棉铃虫酪氨酸羟化酶基因的分子特性及功能分析
|
摘要
【目的】酪氨酸羟化酶(tyrosine hydroxylase, TH)是黑色素形成的关键酶,在昆虫表皮骨化过程中扮演重要角色。本研究旨在获得棉铃虫Helicoverpa armigera TH基因序列,并研究其分子特性、表达模式和功能,为更深入探析该基因作用机理奠定基础。【方法】通过生物信息学和分子生物学技术获得了棉铃虫TH基因序列,利用qRT-PCR分析该基因在棉铃虫不同生长发育阶段的表达模式;利用qRT-PCR技术,分别测定了蜕皮激素20E(400 ng/头)处理不同时间和RNAi成功干扰蜕皮激素受体基因(EcR)前提下再用20E(400 ng/头)处理后,棉铃虫5龄幼虫TH表达量变化;采用生物化学方法检测鞣化激素(30μg/mg组织)和环腺苷酸(cAMP, 200 ng/mg组织)处理后棉铃虫幼虫脂肪体中TH活性。【结果】获得了棉铃虫酪氨酸羟化酶基因TH(GenBank登录号:MF440319) cDNA片段,长2 270 bp,开放阅读框1 686 bp,编码561个氨基酸残基。该基因在棉铃虫整个发育期均表达,其中在卵期第3天、2龄幼虫第1天、3-5龄蜕皮期、预蛹期和成虫羽化第1天表达量相对较高。研究还发现,400 ng/头20E注射能够促进TH的转录;在成功干扰并调低幼虫EcR转录水平的前提条件下(对照仅注射dsGFP)再注射20E,对TH表达量无明显影响;而鞣化激素(30μg/mg组织)和cAMP(200 ng/mg组织)均显著提高了TH的酶活性。【结论】20E在转录水平参与了TH的表达;鞣化激素和cAMP均能够提高TH活性,在蛋白水平上对TH进行调控。
【Aim】 Tyrosine hydroxylase(TH) is a key enzyme of melanin biosynthesis and plays an important role in epidermal sclerotization. This study aims to obtain the cDNA sequence of TH gene of Helicoverpa armigera, and to analyze its molecular characteristics, expression pattern and function, so as to provide a foundation for further exploring its action mechanism. 【Methods】 The full-length cDNA sequence of TH gene was cloned from H. armigera by using bioinformatics and molecular cloning technique. The developmental expression pattern of this gene was assayed with qRT-PCR. The expression of TH gene in the 5 th instar larva of H. armigera after treatment with 20 E(20-hydroxyecdysone)(400 ng/individual) for different time and RNAi of ecdysone receptor gene(EcR) followed by 20 E treatment(400 ng/individual) was analyzed by qRT-PCR, respectively. The TH acitivity in the fat body of H. armigera larvae after treatment with bio-stimulants bursicon(30 μg/mg tissue) and cAMP(200 ng/mg tissue) was determined by biochemical method. 【Results】 We cloned the full-length cDNA sequence of TH(GenBank accession no.: MF440319) from H. armigera, which is 2 270 bp in length with a 1 686 bp open reading frame encoding 561 amino acids. TH is ubiquitously expressed in all the examined developmental stages of H.armigera and relatively highly expressed in day-3 of egg stage, day-1 of the 2 nd instar larval stage, 3 rd-5 th instar molting stages, prepupal stage and 1 day-old adult. In vitro treatment, the steroid hormone 20 E(400 ng/individual) significantly up-regulated the transcription level of TH, but the 20 E treatment after RNAi-mediated knockdown of 20 E receptor gene EcR showed no obvious effect on the transcription of TH as compared with the control(injected with dsGFP only). Enzymatic activity assay demonstrated that both bursicon(30 μg/mg tissue) and cAMP(200 ng/mg tissue) led to significant increase of the TH activity. 【Conclusion】 20 E is involved in the regulation of TH expression at the transcriptional level, and both bursicon and cAMP can promote the TH activity at the protein level.
【Aim】 Tyrosine hydroxylase(TH) is a key enzyme of melanin biosynthesis and plays an important role in epidermal sclerotization. This study aims to obtain the cDNA sequence of TH gene of Helicoverpa armigera, and to analyze its molecular characteristics, expression pattern and function, so as to provide a foundation for further exploring its action mechanism. 【Methods】 The full-length cDNA sequence of TH gene was cloned from H. armigera by using bioinformatics and molecular cloning technique. The developmental expression pattern of this gene was assayed with qRT-PCR. The expression of TH gene in the 5 th instar larva of H. armigera after treatment with 20 E(20-hydroxyecdysone)(400 ng/individual) for different time and RNAi of ecdysone receptor gene(EcR) followed by 20 E treatment(400 ng/individual) was analyzed by qRT-PCR, respectively. The TH acitivity in the fat body of H. armigera larvae after treatment with bio-stimulants bursicon(30 μg/mg tissue) and cAMP(200 ng/mg tissue) was determined by biochemical method. 【Results】 We cloned the full-length cDNA sequence of TH(GenBank accession no.: MF440319) from H. armigera, which is 2 270 bp in length with a 1 686 bp open reading frame encoding 561 amino acids. TH is ubiquitously expressed in all the examined developmental stages of H.armigera and relatively highly expressed in day-3 of egg stage, day-1 of the 2 nd instar larval stage, 3 rd-5 th instar molting stages, prepupal stage and 1 day-old adult. In vitro treatment, the steroid hormone 20 E(400 ng/individual) significantly up-regulated the transcription level of TH, but the 20 E treatment after RNAi-mediated knockdown of 20 E receptor gene EcR showed no obvious effect on the transcription of TH as compared with the control(injected with dsGFP only). Enzymatic activity assay demonstrated that both bursicon(30 μg/mg tissue) and cAMP(200 ng/mg tissue) led to significant increase of the TH activity. 【Conclusion】 20 E is involved in the regulation of TH expression at the transcriptional level, and both bursicon and cAMP can promote the TH activity at the protein level.
引文
An S, Dong S, Wang Q, Li S, Gilbert LI, Stanley D, Song Q, 2012. Insect neuropeptide bursicon homodimers induce innate immune and stress genes during molting by activating the NF-κB transcription factor Relish. PLoS ONE, 7(3): e34510.
An S, Wang S, Gilbert LI, Beerntsen B, Ellersieck M, Song Q, 2008. Global identification of bursicon-regulated genes in Drosophila melanogaster. BMC Genomics, 9(1): 424-437.
Andersen SO, 2012. Cuticular sclerotization and tanning. In: Gilbert LI ed. Insect Molecular Biology and Biochemistry. Academic Press, Oxford. 167-192.
Arakane Y, Mi YN, Asano T, Kramer KJ, 2016. Tyrosine metabolism for insect cuticle pigmentation and sclerotization. In: Cohen E, Moussian B eds. Extracellular Composite Matrices in Arthropods. Springer International Publishing, Cham, Switzerland. 165-205.
Baker J, Truman JW, 2002. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J. Exp. Biol., 205(17): 2555-2565.
Chen CH, Pan J, Di YQ, Liu W, Hou L, Wang JX, Zhao XF, 2017. Protein kinase C delta phosphorylates ecdysone receptor B1 to promote gene expression and apoptosis under 20-hydroxyecdysone regulation. Proc. Natl. Acad. Sci. USA, 114(34): E7121-E7130.
Clark AC, del Campo ML, Ewer J, 2004. Neuroendocrine control of larval ecdysis behavior in Drosophila: complex regulation by partially redundant neuropeptides. J. Neurosci., 24(17): 4283-4292.
Curtis AT, Hori M, Green JM, Wolfgang WJ, Hiruma K, Riddiford LM, 1984. Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae. J. Insect Physiol., 30(8): 597-606.
Davis MM, O′Keefe SL, Primrose DA, Hodgetts RB, 2007. A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster. Development, 134(24): 4395-4404.
Du M, Zhao W, Jurenka R, Liu X, Yin X, Song Q, An S, 2017. Transcriptome analysis of Helicoverpa armigera male hairpencils: alcohol biosynthesis and requirement for mating success. Insect Biochem. Molec. Biol., 87: 154-164.
Gervasi NM, Scott SS, Aschrafi A, Gale J, Vohra SN, MacGibeny MA, Kar AN, Gioio AE, Kaplan BB, 2016. The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons. RNA, 22(6): 883-895.
Gorman MJ, Arakane Y, 2010. Tyrosine hydroxylase is required for cuticle sclerotization and pigmentation in Tribolium castaneum. Insect Biochem. Molec. Biol., 40(3): 267-273.
Han Y, Liu C, Wei LW, Ren JB, Liu PY, Xia QY, 2010. Expression profile and function of tyrosine hydroxylase gene Bmth in the silkworm, Bombyx mori. Acta Entomol. Sin., 53(9): 949-956. [韩宇, 刘春, 魏丽婉, 任静波, 刘品彦, 夏庆友, 2010. 家蚕酪氨酸羟化酶基因Bmth的表达及功能. 昆虫学报, 53(9): 949-956]
Kramer KJ, Morgan TD, Hopkins TL, Christensen A, Schaefer J, 1991. Insect cuticle tanning: enzymes and cross-link structure. In: Hedin PA ed. Naturally Occurring Pest Bioregulators. ACS Symposium Series No. 449. American Chemical Society, Washington DC. 87-105.
Liu X, Dai F, Guo E, Li K, Ma L, Tian L, Cao Y, Zhang G, Palli SR, Li S, 2015. 20-Hydroxyecdysone (20E) primary response gene E93 modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. J. Biol. Chem., 290(45): 27370-27383.
Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4): 402-408.
Lu YH, Jiang YY, Liu J, Zeng J, Yang XM, Wu KM, 2018. Adjustment of cropping structure increases the risk of cotton bollworm outbreaks in China. Chin. J. Appl. Entomol., 55(1): 19-24. [陆宴辉, 姜玉英, 刘杰, 曾娟, 杨现明, 吴孔明, 2018. 种植业结构调整增加棉铃虫的灾变风险. 应用昆虫学报, 55(1): 19-24]
Luo CW, Dewey EM, Sudo S, Ewer J, Hsu SY, Honegger HW, Hsueh AJW, 2005. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. Proc. Natl. Acad. Sci. USA, 102(8): 2820-2825.
Mendive FM, Van Loy TV, Claeysen S, Poels J, Williamson M, Hauser F, Grimmelikhuijzen CJP, Vassart G, Vanden Broeck J, 2005. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2. FEBS Lett., 579(10): 2171-2176.
Nagatsu T, Levitt M, Udenfriend S, 1964. Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. J. Biol. Chem., 239(9): 2910-2917.
Reynolds SE, Taghert P, Truman JW, 1979. Eclosion hormone and bursicon titres and the onset of hormonal responsiveness during the last day of adult development in Manduca sexta (L.). J. Exp. Biol., 78: 77-86.
Song Q, 2012. Bursicon, a neuropeptide hormone that controls cuticle tanning and wing expansion. In: Gilbert LI ed. Insect Endocrinology. Academic Press, Oxford. 93-105.
Wright TRF, 1987. The genetic and molecular organization of the dense cluster of functionally related, vital genes in the DOPA decarboxylase region of the Drosophila melanogaster genome. In: Hennig W ed. Results and Problems in Cell Differentiation, Vol. 14. Structure and Function of Eukaryotic Chromosomes. Springer-Verlag, Berlin, Heidelberg, New York. 95-120.
Yue Z, Liu X, Zhou Z, Hou G, Hua J, Zhao Z, 2016. Development of a novel-type transgenic cotton plant for control of cotton bollworm. Plant Biotechnol. J., 14(8): 1747-1755.
An S, Wang S, Gilbert LI, Beerntsen B, Ellersieck M, Song Q, 2008. Global identification of bursicon-regulated genes in Drosophila melanogaster. BMC Genomics, 9(1): 424-437.
Andersen SO, 2012. Cuticular sclerotization and tanning. In: Gilbert LI ed. Insect Molecular Biology and Biochemistry. Academic Press, Oxford. 167-192.
Arakane Y, Mi YN, Asano T, Kramer KJ, 2016. Tyrosine metabolism for insect cuticle pigmentation and sclerotization. In: Cohen E, Moussian B eds. Extracellular Composite Matrices in Arthropods. Springer International Publishing, Cham, Switzerland. 165-205.
Baker J, Truman JW, 2002. Mutations in the Drosophila glycoprotein hormone receptor, rickets, eliminate neuropeptide-induced tanning and selectively block a stereotyped behavioral program. J. Exp. Biol., 205(17): 2555-2565.
Chen CH, Pan J, Di YQ, Liu W, Hou L, Wang JX, Zhao XF, 2017. Protein kinase C delta phosphorylates ecdysone receptor B1 to promote gene expression and apoptosis under 20-hydroxyecdysone regulation. Proc. Natl. Acad. Sci. USA, 114(34): E7121-E7130.
Clark AC, del Campo ML, Ewer J, 2004. Neuroendocrine control of larval ecdysis behavior in Drosophila: complex regulation by partially redundant neuropeptides. J. Neurosci., 24(17): 4283-4292.
Curtis AT, Hori M, Green JM, Wolfgang WJ, Hiruma K, Riddiford LM, 1984. Ecdysteroid regulation of the onset of cuticular melanization in allatectomized and Black mutant Manduca sexta larvae. J. Insect Physiol., 30(8): 597-606.
Davis MM, O′Keefe SL, Primrose DA, Hodgetts RB, 2007. A neuropeptide hormone cascade controls the precise onset of post-eclosion cuticular tanning in Drosophila melanogaster. Development, 134(24): 4395-4404.
Du M, Zhao W, Jurenka R, Liu X, Yin X, Song Q, An S, 2017. Transcriptome analysis of Helicoverpa armigera male hairpencils: alcohol biosynthesis and requirement for mating success. Insect Biochem. Molec. Biol., 87: 154-164.
Gervasi NM, Scott SS, Aschrafi A, Gale J, Vohra SN, MacGibeny MA, Kar AN, Gioio AE, Kaplan BB, 2016. The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons. RNA, 22(6): 883-895.
Gorman MJ, Arakane Y, 2010. Tyrosine hydroxylase is required for cuticle sclerotization and pigmentation in Tribolium castaneum. Insect Biochem. Molec. Biol., 40(3): 267-273.
Han Y, Liu C, Wei LW, Ren JB, Liu PY, Xia QY, 2010. Expression profile and function of tyrosine hydroxylase gene Bmth in the silkworm, Bombyx mori. Acta Entomol. Sin., 53(9): 949-956. [韩宇, 刘春, 魏丽婉, 任静波, 刘品彦, 夏庆友, 2010. 家蚕酪氨酸羟化酶基因Bmth的表达及功能. 昆虫学报, 53(9): 949-956]
Kramer KJ, Morgan TD, Hopkins TL, Christensen A, Schaefer J, 1991. Insect cuticle tanning: enzymes and cross-link structure. In: Hedin PA ed. Naturally Occurring Pest Bioregulators. ACS Symposium Series No. 449. American Chemical Society, Washington DC. 87-105.
Liu X, Dai F, Guo E, Li K, Ma L, Tian L, Cao Y, Zhang G, Palli SR, Li S, 2015. 20-Hydroxyecdysone (20E) primary response gene E93 modulates 20E signaling to promote Bombyx larval-pupal metamorphosis. J. Biol. Chem., 290(45): 27370-27383.
Livak KJ, Schmittgen TD, 2001. Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods, 25(4): 402-408.
Lu YH, Jiang YY, Liu J, Zeng J, Yang XM, Wu KM, 2018. Adjustment of cropping structure increases the risk of cotton bollworm outbreaks in China. Chin. J. Appl. Entomol., 55(1): 19-24. [陆宴辉, 姜玉英, 刘杰, 曾娟, 杨现明, 吴孔明, 2018. 种植业结构调整增加棉铃虫的灾变风险. 应用昆虫学报, 55(1): 19-24]
Luo CW, Dewey EM, Sudo S, Ewer J, Hsu SY, Honegger HW, Hsueh AJW, 2005. Bursicon, the insect cuticle-hardening hormone, is a heterodimeric cystine knot protein that activates G protein-coupled receptor LGR2. Proc. Natl. Acad. Sci. USA, 102(8): 2820-2825.
Mendive FM, Van Loy TV, Claeysen S, Poels J, Williamson M, Hauser F, Grimmelikhuijzen CJP, Vassart G, Vanden Broeck J, 2005. Drosophila molting neurohormone bursicon is a heterodimer and the natural agonist of the orphan receptor DLGR2. FEBS Lett., 579(10): 2171-2176.
Nagatsu T, Levitt M, Udenfriend S, 1964. Tyrosine hydroxylase. The initial step in norepinephrine biosynthesis. J. Biol. Chem., 239(9): 2910-2917.
Reynolds SE, Taghert P, Truman JW, 1979. Eclosion hormone and bursicon titres and the onset of hormonal responsiveness during the last day of adult development in Manduca sexta (L.). J. Exp. Biol., 78: 77-86.
Song Q, 2012. Bursicon, a neuropeptide hormone that controls cuticle tanning and wing expansion. In: Gilbert LI ed. Insect Endocrinology. Academic Press, Oxford. 93-105.
Wright TRF, 1987. The genetic and molecular organization of the dense cluster of functionally related, vital genes in the DOPA decarboxylase region of the Drosophila melanogaster genome. In: Hennig W ed. Results and Problems in Cell Differentiation, Vol. 14. Structure and Function of Eukaryotic Chromosomes. Springer-Verlag, Berlin, Heidelberg, New York. 95-120.
Yue Z, Liu X, Zhou Z, Hou G, Hua J, Zhao Z, 2016. Development of a novel-type transgenic cotton plant for control of cotton bollworm. Plant Biotechnol. J., 14(8): 1747-1755.