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光裸星虫SERCA基因克隆及在卵母细胞中的表达分析

苏泳霖 钟如卓 郭志诚 王庆恒

苏泳霖, 钟如卓, 郭志诚, 王庆恒. 光裸星虫SERCA基因克隆及在卵母细胞中的表达分析[J]. 南方水产科学. doi: 10.12131/20220226
引用本文: 苏泳霖, 钟如卓, 郭志诚, 王庆恒. 光裸星虫SERCA基因克隆及在卵母细胞中的表达分析[J]. 南方水产科学. doi: 10.12131/20220226
SU Yonglin, ZHONG Ruzhuo, GUO Zhicheng, WANG Qingheng. Cloning and expression analysis of SERCA gene of Sipunculus nudus in oocytes[J]. South China Fisheries Science. doi: 10.12131/20220226
Citation: SU Yonglin, ZHONG Ruzhuo, GUO Zhicheng, WANG Qingheng. Cloning and expression analysis of SERCA gene of Sipunculus nudus in oocytes[J]. South China Fisheries Science. doi: 10.12131/20220226

光裸星虫SERCA基因克隆及在卵母细胞中的表达分析

doi: 10.12131/20220226
基金项目: 广东省科技计划项目 (GDKTP2020046400, 163-2019-XMZC-0009-02-0059);湛江市科技计划项目 (2021A05190)
详细信息
    作者简介:

    苏泳霖 (1998—),女,学士,研究方向为水产养殖。E-mail: syl12342@163.com

    通讯作者:

    王庆恒 (1977—),男,教授,硕士,研究方向为无脊椎动物生物学与养殖技术。E-mail: wangqingheng@163.com

  • 中图分类号: S 917.4

Cloning and expression analysis of SERCA gene of Sipunculus nudus in oocytes

  • 摘要: 细胞内钙离子 (Ca2+) 浓度的变化是介导卵母细胞成熟的重要因素。肌质网/内质网Ca2+-ATP酶 (Sarco/endoplasmic reticulum calcium adenosine triphosphatase, SERCA) 属P型ATP酶家族成员,是细胞内钙离子转运的重要调控蛋白。前期转录组分析显示,光裸星虫 (Sipunculus nudus) SERCA (Sn-SERCA) 的表达量在卵母细胞发育过程中差异显著,为进一步研究SERCA在卵母细胞不同发育阶段中的作用,利用RACE技术得到Sn-SERCA cDNA全长,采用实时荧光定量PCR检测Sn-SERCA在各卵母细胞发育时期的相对表达量。结果表明,Sn-SERCA全长为3 840 bp,5'UTR为196 bp,3'UTR为581 bp,开放阅读框3 060 bp,编码1 020 个氨基酸,Sn-SERCA具有P型ATP酶家族进行催化反应所需的两个保守基序“TGES”和“DKTGT”。多序列比对、Motif分析及三级结构预测结果显示SERCA同源蛋白具有较高保守性。系统进化树分析表明Sn-SERCA与粉正蚓 (Lumbricus rubellus)、鸭嘴海豆芽 (Lingula anatine) 等无脊椎动物同源蛋白序列聚为一支。荧光定量结果显示:卵母细胞在体腔液中发育时,Sn-SERCA在卵黄旺盛合成后期高表达;当卵母细胞进入肾管后,Sn-SERCA的表达量大幅上升,显著高于其他时期 (P<0.05)。结合前期卵母细胞发育的超微结构观察,研究结果表明Sn-SERCA在光裸星虫卵母细胞的卵黄积累和生发泡破裂发生过程起重要作用。
  • 图  1  光裸星虫卵母细胞各时期总RNA(a)和cDNA(b)琼脂糖凝胶电泳图

    Figure  1.  Agarose gel electrophoresis diagram of total RNA (a) and cDNA (b) from oocytes of S. nudus

    图  2  Sn-SERCA cDNA序列全长及推导的氨基酸序列

    Figure  2.  Full length and deduced amino acid sequence of Sn-SERCA cDNA sequence

    图  3  Sn-SERCA蛋白跨膜螺旋

    Figure  3.  Transmembrane helix of Sn-SERCA

    图  4  Sn-SERCA蛋白结构域

    Figure  4.  Protein domains of Sn-SERCA

    图  5  光裸星虫 (a)、粉正蚓 (b)、鸭嘴海豆芽 (c) 和加州海兔 (d) SERCA蛋白三级结构

    Figure  5.  Tertiary structure of protein of SERCA of S. nudus (a), L. rubellus (b), L. anatina (c) and A. californica (d)

    图  6  SERCA同源氨基酸序列多序列比对

    Figure  6.  Multiple sequence alignment of SERCA homologous protein

    图  7  SERCA同源氨基酸序列 Motif 分析

    Figure  7.  Motif analysis of SERCA homologous protein

    图  8  SERCA同源氨基酸序列系统发育树

    Figure  8.  Phylogenetic trees of SERCA homologous protein

    图  9  光裸星虫各发育时期卵母细胞中SERCA的相对表达量

    Figure  9.  Relative expression of Sn-SERCA in different developmental periods of oocytes

    表  1  Sn-SERCA基因克隆及实时荧光定量引物序列

    Table  1.   Primer sequence used in gene cloning and qRT-PCR of Sn-SERCA gene

    引物
    Primer
    序列(5'—3')
    Sequence (5'–3')
    用途
    Function
    5'-outer GTCTGCTGGAACCTTGTC 5'RACE
    5'-inner CTGCCATACTCCAACAACT 5'RACE
    3'-outer CAACTCAACTACTACCAACTG 3'RACE
    3'-inner GTCACTTCTTGTCTCCTCAT 3'RACE
    M13-F CGCCAGGGTTTTCCCAGTCACGAC 菌落PCR
    M13-R AGCGGATAACAATTTCACACAGGA 菌落PCR
    qRT-PCR-S TGCCTTGTAGCCTCCTTCC qRT-PCR
    qRT-PCR-A ACTGTCCTCATCTTCCATTGTG qRT-PCR
    60SL7-S CAGGCTAACAACTTCTTATGG qRT-PCR(内参)
    60SL7-A TGGCTTGACAGATAACACTT qRT-PCR(内参)
    下载: 导出CSV

    表  2  Sn-SERCA序列分析所用软件及在线工具

    Table  2.   Software and online tool for sequence analysis of Sn-SERCA

    名称
    Name
    网址
    Website
    用途
    Function
    DNAMAN 8拼接测序结果及多序列比对
    ORF Finderhttps://www.ncbi.nlm.nih.gov/orffinder预测ORF及氨基酸序列
    ProtParamhttps://web.expasy.org/protparam预测蛋白的理化性质
    NCBI Blasthttps://blast.ncbi.nlm.nih.gov/Blast.cgi搜索同源蛋白
    SMARThttp://smart.embl-heidelberg.de预测保守结构域
    SOPMAhttps://npsa-prabi.ibcp.fr/NPSA/npsa_sopma.html预测蛋白二级结构
    phyre2http://www.sbg.bio.ic.ac.uk/phyre2/html/page.cgi?id=index预测蛋白三级结构
    MEME Suithttp://meme-suite.org/index.htmlMotif搜索
    下载: 导出CSV
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  • 收稿日期:  2022-08-24
  • 修回日期:  2022-10-13
  • 录用日期:  2022-11-18
  • 网络出版日期:  2022-12-03

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