家蚕蜕皮激素合成相关的细胞色素P450基因的鉴定分析

doi:10.3864/j.issn.0578-1752.2014.03.019

摘要/Abstract

摘要： 【目的】昆虫变态发育的启动主要受前胸腺合成分泌的蜕皮激素所调控，而蜕皮激素的合成是由细胞色素P450基因催化完成。家蚕（Bombyx mori）是一种产丝昆虫，蚕业生产中如果能阻断或延迟家蚕蛹变态发育进程，将有利于改进蚕茧处理工艺，提高蚕丝品质。论文旨在鉴定参与家蚕蜕皮激素合成的细胞色素P450基因，从而为人为遗传调节家蚕变态发育提供靶基因。【方法】基于序列同源性比对，筛选家蚕及其他昆虫中参与蜕皮激素合成的P450基因。利用ClustalW软件，分析昆虫蜕皮激素合成相关P450基因的遗传发生关系。通过全基因组表达芯片数据分析及RT-PCR验证，调查家蚕蜕皮激素合成相关P450基因的时空表达特征。利用RNAi技术分析Cyp314a1表达下调对家蚕变态发育的影响。【结果】家蚕基因组中有4个参与家蚕蜕皮激素合成的P450基因，即Cyp306a1、Cyp302a1、Cyp315a1和Cyp314a1。比较分析显示，这4个蜕皮激素合成相关的P450基因在家蚕及其他昆虫中都是单拷贝，而且每个基因的同源体在遗传发生树上能很好地聚成一类，表明昆虫蜕皮激素合成相关的P450基因及其负责的蜕皮激素合成通路非常保守。时空表达谱分析显示，在家蚕幼虫5龄第3天，Cyp302a1、Cyp315a1和Cyp314a1主要在家蚕幼虫卵巢、精巢和头部等组织器官中高表达；在幼虫-蛹-成虫变态发育进程中，Cyp302a1、Cyp315a1和Cyp314a1主要在蛹变态发育后期表达，其中Cyp314a1分别在上簇、化蛹及羽化前高表达，这与蜕皮激素滴度高峰出现的时期基本一致。Cyp314a1的RNAi导致家蚕不能正常化蛹及雌蛾卵巢发育异常，且降低了蜕皮激素信号通路关键基因HR3及Ftz-f1的表达。【结论】家蚕蜕皮激素合成相关的P450基因在进化上非常保守，其表达水平降低能引起家蚕蛹变态发育受阻，暗示蜕皮激素合成相关P450基因可以用作家蚕变态发育控制的靶标基因之一。

关键词: 家蚕 , 蜕皮激素合成 , 细胞色素P450基因 , 变态发育 , RNAi

Abstract: 【Objective】Insect metamorphosis is initiated by ecdysone, which is synthesized in prothoracic gland through cytochrome P450 enzymes-catalyzed enzymatic reactions. Silkworm (Bombyx mori) is a silk-producing insect. Interrupting or delaying the pupa-adult transition in silkworm will help to reform the flow of silkworm cocoon-processing and to further improve the quality of silk. The objective of this study is to identify and characterize P450 genes involving in ecdysteroidogenesis in silkworm and to provide candidate genes for genetic controlling of silkworm metamorphosis. 【Method】 Ecdysteroidogenesis-related P450 genes were identified in silkworm and other insects through sequence homology search. Phylogenetic tree of insect ecdysteroidogenesis- related P450 genes was constructed by ClustalW program. The mRNA expression profiles of ecdysteroidogenesis-related P450 genes from silkworm were checked by microarray and RT-PCR analysis. RNAi experiment was used to examine the effects of down- regulation of expression level of ecdysteroidogenesis-related P450 genes on silkworm metamorphosis. 【Result】Four P450 genes involving in ecdysteroidogenesis, including Cyp306a1, Cyp302a1, Cyp315a1, and Cyp314a1, were identified in the silkworm genome. Comparative analysis revealed that each edysteroidogenesis-related P450 gene existed as a single copy in the surveyed insects and all its orthologs in insects were grouped together well in the phylogenetic tree, indicating edysteroidogenesis-related P450 genes and edysteroidogenesis pathway were very conserved during insect evolution. Spatio-temporal expression profiling found that Cyp302a1, Cyp315a1, and Cyp314a1 showed a high expression in the ovary, testis, and head of silkworm larvae and were continually expressed during the late stages of silkworm metamorphosis. Cyp314a1 expression could be highly detected before wandering, pupation, or eclosion, showing a consistency with the stage-specific changes of ecdysone pulses. RNAi of Cyp314a1 resulted in not only larval death before silkworm pupation but also developmental abnormality of adult ovary. Two key genes involving in ecdysone signaling, HR3 and Ftz-f1, were also down-regulated after Cyp314a1 RNAi.【Conclusion】Silkworm P450 genes involving in ecdysteroidogenesis are evolutionarily conserved and down-regulating the expression level of the selected P450 gene interrupts normal pupation, suggesting that ecdysteroidogenesis-related P450 genes might be used in altering genetically the progress of silkworm metamorphosis.

Key words: silkworm (Bombyx mori) , ecdysteroidogenesis , cytochrome P450 , metamorphosis , RNAi

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[J]. 中国农业科学, 2014, 47(3): 594-604.

CHENG Dao-Jun, LI Zhi-Qing, MENG Meng, PENG Jian, QIAN Wen-Liang, KANG Li-Xia, XIA Qing-You. Characterization of Cytochrome P450 Genes Involving in Ecdysteroidogenesis in Silkworm (Bombyx mori)[J]. Scientia Agricultura Sinica, 2014, 47(3): 594-604.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2014.03.019

https://www.chinaagrisci.com/CN/Y2014/V47/I3/594

参考文献

[1]程道军, 唐林, 孟勐, 康丽霞, 王永虎, 彭健, 马三垣, 夏庆友. 家蚕蛹期特异表达基因BmCP283鉴定及其启动子的克隆分析. 中国农业科学, 2013, 46(11): 2353-2362.

Cheng D J, Tang L, Meng M, Kang L X, Wang Y H, Peng J, Ma S Y, Xia Q Y. Identification of silkworm pupa-specific gene BmCP283 and its promoter. Scientia Agricultura Sinica, 2013, 46(11): 2353-2362. (in Chinese)

[2]Woodard C T, Baehrecke E H, Thummel C S. A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell, 1994, 79(4): 607-615.

[3]Mizoguchi A, Ohashi Y, Hosoda K, Ishibashi J, Kataoka H. Developmental profile of the changes in the prothoracicotropic hormone titer in hemolymph of the silkworm Bombyx mori: correlation with ecdysteroid secretion. Insect Biochemical and Molecular Biology, 2001, 31(4/5): 349-358.

[4]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.

[5]Scott J G, Liu N, Wen Z M. Insect cytochromes P450: diversity, insecticide resistance and tolerance to plant toxins. Comparative Biochemistry and Physiology C: Pharmacology Toxicology and Endocrinology, 1998, 121(1/3): 147-155.

[6]Gilbert L I. Halloween genes encode P450 enzymes that mediate steroid hormone biosynthesis in Drosophila melanogaster. Molecular and Cellular Endocrinology, 2004, 215(1/2): 1-10.

[7]Petryk A, Warren J T, Marques G, Jarcho M P, Gilbert L I, Kahler J, Parvy J P, Li Y T, 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.

[8]Marchal E, Verlinden H, Badisco L, Van Wielendaele P, Vanden Broeck J. RNAi-mediated knockdown of Shade negatively affects ecdysone-20-hydroxylation in the desert locust, Schistocerca gregaria. Journal of Insect Physiology, 2012, 58(7): 890-896.

[9]Ai J, Zhu Y, Duan J, Yu Q, Zhang G, Wan F, Xiang Z H. Genome-wide analysis of cytochrome P450 monooxygenase genes in the silkworm, Bombyx mori. Gene, 2011, 480(1/2): 42-50.

[10]Li B, Xia Q, Lu C, Zhou Z, Xiang Z. Analysis of cytochrome P450 genes in silkworm genome (Bombyx mori). Science in China Series C: Life Science, 2005, 48(4): 414-418.

[11]Altschul S F, Madden T L, Schaffer A A, Zhang J, Zhang Z, Miller W, Lipman D J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Research, 1997, 25(17): 3389-3402.

[12]Thompson J D, Gibson T J, Higgins D G. Multiple sequence alignment using ClustalW and ClustalX//Current Protocols in Bioinformatics, 2002, Chapter 2: Unit 2 3.

[13]Tamura K, Dudley J, Nei M, Kumar S. MEGA4: Molecular evolutionary genetics analysis (MEGA) software version 4.0. Molecular Biology and Evolution, 2007, 24(8): 1596-1599.

[14]Xia Q, Cheng D, Duan J, Wang G, Cheng T, Zha X, Liu C, Zhao P, Dai F, Zhang Z, He N, Zhang L, Xiang Z. Microarray-based gene expression profiles in multiple tissues of the domesticated silkworm, Bombyx mori. Genome Biology, 2007, 8(8): R162.

[15]程道军. 家蚕激素信号通路及其对变态发育期基因表达的调控研究[D]. 2008, 重庆: 西南大学.

Cheng D J. Hormone-related pathway and its regulation on gene expression during metamorphosis in the silkworm, Bombyx mori[D]. Chongqing: Southwest University, 2008. (in Chinese)

[16]Wang G H, Liu C, Xia Q Y, Zha X F, Chen J, Jiang L. Cathepsin B protease is required for metamorphism in silkworm, Bombyx mori. Insect Science, 2008, 15(3): 201-208.

[17]Li Z Q, Cheng D J, Wei L, Zhao P, Shu X, Tang L, Xiang Z H, Xia Q Y. The silkworm homolog of Methoprene-tolerant (Met) gene reveals sequence conservation but function divergence. Insect Science, 2010, 17(4): 313-324.

[18]King-Jones K, Thummel C S. Nuclear receptors-a perspective from Drosophila. Nature Reviews Genetics, 2005, 6(4): 311-323.

[19]Parvy J P, Blais C, Bernard F, Warren J T, Petryk A, Gilbert L I, O'Connor M B, Dauphin-Villemant C. A role for βFTZ-F1 in regulating ecdysteroid titers during post-embryonic development in Drosophila melanogaster. Developmental Biology, 2005, 282(1): 84-94.

[20]Tijet N, Helvig C, Feyereisen R. The cytochrome P450 gene superfamily in Drosophila melanogaster: annotation, intron-exon organization and phylogeny. Gene, 2001, 262(1/2): 189-198.

[21]Strode C, Wondji C S, David J P, Hawkes N J, Lumjuan N, Nelson D R, Drane D R, Karunaratne S H, Hemingway J, Black W C, Ranson H. Genomic analysis of detoxification genes in the mosquito Aedes aegypti. Insect Biochemical and Molecular Biology, 2008, 38(1): 113-123.

[22]Zhu F, Moural T W, Shah K, Palli S R. Integrated analysis of cytochrome P450 gene superfamily in the red flour beetle, Tribolium castaneum. BMC Genomics, 2013, 14: 174.

[23]Gilbert L I, Song Q, Rybczynski R. Control of ecdysteroidogenesis: activation and inhibition of prothoracic gland activity. Invertebrate Neuroscience, 1997, 3(2/3): 205-216.

[24]Tu M P, Yin C M, Tatar M. Impaired ovarian ecdysone synthesis of Drosophila melanogaster insulin receptor mutants. Aging Cell, 2002, 1(2): 158-160.

[25]Hentze J L, Moeller M E, Jorgensen A F, Bengtsson M S, Bordoy A M, Warren J T, Gilbert L I, Andersen O, Rewitz K F. Accessory gland as a site for prothoracicotropic hormone controlled ecdysone synthesis in adult male insects. Plos One, 2013, 8(2): e55131.

[26]Spindler K D, Vanwormhoudt A, Sellos D, Spindler-Barth M. Ecdysteroid levels during embryogenesis in the shrimp, Palaemon serratus (Crustacea, Decapoda) - quantitative and qualitative changes. General and Comparative Endocrinology, 1987, 66(1): 116-122.

[27]Evans S J, Watson S J, Akil H. Evaluation of sensitivity, performance and reproducibility of microarray technology in neuronal tissue. Integrative and Comparative Biology, 2003, 43(6): 780-785.

[28]Lam G, Hall B L, Bender M, Thummel C S. DHR3 is required for the prepupal-pupal transition and differentiation of adult structures during

Drosophila metamorphosis. Developmental Biology, 1999, 212(1): 204-216.

[29]Yamada M, Murata T, Hirose S, Lavorgna G, Suzuki E, Ueda H. Temporally restricted expression of transcription factor beta FTZ-F1: significance for embryogenesis, molting and metamorphosis in Drosophila melanogaster. Development, 2000, 127(23): 5083-5092.

[30]Mane-Padros D, Cruz J, Cheng A, Raikhel A S. A critical role of the nuclear receptor HR3 in regulation of gonadotrophic cycles of the mosquito Aedes aegypti. Plos One, 2012, 7(9): e45019.