中华鳖dct基因克隆及表达分析

doi:10.3969/j.issn.1000-1565.2019.06.009

摘要/Abstract

摘要： 为了阐明中华鳖酪氨酸酶家族成员dct基因的结构与表达特征,根据Ensembl数据库的中华鳖dct基因序列设计引物,运用RT-PCR技术进行开放阅读框(opening reading frame,ORF)克隆,并进行生物信息学分析,进而采用实时荧光定量PCR(quantitative real-time PCR,q-PCR)技术检测正常体色和黄色中华鳖肺、肾脏、裙边和肌肉组织的mRNA表达水平.结果显示中华鳖dct基因ORF区全长1 578 bp,编码525个氨基酸,2种体色鳖dct基因序列完全一致;氨基酸序列分析得C末端无L-亮氨酸基序;N末端含有1个信号肽结构域;预测蛋白(成熟肽)分子质量为56.449 ku,理论等电点为6.67,不稳定指数为38.71,表明DCT蛋白是酸性稳定蛋白;预测DCT蛋白为亲水性蛋白;含有跨膜结构域、类表皮生长因子结构域、层粘连蛋白型类表皮生长因子结构域和2个铜离子结合结构域.系统发育树分析显示,在dct基因进化过程中,中华鳖与绿海龟和锦龟的亲缘关系较近.q-PCR结果表明:dct基因在同一个体各组织中均有表达,肺组织中相对表达量最高,且极显著高于其他3种组织;黄色中华鳖肺、肾脏和裙边组织dct 表达水平与正常体色鳖的相应组织均存在显著性差异.结果表明,中华鳖体色变异与dct基因突变无关,但dct基因表达改变可能对体色变异起了一定的作用.

关键词: 中华鳖, dct基因, 克隆, 生物信息学, q-PCR

Abstract: This work aims to clarify the structure and expression characteristics of dct gene in Pelodiscus sinensis, which belongs to tyrosinase family. Primers were designed according mRNA sequence of dct gene in Ensembl online database. Opening reading frame(ORF )region was cloned using RT-PCR and the bioinformatics analysis of dct ORF sequence was carried out. At the same time, mRNAs expression levels of lung, kidney, calipash and muscle tissue in P. sinensis were detected via q-PCR method. The results showed that the full length of ORF of dct gene in P.sinensis was 1 578 bp, encoding a putative 525 aa; The sequence- DOI:10.3969/j.issn.1000-1565.2019.06.009中华鳖dct基因克隆及表达分析刘文婷¹,许鹏²,张娜¹,管越强¹,张耀红³(1. 河北大学生命科学学院,河北保定 071002;2. 河北省药品检验研究院,河北石家庄 050011;3. 保定水产技术推广站,河北保定 071051)摘要:为了阐明中华鳖酪氨酸酶家族成员dct基因的结构与表达特征,根据Ensembl数据库的中华鳖dct基因序列设计引物,运用RT-PCR技术进行开放阅读框(opening reading frame,ORF)克隆,并进行生物信息学分析,进而采用实时荧光定量PCR(quantitative real-time PCR,q-PCR)技术检测正常体色和黄色中华鳖肺、肾脏、裙边和肌肉组织的mRNA表达水平.结果显示中华鳖dct基因ORF区全长1 578 bp,编码525个氨基酸,2种体色鳖dct基因序列完全一致;氨基酸序列分析得C末端无L-亮氨酸基序;N末端含有1个信号肽结构域;预测蛋白(成熟肽)分子质量为56.449 ku,理论等电点为6.67,不稳定指数为38.71,表明DCT蛋白是酸性稳定蛋白;预测DCT蛋白为亲水性蛋白;含有跨膜结构域、类表皮生长因子结构域、层粘连蛋白型类表皮生长因子结构域和2个铜离子结合结构域.系统发育树分析显示,在dct基因进化过程中,中华鳖与绿海龟和锦龟的亲缘关系较近.q-PCR结果表明:dct基因在同一个体各组织中均有表达,肺组织中相对表达量最高,且极显著高于其他3种组织;黄色中华鳖肺、肾脏和裙边组织dct 表达水平与正常体色鳖的相应组织均存在显著性差异.结果表明,中华鳖体色变异与dct基因突变无关,但dct基因表达改变可能对体色变异起了一定的作用.关键词:中华鳖;dct基因;克隆;生物信息学;q-PCR中图分类号:S917.4 文献标志码:A 文章编号:1000-1565(2019)06-0619-10Cloning and expression analysis of dct gene in Pelodiscus sinensisLIU Wenting¹, XU Peng², ZHANG Na¹, GUAN Yueqiang¹, ZHANG Yaohong³(1. College of Life Sciences,Hebei University,Baoding 071002, China;2. Hebei Institute for Drug Control and Testing, Shijiazhuang 050011, China;3. Baoding Fishery Technology Extension Station, Baoding 071051, China)Abstract: This work aims to clarify the structure and expression characteristics of dct gene in Pelodiscus sinensis, which belongs to tyrosinase family. Primers were designed according mRNA sequence of dct gene in Ensembl online database. Opening reading frame(ORF )region was cloned using RT-PCR and the bioinformatics analysis of dct ORF sequence was carried out. At the same time, mRNAs expression levels of lung, kidney, calipash and muscle tissue in P. sinensis were detected via q-PCR method. The results showed that the full length of ORF of dct gene in P.sinensis was 1 578 bp, encoding a putative 525 aa; The sequence- 收稿日期:2019-03-19 基金项目:河北省自然科学基金资助项目(C2016201207);河北省现代农业产业技术体系淡水养殖创新团队(HBCT2018180203) 第一作者:刘文婷(1990—),女,安徽合肥人,河北大学在读硕士研究生.E-mail: 280408479@qq.com 通信作者:管越强(1973—),男,河北深泽人,河北大学教授,博士,主要从事水生动物营养与免疫研究.E-mail:guanyueqiang@hbu.edu.cn第6期刘文婷等:中华鳖dct基因克隆及表达分析of dct gene was completely identical in two groups of turtles with different body colours of P. sinensis. The amino acid sequence analysis result showed that L-leocine motif was not observed in the C-terminus. A signal peptide domain was located in the N-terminus. The deduced molecular weight of DCT protein(Mature Peptide)was 56.449 ku, the theoretical isoelectric point was 6.67, and the instability index was 38.71, which indicated it was an acid stable protein. Prediction of hydrophilicity showed that DCT protein was a hydrophilic protein. It contain transmembrane domain, EGF-like domain, laminin-type EGF-like(LE)domain and two copper ion-binding domains. Phylogenetic tree of DCT protein showed that P.sinesis, green sea turtle Chelonia mydas, and Western painted turtle Chrysemys picta clustered into one clade, and the relationship among them was close. The results of q-PCR showed that dct gene was expressed in each detected tissue of the same individual and the expression level of lung tissue was highest and was very significantly higher than the other three detected tissues; In the same tissues of two group with different body colours, there were significant differences in lung, kidney and calipash, respectively.The results showed that the body colour variation of P.sinensis was not related to the mutation of dct gene, and the change of dct gene expression may play a role in the body color variation.

Key words: Pelodiscus sinensis, dct gene, cloning, bioinformatics, q-PCR

中图分类号:

S917.4

刘文婷,许鹏,张娜,管越强,张耀红. 中华鳖dct基因克隆及表达分析[J]. 河北大学学报(自然科学版), 2019, 39(6): 619-628.

LIU Wenting, XU Peng, ZHANG Na, GUAN Yueqiang, ZHANG Yaohong. Cloning and expression analysis of dct gene in Pelodiscus sinensis[J]. Journal of Hebei University (Natural Science Edition), 2019, 39(6): 619-628.

参考文献

[1] TSUKAMOTO K, JACKSON I J, URABE K, et al. A second tyrosinase-related protein, TRP-2, is a melanogenic enzyme termed DOPAchrome tautomerase[J]. Embo Journal, 1992, 11(2):519-526. DOI:http://dx.doi.org/10.1111/j.1559-3584.1927.tb04229.x.
[2] JIANG S, LIU X M, DAI X, et al. Regulation of DHICA-mediated antioxidation by dopachrome tautomerase: implication for skin photoprotection against UVA radiation[J]. Free Radical Biology & Medicine, 2010, 48(9):1144-1151. DOI:10.1016/j.freeradbiomed.2010.01.033.
[3] HEARING V J, TSUKAMOTO K, URABE K, et al. Functional properties of cloned melanogenic proteins[J]. Pigment Cell Research, 1992, 5(5):264-270. DOI:10.1111/j.1600-0749.1992.tb00547.x.
[4] POPA I L, MILAC A L, SIMA L E, et al. Cross-talk between dopachrome tautomerase and caveolin-1 is melanoma cell phenotype specific and potentially involved in tumor progression[J]. Journal of Biological Chemistry, 2016, 291(24):12481. DOI:10.1074/jbc.M116.714733.
[5] 刘伟兰, 李祥龙, 周荣艳, 等. 不同物种TYRP2基因完整编码区生物信息学分析[J]. 河南农业科学, 2011, 40(10): 144-148. DOI:10.15933/j.cnki.1004-3268.2011.10.003.
[6] 白春雨,高玉花,庞全海.影响动物毛色的基因[J].国外畜牧学(猪与禽),2008, 28(5):71-73. DOI:10.3969/j.issn.1001-0769.2008.05.026.
[7] HIROBE T, ABE H. The slaty mutation affects the morphology and maturation of melanosomes in the mouse melanocytes[J]. Pigment Cell Research, 2006, 19(5):454-459. DOI:10.1111/j.1600-0749.2006.00335.x.
[8] GUYONNEAU L, MURISIER F, ROSSIER A, et al. Melanocytes and pigmentation are affected in dopachrome tautomerase knockout mice[J]. Molecular & Cellular Biology, 2004, 24(8):3396-3403. DOI:10.1128/MCB.24.8.3396-3403.2004.
[9] APRIL C S, JACKSON I J, KIDSON S H. Molecular cloning and sequence analysis of a chicken cDNA encoding tyrosinase-related protein-2/DOPAchrome tautomerase[J]. Gene, 1998, 219(1/2):45-53. DOI:10.1016/S0378-1119(98)00403-X.
[10] CAMACHO-HÜBNER A, RICHARD C, BEERMANN F. Genomic structure and evolutionary conservation of the tyrosinase gene family from Fugu[J]. Gene, 2002, 285(1/2):59-68. DOI:10.1016/S0378-1119(02)00411-0.
[11] KUMASAKA M, SATO S, YAJIMA I, et al. Isolation and developmental expression of tyrosinase family genes in Xenopus laevis[J]. Pigment Cell Research, 2003, 16(5):455-462. DOI:10.1034/j.1600-0749.2003.00064.x.
[12] 邵庆均. 中华鳖体内Vc合成及其对饲料Vc需求的研究[D]. 杭州: 浙江大学, 2007.
[13] 王爱民,窦超,吴文静,等. 中华鳖幼鳖肌肉营养成分与品质的评价[J]. 江苏农业科学, 2016, 44(1): 269-272. DOI:10.15889/j.issn.1002-1302.2016.01.080.
[14] 张显良,肖放,李书民,等.中国渔业统计年鉴[M].北京:中国农业出版社,2017.
[15] 刘文婷,张贝贝,张耀红,等. 中华鳖实时定量PCR内参基因的筛选与评价[J]. 内蒙古大学学报(自然科学版), 2017, 48(5): 531-537. DOI:10.13484/j.nmgdxxbzk.20170508.
[16] LIVAK K J, SCHMITTGEN T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.DOI:10.1006/meth.2001.1262.
[17] 李闯,徐云明,唐峰,等.小尾寒羊溶菌酶基因的克隆与差异表达分析[J].中国畜牧兽医, 2013, 40(3):19-22. DOI:10.3969/j.issn.1671-7236.2013.03.004.
[18] COSTIN G E, VALENCIA J C, WAKAMATSU K, et al. Mutations in dopachrome tautomerase(Dct)affect eumelanin/pheomelanin synthesis, but do not affect intracellular trafficking of the mutant protein[J]. Biochemical Journal, 2005, 391(2):249-259. DOI:10.1042/bj20042070.
[19] NEGROIU G, DWEK R A, PETRESCU S M. The inhibition of early N-glycan processing targets TRP-2 to degradation in B16 melanoma cells[J]. Journal of Biological Chemistry, 2003, 278(29):27035-27042. DOI:10.1074/jbc.m303167200.
[20] NEGROIU G, DWEK R A, PETRESCU S M. Tyrosinase-related protein-2 and -1 are trafficked on distinct routes in B16 melanoma cells[J]. Biochemical and Biophysical Research Communications, 2005, 328(4):914-921. DOI:10.1016/j.bbrc.2005.01.040.
[21] GASTEL M V, BUBACCO L, GROENEN E J J, et al. EPR study of the dinuclear active copper site of tyrosinase from Streptomyces antibioticus[J]. FEBS Letters, 2000, 474(2/3):228-232. DOI:10.1016/s0014-5793(00)01609-4.
[22] HIMMELWRIGHT R S, EICKMAN N C, LUBIEN C D, et al. Chemical and spectroscopic studies of the binuclear copper active site of Neurospora tyrosinase: comparison to hemocyanins[J]. Journal of the American Chemical Society, 1980, 102(24):7339-7344. DOI:10.1021/ja00544a031.
[23] LINZEN B. Blaues blut: struktur und evolution der hämocyanine[J]. Naturwissenschaften, 1989, 76(5): 206-211. DOI:10.1007/bf00627687.
[24] LANG W H, HOLDE K E V. Cloning and sequencing of Octopus dofleini hemocyanin cDNA: derived sequences of functional units Ode and Odf[J]. Proceedings of the National Academy of Sciences of the United States of America, 1991, 88(1):244-248. DOI:10.2307/2355746.
[25] PALUMBO A, SOLANO F, MISURACA G, et al. Comparative action of dopachrome tautomerase and metal ions on the rearrangement of dopachrome[J]. Biochimica et Biophysica Acta(BBA)- General Subjects, 1991, 1115(1): 1-5. DOI:10.1016/0304-4165(91)90003-Y.
[26] 李康乐. 瓯江彩鲤体色相关基因Sox10、Agouti、Tyrp1、Dct的分子克隆与表达分析[D]. 上海: 上海海洋大学, 2014.
[27] 陈兴婷. 三种不同品系红罗非鱼耐寒性能比较以及体色相关基因表达分析[D]. 南京: 南京农业大学, 2016.
[28] CAO D, GONG S, YANG J, et al. Melanin deposition ruled out as cause of color changes in the red-eared sliders(Trachemys scripta elegans)[J]. Comparative Biochemistry and Physiology B-Biochemistry & Molecular Biology. 2018, 217: 79-85. DOI:10.1016/j.cbpb.2017.12.011.