版纳微型猪近交系FANK1基因CDS克隆、qPCR表达及功能生物信息学分析

摘要/Abstract

摘要： 【目的】获得猪FANK1基因CDS序列、组织表达和蛋白功能信息。【方法】以GenBank下载的猪及近缘物种的FANK1 mRNA序列为参考序列，设计特异引物扩增版纳微型猪近交系（BMI）FANK1基因。应用qPCR技术分析15个重要组织的mRNA表达谱，并对其蛋白质序列进行功能生物信息学分析。【结果】研究获得了BMI FANK1 1041 bp的编码区序列（GenBank登录号：KU705617和KU705618，对应的氨基酸登录号：AOC89035和AOC89036），编码346个氨基酸，蛋白质分子量（Mw）为38.44 kD，等电点（pI）为8.44。基因组结构分析表明FANK1基因定位于猪14号染色体，有11个外显子和10个内含子。多组织荧光定量表达分析表明FANK1基因在睾丸、尿道球腺、精囊腺、脑、脾中呈高表达水平；在其他组织中呈中低表达水平。功能生物信息学分析表明FANK1蛋白质存在2个保守结构域，无跨膜结构，无信号肽序列，N末端和C末端均亲水，有4类功能活性位点。系统进化分析表明，BMI与牛、羊亲缘关系最近。【结论】为进一步研究FANK1基因在猪精细胞分裂过程中及调节信号通路方面的作用及功能奠定基础。

关键词: 纤连蛋白3（FN3）, 锚蛋白重复（ANK）, 版纳微型猪近交系, 组织表达, 生物信息学

Abstract: 【Objective】 In order to obtain the CDS sequence, tissue expression profile and protein function information of swine FANK1 gene.【Methods】 The FANK1 mRNA sequence of pig and related species from GenBank was used as reference sequences, We designed specific primers and amplified Banna mini-pig inbred line (BMI) FANK1 gene. qPCR was applied to analysis the mRNA expression profiles of 15 important tissues. Protein sequence was used to carry out functional bioinformatics analysis and construct FANK1 phylogenetic tree. 【Results】 A coding sequence of 1041 bp (GenBank accession number: KU705617 and KU705618, the corresponding amino acid sequence accession number: AOC89035 and AOC89036) of BMI FANK1 was obtained, which encodes a protein of 346 amino acids, protein molecular weight 38.44 kD, and isoelectric point 8.44. Genomic structural analysis revealed that the FANK1 gene was located on porcine chromosome 14 and consisted of 11 exons and 10 introns. Fluorescence quantitative expression indicated that FANK1 expressed highly in the testis, urethral gland, seminal vesicle, brain, spleen and low expression in the other tissues. Functional bioinformatics analysis indicated that FANK1 protein contained two conserved domains, no transmembrane region, no signal peptide sequences; its N-terminal and C-terminal were hydrophilic; it has four kinds functional active sites. Phylogenetic analysis demonstrated that BMI had the closest relationship with cattle and sheep. 【Conclusion】 The results of the study will lay a foundation for further study of the gene about in the process of spermatocyte division and regulation of signal pathway.

Key words: Fibronectin type Ⅲ (FN3), Ankyrin repeats (ANK), Banna mini-pig inbred line (BMI), Tissue expression, Bioinformatics

王配. 版纳微型猪近交系FANK1基因CDS克隆、qPCR表达及功能生物信息学分析[J]. , 2017, 35(03): 408-432.

参考文献

[1] Eddy E M, O'Brien D A. Gene expression during mammalian meiosis [J]. Current Topics in Developmental Biology, 1998, 37: 141-200.
[2] Eddy E M. Male germ cell gene expression [J]. Recent Progress in Hormone Research, 2002, 57: 103-128.
[3] Hecht N B. Molecular mechanisms of male germ cell differentiation [J]. Bioessays, 1998, 20(7): 555-561.
[4] Zheng Z, Zheng H, Yan W. Fank1 is a testis-specific gene encoding a nuclear protein exclusively expressed during the transition from the meiotic to the haploid phase of spermatogenesis [J]. Gene Expression Patterns, 2007, 7(7): 777-783.
[5] Mosavi L K, Minor D L, Peng Z Y. Consensus-derived structural determinants of the ankyrin repeat motif [J]. Proceedings of the National Academy of Sciences, 2002, 99(25): 16029-16034.
[6] Shalini S, Bansal M P. Role of selenium in spermatogenesis: differential expression of cjun and cfos in tubular cells of mice testis [J]. Molecular and Cellular Biochemistry, 2006, 292(1-2): 27-38.
[7] Wang H, Song W, Hu T, et al. Fank1 interacts with Jab1 and regulates cell apoptosis via the AP-1 pathway [J]. Cellular and Molecular Life Sciences, 2011, 68(12): 2129-2139.
[8] Claret F X, Hibi M, Dhut S, et al. A new group of conserved coactivators that increase the specificity of AP-1 transcription factors [J]. Nature, 1996, 383(6599): 453-457.
[9] Dong W W, Huang H L, Yang W, et al. Testis-specific Fank1 gene in knockdown mice produces oligospermia via apoptosis [J]. Asian Journal of Andrology, 2014, 16(1): 124-130.
[10] Yu P, Zhang L, Li S, et al. Screening and analysis of porcine endogenous retrovirus in Chinese Banna minipig inbred line [J]. Transplant Proceedings, 2004, 36(8): 2485-2487.
[11] Zeng R, Zeng Y Z. Molecular cloning and characterization of SLA-DR genes in the 133-family of the Banna mini-pig inbred line [J]. Animal Genetics, 2005, 36(3): 267-269.
[12] Huo J L, Wang P, Zhao Y, et al. Molecular cloning, mRNA expression and characterization of a novel FAIM1 gene from Chinese Banna mini-pig inbred line (BMI) [J]. Journal of Animal and Veterinary Advances, 2012, 11(8): 1080-1086.
[13] Wang P, Huo H L, Wang S Y, et al. Cloning, sequence characterization, and expression patterns of members of the porcine TSSK family [J]. Genetics and Molecular Research, 2015, 14(4): 14908-14919.
[14] Hwang K C, Park S Y, Park S P, et al. Specific maternal transcripts in bovine oocytes and cleavaged embryos: identification with novel DDRT-PCR methods [J]. Molecular Reproduction and Development, 2005, 71(3): 275-283.
[15] 王镜岩, 朱圣庚, 徐长法. 生物化学 [M]. 第三版. 北京: 高等教育出版社, 2002: 207-220.
[16] Marchler-Bauer A, Lu S, Anderson J B, et al. CDD: a Conserved Domain Database for the functional annotation of proteins [J]. Nucleic Acids Res, 2011, 39: 225-229.
[17] Marchler-Bauer A, Derbyshire M K, Gonzales N R, et al. CDD: NCBI's conserved domain database [J]. Nucleic Acids Res, 2015, 4: 222-226.
[18] Marchler-Bauer A, Anderson J B, Chitsaz F, et al. CDD: specific functional annotation with the Conserved Domain Database [J]. Nucleic Acids Res, 2009, 37: 205-210.
[19] Marchler-Bauer A, Bryant S H. CD-Search: protein domain annotations on the fly [J]. Nucleic Acids Res, 2004, 32(Web Server issue): W327-W331.