水貂DCT基因5'UTR克隆分析与启动子活性探究

doi:10.11843/j.issn.0366-6964.2019.03.007

摘要/Abstract

摘要：

旨在克隆获得水貂DCT基因5'UTR序列并分析其结构特征，预测转录调控元件并检测启动子活性，为探究DCT基因在调控水貂毛皮颜色形成中的作用提供理论依据。本研究利用PCR扩增黑貂、白貂和咖啡貂DCT基因5'UTR，构建咖啡貂DCT基因5'UTR的pGL3-1~pGL3-7和黑貂pGL3-4~pGL3-6缺失片段的荧光素酶报告基因重组质粒，检测各片段的启动子活性；利用亚硫酸氢盐法检测3种毛色水貂DCT基因启动子区CpG岛甲基化水平。结果，克隆获得水貂DCT基因长8 203 bp的5'UTR序列，发现g.7133-7336为长204 bp的转座元件，与其高相似度的100条序列中，一条为蜕皮动物总门线虫纲的索巴利吸虫，其他均来自犬形亚目。P3和P4片段具有显著的启动子活性（P<0.05）；咖啡貂的CpG岛甲基化水平显著高于黑貂和白貂（P<0.05）；咖啡貂CC单倍型启动子活性显著低于黑貂的TT单倍型片段（P<0.05）。结果表明，水貂DCT基因5'UTR长204 bp的犬形亚目特异短散在元件Can-SINEs由蜕皮动物门的索巴利吸虫侵入动物基因组形成；基因上游32 bp元件和近端域共同作用发挥启动子活性，而GC-box和CpG岛结构沉默水貂DCT基因启动；g.-684和g.-621位点的T > C突变形成的CC单倍型导致咖啡貂DCT基因的高甲基化与低启动子活性，从而抑制真黑素合成，产生咖啡色被毛特征。

Abstract:

The research aimed to get 5'UTR sequence of mink DCT gene, characterize its structure, predict the transcriptional regulation elements, detect its promoter activity, and provide a theoretical basis for exploring the role of DCT gene in regulating the fur color formation of mink. 5'UTR of DCT gene of black mink, white mink and coffee mink were amplified and compared. Serial fragments deleted in 5'UTR, pGL3-1-pGL3-7 for coffee mink and pGL3-4-pGL3-6 for black mink, were amplified to construct the recombined luciferase reporter gene plasmid and to measure the promoter activity. The three kinds of minks with different coat colors were detected for the methylation level of CpG island in promoter of DCT gene by bisulfite sequencing PCR. 8 203 bp sequence of 5'UTR in DCT gene was cloned and a transposon with 204 bp in length in g.7133-7336 region was discovered. Among the 100 items with high similarity, one was from Soboliphyme baturini in Ecdysozoa and the others were all from Caniformia. P3 and P4 fragments had significant promoter activity (P<0.05). The methylation level in CpG island of coffee mink was significantly higher than that of black mink and white mink (P<0.05), and the promoter activity of individuals with CC haplotype in coffee mink was significantly lower than those with TT haplotype in black mink (P<0.05). The 204 bp transposable element in 5'UTR of mink DCT gene, special Can-SINEs of Caniformia, was inserted into the genome from Soboliphyme baturini into Ecdysozoa. The 32 bp element and the proximal domain in DCT co-activated the promoter, while the GC-box and CpG islands silenced the promoter activity of mink DCT gene. The CC haplotype, formed from the 2 SNPs of T > C mutation at locus of g.-684 and g.-621, resulted in the higher methylation level and the lower promoter activity in coffee mink, and might ultimately decrease the synthesis of eumelanin and form the coffee coat character.

中图分类号:

S829.9

李兰会, 杜小龙, 王麒, 葛琳涵, 张乐超, 李雪梅, 李祥龙. 水貂DCT基因5'UTR克隆分析与启动子活性探究[J]. 畜牧兽医学报, 2019, 50(3): 524-533.

LI Lanhui, DU Xiaolong, WANG Qi, GE Linhan, ZHANG Lechao, LI Xuemei, LI Xianglong. Exploration of Promoter Activity and Analysis of 5'UTR Sequence of Mink DCT Gene[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2019, 50(3): 524-533.

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https://www.xmsyxb.com/CN/Y2019/V50/I3/524

参考文献

[1] BUDD P S,JACKSON I J.Structure of the mouse tyrosinase-related protein-2/dopachrome tautomerase (Tyrp2/DCT) gene and sequence of two novel slaty alleles[J].Genomics,1995,29(1):35-43.
[2] HIROBE T,ITO S,WAKAMATSU K.The slaty (slt/Dct^slt) allele decreases the content of eumelanin,but not pheomelanin in the mouse hair[J].Pigment Cell Melanoma Res,2016,29(1):110-112.
[3] CAI Z X,PETERSEN B,SAHANA G,et al.The first draft reference genome of the American mink (Neovison vison)[J].Sci Rep,2017,7:Article number:14564.
[4] SONG X C,XU C,LIU Z Y,et al.Comparative transcriptome analysis of mink (Neovison vison) skin reveals the key genes involved in the melanogenesis of black and white coat colour[J].Sci Rep,2017,7:Article number:12461.
[5] XUE L L,LI Y N,ZHAO B L,et al.TRP-2 mediates coat color pigmentation in sheep skin[J].Mol Med Rep, 2018, 17(4):5869-5877.
[6] DENG W D,TAN Y W,WANG X Y,et al.Molecular cloning,sequence characteristics,and polymorphism analyses of the tyrosinase-related protein 2/DOPAchrome tautomerase gene of black-boned sheep (Ovis aries)[J].Genome, 2009, 52(12):1001-1011.
[7] LI L C,DAHIYA R.MethPrimer:designing primers for methylation PCRs[J].Bioinformatics,2002,18(11):1427-1431.
[8] SHAHMURADOV I A,SOLOVYEV V V.Nsite,NsiteH and NsiteM Computer tools for studying transcription regulatory elements[J].Bioinformatics,2015,31(21):3544-3545.
[9] 刘小辉,彭永东,周荣艳,等.坝上长尾鸡MITF基因核心启动子鉴定与分析[J].畜牧兽医学报,2018,49(6):1116-1123.
LIU X H,PENG Y D,ZHOU R Y,et al.Identification and analysis of the core promoter of MITF gene in Bashang long-tail chicken[J].Acta Veterinaria et Zootechnica Sinica,2018,49(6):1116-1123.(in Chinese)
[10] 王涵,陈烨,周荣艳,等.蛋鸡胱硫醚β合酶基因启动子鉴定与分析[J].畜牧兽医学报,2018,49(1):211-217.
WANG H,CHEN Y,ZHOU R Y,et al.Identification and analysis of promoter of Cystathionine beta synthase in layer[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):211-217.(in Chinese)
[11] YOKOYAMA K,YASUMOTO K,SUZUKI H,et al.Cloning of the human DOPAchrome tautomerase/tyrosinase-related protein 2 gene and identification of two regulatory regions required for its pigment cell-specific expression[J].J Biol Chem, 1994, 269(43):27080-27087.
[12] 刘春杨,张乐超,王麒,等.山羊DCT基因启动子活性区及其转录因子调控探究[J].畜牧兽医学报,2018,49(1):55-64.
LIU C Y,ZHANG L C,WANG Q,et al.The exploration of the promoter activity area and regulation by transcription factors of goat DCT gene[J].Acta Veterinaria et Zootechnica Sinica,2018,49(1):55-64.(in Chinese)
[13] JOSHI S S,TANDUKAR B,CASTANEDA M,et al.Characterization of a new,inducible transgenic mouse model with GFP expression in melanocytes and their precursors[J].Gene Expr Patterns,2018,27:76-84.
[14] JIAO Z X,MOLLAAGHABABA R,PAVAN W J,et al.Direct interaction of Sox10 with the promoter of murine Dopachrome Tautomerase (Dct) and synergistic activation of Dct expression with Mitf[J].Pigment Cell Res,2004,17(4):352-362.
[15] ARAKI R,NISHIDA S,HIRAKI Y,et al.DNA methylation of the GC box in the promoter region mediates isolation rearing-induced suppression of srd5a1 transcription in the prefrontal cortex[J].Neurosci Lett,2015,606:135-139.
[16] NAGASAWA T,TAKEDA T,MINEMURA K,et al.Oct-1,silencer sequence,and GC box regulate thyroid hormone receptor β1 promoter[J].Mol Cell Endocrinol,1997,130(1-2):153-165.
[17] 黎川,李宏江,陈璩,等.侵袭性乳腺癌ASPH基因表达与启动子甲基化的临床意义[J].四川大学学报:医学版, 2018, 49(1):54-58.
LI C,LI H J,CHEN Q,et al.Expression of ASPH gene in invasive breast cancer and its clinical significance in promoter methylation[J].Journal of Sichuan University:Medical Science Edition,2018,49(1):54-58.(in Chinese)
[18] 谷淑华,孙文星,褚维伟,等.猪NCOA3启动子甲基化对其在肌内和皮下脂肪组织差异表达的影响[J].南京农业大学学报,2016,39(1):120-126.
GU S H,SUN W X,CHU W W,et al.Effect of NCOA3 promotor methylation on its differential expression profile in intramuscular and subcutaneous adipose tissue in porcine[J].Journal of Nanjing Agricultural University,2016,39(1):120-126.(in Chinese)
[19] 潘金春,闵凡贵,王希龙.五指山小型猪PPARγ2基因启动子多态性分析[J].中国比较医学杂志,2018,28(9):21-26.
PAN J C,MIN F G,WANG X L.Analysis of polymorphisms in the promoter region of the PPARγ2 gene in Wuzhishan minipigs[J].Chinese Journal of Comparative Medicine,2018,28(9):21-26.(in Chinese)
[20] 刘子记,朱婕,牛玉,等.苦瓜α-苦瓜素基因启动子克隆与单倍型分析[J].浙江农业学报,2018,30(1):58-64.
LIU Z J,ZHU J,NIU Y,et al.Cloning and haplotype analysis of α-momorcharin promoter in bitter gourd[J].Acta Agriculturae Zhejiangensis, 2018,30(1):58-64.(in Chinese)
[21] WALTERS-CONTE K B,JOHNSON D L E,ALLARD M W,et al.Carnivore-specific SINEs (Can-SINEs):Distribution, evolution, and genomic impact[J].J Hered,2011,102(S1):S2-S10.
[22] DAS M,CHU L L,GHAHREMANI M,et al.Characterization of an abundant short interspersed nuclear element (SINE) present in Canis familiaris[J].Mamm Genome,1998,9(1):64-69.
[23] ZEHR S M,NEDBAL M A,FLYNN J J.Tempo and mode of evolution in an orthologous Can SINE[J].Mamm Genome,2001, 12(1):38-44.
[24] PENG C J,NIU L L,DENG J B,et al.Can-SINE dynamics in the giant panda and three other Caniformia genomes[J].Mob DNA, 2018, 9:32.
[25] GELALETI G B,GRANZOTTO A,LEONEL C,et al.Short interspersed CAN SINE elements as prognostic markers in canine mammary neoplasia[J].Oncol Rep,2014,31(1):435-441.