亚洲玉米螟幼虫膜结合海藻糖酶基因RNAi效应

doi:10.16409/j.cnki.2095-039x.2020.03.023

摘要/Abstract

摘要： 海藻糖是昆虫的血糖，为昆虫提供能量；海藻糖酶催化海藻糖分解为葡萄糖，是几丁质生物合成的原料。围食膜（peritrophic membranes，PMs）是昆虫消化道特有结构，对昆虫消化食物、保护肠道表皮细胞具有重要作用；几丁质是PMs的重要组分。海藻糖酶（trehalase，Tre）和几丁质合成酶（chitin synthase，CHS）是几丁质合成途径的第1个和最后1个酶。本研究通过饲喂亚洲玉米螟（Orstrinia furnacalis，Asian corn borer，ACB）膜结合海藻糖酶（OfMT）基因特异的干扰dsRNA，研究RNAi对ACB幼虫中肠CHSB（OfCHSB）基因表达及幼虫发育的影响。发现处理48 h后，OfMT基因和OfCHSB基因表达量分别下降了52%和53%，幼虫发育迟缓。通过对处理及对照ACB幼虫中肠石蜡切片进行苏木精-伊红染色（hematoxylin-ethanol，HE染色）和几丁质标记，发现血腔内脂肪体组织减小、中肠围食膜组织中几丁质含量减少。推测OfMT基因有可能成为ACB的生物防治的靶标基因。

关键词: 亚洲玉米螟, 膜结合海藻糖酶, RNAi, 几丁质合成酶, 围食膜

Abstract: Trehalose is the blood sugar component. The catabolism of trehalose is catalyzed by trehalase, providing energy for insects and the metabolite, glucose, as substrate for chitin synthesis. Chitin is an important component of peritrophic membranes (PMs), which play important roles in facilitating food digestion and providing protection to the gut epithelium. Trehalase and chitin synthase are the first and the final enzymes in the chitin synthesis pathway. In this study, RNAi was performed by means of oral delivery of double-stranded RNA of OfMT gene. The results show that the expression of both the OfMT and OfCHSB genes were interfered (52% and 53% down-regulated) in 48 h. Meanwhile, the growth of Asia corn borer larvae were inhibited. Further findings by hematoxylin-ethanol staining and chitin labeling show that the fat body tissue and chitin content were reduced in hamocoel and midgut PMs. The results suggest that it is feasible to silence OfMT by feeding dsRNA to the corn borer larvae and that OfMT can be the target gene for biocontrol of the borer.

Key words: Ostrinia furnacalis, membrane-bound trehalase, RNAi, chitin synthase, peritrophic membranes

中图分类号:

S433.4

靳婷婷, 戈峰, 吴杰. 亚洲玉米螟幼虫膜结合海藻糖酶基因RNAi效应[J]. 中国生物防治学报, 2020, 36(3): 452-457.

JIN Tingting, GE Feng, WU Jie. RNAi Effects on Membrane-Bound Trehalase Gene in Ostrinia furnacalis Larvae[J]. Chinese Journal Of Biological Control, 2020, 36(3): 452-457.

参考文献

[1] Shukla E, Thorat L J, Nath B B, et al. Insect trehalase:physiological significance and potential applications[J]. Glycobiology, 2015, 25(4):357-367.
[2] Kalra B, Tamang A M, Parkash R. Cross-tolerance effects due to adult heat hardening, desiccation and starvation acclimation of tropical drosophilid-Zaprionus indianus[J]. Comparative Biochemistry and Physiology Part A:Molecular & Integrative Physiology. 2017, 209:65-73.
[3] Reina-Bueno M, Argandona M, Nieto J J, et al. Role of trehalose in heat and desiccation tolerance in the soil bacterium Rhizobium etli[J]. BMC Microbiology, 2012, 12:207.
[4] Chen Q, Ma E, Behar K L, et al. Role of trehalose phosphate synthase in anoxia tolerance and development in Drosophila melanogaster[J]. The Journal of Biological Chemistry, 2002, 277(5):3274-3279.
[5] Storey K M. Storey J. Insect cold hardiness:Metabolic, gene, and protein adaptation 1[J]. Canadian Journal of Zoology, 2012, 90:456-475.
[6] Sinclair B J. Linking energetics and overwintering in temperate insects[J]. Journal of Thermal Biology, 2015, 54:5-11.
[7] Kong X, Liu Y, Gou X, et al. Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli[J]. Biochemical and Biophysical Research Communications, 2001, 280(1):396-400.
[8] Merzendorfer H, Zimoch L. Chitin metabolism in insects:structure, function and regulation of chitin synthases and chitinases[J]. The Journal of Experimental Biology 2003, 206(24):4393-4412.
[9] Arakane Y, Muthukrishnan S, Kramer K J, et al. The Tribolium chitin synthase genes TcCHS1 and TcCHS2 are specialized for synthesis of epidermal cuticle and midgut peritrophic matrix[J]. Insect Molecular Biology, 2005, 14(5):453-463.
[10] Zhuo W, Chu F, Kong L, et al. Chitin synthase B:a midgut-specific gene induced by insect hormones and involved in food intake in Bombyx mori larvae[J]. Archives of Insect Biochemistry and Physiology, 2014, 85(1):36-47.
[11] Tian H, Peng H, Yao Q, et al. Developmental control of a lepidopteran pest Spodoptera exigua by ingestion of bacteria expressing dsRNA of a non-midgut gene[J]. PLoS ONE, 2009, 4(7):e6225.
[12] Zhai Y, Fan X, Yin Z, et al. Identification and functional analysis of chitin synthase A in Oriental Armyworm, Mythimna separata[J]. Proteomics, 2017, 17(21):1700165.
[13] Shi J F, Mu L L, Chen X, et al. RNA interference of chitin synthase genes inhibits chitin biosynthesis and affects larval performance in Leptinotarsa decemlineata (Say)[J]. International Journal of Biological Sciences, 2016, 12(11):1319-1331.
[14] 唐斌, 魏苹, 陈洁, 等. 昆虫海藻糖酶的基因特性及功能研究进展[J]. 昆虫学报, 2012(11):1315-1321.
[15] Chen J, Tang B, Chen H, et al. Different functions of the insect soluble and membrane-bound trehalase genes in chitin biosynthesis revealed by RNA interference[J]. PLoS ONE, 2010, 5(4):e10133.
[16] 张倩, 鲁鼎浩, 蒲建, 等. 灰飞虱海藻糖酶基因的克隆及RNA干扰效应[J]. 昆虫学报, 2012(8):911-920.
[17] 魏苹. 赤拟谷盗海藻糖酶基因调控几丁质合成及能量代谢的功能研究[D]. 杭州:杭州师范大学, 2013.
[18] Zhao L, Yang M, Shen Q, et al. Functional characterization of three trehalase genes regulating the chitin metabolism pathway in rice brown planthopper using RNA interference[J]. Scientific Reports, 2016, 6(1):27841.
[19] 宋彦英, 周大荣, 何康来. 亚洲玉米螟无琼脂半人工饲料的研究与应用[J]. 植物保护学报, 1999, 26(4):324-328.
[20] Wu K, Hoy M A. Oral delivery of double-stranded RNA induces prolonged and systemic gene knockdown in Metaseiulus occidentalis only after feeding on Tetranychus urticae[J]. Experimental & Applied Acarology, 2014, 63(2):171-187.
[21] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method[J]. Methods, 2001, 25(4):402-408.
[22] Liu Y, Shen D, Zhou F, et al. Identification of immunity-related genes in Ostrinia furnacalis against entomopathogenic fungi by RNA-seq analysis[J]. PLoS ONE, 2014, 9(1):e86436.
[23] Farnesi L C, Brito J M, Linss J G, et al. Physiological and morphological aspects of Aedes aegypti developing larvae:effects of the chitin synthesis inhibitor novaluron[J]. PLoS ONE, 2012, 7(1):e30363.
[24] Merzendorfer H. Insect chitin synthases:a review[J]. Journal of Comparative Physiology B, Biochemical, Systemic, and Environmental Physiology, 2006, 176(1):1-15.
[25] Chen X, Yang X, Senthil Kumar N, et al. The class A chitin synthase gene of Spodoptera exigua:molecular cloning and expression patterns[J]. Insect Biochemistry and Molecular Biology, 2007, 37(5):409-417.
[26] Lehane M J. Peritrophic matrix structure and function[J]. Annual Review of Entomology, 1997, 42:525-550.
[27] Cohen E. Chitin synthesis and inhibition:a revisit[J]. Pest Management Science, 2001, 57(10):946-950.
[28] Peters W. Peritrophic Membranes[M]. Springer Berlin Heidelberg, 1992.
[29] Kramer K J, Hopkins T L, Schaefer J. Applications of solids NMR to the analysis of insect sclerotized structures[J]. Insect Biochemistry & Molecular Biology, 1995, 25(10):1067-1080.
[30] Hegedus D, Erlandson M, Gillott C, et al. New insights into peritrophic matrix synthesis, architecture, and function[J]. Annual Review of Entomology, 2009, 54:285-302.
[31] Shen Z, Jacobs-Lorena M. Characterization of a novel gut-specific chitinase gene from the human malaria vector Anopheles gambiae[J]. The Journal of Biological Chemistry, 1997, 272(46):28895-28900.32] Khajuria C, Buschman L L, Chen M S, et al. A gut-specific chitinase gene essential for regulation of chitin content of peritrophic matrix and growth of Ostrinia nubilalis larvae[J]. Insect Biochemistry and Molecular Biology 2010, 40(8):621-629.
[33] 张文庆, 陈晓菲, 唐斌, 等. 昆虫几丁质合成及其调控研究前沿[J]. 应用昆虫学报, 2011(3):475-479.