|
|
|
Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain |
ZHOU Zi-shan, YANG Su-juan, SHU Chang-long, SONG Fu-ping, ZHOU Xue-ping, ZHANG Jie |
1、State Key Laboratory of Rice Biology, Institute of Biotechnology, Zhejiang University, Hangzhou 310012, 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 |
|
|
摘要 Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene. Because Cry1Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops. The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments. The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin. In this paper, three media for crystal production and the method for Cry1Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1Ac protoxin. The results showed that the 1/2 LB (Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1Ac protoxin in 300 mL 1/2 LB medium was (112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method. It will be a reference for other Cry protoxin preparation, especially for larger number.
Abstract Bacillus thuringiensis is one of the most widely used bioinsecticides, and cry gene is the major insecticidal gene. Because Cry1Ac protein shows strong toxicity against many lepidopteran species, it has been applied widely in spraying products and transgenic Bt-crops. The preparation of Cry protoxin is the first step in the very important processes of understanding the insecticidal mechanism, resistance screening, and biosafety assessments. The media for crystal production and the method for Cry protoxin preparation were varied, however, it was not clear which was better for preparing a larger amount of Cry protoxin. In this paper, three media for crystal production and the method for Cry1Ac protoxin preparation from HD73 strain were compared to find an efficacious way to prepare a large number of Cry1Ac protoxin. The results showed that the 1/2 LB (Luria-Bertani) medium was the ideal medium for crystal production, because the total yield of Cry1Ac protoxin in 300 mL 1/2 LB medium was (112.38±5.64) mg, the highest one among three media; the repeated crystal solubilization method was better for the preparation of the Cry protoxin comparing with the continuous crystal solubilization method. It will be a reference for other Cry protoxin preparation, especially for larger number.
|
Received: 23 September 2014
Accepted:
|
Fund: This study was supported by the National Natural Science Foundation of China (31272115), the National High-Tech R&D Program (863 Program 2011AA10A203), and the China Postdoctoral Science Foundation (2014M560487). |
Corresponding Authors:
ZHANG Jie, Tel: +86-10-62815921,E-mail: jzhang@ippcaas.cn
E-mail: jzhang@ippcaas.cn
|
About author: ZHOU Zi-shan, Tel: +86-10-62815612, E-mail: zishanzhou@126.com; |
Cite this article:
ZHOU Zi-shan, YANG Su-juan, SHU Chang-long, SONG Fu-ping, ZHOU Xue-ping, ZHANG Jie.
2015.
Comparison and optimization of the method for Cry1Ac protoxin preparation in HD73 strain. Journal of Integrative Agriculture, 14(8): 1598-1603.
|
Anilkumar K J, Rodrigo-Simón A , Ferré J, Pusztai-Carey M,Sivasupramaniam S, Moar W J. 2008. Production andcharacterization of Bacillus thuringiensis Cry1Ac-resistantcotton bollworm Helicoverpa zea (Boddie). Applied andEnvironmental Microbiology, 74, 462-469Chen M, Zhao J Z, Collins H L, Earle E D, Cao J, Shelton A M2008. A critical assessment of the effects of Bt transgenicplants on parasitoids. PLoS One, 3, e2284.Dai P L, Zhou W, Zhang J, Jiang W Y, Wang Q, Cui H J, Sun JH, Wu Y Y, Zhou T. 2012. The effects of Bt Cry1Ah toxin onworker honeybees (Apis mellifera ligustica and Apis ceranacerana). Apidologie, 43, 384-391Estela A, Escriche B, Ferré J. 2004. Interaction of Bacillusthuringiensis toxins with larval midgut binding sites ofHelicoverpa armigera (Lepidoptera: Noctuidae). Appliedand Environmental Microbiology, 70, 1378-1384Guo S Y, Ye S, Liu Y F, Wei L, Xue J, Wu H F, Song F P, ZhangJ, Wu X A, Huang D F, Rao Z H. 2009. Crystal structure ofBacillus thuringiensis Cry8Ea1: An insecticidal toxin toxicto underground pests, the larvae of Holotrichia parallela.Journal of Structural Biology, 168, 259-266Gahan L J, Pauchet Y, Vogel H, Heckel D G. 2010. An ABCtransporter mutation is correlated with insect resistanceto Bacillus thuringiensis Cry1Ac toxin. PLoS Genetics, 6,e1001248.Gilliland A, Chambers C E, Bone E J, Ellar D J. 2002. Roleof Bacillus thuringiensis Cry1 δ endotoxin binding indetermining potency during lepidopteran larval development.Applied and Environmental Microbiology, 68, 1509-1515Ibrahim M A, Griko N, Junker M, Bulla L A. 2010. Bacillusthuringiensis: A genomics and proteomics perspective.Bioengineered Bugs, 1, 31-50Iracheta M M, Pereyra-Alférez B, Galán-Wong L, Ferré J. 2000.Screening for Bacillus thuringiensis crystal proteins activeagainst the cabbage looper, Trichoplusia ni. Journal ofInvertebrate Pathology, 76, 70-75Kain W C, Zhao J Z, Janmaat A F, Myers J, Shelton A M, WangP. 2004. Inheritance of resistance to Bacillus thuringiensisCry1Ac toxin in a greenhouse-derived strain of cabbagelooper (Lepidoptera: Noctuidae). Journal EconomicEntomology, 97, 2073-2078Lemeshko V V, Orduz S. 2013. Electrical hypothesis of toxicityof the Cry toxins for mosquito larvae. Bioscience Report,33, 125-136Liao C Y, Heckel D G, Akhurst R. 2002. Toxicity of Bacillusthuringiensis insecticidal proteins for Helicoverpa armigeraand Helicoverpa punctigera (Lepidoptera: Noctuidae),major pests of cotton. Journal of Invertebrate Pathology,80, 55-63Luo K, Banks D, Adang M J. 1999. Toxicity, binding, andpermeability analyses of four Bacillus thuringiensis Cry1δ-endotoxins using brush border membrane vesicles ofSpodoptera exigua and Spodoptera frugiperda. Appliedand Environmental Microbiology, 65, 457-464Luttrell R G, Wan L, Knighten K. 1999. Variation in susceptibilityof noctuid (Lepidoptera) larvae attacking cotton andsoybean to purified endotoxin proteins and commercialformulations of Bacillus thuringiensis. Journal of EconomicEntomology, 92, 21-32Ohsawa M, Tanaka M, Moriyama K, Shimazu M, Asano S I,Miyamoto K, Haginoya K, Mitsui T, Kouya T, TaniguchiM, Hori H. 2012. A 50-kilodalton Cry2A peptide is lethalto Bombyx mori and Lymantria dispar. Applied and Environmental Microbiology, 78, 4755-4757Pardo-López L, Soberón M, Bravo A. 2013. Bacillusthuringiensis insecticidal three-domain Cry toxins: Modeof action, insect resistance and consequences for cropprotection. FEMS Microbiology Reviews, 37, 3-22Rodrigo-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. Applied andEnvironmental Microbiology, 74, 1710-1716Schaeffer P, Millet J, Aubert J P. 1956. Catabolic repression ofbacterial sporulation. Proceedings of the National Academyof Sciences of the United States of America, 54, 704-711Sharif F A, Alaeddino?lu N G. 1988. A rapid and simple methodfor staining of the crystal protein of Bacillus thuringiensis.Journal of Industrial Microbiology, 3, 227-229Shu C L, Liu D M, Zhou Z S, Cai J L, Peng Q, Gao J G, SongF P, Zhang J. 2013. An improved PCR-restriction fragmentlength polymorphism (RFLP) method for the identification ofcry1-type genes. Applied and Environmental Microbiology,79, 6706-6711Smirnoff W A. 1962. A staining method for differentiating spores,crystals, and cells of Bacillus thuringiensis (Berliner).Journal of Insect Pathology, 4, 384-386Tabashnik B E, Unnithan G C, Masson L, Crowder D W, Li X,Carrière Y. 2009. Asymmetrical cross-resistance betweenBacillus thuringiensis toxins Cry1Ac and Cry2Ab in pinkbollworm. Proceedings of the National Academy of Sciencesof the United States of America, 106, 11889-11894Tiewsiri K, Wang P. 2011. Differential alteration of twoaminopeptidases N associated with resistance to Bacillusthuringiensis toxin Cry1Ac in cabbage looper. Proceedingsof the National Academy of Sciences of the United Statesof America, 108, 14037-14042Upadhyay S K, Singh P K. 2011. Role of alkaline phosphatase ininsecticidal action of Cry1Ac against Helicoverpa armigeralarvae. Biotechnology Letters, 33, 2027-2036Xue J, Liang G M, Crickmore N, Li H T, He K L, Song F P, HuangD F, Zhang J. 2008. Cloning and characterization of a novelCry1A toxin from Bacillus thuringiensis with high toxicity tothe Asian corn borer and other lepidopteran insects. FEMSMicrobiology Letters, 280, 95-101 |
No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
|
Shared |
|
|
|
|
|
Discussed |
|
|
|
|