三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析

doi:10.3969/j.issn.1004-1524.2022.06.07

浙江农业学报 ›› 2022, Vol. 34 ›› Issue (6): 1162-1174.DOI: 10.3969/j.issn.1004-1524.2022.06.07

三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析

刘凯(), 谢楠, 郭炜, 马恒甲

杭州市农业科学研究院水产研究所,浙江杭州 310024

收稿日期:2021-08-26 出版日期:2022-06-25 发布日期:2022-06-30
作者简介:刘凯(1982—),男,安徽马鞍山人,硕士,高级工程师,主要从事鱼类遗传与免疫研究。E-mail: liukai0106@email.cn
基金资助:
杭州市农科院创新基金(2019HNCT-01);杭州市农业科研主动设计项目(20162012A03);现代农业产业技术体系专项(CARS-45-39)

Full-length cDNA cloning and bioinformatic analysis of MHCⅠα gene from black amur bream (Megalobrama terminalis)

LIU Kai(), XIE Nan, GUO Wei, MA Hengjia

Institute of Fishery Science, Hangzhou Academy of Agricultural Sciences, Hangzhou 310024, China

Received:2021-08-26 Online:2022-06-25 Published:2022-06-30

摘要/Abstract

摘要：

主要组织相容复合体（major histocompatibility complex,MHC）是一类具有高度多态性的分子,在脊椎动物的先天免疫应答中起到重要作用。根据已报道鱼类的MHCⅠα基因序列设计特异性引物,通过RT-PCR和RACE技术克隆三角鲂MHCⅠα基因cDNA序列全长,命名为Mete-UAA,并对其进行生物信息学分析。结果显示,Mete-UAA全长为2 102 bp,包含1 044 bp的开放阅读框,编码347个氨基酸。编码的MHCⅠα蛋白质预测分子量为38 699.18,等电点5.23,含有16个氨基酸组成的信号肽,具有亲水性,定位于细胞膜上,具有1个N-糖基化、3个O-糖基化和39个磷酸化的潜在位点。氨基酸序列同源性比对发现,Mete-UAA氨基酸序列与团头鲂同源性最高为75.79%,与草鱼同源性稍低于团头鲂,为75.25%。Mete-UAA具有MHCⅠα的典型结构特征,包含1个前导肽、3个胞外结构域、1个跨膜区和1个胞质区。二级结构分析显示,Mete-UAA蛋白质中α-螺旋、β-转角、延伸链和无规则卷曲所占的比例分别为25.94%、9.22%、26.80%和38.04%。基于转录组学分析表明,感染嗜水气单胞菌(Aeromonas hydrophila)后三角鲂MHCⅠα基因表达呈总体下降趋势。本研究从三角鲂肝中成功克隆MHCⅠα基因,分析了其生物信息学特征,为后续研究MHCⅠα参与调控三角鲂先天免疫应答的机制提供理论基础。

关键词: 三角鲂, 主要组织相容复合体, 基因克隆, 生物信息学分析

Abstract:

Major histocompatibility complex (MHC) is a highly polymorphic molecule that plays an essential role in vertebrates' innate immune response. Specific primers were designed according to the reported MHCⅠα sequence of fish. Finally, the full-length MHCⅠα cDNA sequence of Megalobrama terminalis was obtained by RT-PCR and RACE. The cDNA sequence was named Mete-UAA and conducted bioinformatics analysis. The results showed that the Mete-UAA was 2 102 bp in length, containing 1 044 bp open reading frame, encoding 347 amino acids. The Mete-UAA encoded a hydrophilic protein that contains 16 amino acid signal peptides. It was localized on the cell membrane and had one N-glycosylation, three O-glycosylation, and 39 potential phosphorylation sites. The encoded MHCⅠα protein had a predicted relative molecular mass of 38 699.18 and an isoelectric point of 5.23. Amino acid sequence homology comparison showed that the homology of the Mete-UAA amino acid sequence was up to 75.79% with M. amblycephala (AEE87250) and 75.25% with Ctenopharyngodon idella (BAD01521) slightly lower than M. amblycephala (AEE87250). The Mete-UAA possessed the typical structural features of MHCⅠα, including a leader peptide, three extracellular domains, one transmembrane region, and one cytoplasmic region. The secondary structure analysis of the Mete-UAA protein showed that the proportions of the α-helix, β-turn, extended chain and random coil were 25.94%, 9.22%, 26.80%, and 38.04%, respectively. Based on the transcriptome analysis, expression of the MHCⅠα gene in M. terminalis infected with Aeromonas hydrophila showed an overall decreasing trend. In this study, the MHCⅠα gene was successfully cloned from the liver of M. terminalis. Its bioinformatics characteristics were analyzed, which would provide a theoretical basis for further research on the mechanism of MHCⅠα involved in regulating the innate immune response in M. terminalis.

Key words: Megalobrama terminalis, major histocompatibility complex, gene cloning, bioinformatic analysis

中图分类号:

S917.4

刘凯, 谢楠, 郭炜, 马恒甲. 三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析[J]. 浙江农业学报, 2022, 34(6): 1162-1174.

LIU Kai, XIE Nan, GUO Wei, MA Hengjia. Full-length cDNA cloning and bioinformatic analysis of MHCⅠα gene from black amur bream (Megalobrama terminalis)[J]. Acta Agriculturae Zhejiangensis, 2022, 34(6): 1162-1174.

图/表 9

表1 三角鲂MHCⅠα基因cDNA全长克隆使用的引物序列

Table 1 Primer sequences used in full-length cDNA cloning of MHCⅠα from Megalobrama terminalis

引物名称 Primer name	引物序列 Primer Sequences (5'-3')
MHCⅠ-Z-NF1	CTWCACTGSTGTGTCTGGAG
MHCⅠ-Z-NR1	GGWGATTGTTACTCCACTGGG
MHCⅠ-3-GSP1	ATCCTCAAGCTACGATTACCAAGG
MHCⅠ-3-GSP2	GTCTCTCCTCAGGTGTCTCTGTTG
MHCⅠ-5-GSP1	GGGTGCTCTTATCCAGGGCGAGGAAG
MHCⅠ-5-GSP2	GCTCCCTCATTCTGTCTGATCCACTCTG
Outer primer	TACCGTCGTTCCACTAGTGATTT
Inner primer	CGCGGATCCTCCACTAGTGATTTCACTATAGG
Univers primer	TAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT
Short primer	CTAATACGACTCACTATAGGGC

图1 三角鲂MHCⅠα基因片段克隆电泳图 A,MHCⅠα基因核心片段;B,3' RACE第二轮扩增后片断;C,5' RACE第二轮扩增后片断。Marker,DNA标准物质。

Fig. 1 Electrophoresis of amplified MHCⅠα fragment from Megalobrama terminalis A, MHCⅠα core fragment; B, 3'RACE amplified fragment; C, 5'RACE amplified fragment. Marker, DNA marker.

图2 三角鲂MHCⅠα基因cDNA全长及其编码的氨基酸序列

Fig. 2 Complete cDNA and encoded amino acid sequence of MHCⅠα from Megalobrama terminalis

图3 三角鲂MHCⅠα蛋白质的保守结构域(A)、二级结构(B)和卷曲螺旋结构(C)预测图B中蓝色代表α-螺旋,绿色代表β-转角,紫色代表延伸链,红色代表无规则卷曲。

Fig. 3 Predicted conservative domain (A), secondary structure (B), and coiled-coil structure (C) of MHCⅠα protein from Megalobrama terminalis In figure B, Blue represented α-helix, green represented β-turn, purple represented extended chain, red represented random coil.

图 4 MHCⅠα蛋白质的三级结构预测模型 A,三角鲂Mete-UAA编码蛋白的三级结构预测模型;B,团头鲂Meam-UBA*F8-0102编码蛋白的三级结构预测模型。

Fig. 4 Predicted tertiary structure model of MHCⅠαprotein A, Prediction model of tertiary structure of Mete-UAA coding protein of Megalobrama terminalis; B, Prediction model of tertiary structure of Meam-UBA*F8-0102 coding protein of Megalobrama amblycephala.

图5 三角鲂MHCⅠα蛋白质信号肽(A)、磷酸化位点(B)、N-糖基化位点(C)和蛋白跨膜区(D)预测

Fig. 5 Predicted signal peptide (A), phosphorylation sites (B), N-glycosylated sites (C), and protein transmembrane (D) of MHCⅠα protein from Megalobrama terminalis

图6 基于MHCⅠα氨基酸序列的三角鲂和其他物种多重序列比对每列上的氨基酸残基一致性越高则该列的颜色越深,圈中字符为高度保守的半胱氨酸残基。

Fig. 6 Multiple alignments of MHCⅠα’s amino acid sequence from Megalobrama terminalis and other animals The higher the identity of the amino acid residues on each column, the darker the column, the characters in the circle were highly conservative cysteine residues.

图7 基于MHCⅠα氨基酸序列的三角鲂和其他物种最大似然系统发育分析进化树节点上的数字分别表示SH-aLRT检验和Ultrafast Bootstrap校验的百分数。

Fig. 7 Phylogenetic analysis of MHCⅠα's amino acid sequence from Megalobrama terminalis and other fish by Maximum Likelihood method Values shown at each node of the tree correspond to the SH-aLRT test values and Ultrafast Bootstrap value given in percentages.

图 8 三角鲂MHCⅠα基因表达的转录组分析

Fig. 8 Transcriptome analysis of expression of MHCⅠα gene in Megalobrama terminalis

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三角鲂MHCⅠα基因全长cDNA克隆与生物信息学分析

Full-length cDNA cloning and bioinformatic analysis of MHCⅠα gene from black amur bream (Megalobrama terminalis)

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