ni is conferred by a novel genetic mechanism[J]. Applied and Environmental Microbiology, 2015, 81(15): 5184-5195.
[88] Coates B S, Siegfried B D. Linkage of an ABCC transporter to a single QTL that controls Ostrinia nubilalis larval resistance to the Bacillus thuringiensis Cry1Fa toxin[J]. Insect Biochemistry and Molecular Biology, 2015, 63: 86-96.