金川牦牛BCL2A1基因克隆及生物信息学分析

doi:10.16431/j.cnki.1671-7236.2018.06.005

摘要/Abstract

摘要：

为探讨金川牦牛抗凋亡因子——B淋巴细胞瘤2相关蛋白A1（B cell lymphoma 2 related A1，BCL2A1）基因特点，本试验采用PCR方法以金川牦牛血液DNA为模板扩增BCL2A1基因，用DNAStar、ExPASy、ABCpred等生物信息学软件分析基因序列和蛋白质结构。结果显示，克隆获得的片段长622 bp，GenBank登录号：MG459158，开放阅读框为516 bp，共编码171个氨基酸，其中缬氨酸（V）、赖氨酸（K）的含量较多，分别为9.4%和8.8%。BCL2A1分子质量为19.46 ku，理论等电点为4.99，不稳定系数为17.44，具有跨膜螺旋结构域（Scores>500）。二级结构主要为α-螺旋，占60.82%，有一个BCL2结构域，三级结构模型与人BCL2A1同源性最高（72.79%），BCL2A1存在潜在的B细胞抗原表位。与野牦牛氨基酸序列进行比对显示，金川耗牛BCL2A1存在5个氨基酸差异位点。NJ法系统进化分析显示，金川牦牛与野牦牛同源性为98.5%，亲缘关系最近。本试验成功克隆了金川牦牛BCL2A1基因，其在哺乳动物中具有较高的保守性，为深入研究牦牛BCL2A1基因功能提供理论依据。

关键词: BCL2A1基因; 金川牦牛; 克隆; 蛋白质分析; 系统进化分析

Abstract:

This study was aimed to explore the characteristics of B cell lymphoma 2 related A1 (BCL2A1) gene in Jinchuan yak.BCL2A1 gene was amplified by PCR using Jinchuan yak blood DNA as a template,and the gene sequences and protein structures were analyzed using DNAStar,ExPASy,ABCpred and other bioinformatics softwares.The results showed that the length of BCL2A1 gene was 622 bp,GenBank accession number:MG459158.The open reading frame was 516 bp,which could encode 171 amino acids with molecular weight of 19.46 ku.The higher proportions of amino acids were Val (V) and Lys (K),up to 9.4% and 8.8%,respectively.The theoretical isoelectric point was 4.99.The unstability index was 17.44,with transmembrane helices(Scores>500).The main secondary structure was alpha helix of 60.82%.There was a BCL2 domain,the tertiary structure model had the highest homology with human BCL2A1 (72.79%).BCL2A1 had a B cell potential epitope.Alignment with Bos mutus amino acid sequence showed that there were five amino acids mutations.Phylogenetic relationships revealed that the genetic relationship was the closest between Jinchuan yak and Bos mutus,and the homology was 98.5%.The BCL2A1 gene of Jinchuan yak was cloned successfully,which was highly conserved in mammals.It provided a theoretical evidence for further study on the function of BCL2A1 gene in yak.

Key words: BCL2A1 gene; Jinchuan yak; cloning; protein analysis; phylogenetic analysis

中图分类号:

S823.8⁺5

吴开年, 王利, 李晨阳. 金川牦牛BCL2A1基因克隆及生物信息学分析[J]. 《中国畜牧兽医》, 2018, 45(6): 1463-1470.

WU Kainian, WANG Li, LI Chenyang. Cloning and Bioinformatics Analysis of BCL2A Gene in Jinchuan Yak[J]. , 2018, 45(6): 1463-1470.

参考文献

[1] SMYTH L A,MEADER L,XIAO F,et al.Constitutive expression of the anti-apoptotic Bcl-2 family member A1 in murine endothelial cells leads to transplant tolerance[J].Clinical and Experimental Immunology,2017,188(2):219-225.
[2] 车江雁,王宝宏.BCL2、BCL6、MUM1蛋白表达及其与弥漫性大B细胞瘤预后的关系[J].现代医药卫生,2016,32(1):25-30. CHEN J Y,WANG B H.Expressions of BCL2,BCL6 and MUM1 protein and their relationship with prognosis of diffuse large B cell lymphoma[J].Journal of Modern Medicine & Health,2016,32(1):25-30.(in Chinese)
[3] 王静,崔玉东.BCL-2家族蛋白对线粒体膜通透作用的调控和caspases的影响[J].中国畜牧兽医,2006,33(4):28-30. WANG J,CUI Y D.Regulation of BCL-2 family protein on mitochondrial membrane permeability and effects of caspases[J]. China Animal Husbandry & Veterinary Medicine,2006,33(4):28-30.(in Chinese)
[4] SOCHALSKA M,SCHULER F,WEISS J G,et al.MYC selects against reduced BCL2A1/A1 protein expression during B cell lymphomagenesis[J].Oncogene,2017,36(15):2066-2073.
[5] 卢玉婷,李月红,吉尚雷,等.B细胞淋巴瘤-2家族蛋白与细胞凋亡[J].中国畜牧兽医,2014,41(1):123-126. LU Y T,LI Y H,JI S L,et al.B-cell lymphoma-2 family proteins and cell apoptosis[J].China Animal Husbandry & Veterinary Medicine,2014,41(1):123-126.(in Chinese)
[6] WERNER A B,DE VRIES E,TAIT S W,et al.Bcl-2 family member Bfl-1/A1 sequesters truncated bid to inhibit is collaboration with pro-apoptotic Bak or Bax[J].The Journal of Biological Chemistry,2002,277(25):22781-22788.
[7] 陈礼仪,田金举,任婧,等.植物花色苷对血小板凋亡通路中BCL2家族影响[J].中国公共卫生,2014,30(1):67-69. CHEN L Y,TIAN J J,REN J,et al.Effects of anthocyanin on BCL2 family of platelet apoptosis pathway[J].Chinese Journal of Public Health,2014,30(1):67-69.(in Chinese)
[8] IGLESIAS M,AUGUSTIN JJ,ALVAREZ P,et al.Selective impairment of Th17-differentiation and protection against autoimmune arthritis after overexpression of BCL2A1 in T lymphocytes[J].PLoS One,2016,11(7):e0159714.
[9] 郝志敏.Wnt11和BCL2A1在完全性葡萄胎中的表达及临床意义[D].杭州:浙江大学,2016. HAO Z M.Immunohistochemical expression and clinical significance of Wnt11 and BCL2A1 in complete moles[J].Hangzhou:Zhejiang University,2016.(in Chinese)
[10] 文勇立,李昌平,钟光辉,等.牦牛的泌乳性能及提高产奶量的途径[J].四川草原,2003,1:1-12. WEN Y L,LI C P,ZHONG G H,et al.Milk performance of the yak and methods for improvement[J].Journal of Sichuan Grassland,2003,1:1-12.(in Chinese)
[11] 艾鹥,文勇立,傅昌秀,等.金川多胸椎牦牛宰后肌肉矿物质、脂肪酸及肉色分析[J].食品科学,2013,34(16):251-255. AI Y,WEN Y L,FU C X,et al.Analysis of muscle trace elements,fatty acid composition and color parameters of Jinchuan multi-vertebrate yak[J].Food Science,2013,34(16):251-255.(in Chinese)
[12] 李善蓉,文勇立,马定惠,等.金川多肋牦牛种群调查[J].家畜生态学报,2014,35(1):65-70. LI S R,WEN Y L,MA D H,et al.The population survey of Jinchuan multi-rib yak[J]. Journal of Domestic Animal Ecology,2014,35(1):65-70.(in Chinese)
[13] 钟金城,赵素君,陈智华,等.牦牛品种的遗传多样性及其分类研究[J].中国农业科学,2006,39(2):389-397. ZHONG J C,ZHAO S J,CHEN Z H,et al.Study on genetic diversity and classification of the yak[J].Scientia Agricultura Sinica,2006,39(2):389-397.(in Chinese)
[14] SOCHALSKA M,OTTINA E,TUZLAK S,et al.Conditional knockdown of BCL2A1 reveals rate-limiting roles in BCR-dependent B-cell survival[J].Cell Death and Differentiation,2016,23(4):628-639.
[15] 周玲玲,李岩,梁杜,等.鹌鹑早期胚胎bcl-2基因表达的差异及发育性变化[J].中国畜牧兽医,2010,37(4):56-59. ZHOU L L,LI Y,LIANG D,et al.The Expresstional differences and developmental changes of bcl-2 gene in early embryos of quail[J].China Animal Husbandry & Veterinary Medicine,2010,37(4):56-59.(in Chinese)
[16] 张梅,周玲玲,张苗苗,等.Bcl-2、PCNA基因在鸡、鹌鹑及其杂交种睾丸组织中的表达研究[J].中国畜牧兽医,2017,44(7):2050-2056. ZHANG M,ZHOU L L,ZHANG M M,et al.Study on expression of Bcl-2 and PCNA genes in testicular tissue of chickens,quails and chicken-quail hybrids[J].China Animal Husbandry & Veterinary Medicine,2017,44(7):2050-2056.(in Chinese)
[17] 高薇,侯微,李伟,等.细胞凋亡机制研究进展[J].中国畜牧兽医,2014,41(10):150-156. GAO W,HOU W,LI W,et al.Research progress on cell apoptosis mechanism[J].China Animal Husbandry & Veterinary Medicine,2014,41(10):150-156.(in Chinese)
[18] AKATSUKA Y,NISHIDA T,KONDO E,et al.Identification of a polymorphic gene,BCL2A1,encoding two novel hematopoietic lineage-specific minor histocompatibility antigens[J].The Journal of Experimental Medicine,2003,197(11):1489-1500.
[19] USHIZAWA K,TAKAHASHI T,KANEYAMA K,et al.Cloning of the bovine antiapoptotic regulator,BCL2-related protein A1,and its expression in trophoblastic binucleate cells of bovine placenta[J].Biology of Reproduction,2006,74(2):344-351.
[20] CROSIO C,CASCIATI A,IACCARINO C,et al.Bcl2a1 serves as a switch in death of motor neurons in amyotrophic lateral sclerosis[J].Cell Death and Differentiation,2006,13(12):2150-2153.
[21] 王美兰.免疫疾病中调节性B细胞调控机制分析[J].四川医学,2013,34(11):1770-1771. WANG M L.Regulatory mechanism analysis of regulatory B cells in immune diseases[J].Sichuan Medical Journal,2013,34(11):1770-1771.(in Chinese)
[22] MÉTAIS J Y,WINKLER T,GEYER J T,et al.BCL2A1a over-expression in murine hematopoietic stem and progenitor cells decreases apoptosis and results in hematopoietic transformation[J].PLoS One,2012,7(10):e48267.
[23] CRUZ-MUÑOZ W,JARAMILLO M L,MAN S,et al.Roles for endothelin receptor B and BCL2A1 in spontaneous CNS metastasis of melanoma[J]. Cancer Research,2012,72(19):4909-4919.
[24] OTTINA E,LYBERG K,SOCHALSKA M,et al.Knockdown of the antiapoptotic Bcl-2 family member A1/Bfl-1 protects mice from anaphylaxis[J].Journal of Immunology,2015,194(3):1316-1322.
[25] TORIKAI H,AKATSUKA Y,YATABE Y,et al.Aberrant expression of BCL2A1-restricted minor histocompatibility antigens in melanoma cells:Application for allogeneic transplantation[J].International Journal of Hematology,2008,87(5):463-473.