水貂AANAT基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2023.04.003

摘要/Abstract

摘要： 【目的】试验旨在对美洲水貂(Neovison vison)褪黑素(N-acetyl-5-methoxytryptamine,MT)合成的限速酶5-羟色胺-N-乙酰基转移酶(aralkylamine N-acetyltransferase,AANAT)基因进行克隆和生物信息学分析,为探究AANAT基因在水貂中的生物学功能提供参考。【方法】对水貂尾静脉采血后提取DNA,利用同源重组的方法克隆AANAT基因并分析其编码区(CDS)序列,推导成氨基酸序列后进行相似性比对及系统进化树构建,并对AANAT蛋白进行生物信息学分析。【结果】水貂AANAT基因序列长约1631 bp,CDS区长504 bp,可编码167个氨基酸。水貂AANAT氨基酸序列与雪貂相似性最高(98.2%),系统进化树分析也显示其与雪貂亲缘关系最近。生物信息学分析结果表明,水貂AANAT蛋白为疏水性蛋白,无跨膜结构域和信号肽,主要分布于细胞质中,有5个抗原结合位点为非分泌型蛋白,该蛋白含有多个磷酸化、糖基化等翻译后修饰位点。AANAT蛋白含有1个保守的N-酰基转移酶超家族结构域,该家族与哺乳动物的昼夜节律密切相关。AANAT蛋白二级结构以无规则卷曲为主,三级结构含有多个保守的催化残基。蛋白互作网络分析表明,AANAT蛋白可与多个5-羟色胺合成相关的蛋白相互作用。【结论】试验成功获得水貂AANAT基因序列,AANAT蛋白结构和特异性修饰位点可能通过定位乙酰辅酶A和5-羟色胺参与调控MT的生物合成,结果可为AANAT基因对水貂胚胎滞育的调控机制及提高繁殖力等研究提供参考。

关键词: 水貂; AANAT基因; 克隆; 生物信息学分析; 蛋白结构

Abstract: 【Objective】 The purpose of this experiment was to clone and bioinformatically analyze the gene of the rate-limiting enzyme aralkylamine-N-acetyltransferase (AANAT) for N-acetyl-5-methoxytryptamine (MT) synthesis in American mink (Neovison vison),so as to provide a reference for biological functions in mink.【Method】 DNA was extracted from the caudal vein of mink,the sequence of AANAT gene was cloned by homologous recombination,the sequence of CDS region was analyzed,the amino acid sequence of AANAT gene was deduced for similarity alignment and phylogenetic tree construction,and the AANAT protein was analyzed by bioinformatics.【Result】 The sequence of AANAT gene in mink was about 1 631 bp,and the sequence of CDS region was 504 bp,which could encode 167 amino acids.The amino acid sequence similarity of AANAT gene in mink was 98.2% with Mustela putorius furo,and the phylogenetic tree analysis also showed that it was the closest relative to Mustela putorius furo.Bioinformatics analysis results showed that AANAT protein in mink was a hydrophobic protein without transmembrane domain and signal peptide,mainly distributed in the cytoplasm,and had 5 antigen-binding sites as a non-secreted protein.AANAT protein contained multiple post-translational modification sites,such as phosphorylation and glycosylation.AANAT protein contained a conserved domain of the N-acyltransferase superfamily,which was closely related to mammalian circadian rhythms.The secondary structure of AANAT protein was dominated by random coil,and the tertiary structure contained several conserved catalytic residues.Protein network interaction analysis results showed that AANAT protein could interact with multipleserotonin synthesis-related proteins.【Conclusion】 The sequence of AANAT gene in mink was successfully obtained.The structure and specific modification sites of AANAT protein might participate in the regulation of MT biosynthesis by localizing acetyl-CoA and 5-hydroxytryptamine.The reults provided a reference for research on the regulation mechanism of AANAT gene on embryonic diapause and improving fecundity in mink.

Key words: mink; AANAT gene; cloning; bioinformatics analysis; protein structure

中图分类号:

范冰峰, 李文, 刘理想, 赵向远, 邵静, 林乐丰, 孙慧敏, 张旭林, 许保增. 水貂AANAT基因克隆及生物信息学分析[J]. 中国畜牧兽医, 2023, 50(4): 1307-1318.

FAN Bingfeng, LI Wen, LIU Lixiang, ZHAO Xiangyuan, SHAO Jing, LIN Lefeng, SUN Huimin, ZHANG Xulin, XU Baozeng. Cloning and Bioinformatics Analysis of AANAT Gene in Mink[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(4): 1307-1318.

参考文献

[1] WANG C,LI F,ZHANG Z,et al.Recent research progress in China on Haemonchus contortus[J].Frontiers in Microbiology,2017,8:1509.
[2] 郝丹,苏国生,吴晓平,等.世界水貂遗传育种的研究进展[J].黑龙江畜牧兽医,2021,7:43-47. HAO D,SU G S,WU X P,et al.Progress in research on genetic breeding of worldwide mink[J].Heilongjiang Animal Science and Veterinary Medicine,2021,7:43-47.(in Chinese)
[3] HICKMAN A B,KLEIN D C,DYDA F.Melatonin biosynthesis:The structure of serotonin N-acetyltransferase at 2.5 A resolution suggests a catalytic mechanism[J].Molecular Cell,1999,3(1):23-32.
[4] SZEWCZYK-GOLEC K,WOZNIAK A,REITER R J.Inter-relationships of the chronobiotic,melatonin,with leptin and adiponectin:Implications for obesity[J].Journal of Pineal Research,2015,59(3):277-291.
[5] KAMRUZZAMAN A S M,HIRAGAKI S,WATARI Y,et al.Clock-controlled arylalkylamine N-acetyltransferase (aaNAT) regulates circadian rhythms of locomotor activity in the American cockroach,Periplaneta americana,via melatonin/MT2-like receptor[J].Journal of Pineal Research,2021,71(2):e12751.
[6] KLEIN D C.Photoneural regulation of the mammalian pineal gland[J].Ciba Foundation Symposium,1985,117:38-56.
[7] MARONDE E,SCHOMERUS C,STEHLE J H,et al.Control of CREB phosphorylation and its role for induction of melatonin synthesis in rat pinealocytes[J].Biology of the Cell,1997,89(8):505-511.
[8] HO A K,CHIK C L.Modulation of aanat gene transcription in the rat pineal gland[J].Journal of Neurochemistry,2010,112(2):321-331.
[9] FAILLACE M P,CUTRERA R,SARMIENTO M I,et al.Evidence for local synthesis of melatonin in golden hamster retina[J].Neuroreport,1995,6(15):2093-2095.
[10] TAGLIAFERRI V,ROMUALDI D,SCARINCI E,et al.Melatonin treatment may be able to restore menstrual cyclicity in women with PCOS:A pilot study[J].Reproductive Sciences,2018,25(2):269-275.
[11] ZHANG W L,ZHANG Z L,PENG J,et al.Expression of arylalkylamine N-acetyltransferase (AANAT) and acetylserotonin O-methyltransferase (ASMT) in the corpus luteum of pregnant sows and synthesis of melatonin in luteal cells[J].Cell and Tissue Research,2022,388(1):167-179.
[12] RENFREE M B,SHAW G.Diapause[J].Annual Review of Physiology,2000,62:353-375.
[13] 韩玉萍,赵向远,范冰峰,等.基于高通量测序的水貂胚胎滞育期和激活期卵巢转录组分析[J].中国畜牧兽医,2021,48(11):4035-4046. HAN Y P,ZHAO X Y,FAN B F,et al.Transcriptomic analysis of mink ovaries at embryonic diapause and activation stages based on high-throughput sequence technology[J].China Animal Husbandry & Veterinary Medicine,2021,48(11):4035-4046.(in Chinese)
[14] 韩玉萍,李文,范冰峰,等.胚胎滞育的研究进展[J].特产研究,2020,42(6):60-67. HAN Y P,LI W,FAN B F,et al.Research progress on embryo diapause[J].Special Wild Economic Animal and Plant Research, 2020,42(6):60-67.(in Chinese)
[15] TAKEDA M,SUZUKI T.Circadian and neuroendocrine basis of photoperiodism controlling diapause in insects and mites:A review[J].Frontiers in Physiology,2022,13:867621.
[16] CARLOMAGNO G,MININI M,TILOTTA M,et al.From implantation to birth:Insight into molecular melatonin functions[J].International Journal of Molecular Sciences,2018,19(9):2802.
[17] DENG L,LI C,CHEN L,et al.Research advances on embryonic diapause in mammals[J].Animal Reproduction Science,2018,198:1-10.
[18] TAN D X,MANCHESTER L C,QIN L,et al.Melatonin:A mitochondrial targeting molecule involving mitochondrial protection and dynamics[J].International Journal of Molecular Sciences,2016,17(12):2124.
[19] KLEIN D C,COON S L,ROSEBOOM P H,et al.The melatonin rhythm-generating enzyme:Molecular regulation of serotonin N-acetyltransferase in the pineal gland[J].Recent Progress in Hormone Research,1997,52:307-357.
[20] VETTING M W,DE CARVALHO L P S,YU M,et al.Structure and functions of the GNAT superfamily of acetyltransferases[J].Archives of Biochemistry and Biophysics,2005,433(1):212-226.
[21] VETTING M W,RODERICK S L,YU M,et al.Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases[J].Protein Science:A Publication of the Protein Society,2003,12(9):1954-1959.
[22] VETTING M W,HEGDE S S,JAVID-MAJD F,et al.Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates[J].Nature Structural Biology,2002,9(9):653-658.
[23] SUGDEN D,VANECEK J,KLEIN D C,et al.Activation of protein kinase C potentiates isoprenaline-induced cyclic AMP accumulation in rat pinealocytes[J].Nature,1985,314(6009):359-361.
[24] COON S L,ROSEBOOM P H,BALER R,et al.Pineal serotonin N-acetyltransferase:Expression cloning and molecular analysis[J].Science,1995,270(5242):1681-1683.
[25] POIREL V J,BOGGIO V,DARDENTE H,et al.Contrary to other non-photic cues,acute melatonin injection does not induce immediate changes of clock gene mRNA expression in the rat suprachiasmatic nuclei[J].Neuroscience,2003,120(3):745-755.
[26] EICHLER J.Protein glycosylation[J].Current Biology,2019,29(7):R229-R231.
[27] SCHJOLDAGER K T,NARIMATSU Y,JOSHI H J,et al.Global view of human protein glycosylation pathways and functions[J].Nature Reviews Molecular Cell Biology,2020,21(12):729-749.
[28] MACLEA K S,KRIESER R J,EASTMAN A.Structural requirements of human DNase Ⅱ alpha for formation of the active enzyme:The role of the signal peptide,N-glycosylation,and disulphide bridging[J].Biochemical Journal,2003,371:867-876.
[29] WANG B,DAI T,SUN W,et al.Protein N-myristoylation:Functions and mechanisms in control of innate immunity[J].Cellular and Molecular Immunology,2021,18(4):878-888.
[30] FARAZI T A,WAKSMAN G,GORDON J I.The biology and enzymology of protein N-myristoylation[J].The Journal of Biological Chemistry,2001,276(43):39501-39504.
[31] 杨爱玲,李广栋,吴昊,等.褪黑素合成酶AANAT/ASMT基因过表达绵羊生物安全评价研究[J].畜牧兽医学报,2020,51(7):1563-1572. YANG A L,LI G D,WU H,et al.Study of biosafety evaluation melatonin AANAT/ASMT overexpressed sheep[J].Acta Veterinaria et Zootechnica Sinica,2020,51(7):1563-1572.(in Chinese)
[32] GUENTER J,LENARTOWSKI R.Molecular characteristic and physiological role of DOPA-decarboxylase[J].Postepy Higieny I Medycyny Doswiadczalnej, 2016,70:1424-1440.
[33] SON S Y,MA J,KONDOU Y,et al.Structure of human monoamine oxidase A at 2.2-A resolution:The control of opening the entry for substrates/inhibitors[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(15):5739-5744.
[34] THOMPSON M A,MOON E,KIM U J,et al.Human indolethylamine N-methyltransferase:cDNA cloning and expression,gene cloning,and chromosomal localization[J].Genomics,1999,61(3):285-297.