The Construction, Expression and Insecticidal Activity Determination of Cry1Ac Toxin Mutant

doi:10.11924/j.issn.1000-6850.casb16110021

Abstract

Abstract: The aim is to construct the toxin with high insecticidal activity for resistant insects with sensitive resistance and cadherin-like protein mutant. α-chymotrypsin site was inserted between α1 and α2 of domain I of Cry1Ac toxin, the expression of target protein and the formation of parasporal crystal from mutant toxin in Bt was analyzed, the insecticidal activity of mutant toxin was determined. The results showed that, the four mutant proteins could express about 130 kDa target protein in Bacillus thuringiensis, but these mutants did not form regular parasporal crystal. The reason might be that the mutant protein reached the top at the stationary stage of Bacillus thuringiensis, and then it was degraded at the beginning of the decline stage. In the four mutants, Cry1Acm4 had the highest yield at 48 h, and its insecticidal activity for Spodoptera exigua was 1.6 times higher than that of the wild type, but the yield was only one tenth of toxin yield of wild type. Although it had little value in application for Cry1Acm4 because of the low expression, the construction method provided a new approach for researching Cry1Ac toxin with high insecticidal activity.

袁晓曼,, and . The Construction, Expression and Insecticidal Activity Determination of Cry1Ac Toxin Mutant[J]. Chinese Agricultural Science Bulletin, 2017, 33(36): 139-144.

References

[1].Schnepf E, Crickmore N, Van RJ, et al. Bacillus thuringiensis and its pesticidal crystal proteins [J]. Microbiology and Molecular Biology Reviews, 1998(62):775-806.
[2].Bravo A, Likitvivatanavong S, Gill SS, et al. Bacillus thuringiensis: A story of a successful bioinsecticide [J]. Insect Biochemistry and Molecular Biology, 2011, 41(7):423-431.
[3].Tabashnik B. Evolution of resistance to Bacillus thurinigienis[J]. Annual Review of Entomology, 1994, 39:47-79.
[4].Janmaat A, Myers J. Rapid evolution and the cost of resistance to Bacillus thuringiensis in greenhouse populations of cabbage loopers, Trichoplusia ni[J]. Proceedings of the Royal Society B: Biological Sciences, 2003, 270: 2263-2270.
[5].Pardo-López L, Soberón 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.
[6].Gómez I, Sánchez J, Miranda R, et al. Cadherin-like receptor binding facilitates proteolytic cleavage of helix alpha-1 in domain I and oligomer pre-pore formation of Bacillus thuringiensis Cry1Ab toxin [J]. FEBS Letter, 2002, 513(2-3):242-246.
[7].Jiménez-Juárez N, Mu?oz-Garay C, Gómez I, et al. Bacillus thuringiensis Cry1Ab mutants affecting oligomer formation are non-toxic to Manduca sexta larvae [J]. The Journal of Biological Chemistry, 2007, 282(29):21222-21229.
[8].Mu?óz-Garay C, Portugal L, Pardo-López L, et al. Characterization of the mechanism of action of the genetically modified Cry1AbMod toxin that is active against Cry1Ab-resistant insects [J]. Biochimica et Biophysica Acta, 2009, 1788(10):2229-2237.
[9].Bravo A, Gómez 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.
[10].Chen W, Liu C, Xiao Y, et al. A toxin-binding alkaline phosphatase fragment synergizes Bt toxin Cry1Ac against susceptible and resistant Helicoverpa armigera. PLoS One, 2015, 10(4):e0126288. doi: 10.1371/journal.pone.0126288. eCollection 2015.
[11].Zhuang M, Oltean DI, Gómez I, et al. Heliothis virescens and Manduca sexta lipid rafts are involved in Cry1A toxin binding to the midgut epithelium and subsequent pore formation [J]. The Journal of Biological Chemistry, 2002, 277(16):13863-13872.
[12].Caccia S, Di Lelio I, La Storia A, et al. Midgut microbiota and host immunocompetence underlie Bacillus thuringiensis killing mechanism [J]. Proceedings of the National Academy of Sciences USA, 2016, 113(34):9486-9491.
[13].Liu C, Li Y, Gao Y, Ning C, et al. Cotton bollworm resistance to Bt transgenic cotton: a case analysis [J]. Science China Life Sciences, 2010, 53(8):934-941.
[14].Xiao Y, Zhang T, Liu C, et al. Mis-splicing of the ABCC2 gene linked with Bt toxin resistance in Helicoverpa armigera [J]. Scientific Reports, 2014, 4:6184. doi: 10.1038/srep06184.
[15].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.
[16].Soberón M, Pardo-López L, López I, et al. Engineering modified Bt toxins to counter insect resistance [J]. Science, 2007, 318(5856):1640-1642.
[17].Liu G, Song L, Shu C, et al. Complete genome sequence of Bacillus thuringiensis subsp. kurstaki strain HD73 [J]. Genome Announcements, 2013, 1(2):e0008013. doi: 10.1128/genomeA.00080-13.
[18].刘晶晶, 束长龙, 张杰, 等. 苏云金芽孢杆菌内生质粒提取方法的改进[J]. 生物技术通报, 2008 (6):120-123。
[19].萨姆布鲁克, 拉塞尔(2005). 分子克隆实验指南. 第3版. 北京: 科学出版社。
[20].Nickerson KW, Julian St G, Bulla LA. Physiology of spore forming bacteria associated with insects: radiorespirometric survey of carbohydrate metabolism in the 12 serotypes of Bacillus thuringiensis [J]. Journal of Applied Microbiology, 1974, 28: 129-132．
[21].丁照鑫,王涵祎,曹利娟,等. 苏云金芽孢杆菌Vip3Aa10 毒素结合肽的筛选及活性测定[J]。中国农业科学, 2015, 48(18):3627-3634.
[22].Melo AL, Soccol VT, Soccol CR. Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review [J]. Critical Reviews in Biotechnology, 2016; 36(2):317-326.
[23].Boonserm P, Davis P, Ellar DJ, et al. Crystal structure of the mosquito-larvicidal toxin Cry4Ba and its biological implications [J]. Journal of Molecular Biology, 2005, 348(2):363-382.
[24].Carroll J, Convents D, Van Damme J, et al. Intramolecular proteolytic cleavage of Bacillus thuringiensis Cry3A δ-endotoxin may facilitate its Coleopteran Toxicity [J]. Journal of invertebrate pathology, 1997, 70:41-49.
[25].Yamaguchi T, Sahara K, Bando H, et al. Intramolecular proteolytic nicking and binding of Bacillus thuringiensis Cry8Da toxin in BBMVs of Japanese beetle [J]. Journal of invertebrate pathology, 2010, 105(3):243-247.