|
Special Issue:
昆虫合辑Plant Protection—Entomolgy
害虫抗药性和毒理学合辑Pest Toxicology
|
|
|
|
| Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) |
| YANG Ya-jun, XU Hong-xing, WU Zhi-hong, LU Zhong-xian |
| State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Institute of Plant Protection and Microbiology, Zhejiang Academy of Agricultural Sciences, Hangzhou 310021, P.R.China |
|
|
|
|
摘要
昆虫中肠液在食物消化与解毒过程中发挥重要作用，为了明确稻纵卷叶螟中肠液对Bt蛋白潜在降解能力，本文利用蛋白酶抑制剂处理研究了稻纵卷叶螟中肠液酶活特性和中肠液对Bt蛋白（Cry2A，Cry1C，Cry1Aa和Cry1Ac）的降解能力。PMSF、TLCK、Pepstatin A和Leupeptin显著抑制稻纵卷叶螟中肠总蛋白酶活性；PMSF对胰凝乳蛋白酶活性有较强的抑制作用；EDTA、PMSF、TPCK、TLCK和E-64对胰蛋白酶活性有较强的抑制作用。EDTA对稻纵卷叶螟降解Cry2A的能力具有明显的抑制作用；稻纵卷叶螟对Cry1C和Cry1Aa的降解能力不同程度地受EDTA、PMSF、TPCK、TLCK抑制；稻纵卷叶螟对Cry1Ac降解能力不同程度地受EDTA、PMSF、TPCK、TLCK、E64抑制。研究结果表明，一些蛋白酶抑制剂可以抑制稻纵卷叶螟中肠蛋白酶活性，降低昆虫降解Bt蛋白的能力。本文研究结果有助于今后开发基于蛋白酶抑制剂的害虫防控新技术。
Abstract Midgut juice plays an important role in food digestion and detoxification in insects. In order to understand the potential of midgut juice of Cnaphalocrocis medinalis (Guenée) to degrade Bt proteins, the enzymatic activity of midgut juice and its degradation of Bt proteins (Cry2A, Cry1C, Cry1Aa, and Cry1Ac) were evaluated in this study through protease inhibitor treatments. The activities of total protease in midgut juices were significantly inhibited by phenylmethylsulfonyl fluoride (PMSF), tosyl-L-lysine chloromethyl ketone (TLCK), pepstatin A and leupeptin. The enzymatic activity of chymotrypsin was significantly inhibited by PMSF, and enzymatic activity of trypsin was significantly inhibited by ethylenediaminetetraacetic acid (EDTA), PMSF, tosyl phenylalanine chloromethyl ketone (TPCK), TLCK and trans-epoxysuccinyl-L-leucylamido-(4-guanidino) butane (E-64). EDTA could significantly inhibit the degradation of Cry2A by C. medinalis. EDTA, PMSF, TPCK, and TLCK could inhibit the degradation of Cry1C and Cry1Aa. EDTA, PMSF, TPCK, TLCK, and E-64 could inhibit the degradation of Cry1Ac. Our results indicated that some protease inhibitors hindered various enzymatic activities in the larval midgut of C. medinalis, which may reduce the insect’s ability to degrade Bt toxins. These findings may aid the application of protease inhibitors in the management of this insect pest in the future.
|
|
Received: 02 April 2020
Accepted:
|
| Fund: This study was supported by the Zhejiang Provincial Natural Science Foundation of China (LY20C140004), the National Science and Technology Major Project of China (2016ZX08001001), the National Natural Science Foundation of China (31501669) and the earmarked fund for China Agriculture Research System (CARS-01-36). |
|
Corresponding Authors:
Correspondence LU Zhong-xian, E-mail: luzxmh@163.com
|
| About author: YANG Ya-jun, E-mail: yargiuneyon@163.com; |
Cite this article:
YANG Ya-jun, XU Hong-xing, WU Zhi-hong, LU Zhong-xian.
2021.
Effects of inhibitors on the protease profiles and degradation of activated Cry toxins in larval midgut juices of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Journal of Integrative Agriculture, 20(8): 2195-2203.
|
Alfonso-Rubí J, Ortego F, Castañera P, Carbonero P, Díaz I. 2003. Transgenic expression of trypsin inhibitor CMe from barley in indica and japonica rice, confers resistance to the rice weevil Sitophilus oryzae. Transgenic Research, 12, 23–31.
Applebaum S W. 1985. Biochemistry of digestion. In: Kerkot G A, Gillbert L I, eds., Comprehensive Insect Physiology, Biochemistry and Pharmacology. Peragmon Press, NY. pp. 279–311.
Berenbaum M. 1980. Adaptive significance of midgut pH in larval Lepidoptera. American Naturalist, 115, 138–146.
Bigham M, Hosseininaveh V, Darvishzadeh A, Ghazizadeh M, Ghamari M. 2013. Activity of digestive proteinases and carbohydrases in the alimentary tract of the fig leaf roller, Choreutis nemorana Hübner (Lepidoptera: Choreutidae). Archives of Phytopathology and Plant Protection, 46, 1–8.
Bradford M M. 1976. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of dye-protein binding. Analytical Biochemistry, 72, 248–254.
Bravo A, Gómez I, Porta H, García-Gómez B I, Rodriguez-Almazan C, Pardo L, Soberón M. 2013. Evolution of Bacillus thuringiensis Cry toxins insecticidal activity. Microbial Biotechnology, 6, 17–26.
Chen M, Shelton A, Ye G Y. 2011. Insect-resistant genetically modified rice in China: From research to commercialization. Annual Review of Entomology, 56, 81–101.
Chen Y J, Huang F D, Li B. 2015. Control effect of biochemical Bt against Lepidopterous pests in rice. Journal of Anhui Agricultural Science, 43, 129–130, 163. (in Chinese)
Cheng J A. 1996. Rice Pests. China Agricultural Press, China. (in Chinese)
Duan X, Li X, Xue Q, Abo-EI-Saad M, Xu D, Wu R. 1996. Transgenic rice plants harboring an introduced potato proteinase inhibitor II gene are insect resistant. Nature Biotechnology, 14, 494.
Dunse K M, Kaas Q, Guarino R F, Barton P A, Craik D J, Anderson M A. 2010. Molecular basis for the resistance of an insect chymotrypsin to a potato type II proteinase inhibitor. Proceedings of the National Academy of Sciences of the United States of America, 107, 15016–15021.
Gatehouse J A. 2011. Prospects for using proteinase inhibitors to protect transgenic plants against attack by herbivorous insects. Current Protein and Peptide Science, 12, 409–416.
Green T R, Ryan C A. 1972. Wound-induced proteinase inhibitor in plant leaves: A possible defense mechanism against insects. Science, 175, 776–777.
Jamal F, Singh D, Pandey P K. 2014. Negative effects of a nonhost proteinase inhibitor of ~19.8 kDa from Madhuca indica seeds on developmental physiology of Helicoverpa armigera (Hübner). BioMed Research International, 1, 202398.
Jin L, Zhang H N, Lu Y H, Yang Y H, Wu K M, Tabashnik B E, Wu Y D. 2015. Large-scale test of the natural refuge strategy for delaying insect resistance to transgenic Bt crops. Nature Biotechnology, 33, 169–174.
Karim S, Dean D H. 2000. Toxicity and receptor binding properties of Bacillus thuringiensis δ-endotoxins to the midgut brush border membrane vesicles of the rice leaf folders, Cnaphalocrocis medinalis and Marasmia patnalis. Current Microbiology, 41, 276–283.
Keller M, Sneh B, Strizhov N, Prudovsky E, Regev A, Koncz C, Schell J, Zilberstein A. 1996. Digestion of δ-entotoxin by gut proteases may explain reduced sensitivity of advanced instar larvae of Spodoptera littoralis to CryIC. Insect Biochemistry and Molecular Biology, 26, 365–373.
Kipgen L, Aggarwal K K. 2014. Gut protease profiles of different instars of Helicoverpa armigera (Lepidoptera: Noctuidae). International Journal of Tropical Insect Science, 34, 172–178.
Kirouac M, Vachon V, Quievy D, Schwartz K L, Laprade R. 2006. Protease inhibitors fail to prevent pore formation by the activated Bacillus thuringiensis toxin Cry1Aa in insect brush border membrane vesicles. Applied and Environmental Microbiology, 72, 506–515.
Kumar R, Bhardwaj U, Kumar P, Mazumdar-Leighton S. 2015. Midgut serine proteases and alternative host plant utilization in Pieris brassicae L. Frontier in Physiology, 6, 95.
Lightwood D J, Ellar D J, Jarrett P. 2000. Role of proteolysis in determining potency of Bacillus thuringiensis Cry1Ac δ-endotoxin. Applied and Environmental Microbiology, 66, 5174–5181.
Linser P J, Dinglasan R R. 2014. Chapter one - insect gut structure, function, development, and target of biological toxins. In: Dhadialla T S, Gill S S, eds., Advances in Insect Physiology. Academic Press, Oxford. pp. 1–13.
Lu Z X. 2017. Pictorial Handbook on Sustainable Management of Rice Leaffolder. China Agricultural Press, Beijing, China. (in Chinese)
MacIntosh S C, Kishore G M, Perlak F J, Marrone P G, Stone T B, Sims S R, Fuchs R. 1990. Potentiation of Bacillus thuringiensis insecticidal activity by serine protease inhibitor. Journal of Agricultural and Food Chemistry, 8, 1145–1152.
Miranda R, Zamudio F Z, Bravo A. 2001. Processing of Cry1Ab delta-endotoxin from Bacillus thuringiensis by Manduca sexta and Spodoptera frugiperda midgut proteases: Role in protoxin activation and toxin inactivation. Insect Biochemistry and Molecular Biology, 31, 1155–1163.
Melo A L, Soccol V T, Soccol C R. 2016. Bacillus thuringiensis: mechanism of action, resistance, and new applications: a review. Critical Reviews in Biotechnology, 36, 317–326.
Oppert K, Kramer J, Beeman R, Johnson D, McGaughey W H. 1997. Proteinase-mediated insect resistance to Bacillus thuringiensis toxins. Journal of Biological Chemistry, 272, 23473–23476.
Pang A S D, Gringorten J L. 1998. Degradation of Bacillus thuringiensis δ-endotoxin in host insect gut juice. FEMS Microbiology Reviews, 167, 281–285.
Pardo-López L, Soberón M, Bravo A. 2013. Bacillus thuringiensis insecticidal three-domain Cry toxins: mode of action, insect resistance and consequences for crop protection. FEMS Microbiology Reviews, 37, 3–22.
Rukmini V, Reddy C Y, Venkateswerlu J. 2000. Bacillus thuringiensis crystal δ-endotoxin: Role of proteases in the conversion of protoxin to toxin. Biochimie, 82, 109−116.
Saadati F, Bandani A R. 2011. Effects of serine protease inhibitors on growth and development and digestive serine proteinases of the Sunn Pest, Eurygaster integriceps. Journal of Insect Science, 11, 1–12.
Singh S, Singh A, Kumar S, Mittal P, Singh I K. 2020. Protease inhibitors: Recent advancement in its usage as a potential biocontrol agent for insect pest management. Insect Science, 27, 186–201.
Srinivasan A, Girim A P, Gupta V S. 2006. Structural and functional diversities in lepidopteran serine proteases. Cellular and Molecular Biology Letters, 11, 132–154.
Sugimura M, Iwahana H, Sato R. 1997. Unusual proteolytic processing of a δ-endotoxin from Bacillus thuringiensis strain Buibui by larval midgut-juice of Anomala cuprea Hope (Coleoptera: Scarabaeidae). Applied Entomology and Zoology, 32, 533–540.
Talaei-Hassanloui R, Bakhshaei R, Hosseininaveh V, Khorramnezhad A. 2014. Effect of midgut proteolytic activity on susceptibility of lepidopteran larvae to Bacillus thuringiensis subsp. Kurstaki. Frontier in Physiology, 4, 406.
Teo L H, Hammond A M, Woodring J P. 1990. Digestive enzymes of the velvet bean caterpillar (Lepidoptera: Noctuidae). Annals of the Entomological Society of America, 83, 820–826.
Terra W R, Ferriera C. 2012. Biochemistry of digestion. In: Gilbert L I, ed., Insect Molecular Biology and Biochemistry. Elsevier, New York. pp. 365–418.
Tetreau G, Stalinski R, David J P, Després L. 2013. Increase in larval gut proteolytic activities and Bti resistance in the dengue fever mosquito. Archives of Insect Biochemistry and Physiology, 82, 71–83.
Wu G Z, Zhu K Y, Zeng R S. 2013. Effects of soybean trypsin inhibitor on growth and development phase of Spodoptera litura (F.). Ecology and Environmental Sciences, 22, 1335–1340. (in Chinese)
Wu Y D. 2014. Detection and mechanisms of resistance evolved in insects to Cry toxins from Bacillus thuringiensis. Advances in Insect Physiology, 47, 297–342.
Wu Z H, Yang Y J, Xu H X, Zheng X S, Tian J C, Lu Y H, Lu Z X. 2015. Changes in growth and development and main enzyme activities in midgut of Cnaphalocrocis medinalis intermittently treated with low amount of Bt rice leaves over generations. Chinese Journal of Rice Science, 29, 417–423. (in Chinese)
Xu D, Xue Q, McElroy D, Mawal Y, Hilder V A, Wu R. 1996. Constitutive expression of a cowpea trypsin inhibitor gene, CpTi, in transgenic rice plants confers resistance to two major rice insect pests. Molecular Breeding, 2, 167–173.
Xu L, Pan Z Z, Zhang J, Liu B, Zhu Y J, Chen Q X. 2016. Proteolytic activation of Bacillus thuringiensis Cry2Ab through a belt-and-braces approach. Journal of Agricultural and Food Chemistry, 64, 7195–7200.
Yamazaki T, Ishikawa T, Pandian G N, Okazaki K, Haginoya K, Tachikawa Y, Mitsui T, Miyamoto K, Angusthanasombat C, Hori H. 2011. Midgut juice of Plutella xylostella highly resistant to Bacillus thuringiensis Cry1Ac contains a three times larger amount of glucosinolatesulfatase which binds to Cry1Ac compared to that of susceptible strain. Pesticide Biochemistry and Physiology, 101, 125–131.
Yang Y J, Chen H Y, Wu Y D, Yang Y H, Wu S W. 2007. Mutated cadherin alleles from field population of Helicoverpa armigera confer resistance to Bacillus thuringiensis toxin Cry1Ac detected in the field. Applied and Environmental Microbiology, 73, 6939–6944.
Yang Y J, Liu K, Han H L, Xu H X, Zhang F C, Zheng X S, Tian J C, Wang G Y, Chen G H, Lu Z X. 2016. Impacts of nitrogen fertilizer on major insect pests and their predators in transgenic Bt rice lines T2A-1 and T1C-19. Entomologia Experimentalis et Applicata, 160, 281–291.
Yang Y J, Wu Z H, Xu H X, Lu Z X. 2020. pH influences the profiles of midgut extracts in Cnaphalocrocis medinalis (Guenée) and its degradation of activated Cry toxins. Journal of Integrative Agriculture, 19, 775–784.
Yang Y J, Xu H X, Lu Y H, Wang C Y, Lu Z X. 2018. Midgut transcriptomal response of the rice leaffolder, Cnaphalocrocis medinalis (Guenée) to Cry1C toxin. PLoS ONE, 13, e0191686.
Yang Y J, Xu H X, Zheng X S, Lu Z X. 2012. Susceptibility and selectivity of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae) to different Cry toxins. Journal of Economic Entomology, 105, 2122–2128.
Yang Y J, Xu H X, Zheng X S, Tian J C, Lu Y H, Lu Z X. 2015. Progresses in management technology of rice leaffolders in China. Journal of Plant Protection, 42, 691–701. (in Chinese)
Yang Z, Chen H, Tang W, Hua H, Lin Y. 2011. Development and characterisation of transgenic rice expressing two Bacillus thuringiensis genes. Pest Management Science, 67, 414–422.
Yu F, Zeng X N, Xiong Z H, Du L X. 2004. Study and application of proteinase inibitors on pest control. Plant Protection, 30, 13–17. (in Chinese)
Zhao A, Li Y, Leng C, Wang P, Li Y. 2019. Inhibitory effect of protease inhibitors on larval midgut protease activities and the performance of Plutella xylostella (Lepidoptera: Plutellidae). Frontier in Physiology, 9, 1963.
Zhao A P, Sun C, Zhan E L, Wu J X, Li Y P. 2016. Effects of protease inhibitors on the activities of midgut proteases of larval Grapholitha molesta (Lepidoptera: Tortricidae). Acta Entomologia Sinica, 59, 1069–1078. (in Chinese)
Zheng X S, Lu T, Xu H X, Guo W Q, Gao G C, Lu Z X. 2010. A new, simple and efficient method of collecting the eggs of rice leaffolder, Cnaphalocrocis medinalis. Chinese Bulletin Entomology, 47, 1253–1256. (in Chinese)
Zheng X S, Yang Y J, Xu H X, Chen H, Wang B J, Lin Y J, Lu Z X. 2011. Resistance performances of transgenic Bt rice lines T2A-1 and T1c-19 against Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Journal of Economic Entomology, 104, 1730–1735.
|
| No Suggested Reading articles found! |
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
