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三疣梭子蟹(Portunus trituberculatusfoxl2基因功能初探及相关miRNA分析

张梦倩 张景琰 葛红星 刘萍 李健 孟宪亮

张梦倩,张景琰,葛红星,等. 三疣梭子蟹(Portunus trituberculatus)foxl2基因功能初探及相关miRNA分析[J]. 海洋学报,2022,44(4):85–94 doi: 10.12284/hyxb2022060
引用本文: 张梦倩,张景琰,葛红星,等. 三疣梭子蟹(Portunus trituberculatusfoxl2基因功能初探及相关miRNA分析[J]. 海洋学报,2022,44(4):85–94 doi: 10.12284/hyxb2022060
Zhang Mengqian,Zhang Jingyan,Ge Hongxing, et al. Preliminary functional study of foxl2 in Portunus trituberculatus and analysis of its related miRNA[J]. Haiyang Xuebao,2022, 44(4):85–94 doi: 10.12284/hyxb2022060
Citation: Zhang Mengqian,Zhang Jingyan,Ge Hongxing, et al. Preliminary functional study of foxl2 in Portunus trituberculatus and analysis of its related miRNA[J]. Haiyang Xuebao,2022, 44(4):85–94 doi: 10.12284/hyxb2022060

三疣梭子蟹(Portunus trituberculatusfoxl2基因功能初探及相关miRNA分析

doi: 10.12284/hyxb2022060
基金项目: 国家自然科学基金(41976106,41306178);青岛市市南区科技计划(2020-2-001-QT);国家现代农业产业技术体系(CARS-48);中国水产科学研究院基本科研业务费(2020TD46,2018HY-ZD0201)。
详细信息
    作者简介:

    张梦倩(1997-),女,安徽省马鞍山市人,研究方向为甲壳动物繁殖生物学。E-mail:2080855236@qq.com

    通讯作者:

    孟宪亮,男,副研究员,主要从事甲壳动物生理学研究。E-mail:xlmeng@ysfri.ac.cn

  • 中图分类号: S917.4

Preliminary functional study of foxl2 in Portunus trituberculatus and analysis of its related miRNA

  • 摘要: foxl2在脊椎动物卵巢分化、发育和功能维持等方面具有重要作用,然而其在三疣梭子蟹(Portunus trituberculatus)卵巢发育中的功能尚不明确。本研究首先克隆了三疣梭子蟹foxl2Ptfoxl2)基因cDNA全长序列,该基因5′和3′非编码区域(UTR)长度分别为701 bp和211 bp,开放阅读框的长度为1 590 bp。基因表达分析结果显示,foxl2在三疣梭子蟹不同组织中均有表达,但在卵巢中表达量最高;其在卵巢发育不同时期的表达存在显著差异,在V期表达量最高;切除眼柄后,该基因的表达出现显著下降;干扰该基因表达后,卵巢vtg基因的表达显著上调。上述结果表明,foxl2可能在三疣梭子蟹卵巢发育调控中发挥重要功能,能够抑制卵巢组织中卵黄蛋白的合成。为进一步分析该基因的表达调控方式,利用生物信息学方法,预测了靶向foxl2的miRNA,并通过双荧光素酶报告基因检测实验,从细胞水平验证了这些miRNA对Ptfoxl2的调控作用;分析了其在卵巢发育不同时期以及切除眼柄后的表达模式。结果显示,共转染miR-9类似物和包含foxl2 3′UTR的pmirGLO质粒组,萤火虫酶与海肾荧光素酶活性比值出现显著下降,且在卵巢发育过程及切除眼柄后与foxl2表达模式相反。该结果初步证实miR-9可以从转录后水平调控三疣梭子蟹foxl2基因的表达。
  • 图  1  Ptfoxl2基因cDNA全长序列以及推导的氨基酸序列

    FH结构域用阴影表示;起始密码子和终止密码子用方框标出;下划线标注区域依次分别为miR-9、novel-68和novel-52的预测结合位点;左侧数字为核苷酸和氨基酸位置

    Fig.  1  Nucleotide sequence and deduced amino acid sequence of Ptfoxl2 gene

    FH domain was marked with shadow; initiation codon and stop codon were marked with black box; the underlined regions were the predicted binding sites for miR-9, novel-68, and novel-52 in turn; the numbers on the left indicate the positions of nucleotide and amino acid

    图  2  4种甲壳动物foxl2氨基酸序列多重比对

    右侧数字为氨基酸位置;各物种foxl2基因序列GenBank登录号:三疣梭子蟹(OK413951)、拟穴青蟹(MN412580.1)、中华绒螯蟹(KF806733.1)、斑节对虾(XM_037939236.1);黑色代表氨基酸残基同源性100%;红色代表氨基酸残基同源性75%;蓝色代表氨基酸残基同源性50%

    Fig.  2  Multiple alignments of the amino acid sequences of foxl2 in four crustacean species

    The numbers on the right indicate the positions of the amino acids; the GenBank accession numbers of foxl2 gene were as follows: Portunus trituberculatus (OK413951), Scylla paramamosain (MN412580.1), Eriocheir sinensis (KF806733.1), Penaeus monodon (XM_037939236.1); black represents 100% homology of amino acid residues; red represents 75% homology of amino acid residues; blue represents 50% homology of amino acid residues

    图  3  Ptfoxl2基因在三疣梭子蟹不同组织(A)、不同卵巢发育时期(B)以及切除眼柄后(C)的表达

    不同字母代表数据差异显著(p<0.05);*表示对照组和切除眼柄组表达存在显著差异(p<0.05)

    Fig.  3  The expression of Ptfoxl2 in different tissues (A), in different ovarian developmental stages (B) and after eyestalk ablation (C)

    Different letters indicate significant difference (p<0.05); * indicates a significant difference between the control group and eyestalk ablation group (p<0.05)

    图  4  注射Ptfoxl2 dsRNA 和GFP dsRNA后卵巢Ptfoxl2(A)和vtg(B)基因的表达

    *表示存在显著差异(p<0.05)

    Fig.  4  Relative expression of Ptfoxl2 (A) and vtg (B) in ovary after injecting Ptfoxl2 dsRNA and GFP dsRNA

    * indicates a significant difference (p<0.05)

    图  5  共转染野生型和突变型质粒和miRNA类似物后荧光素酶的相对活性

    +表示实验体系中存在该miRNA类似物或质粒;−表示实验体系中不存在该miRNA类似物或质粒;不同字母代表数据差异显著(p<0.05)

    Fig.  5  Luciferase activity of the reporter plasmid containing wild-type or mutant target site after co-transfection of plasmid and miRNA mimics

    + indicates that the miRNA mimics or plasmid exists in the reaction system; − indicates that the miRNA mimics or plasmid doesn’t exist in the reaction system; different letters indicate significant difference (p<0.05)

    图  6  miR-9在三疣梭子蟹不同组织(A)、不同卵巢发育时期(B)以及切除眼柄后(C)的表达

    不同字母代表数据差异显著(p<0.05);*表示对照组和切除眼柄组表达存在显著差异(p<0.05)

    Fig.  6  The expression of miR-9 in different tissues (A), in different ovarian developmental stages (B) and after eyestalk ablation (C)

    Different letters indicate significant difference (p<0.05) ; * indicates a significant difference between the control group and eyestalk ablation group (p<0.05)

    表  1  实验所用引物的序列

    Tab.  1  The sequences of the primers used in this study

    引物名称序列(5′-3′)用途
    foxl2-3′CGGGAACTTCGCAAGCTACACACAGRACE
    foxl2-5′GGTAGTCGTCTTTCATCGTGCCGTAGGRACE
    UPMCTAATACGACTCACTATAGGGCRACE
    foxl2-FCGTTGTCCTGATCTCACTGCqRT-PCR
    foxl2-RCGTCTTTCATCGTGCCGTAGqRT-PCR
    β-actin-FCGAAACCTTCAACACTCCCGqRT-PCR
    β-actin-RGGGACAGTGTGTGAAACGCCqRT-PCR
    dsRNA-foxl2-T7-FGGATCCTAATACGACTCACTATAGGGCAGATGCAAAGCGGGAACTTRNAi
    dsRNA-foxl2-T7-RGGATCCTAATACGACTCACTATAGGGGGAAAGCGTCTCCAGTCATCRNAi
    dsRNA-GFP-T7-FGGATCCTAATACGACTCACTATAGGCGACGTAAACGGCCACAAGTTRNAi
    dsRNA-GFP-T7-RGGATCCTAATACGACTCACTATAGGATGGGGGTGTTCTGCTGGTAGRNAi
    miR-9GCCTCTTTGGTTATCTAGCTGTATqRT-PCR
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  • 收稿日期:  2021-10-18
  • 修回日期:  2021-11-16
  • 刊出日期:  2022-04-15

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