Creation of Cry1Ab/Cry1Ah Hybrid Proteins and Its Functional Analysis

doi:10.13560/j.cnki.biotech.bull.1985.2015.09.012

Abstract

Abstract: Cry1A is the most widely used insecticidal protein, and the domain swapping in the reported Cry1A family frequently occurs. In this paper, we investigated the Lepidoptera pests’ highly active protein Cry1Ab and Cry1Ah, and constructed the hybrid protein AhAhAb from Cry1Ab/Cry1Ah and bioassayed the activities. The results showed that the domain swapping of two proteins Cry1Ab and Cry1Ah resulted in the insecticidal activities significantly changed. Compared with the origin proteins, hybrid protein AhAhAb lost the insecticidal activity to Heliothis armigera, caused the insecticidal activity to Ostrinia nubilalis, Plutella xylostella reduced. We then modeled Cry1Ah domain I, and compared the structure and the surface properties with other Cry1A proteins by bioinformatics methods. The results indicated that domain I of Cry1Ah and Cry1Ab had identical carbon skeleton and secondary structure but varied surface and potential distribution. The further studies on the reasons of the activity’s difference between hybrid protein AhAhAb and Cry1Ab or Cry1Ah will give enlightenment for unclosing the evolution pattern of insecticidal specificity of Cry1A family proteins.

Key words: Cry1Ah, domain I, insecticidal activity

Xu Man, Jiang Jian, Shu Changlong, Zhang Jie, Song Fuping. Creation of Cry1Ab/Cry1Ah Hybrid Proteins and Its Functional Analysis[J]. Biotechnology Bulletin, 2015, 31(9): 91-96.

References

[1]de Maagd RA, Bravo A, Crickmore N. How Bacillus thuringiensis has evolved specific toxins to colonize the insect world[J]. Trends Genet, 2001, 17(4)：193-199.
[2]Bravo A, Gomez I, Porta H, et al. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity[J]. Microb Biotechnol, 2013, 6(1)：17-26.
[3]Xue J, Zhou Z, Song F, et al. Identifica tion of the minimal active fragment of the Cry1Ah toxin[J]. Biotechnol Lett, 2011, 33(3)：531-537.
[4]Xue J, Liang G, Crickmore N, et al. Cloning and characterization of a novel Cry1A toxin from Bacillus thuringiensis with high toxicity to the Asian corn borer and other lepidopteran insects[J]. FEMS Microbiol Lett, 2008, 280(1)：95-101.
[5]Song Y, Liang C, Wang W, et al. Immuno toxicological evaluation of corn genetically modified with Bacillus thuringiensis Cry1Ah gene by a 30-day feeding study in BALB/c mice[J]. PLoS One, 2014, 9(2)：78566-78674.
[6]Sun H, Lang Z, Zhu L, et al. Acquiring transgenic tobacco plants with insect resistance and glyphosate tolerance by fusion gene transformation[J]. Plant Cell Rep, 2012, 31(10)：1877-1887.
[7]Dai PL, Zhou W, Zhang J, et al. The effects of Bt Cry1Ah toxin on worker honeybees(Apis mellifera ligustica and Apis cerana cerana)[J]. Apidologie, 2012, 43(4)：384-391.
[8]Shu C, Liu D, Zhou Z, et al. An improved PCR-restriction fragment length polymorphism(RFLP)method for the identification of cry1-type genes[J]. Appl Environ Microbiol, 2013, 79(21)：6706-6711.
[9]Kotik M, Ko?anová M, Mare?ová H, et al. High-level expression of a fungal pyranose oxidase in high cell-density fed-batch cultivations of Escherichia coli using lactose as inducer[J]. Protein Expression and Purification, 2004, 36(1)：61-69.
[10]Wu K, Guo Y, Lv N. Geographic variation in susceptibility of Helicoverpa armigera(Lepidoptera：Noctuidae)to Bacillus thuringiensis insecticidal protein in China[J]. Journal of Economic Entomology, 1999, 92(2)：273-278.
[11]Tabashnik BE, Finson N, Chilcutt CF, et al. Increasing efficiency of bioassays：evaluating resistance to Bacillus thuringiensis in diamondback moth(Lepidoptera：Plutellidae)[J]. Journal of economic entomology, 1993, 86(3)：635-644.
[12]Shu C, Su H, Zhang J, et al. Characterization of cry9Da4, cry9Eb2, and cry9Ee1 genes from Bacillus thuringiensis strain T03B001[J]. Appl Microbiol Biotechnol, 2013, 97(22)：9705-9713.
[13]Grochulski P, Masson L, Borisova S, et al. Bacillus thuringiensis CryIA(a)insecticidal toxin：crystal structure and channel formation[J]. J Mol Biol, 1995, 254(3)：447-464.
[14]Morse RJ, Yamamoto T, Stroud RM. Structure of Cry2Aa suggests an unexpected receptor binding epitope[J]. Structure, 2001, 9(5)：409-417.
[15]Galitsky N, Cody V, Wojtczak A, et al. Structure of the insecticidal bacterial delta-endotoxin Cry3Bb1 of Bacillus thuringiensis[J]. Acta Crystallogr D Biol Crystallogr, 2001, 57(Pt8)：1101-1109.
[16]Boonserm P, Davis P, Ellar DJ, et al. Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications[J]. J Mol Biol, 2005, 348(2)：363-382.
[17]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]. J Struct Biol, 2009, 168(2)：259-266.
[18]de Maagd RA, Weemen-Hendriks M, Stiekema W, et al. Bacillus thuringiensis delta-endotoxin Cry1C domain III can function as a specificity determinant for Spodoptera exigua in different, but not all, Cry1-Cry1C hybrids[J]. Appl Environ Microbiol, 2000, 66(4)：1559-1563.
[19]Rang C, Vachon V, de Maagd RA, et al. Interaction between functional domains of Bacillus thuringiensis insecticidal crystal proteins[J]. Appl Environ Microbiol, 1999, 65(7)：2918-2925.
[20]Gouffon C, Van Vliet A, Van Rie J, et al. Binding sites for Bacillus thuringiensis Cry2Ae toxin on heliothine brush border membrane vesicles are not shared with Cry1A, Cry1F, or Vip3A toxin[J]. Appl Environ Microbiol, 2011, 77(10)：3182-3188.
[21]Hibi M, Kawashima T, Kodera T, et al. Characterization of Bacillus thuringiensis L-isoleucine dioxygenase for production of useful amino acids[J]. Appl Environ Microbiol, 2011, 77(19)：6926-6930.