斑马鱼E3泛素连接酶基因的生物信息学分析和mRNA表达

doi:10.12160/j.issn.1672-5190.2019.08.003

摘要/Abstract

摘要： 目的对斑马鱼E3泛素连接酶基因进行生物信息学分析,并研究其mRNA表达情况。方法利用生物信息学方法分析斑马鱼E3泛素连接酶基因的cDNA序列、蛋白质二级结构、结构域、氨基酸序列同源性,并构建进化树;利用整胚原位杂交技术研究目的基因在斑马鱼体内的mRNA表达模式。结果在NCBI基因库中找出TRIM54、TRIM55a、TRIM55b、TRIM63a、TRIM63b和TRIM101 6个斑马鱼E3泛素连接酶基因。这些基因之间有较高的同源性,二级结构以α-螺旋和无规卷曲为主,编码的蛋白质均含有3个保守结构域：环锌指、B-box锌指和卷曲螺旋。进化树分析显示,所有基因聚为4组：各物种TRIM54基因聚为一组;各物种TRIM55基因聚为一组,但靠近于斑马鱼TRIM101基因分支;TRIM63基因分为一组。整胚原位杂交结果显示,这些TRIM基因的mRNA均在受精后24 h的斑马鱼的肌节中表达,且TRIM55a、TRIM63a和TRIM101基因表达量较高。这些TRIM基因在肌肉发育和肌肉功能中可能发挥作用。结论斑马鱼的6个E3泛素连接酶基因有较高的保守性,其mRNA特异性表达于肌节部位。结果对深入研究这些基因在鱼类肌肉中的作用和指导鱼类生产具有一定的意义。

关键词: 斑马鱼, E3泛素连接酶, 生物信息学, mRNA表达

Abstract: [objective] To bioinformatically characterize the E3 ubiquitin ligase genes in zebrafish and to analyze their mRNA expression profile. [Method] The cDNA sequences analysis, protein secondary structure and structural domain prediction, amino acid sequences homology alignment and phylogenetic tree establishment of E3 ubiquitin ligase genes were carried out with bioinformatical methods. The mRNA expression pattern of the target genes in zebrafish was assessed by using whole mount in situ hybridization. [Result] Six E3 ubiquitin ligase genes were found in NCBI GenBank, including TRIM54, TRIM55a, TRIM55b, TRIM63a, TRIM63b and TRIM101. There are high homologies between these genes. The secondary structure of E3 ubiquitin ligases was mainly composed of alpha helix and random coil. The conserved structural domains were RING zinc finger, B-box zinc finger and coiled coils. Phylogenetic tree analysis showed the E3 ubiquitin ligase genes were divided into four groups TRIM54 gene of different species belonged to one branch; TRIM55 were grouped together, nearing the branch of TRIM101; TRIM63 made up the fourth branch. The mRNA expression of these genes was detected in sarcomeres of zebrafish 24 h after fertilization. TRIM55a, TRIM63a and TRIM101 showed a stronger expression than the other TRIM genes did. These TRIM genes might play essential role in muscular development and function in zebrafish. [Conclusion] The E3 ubiquitin ligase genes had high conservation and their mRNA were specifically expressed in sarcomeres in zebrafish. This study provides a theoretical basis for exploring the roles of E3 ubiquitin ligase genes in fish muscle and an important guidance for fish production.

Key words: zebrafish, E3 ubiquitin ligase, bioinformatics, mRNA expression

中图分类号:

李宝钧, 王伟伟, 安丽霞. 斑马鱼E3泛素连接酶基因的生物信息学分析和mRNA表达[J]. 畜牧与饲料科学, 2019, 40(8): 12-16.

LI Bao-jun, WANG Wei-wei, AN Li-xia. Bioinformatical Analysis and mRNA Expression of E3 Ubiquitin Ligase Genes in Zebrafish[J]. Animal Husbandry and Feed Science, 2019, 40(8): 12-16.

参考文献

[1] LAWRENCE C.Advances in zebrafish husbandry and management[J].Methods in Cell Biology,2011,104(1):429-451.
[2] 吴唯唯,李盼,柯翎,等.斑马鱼在鱼类病原免疫学研究中的应用[J].福建农业学报,2014,29(6):602-607.
[3] 刘辉,戴家银.斑马鱼在生态毒理学研究及环境监测中的应用[J].中国实验动物学报,2015,23(5):529-534.
[4] DAHM R,GEISLER R.Learning from small fry: the zebrafish as a genetic model organism for aquaculture fish species[J].Marine Biotechnology,2006,8(4):329-345.
[5] 董忠典,张宁,刘志刚.斑马鱼在水产动物育种学课程教学实践应用初探[J].教育现代化,2018,5(30):216-217.
[6] FRANKE B,GASCH A,RODRIGUEZ D.Molecular basis for the fold organization and sarcomeric targeting of the muscle atrogin MuRF1[J].Open Biology,2014,4(3):130172.
[7] MACQUEEN D J,FUENTES E N,VALDÉS J A,et al. The vertebrate muscle-specific RING finger protein family includes MuRF4—a novel, conserved E3-ubiquitin ligase[J].FEBS Letters,2014,588(23):4390-4397.
[8] PERERA S,MANKOO B,GAUTEL M.Developmental regulation of MURF E3 ubiquitin ligases in skeletal muscle[J].Journal of Muscle Research and Cell Motility,2012,33(2):107-122.
[9] SPENCER J A,ELIAZER S,ILARIA RL J R,et al. Regulation of microtubule dynamics and myogenic differentiation by MURF, a striated muscle RING-finger protein[J].Journal of Cell Biology,2000,150(4):771-784.
[10] MURTON A J,CONSTANTIN D,GREENHAFF P L.The involvement of the ubiquitin proteasome system in human skeletal muscle remodeling and atrophy[J]. Biochimica et Biophysica Acta,2008,1782(12):730-743.
[11] CHEN S N,CZERNUSZEWICZ G,TAN Y,et al.Human molecular genetic and functional studies identify TRIM63, encoding muscle RING finger protein 1, as a novel gene for human hypertrophic cardiomyopathy[J]. Circulation Research,2012,111(7):907-919.
[12] MORISCOT A S,BAPTISTA I L,BOGOMOLOVAS J,et al.MuRF1 is a muscle fiber-type Ⅱ associated factor and together with MuRF2 regulates type-Ⅱ fiber trophicity and maintenance[J].Journal of Structural Biology,2010,170(2):344-353.
[13] 沈鹤. 骨骼肌发育相关的猪新基因家族MURFs的分子特征与功能初步研究[D].武汉:华中农业大学,2011.
[14] THISSE C,THISSE B.High-resolution in situ hybridization to whole-mount zebrafish embryos[J].Nature Protocols,2008,3(1):59-69.
[15] BOUDINOT P,Van Der A A L M,JOUNEAU L,et al. Origin and evolution of TRIM proteins: new insights from the complete TRIM repertoire of zebrafish and pufferfish[J].PLoS One,2011,6(7):e22022.
[16] PASQUIER J,CABAU C,NGUYEN T,et al.Gene evolution and gene expression after whole genome duplication in fish: the PhyloFish database[J].BMC Genomics,2016,17(1):368.
[17] FAGERBERG L,HALLSTRÖM B M,OKSVOLD P,et al. Analysis of the human tissue-specific expression by genome-wide integration of transcriptomics and antibody-based proteomics[J].Molecular and Cellular Proteomics,2014, 13(2):397-406.
[18] YUE F,CHENG Y,BRESCHI A,et al.A comparative encyclopedia of DNA elements in the mouse genome[J].Nature,2014,515(7527):355-364.
[19] KEDAR V,MCDONOUGH H,ARYA R, et al.Muscle-specific RING finger1 is a bona fide ubiquitin ligase that degrades cardiac troponin Ⅰ[J].Proceedings of the National Academy of Sciences of the United States of America,2004,101(52):18135-18140.