Cry1Ac抗、感棉铃虫碱性磷酸酯酶（ALP1）的表达量比较

doi:10.3864/j.issn.0578-1752.2013.17.006

摘要/Abstract

摘要： 【目的】通过比较Cry1Ac抗、感棉铃虫昆虫中肠碱性磷酸酶（ALP1）表达量的差异及抗性棉铃虫取食Cry1Ac蛋白对ALP1表达量的影响，分析ALP1表达量变化与抗性的关系，为进一步明确Bt抗性机制、制定抗性治理策略提供理论依据。【方法】利用实时荧光定量PCR技术，比较敏感棉铃虫、Cry1Ac抗性棉铃虫取食含Cry1Ac蛋白的饲料和正常饲料后ALP1表达量的差异。【结果】ALP1在棉铃虫整个发育期都表达，幼虫的表达量最高，蛹期表达量最低，在成虫体内也有较高的表达；ALP1在幼虫中肠表达量最高，其次是后肠、前肠、马氏管，表皮中的表达量最低；与敏感品系相比，对Cry1Ac具有中等水平抗性的棉铃虫ALP1表达量明显增加，尤其是取食含Cry1Ac蛋白饲料的抗性棉铃虫幼虫的ALP1的表达量显著升高，但抗性棉铃虫取食正常饲料后，2、3龄幼虫的ALP1的表达量与敏感棉铃虫差异不显著；所有的抗性品系4龄棉铃虫幼虫中肠ALP1的表达量都显著高于敏感品系，而且随着棉铃虫抗性倍数的升高，ALP1的表达量呈逐渐降低的趋势。【结论】抗性棉铃虫中肠ALP1表达量的改变可能与Cry1Ac抗性、Cry1Ac代谢有一定的关系。

关键词: 棉铃虫 , Bt , 抗性 , 碱性磷酸酶 , 表达量

Abstract: 【Objective】 The objective of this study is to provide a theoretical basis for further clarifying Cry1Ac resistance mechanism and constituting reasonable resistance management strategy, the difference of ALP1 expression level between Cry1Ac-resistant and susceptible Helicoverpa armigera, the effect of Cry1Ac toxin on ALP1 expression level in Cry1Ac-resistant larvae and the relationship between ALP1 and Cry1Ac-resistant strains were analyzed. 【Method】 The expression levels of ALP1 in susceptible and Cry1Ac-resistant H. armigera larvae feeding on artificial diet containing Cry1Ac and no-Cry1Ac were compared using real-time quantitative PCR analysis. 【Result】 The ALP1 could express in whole development period of H. armigera, the highest expression level occurred in larvae, while the lowest was in pupae, and there was also relatively high expression level in moth. The highest expression level of ALP1 was in midgut of H. armigera larvae, followed by hindgut, foregut, malpighian tubules and epidermis. Compared with the susceptible H. armigera, the expression of ALP1 obviously increased in resistant larvae which had moderate resistance to Cry1Ac. The ALP1 expression of the resistant larvae significantly increased compared with the susceptible strain after they were fed on the artificial diet containing Cry1Ac, while the difference of ALP1 expression in 2nd and 3rd instar larvae was not significant between resistant strain and susceptible strain after they were fed on the normal artificial diet. The expression of ALP1 in the 4th instar larval midgut of all resistant strains were obviously higher than that in susceptible strain, and the expression level of ALP1 appeared downtrend along with the resistance ration increased. 【Conclusion】 The changes of ALP1 expression in midgut of resistant H. armigera may have a relationship with its resistance to Cry1Ac and metabolism of Cry1Ac.

Key words: Helicoverpa armigera , Bt , resistance , alkaline phosphatase , expression level

张涛12, 张丽丽2, 魏纪珍2, 肖玉涛2, 梁革梅2, 周瑞阳1. Cry1Ac抗、感棉铃虫碱性磷酸酯酶（ALP1）的表达量比较[J]. 中国农业科学, 2013, 46(17): 3580-3586.

ZHANG Tao-12, ZHANG Li-Li-2, WEI Ji-Zhen-2, XIAO Yu-Tao-2, LIANG Ge-Mei-2, ZHOU Rui-Yang-1. The Expression Level of Alkaline Phosphatase (ALP1) in Cry1Ac-Resistant and Susceptible Cotton Bollworm Helicoverpa armigera Hübner[J]. Scientia Agricultura Sinica, 2013, 46(17): 3580-3586.

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参考文献

[1]Federici B A. Insecticidal bacteria: an overwhelming success for invertebrate pathology. Journal of Invertebrate Pathology, 2005, 89(1): 30-38.

[2]Bravo A, Soberon M. How to cope with insect resistance to Bt toxins? Trends in Biotechnology, 2008, 26(10): 573-579.

[3]Wu K M, Lu Y H, Feng H Q, Jiang Y Y, Zhao J Z. Suppression of cotton bollworm in multiple crops in China in areas with Bt toxin-containing cotton. Science, 2008, 321(5896): 1676-1678.

[4]Pigott C R, Ellar D J. Role of receptors in Bacillus thuringiensis crystal toxin activity. Microbiology and Molecular Biology Reviews, 2007, 71(2): 255-281.

[5]Griffitts J S, Aroian R V. Many roads to resistance: how invertebrates adapt to Bt toxins. BioEssays, 2005, 27(6): 614-624.

[6]Bravo A, Likitvivatanavong S, Gill S S, Soberon M. Bacillus thuringiensis: A story of a successful bioinsecticide. Insect Biochemistry and Molecular Biology, 2011, 41(7): 423-431.

[7]McNall R J, Adang M J. Identification of novel Bacillus thuringiensis Cry1Ac binding proteins in Manduca sexta midgut through proteomic analysis. Insect Biochemistry and Molecular Biology, 2003, 33(10): 999-1010.

[8]Jurat-Fuentes J L, Adang M J. Characterization of a Cry1Ac-receptor alkaline phosphatase in susceptible and resistant Heliothis virescens larvae. European Journal of Biochemistry, 2004, 271(15): 3127-3135.

[9]Fernandez L E, Aimanova K G, Gill S S, Bravo A, Soberon M. A GPI-anchored alkaline phosphatase is a functional midgut receptor of Cry11Aa toxin in Aedes aegypti larvae. Biochemical Journal, 2006, 394(1): 77-84.

[10]Hua G, Zhang R, Bayyareddy K, Adang M J. Anopheles gambiae alkaline phosphatase is a functional receptor of Bacillus thuringiensis jegathesan Cry11Ba toxin. Biochemistry, 2009, 48(41): 9785-9793.

[11]Martins E S, Monnerat R G, Queiroz P R, Dumas V F, Braz S V, de Souza Aguiar R W, Gomes A C M M, Sánchez J, Bravo A, Ribeiro B M. Midgut GPI-anchored proteins with alkaline phosphatase activity from the cotton boll weevil (Anthonomus grandis) are putative receptors for the Cry1B protein of Bacillus thuringiensis. Insect Biochemistry and Molecular Biology, 2010, 40(2): 138-145.

[12]Ning C, Wu K, Liu C, Gao Y, Jurat-Fuentes J L, Gao X. Characterization of a Cry1Ac toxin-binding alkaline phosphatase in the midgut from Helicoverpa armigera (Hubner) larvae. Journal of Insect Physiology, 2010, 56(6): 666-672.

[13]Jurat-Fuentes J L, Adang M J. A proteomic approach to study Cry1Ac binding proteins and their alterations in resistant Heliothis virescens larvae. Journal of Invertebrate Pathology, 2007, 95(3): 187-191.

[14]Jurat-Fuentes J L, Karumbaiah L, Jakka S R, Ning C, Liu C, Wu K, Jackson J, Gould F, Blanco C, Portilla M, Perera O, Adang M. Reduced levels of membrane-bound alkaline phosphatase are common to lepidopteran strains resistant to Cry toxins from Bacillus thuringiensis. PLoS One, 2011, 6(3): e17606.

[15]Yang Y, Zhu Y C, Ottea J, Husseneder C, Leonard B R, Abel C, Luttrell R, Huang F. Down regulation of a gene for cadherin, but not alkaline phosphatase, associated with Cry1Ab resistance in the sugarcane borer Diatraea saccharalis. PLoS One, 2011, 6(10): e25783.

[16]梁革梅, 谭维嘉, 郭予元. 棉铃虫对转Bt基因棉的抗性筛选及遗传方式的研究. 昆虫学报, 2000, 43(增刊): 57-62.

Liang G M, Tan W J, Guo Y Y. Study on screening and inheritance mode of resistance to Bt transgenic cotton in cotton bollworm. Acta Entomologica Sinica, 2000, 43(Suppl.): 57-62. (in Chinese)

[17]粱革梅, 谭维嘉, 郭予元. 棉铃虫人工饲料技术的改进. 植物保护, 1999, 25(2): 15-17.

Liang G M, Tan W J, Guo Y Y. An improvement in the technique of artificial rearing cotton bollworm. Plant Protection, 1999, 25(2): 15-17. (in Chinese)

[18]Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2-△△CT method. Methods, 2001, 25(4): 402-408.

[19]严盈, 彭露, 刘万学, 万方浩. 昆虫碱性磷酸酶的研究进展. 昆虫学报, 2009, 52(1): 95-105.

Yan Y, Peng L, Liu W X, Wan F H. Research progress in insect alkaline phosphatases. Acta Entomologica Sinica, 2009, 52(1): 95-105. (in Chinese)

[20]Eguchi M. Alkaline phosphatase isozymes in insects and comparison with mammalian enzyme. Comparative Biochemistry and Physiology B: Biochemistry and Molecular Biology, 1995, 111(2): 151-162.

[21]Chang W S, Zachow K R, Bentley D. Expression of epithelial alkaline phosphatase in segmentally iterated bands during grasshopper limb morphogenesis. Development, 1993, 118(2): 651-663.

[22]Itoh M, Kanamori Y, Takao M, Eguchi M. Cloning of soluble alkaline phosphatase cDNA and molecular basis of the polymorphic nature in alkaline phosphatase isozymes of Bombyx mori midgut. Insect Biochemistry and Molecular Biology, 1999, 29(2): 121-129.

[23]Jurat-Fuentes J L, Adang M J. Cry toxin mode of action in susceptible and resistant Heliothis virescens larvae. Journal of Invertebrate Pathology, 2006, 92(3): 166-171.

[24]Likitvivatanavong S, Chen J, Bravo A, Soberon M, Gill S S. Cadherin, alkaline phosphatase, and aminopeptidase N as receptors of Cry11Ba toxin from Bacillus thuringiensis subsp. jegathesan in Aedes aegypti. Applied and Environmental Microbiology, 2011, 77(1): 24-31.

[25]Bravo A, Gomez I, Conde J, Munoz-Garay C, Sanchez J, Miranda R, Zhuang M, Gill S S, Soberon M. Oligomerization triggers binding of a Bacillus thuringiensis Cry1Ab pore-forming toxin to aminopeptidase N receptor leading to insertion into membrane microdomains. Biochimica et Biophysica Acta, 2004, 1667(1): 38-46.

[26]Bravo A, Miranda R, Gomez I, Soberon M. Pore formation activity of Cry1Ab toxin from Bacillus thuringiensis in an improved membrane vesicle preparation from Manduca sexta midgut cell microvilli. Biochimica et Biophysica Acta, 2002, 1562(1/2): 63-69.

[27]Caccia S, Moar W J, Chandrashekhar J, Oppert C, Anilkumar K J, Jurat-Fuentes J L, Ferre J. Association of Cry1Ac toxin resistance in Helicoverpa zea (Boddie) with increased alkaline phosphatase levels in the midgut lumen. Applied and Environment Microbiology, 2012, 78(16): 5690-5698.

[28]Liang G M, Wu K M, Yu H K, Li K K, Feng X, Guo Y Y. Changes of inheritance mode and fitness in Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) along with its resistance evolution to Cry1Ac toxin. Journal of Invertebrate Pathology, 2008, 97(2): 142-149.