橘小实蝇蜕皮激素合成通路基因鉴定分析及饥饿对幼虫发育的影响

doi:10.3864/j.issn.0578-1752.2015.22.008

摘要/Abstract

摘要： 【目的】蜕皮激素（ecdysone）是昆虫体内一种重要的激素，参与调控昆虫的生长发育，并且能对环境胁迫进行响应。研究旨在明确橘小实蝇（Bactrocera dorsalis Hendel）蜕皮激素合成通路基因（ecdysone synthesis pathway gene）的分布和特定条件下的表达特性，为进一步开展橘小实蝇变态发育与抗逆胁迫机制的研究提供理论支持。【方法】采用RT-PCR和RACE技术克隆蜕皮激素合成通路基因BdCyp302a1、BdCyp315a1和BdCyp314a1，利用实时定量PCR（qPCR）技术检测该合成通路基因BdNvd、BdCyp306a1、BdCyp302a1、BdCyp315a1和BdCyp314a1在幼虫不同发育阶段（幼虫1—8日龄）、不同组织（前胸腺复合体、脂肪体、中肠、马氏管、表皮和气管）和饥饿条件下的表达模式，明确饥饿对幼虫发育的影响。【结果】克隆获得了3个蜕皮激素合成通路基因序列的开放阅读框，分别为BdCyp302a1（GenBank登录号：JQ027284）、BdCyp315a1（GenBank登录号：KC515377）和BdCyp314a1（GenBank登录号：JQ229645），且上述序列均具备P450典型结构域：Helix-C/I/K、PERF基序、血红蛋白结合位点以及脯氨酸/甘氨酸富集区，其氨基酸序列较为保守。qPCR检测结果显示蜕皮激素合成通路基因BdNvd、BdCyp306a1和BdCyp314a1幼虫末龄阶段表达量显著升高，其最高表达量分别是最低点的7.33、10.89和7.82倍，但在幼虫前4 d表达量差异不显著。而BdCyp302a1和BdCyp315a1在整个幼虫阶段表达量保持相对稳定。对不同组织的研究发现，蜕皮激素合成通路基因在所选的6个组织中均有表达，其中BdNvd、BdCyp306a1和BdCyp315a1在幼虫前胸腺表达量最高，在其他组织中表达量差异不显著；BdCyp302a1在各组织中的表达量由高到低为脂肪体>前胸腺>表皮/马氏管>气管/中肠，该基因在脂肪体的表达量是中肠的30倍；BdCyp314a1在中肠、马氏管和脂肪体内的表达量依次降低，但均极显著高于其他3个组织。橘小实蝇幼虫在饥饿处理12 h后即出现化蛹现象，其化蛹比例随饥饿时间延长而上升。对蛹宽和蛹长的测量中发现，饥饿导致蛹个体显著缩小，但未影响其存活率。同时，饥饿导致蜕皮激素合成通路基因BdNvd、BdCyp302a1和BdCyp314a1表达量在饥饿处理6 h后出现显著上调，并在处理48 h后出现显著下调；但饥饿处理并未影响BdCyp315a1的表达水平。【结论】蜕皮激素合成通路基因在橘小实蝇幼虫组织的表达具有差异性，并参与介导幼虫-蛹的变态过程和对营养胁迫的响应。

关键词: 橘小实蝇, 蜕皮激素合成通路, 饥饿胁迫, 幼虫发育, 基因表达

Abstract: 【Objective】 As one of the most important endohormones, ecdysone plays an important role in regulating the development of insects and responding to environmental factors. The objective of this study is to investigate the expression profiles of ecdysone synthesis pathway genes in Bactrocera dorsalis Hendel in various tissues and different conditions, providing theoretical knowledge to understand the mechanisms of metamorphosis and the stress response. 【Method】RT-PCR and RACE technologies were applied to clone the ecdysone synthesis pathway genes BdCyp302a1, BdCyp315a1 and BdCyp314a1. And quantitative PCR (qPCR) was carried to evaluate the mRNA expression patterns of ecdysone synthesis pathway genes BdNvd, BdCyp306a1, BdCyp302a1, BdCyp315a1 and BdCyp314a1 at different larval developmental stages (1- to 8-day-old larvae), tissues (prothoracic gland mixture, fat body, midgut, Malpighian tubes, integument and trachea), and starvation condition. Further, the impacts of food deprivation on larvae development were also examined in this research.【Result】The open reading frames (ORF) of BdCyp302a1 (GenBank accession number: JQ027284), BdCyp315a1 (GenBank accession number: KC515377) and BdCyp314a1 (GenBank accession number: JQ229645) were finally obtained. The analysis indicated that the protein sequences were highly conserved, which harbored typical and P450 motifs, such as Helix-C/I/K, PERF motifs, heme-binding domains and prolin/glycine rich domain. The results of qPCR showed that the expression levels of BdNvd, BdCyp306a1 and BdCyp314a1 were stable in the first four days at the larval stage, but significantly elevated in the last two days, which were up-regulated for 7.33, 10.89 and 7.82 folds comparing to the lowest level, respectively. And the expression levels BdCyp302a1 and BdCyp315a1 were stable at the larval stage. The expression profiles showed that all of the ecdysone synthesis pathway genes were traceable in the selected tissues. The highest relative expression levels of BdNvd, BdCyp306a1 and BdCyp315a1 were observed in the prothoracic glands, and no significant differences among other tissues. The expression levels of BdCyp302a1 followed the precedence order: fat body > prothoracic glands > integument/Malpighian tubes > trachea/midgut. The expression level of BdCyp302a1 in the fat body was 30 folds greater than that in midgut. And the expression abundances of BdCyp314a1 were extremely higher in the midgut, Malpighian tubes and fat body than in other tissues. Starvation could shorten the larval duration by prepupation. The larvae began to pupate in 6 h after food deprivation and the pupation rate increased with time extended. And the measurements indicated that the length and width of pupa were affected by starvation, which were significantly decreased in the treated group. However, no significant difference was detected in the survival rate. The expression levels of BdNvd, BdCyp302a1 and BdCyp314a1 were significantly elevated at 6 h post food deprivation, and down-regulated at 48 h. However, the expression levels of BdCyp315a1 were not relevant to nutrition condition.【Conclusion】Ecdysone synthesis pathway genes were transcribed differently in larval tissues, and played a vital role in mediating larva-pupa metamorphosis and nutrition stress.

Key words: Bactrocera dorsalis, ecdysone synthesis pathway genes, food deprivation, larvae development, gene expression

丛林，蒋玄赵，杨文佳，许抗抗，豆威，冉春，王进军. 橘小实蝇蜕皮激素合成通路基因鉴定分析及饥饿对幼虫发育的影响[J]. 中国农业科学, 2015, 48(22): 4469-4482.

CONG Lin, JIANG Xuan-zhao, YANG Wen-jia, XU Kang-kang, DOU Wei, RAN Chun, WANG Jin-jun. Identification of Ecdysone Synthesis Pathway Genes and Analysis on the Impact of Food Deprivation on Larvae Development of Bactrocera dorsalis Hendel[J]. Scientia Agricultura Sinica, 2015, 48(22): 4469-4482.

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参考文献

[1] Henrich V C, Rybczynski R, Gilbert L I. Peptide hormones, steroid hormones, and puffs: mechanisms and models in insect development. Vitamins & Hormones, 1998, 55: 73-125.

[2] Yoshiyama T, Namiki T, Mita K, Kataoka H, Niwa R. Neverland is an evolutionally conserved Rieske-domain protein that is essential for ecdysone synthesis and insect growth. Development, 2006, 133(13): 2565-2574.

[3] Gilbert L I. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Molecular and Cellular Endocrinology, 2004, 215: 1-10.

[4] Lee C Y, Cooksey B A, Baehrecke E H. Steroid regulation of midgut cell death during Drosophila development. Developmental Biology, 2002, 250(1): 101-111.

[5] Winbush A, Weeks J C. Steroid-triggered, cell-autonomous death of a Drosophila motoneuron during metamorphosis. Neural Development, 2011, 6: 15.

[6] Terashima J, Takaki K, Sakurai S, Bownes M. Nutritional status affects 20-hydroxyecdysone concentration and progression of oogenesis in Drosophila melanogaster. Journal of Endocrinology, 2005, 187(1): 69-79.

[7] Conlon I, Raff M. Size control in animal development. Cell, 1999, 96(2): 235-244.

[8] Kappler C, Kabbouh M, Durst F, Hoffmann J A. Studies on the C-2 hydroxylation of 2-deoxyecdysone in Locusta migratoria. Insect Biochemistry, 1986, 16(1): 25-32.

[9] Kappler C, Kabbouh M, Hetru C, Durst F, Hoffmann J A. Characterization of three hydroxylases involved in the final steps of biosynthesis of the steroid hormone ecdysone in Locusta migratoria (Insecta, Orthoptera). Journal of Steroid Biochemistry, 1988, 31(6): 891-898.

[10] Grieneisen M L, Warren J T, Gilbert L I. Early steps in ecdysteroid biosynthesis: evidence for the involvement of cytochrome-P-450 enzymes. Insect Biochemistry and Molecular Biology, 1999, 23(1): 13-23.

[11] Chávez V M, Marques G, Delbecque J P, Kobayashi K, Hollingsworth M, Burr J, Natzle J E, O’Connor M B. The Drosophila disembodied gene controls late embryonic morphogenesis and codes for a cytochrome P450 enzyme that regulates embryonic ecdysone levels. Development, 2000, 127(19): 4115-4126.

[12] Nijhout H F. The control of growth. Development, 2003, 130(24): 5863-5867.

[13] Mirth C K, Riddiford L M. Size assessment and growth control: how adult size is determined in insects. BioEssays, 2007, 29(4): 344-355.

[14] Stern D. Body-size evolution: how to evolve a mammoth moth. Current Biology, 2001, 11(22): 917-919.

[15] Colombani J, Bianchini L, Layalle S, Pondeville E, Dauphin- Villemant C, Antoniewski C, Carré C, Noselli S, Léopold P. Antagonistic actions of ecdysone and insulins determine final size in Drosophila. Science, 2005, 310(5748): 667-670.

[16] Colombani J, Raisin S, Pantalacci S, Radimerski T, Montagne J, Léopold P. A nutrient sensor mechanism controls Drosophila growth. Cell, 2003, 114(6): 739-749.

[17] Gilbert L I, Warren J T. A molecular genetic approach to the biosynthesis of the insect steroid molting hormone. Vitamins & Hormones, 2005, 73: 31-57.

[18] Spit J, Zels S, Dillen S, Holtof M, Wynant N, Broeck J V. Effects of different dietary conditions on the expression of trypsin- and chymotrypsin-like protease genes in the digestive system of the migratory locust, Locusta migratoria. Insect Biochemistry and Molecular Biology, 2014, 48: 100-109.

[19] Tang B, Qin Z, Shi Z K, Wang S, Guo X J, Wang S G, Zhang F. Trehalase in Harmonia axyridis (Coleoptera: Coccinellidae): effects on beetle locomotory activity and the correlation with trehalose metabolism under starvation conditions. Applied Entomology and Zoology, 2014, 49(2): 255-264.

[20] Ellison H E, Estévez-Lao T Y, Steven M C, Hillyer J F. Deprivation of both sucrose and water reduces the mosquito heart contraction rate while increasing the expression of nitric oxide synthase. Journal of Insect Physiology, 2015, 74: 1-9.

[21] Reisenman C E. Hunger is the best spice: effects of starvation in the antennal responses of the blood-sucking bug Rhodnius prolixus. Journal of Insect Physiology, 2014, 71: 8-13.

[22] Wang S P, Guo W Y , Muhammad S A, Chen R R, Mu L L, Li G Q. Mating experience and food deprivation modulate odor preference and dispersal in Drosophila melanogaster males. Journal of Insect Science, 2014, 14: Article 131.

[23] 罗育发, 钟八莲. 巴氏新小绥螨和尼氏真绥螨的耐饥能力及饥饿对其雌成虫捕食作用的影响. 中国南方果树, 2012, 41(1): 1-6.

Luo Y F, Zhong B L. Starvation tolerance of Neoseiulus barkeri and Euseius nicholsi and the influence of starvation on predation function of their female adults. South China Fruits, 2012, 41(1): 1-6. (in Chinese)

[24] Himuro C, Fujisaki K. Sexual size dimorphism and aggressive interactions under starvation conditions in the seed bug Togo hemipterus (Heteroptera: Lygaeidae). Journal of Insect Behavior,2012, 25: 242-253.

[25] Nijhout H F. A threshold size for metamorphosis in the tobacco hornworm, Manduca sexta (L.). Biological Bulletin, 1975, 149(1): 214-225.

[26] Shafiei M, Moczek A P, Nijhout H F. Food availability controls the onset of metamorphosis in the dung beetle Onthophagus taurus (Coleoptera: Scarabaeidae). Physiological Entomology, 2001, 26(2): 173-180.

[27] Telang A, Peterson B, Frame L, Baker E, Brown M R. Analysis of molecular markers for metamorphic competency and their response to starvation or feeding in the mosquito, Aedes aegypti (Diptera: Culicidae). Journal of Insect Physiology, 2010, 56(12): 1925-1934.

[28] 鞠珍, 赵静, 丁福波, 曲建军, 李明贵, 许永玉. 饥饿程度对美国白蛾生长发育和繁殖的影响. 应用昆虫学报, 2008, 45(3): 437-440.

Ju Z, Zhao J, Ding F B, Qu J J, Li M G, Xu Y Y. Effects of starvation on the development and reproduction of Hyphantria cunea. Chinese Bulletin of Entomology, 2008, 45(3): 437-440. (in Chinese)

[29] 郭文超, 吐尔逊, 郭利娜, 何江, 许建军. 营养对马铃薯甲虫迁飞能力的影响. 新疆农业科学, 2012, 49(3): 461-469.

Guo W C, Tuerxun, Guo L N, He J, Xu J J. Effects of different nutrients on flight capacity in Colorado potato beetles. Xinjiang Agricultural Sciences, 2012, 49(3): 461-469. (in Chinese)

[30] Clarke A R, Armstrong K F, Carmichael A E, Milne J R, Raghu S, Roderick G K, Yeates D K. Invasive phytophagous pests arising through a recent tropical evolutionary radiation: the Bactrocera dorsalis complex of fruit flies. Annual Review of Entomology, 2005, 50: 293-319.

[31] Liu J H, Shi W, Ye H. Population genetics analysis of the origin of the oriental fruit fly, Bactrocera dorsalis Hendel (Diptera: Tephritidae), in northern Yunnan Province, China. Entomological Science, 2007, 10(1): 11-19.

[32] Chen S L, Dai S M, Lu K H, Chang C. Female-specific doublesex dsRNA interrupts yolk protein gene expression and reproductive ability in oriental fruit fly, Bactrocera dorsalis (Hendel). Insect Biochemistry and Molecular Biology, 2008, 38(2): 155-165.

[33] Suganya R, Chen S L, Lu K H. Target of rapamycin in the oriental fruit fly Bactrocera dorsalis (Hendel): its cloning and effect on yolk protein expression. Archives of Insect Biochemistry and Physiology, 2010, 75(1): 45-56.

[34] Yang W J, Xu K K, Cong L, Wang J J. Identification, mRNA expression, and functional analysis of chitin synthase 1 gene and its two alternative splicing variants in oriental fruit fly, Bactrocera dorsalis. International Journal of Biological Sciences, 2013, 9(4): 331-342.

[35] Cong L, Yang W J, Shen G M, Dou W, Wang J J. Molecular characterization of the cDNA encoding ecdysone receptor isoform B1 and its expression in the oriental fruit fly, Bactrocera dorsalis (Diptera: Tephritidae). Florida Entomologist, 2012, 95(3): 650-658.

[36] Shen G M, Dou W, Niu J Z, Jiang H B, Yang W J, Jia F X, Hu F, Cong L, Wang J J. Transcriptome analysis of the oriental fruit fly (Bactrocera dorsalis). PLoS ONE, 2011, 6(12): e29127.

[37] Shen G M, Jiang H B, Wang X N, Wang J J. Evaluation of endogenous references for gene expression profiling in different tissues of the oriental fruit fly Bactrocera dorsalis (Diptera: Tephritidae). BMC Molecular Biology, 2010, 11: 76.

[38] Pfaffl M. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Research, 2001, 29(9): e45.

[39] Rewitz K F, O’Connor M B, Gilbert L I. Molecular evolution of the insect Halloween family of cytochrome P450s: phylogeny, gene organization and functional conservation. Insect Biochemistry and Molecular Biology, 2007, 37(8): 741-753.

[40] Petryk A, Warren J T, Marqués G, Jarcho M P, Gilbert L I, Kahler J, Parvy J P, Li Y, Dauphin-Villemant C, O’Connor M B. Shade is the Drosophila P450 enzyme that mediates the hydroxylation of ecdysone to the steroid insect molting hormone 20-hydroxyecdysone. Proceedings of the National Academy of Sciences of the United States of America, 2003, 100(24): 13773-13778.

[41] Iga M, Smagghe G. Identification and expression profile of Halloween genes involved in ecdysteroid biosynthesis in Spodoptera littoralis. Peptides, 2010, 31(3): 456-467.

[42] Feyereisen R, Durst F. Ecdysterone biosynthesis: a microsomal cytochrome-P-450-linked ecdysone 20-monooxygenase from tissues of the African migratory locust. European Journal of Biochemistry, 1978, 88(1): 37-47.

[43] Smith S L, Bollenbacher W E, Gilbert L I. Ecdysone 20- monooxygenase activity during larval-pupal development of Manduca sexta. Molecular and Cellular Endocrinology, 1983, 31(2): 227-251.

[44] Dubrovsky E B. Hormonal cross talk in insect development. Trends in Endocrinology and Metabolism, 2005, 16(1): 6-11.

[45] Warren J T, Petryk A, Marqués G, Jarcho M, Parvy, J P, Dauphin-Villemant C, O’Connor M B, Gilbert L I. Molecular and biochemical characterization of two P450 enzymes in the ecdysteroidogenic pathway of Drosophila melanogaster. Proceedings of the National Academy of Sciences of the United States of America, 2002, 99(17): 11043-11048.

[46] Rewitz K F, Rybczynski R, Warren J T, Gilbert L I. Developmental expression of Manduca shade, the P450 mediating the final step in molting hormone synthesis. Molecular and Cellular Endocrinology, 2006, 247(1): 166-174.

[47] Rewitz K F, Rybczynski R, Warren J T, Gilbert L I. Identification, characterization and developmental expression of Halloween genes encoding P450 enzymes mediating ecdysone biosynthesis in the tobacco hornworm, Manduca sexta. Insect Biochemistry and Molecular Biology, 2006, 36(3): 188-199.

[48] Iga M, Blais C, Smagghe G. Study on ecdysteroid levels and gene expression of enzymes related to ecdysteroid biosynthesis in the larval testis of Spodoptera littoralis. Archives of Insect Biochemistry and Physiology, 2013, 82(1): 14-28.

[49] 程道军, 李志清, 孟勐, 彭健, 钱文良, 康丽霞, 夏庆友. 家蚕蜕皮激素合成相关的细胞色素P450基因的鉴定分析. 中国农业科学, 2014, 47(3): 594-604.

Cheng D J, Li Z Q, Meng M, Peng J, Qian W L, Kang L X, Xia Q Y. Characterization of the cytochrome P450 genes involoving in ecdysteroidogenesis in silkworm (Bombyx moris). Scientia Agricultura Sinica, 2014, 47(3): 594-604. (in Chinese)

[50] Warren J T, Petryk A, Marqués G, Parvy J P, Shinoda T, Itoyama K, Kobayashi J, Jarcho M, Li Y, O’Connor M B, Dauphin-Villemant C, Gilbert L I. Phantom encodes the 25-hydroxylase of Drosophila melanogaster and Bombyx mori: a P450 enzyme critical in ecdysone biosynthesis. Insect Biochemistry and Molecular Biology, 2004, 34(9): 991-1010.

[51] Warren J T, Yerushalmi Y, Shimell M J, O’Connor M B, Restifo L L, Gilbert L I. Discrete pulses of molting hormone, 20-hydroxyecdysone, during late larval development of Drosophila melanogaster correlations with changes in gene activity. Developmental Dynamics, 2006, 235(2): 315-326.

[52] Yang F, Hu G, Shi J J, Zhai B P. Effects of larval density and food stress on life-history traits of Cnaphalocrocis medinalis (Lepidoptera: Pyralidae). Journal of Applied Entomology, 2015, 139(5): 370-380.

[53] Oliver S V, Brooke B D. The effect of larval nutritional deprivation on the life history and DDT resistance phenotype in laboratory strains of the malaria vector Anopheles arabiensis. Malaria Journal, 2013, 12: 44.

[54] Barrera R. Competition and resistance to starvation in larvae of container-inhabiting Aedes mosquitoes. Ecological Entomology, 1996, 21: 117-127.

[55] Araújo D S, Gil L H S, e-Silva D A. Larval food quantity affects development time, survival and adult biological traits that influence the vectorial capacity of Anopheles darlingi under laboratory conditions. Malaria Journal, 2012, 11: 261.

[56] Knapp M, Uhnavá K. Body size and nutrition intake effects on fecundity and overwintering success in Anchomenus dorsalis (Coleoptera: Carabidae). Journal of Insect Science, 2014, 14(240): doi: 10.1093/jisesa/ieu102.

[57] Cooper L C, Desjonqueres C, Leather S R. Cannibalism in the pea aphid, Acyrthosiphon pisum. Insect Science, 2014, 21(6): 750-758.

[58] Brodschneider R, Crailsheim K. Nutrition and health in honey bees. Apidologie, 2010, 41(3): 278-294.

[59] Britton J S, Edgar B A. Environmental control of the cell cycle in Drosophila: nutrition activates mitotic and endoreplicative cells by distinct mechanisms. Development, 1998, 125(11): 2149-2158.

[60] Flores Fernández J M, Gutiérrez Ortega A, Rosario Cruz R, Padilla Camberos E, Alvarez A H, Martínez Velazquez M. Molecular cloning and characterization of two novel autophagy-related genes belonging to the ATG8 family from the cattle tick Rhipicephalus (Boophilus) microplus (Acari: Ixodidae). Experimental and Applied Acarology, 2014, 64(4): 533-542.

[61] Lipovsek S, Janzekovit F, Novak T. Autophagic activity in the midgut gland of the overwintering harvestman Gyas annulatus (Phalangiidae, Opiliones). Arthropod Structure and Development, 2014, 43(5): 493-500.

[62] Chen C H, Gu S H. Stage-dependent effects of starvation on the growth, metamorphosis, and ecdysteroidogenesis by the prothoracic glands during the last larval instar of the silkworm, Bombyx mori. Journal of Insect Physiology, 2006, 52(9): 968-974.

[63] Munyiri F N, Asano W, Shintani Y, Ishikawa Y. Threshold weight for starvation-triggered metamorphosis in the yellow-spotted longicorn beetle, Psacothea hilaris (Coleoptera: Cerambycidae). Applied Entomology and Zoology, 2003, 38(4): 509-515.

[64] Gong J, Yu K, Shu L, Ye H H, Li S J, Zeng C S. Evaluating the effects of temperature, salinity, starvation and autotomy on molting success, molting interval and expression of ecdysone receptor in early juvenile mud crabs, Scylla paramamosain. Journal of Experimental Marine Biology and Ecology, 2015, 464: 11-17.

[65] Telang A, Frame L, Brown M R. Larval feeding duration affects ecdysteroid levels and nutritional reserves regulating pupal commitment in the yellow fever mosquito Aedes aegypti (Diptera: Culicidae). The Journal of Experimental Biology, 2007, 210(5): 854-864.

[66] Walsh A L, Smith W A. Nutritional sensitivity of fifth instar prothoracic glands in the tobacco hornworm, Manduca sexta. Journal of Insect Physiology, 2011, 57: 809-818.