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黄鳍棘鲷IGF-2基因克隆及IGF-1/2在胚胎发育过程中的表达特性分析

崔淼 李钰杰 杨永春 张佳颖 许鲜姬 林李泉 林国荣 张其中 许德麟

崔淼,李钰杰,杨永春,等. 黄鳍棘鲷IGF-2基因克隆及IGF-1/2在胚胎发育过程中的表达特性分析[J]. 海洋学报,2022,44(x):1–10 doi: 10.12284/hyxb2022158
引用本文: 崔淼,李钰杰,杨永春,等. 黄鳍棘鲷IGF-2基因克隆及IGF-1/2在胚胎发育过程中的表达特性分析[J]. 海洋学报,2022,44(x):1–10 doi: 10.12284/hyxb2022158
Cui Miao,Li Yujie,Yang Yongchun, et al. Molecular cloning of insulin-like growth factor-2 and differential expression of IGF-1/2 during embryonic development in Acanthopagrus latus[J]. Haiyang Xuebao,2022, 44(x):1–10 doi: 10.12284/hyxb2022158
Citation: Cui Miao,Li Yujie,Yang Yongchun, et al. Molecular cloning of insulin-like growth factor-2 and differential expression of IGF-1/2 during embryonic development in Acanthopagrus latus[J]. Haiyang Xuebao,2022, 44(x):1–10 doi: 10.12284/hyxb2022158

黄鳍棘鲷IGF-2基因克隆及IGF-1/2在胚胎发育过程中的表达特性分析

doi: 10.12284/hyxb2022158
基金项目: 广东基础与应用基础研究基金(2019A1515012112, 2019A1515011791);广东省科技计划([2019]170,KTP20210324);广东省自然科学基金(2018A030313578);广州市科技项目(GZKTP202032)。
详细信息
    作者简介:

    崔淼(1979-),男,安徽阜阳人,副研究员,研究方向为水生动物遗传育种。E-mail: cuisanshui@163.com

    通讯作者:

    崔淼(1979-),男,副研究员,研究方向为水生动物遗传育种。E-mail: cuisanshui@163.com

    许德麟(1980-),女,副研究员,研究方向为分子微生物学。E-mail: xudelin@live.com

  • 中图分类号: S965.3

Molecular cloning of insulin-like growth factor-2 and differential expression of IGF-1/2 during embryonic development in Acanthopagrus latus

  • 摘要: 类胰岛素样生长因子(Insulin-like Growth Factors,IGFs)是生物生长轴下游的关键调控因子,其对促进细胞分化与机体生长具有重要的作用。为深入研究IGF-1IGF-2基因在黄鳍棘鲷(Acanthopagrus latus)胚胎发育过程中表达机制及可能发挥的作用,本文利用分子克隆技术分离鉴定了黄鳍棘鲷IGF-2基因cDNA序列,并对其进行了生物信息学分析。同时,在对其胚胎发育过程连续观察基础上,利用Real time qPCR的方法,测定并分析了黄鳍棘鲷胚胎发育不同时期IGF-1IGF-2 mRNA的表达情况。实验结果表明,IGF-2基因cDNA序列全长为1 736 bp,其中开放阅读框648 bp,共编码215个氨基酸。多重序列比对分析发现,黄鳍棘鲷IGF-2氨基酸序列与同属鲷科鱼类的大西洋鲷相似性为99.95%,与三长棘刺鲷相似性为98.14%,与其他硬骨鱼类的IGF-2也有较高的相似性,显示出IGF-2在硬骨鱼类进化关系上的保守性。在水温为(26.5±0.5)℃,pH为8.0和盐度为28的条件下,黄鳍棘鲷由受精卵至孵化出膜历时为25.5 h。实时荧光定量PCR结果显示:IGF-1IGF-2 mRNA 在黄鳍棘鲷胚胎发育不同时期均有表达。IGF-1表达量呈现先升高后降低的趋势,在肌肉效应期达到最高表达量;IGF-2表达量呈现先升高后降低再升高的趋势,在原肠期、肌肉效应期与出膜后3天表现高表达量,这表明IGF-1IGF-2在胚胎发育时期可能发挥重要作用。
  • 图  1  黄鳍棘鲷IGF-2基因全长序列及其编码的氨基酸序列

    *代表氨基酸终止密码子;代表poly(A)加尾信号

    Fig.  1  Complete sequence of IGF-2 cDNA and deduced amino acid sequence from A. latus

    *represents stop codon TGA; represents poly(A) signal

    图  2  黄鳍棘鲷IGF-2 氨基酸序列多重序列比对分析

    Fig.  2  Amino acid sequence multiple alignment of IGF-2 from A. latus

    图  3  黄鳍棘鲷和其他物种IGF-2蛋白的系统进化树分析

    Fig.  3  Phylogenetic analysis of IGF-2 amino acid sequence from A. latus and other animals

    图  4  黄鳍棘鲷的胚胎发育

    a. 2细胞期;b. 4细胞期;c. 8细胞期;d. 16细胞期;e. 32细胞期;f. 64细胞期;g. 多细胞期;h. 桑葚胚期;i. 高囊胚期;j. 低囊胚期;k. 原肠前期;l. 原肠中期;m. 原肠晚期;n. 胚孔封闭期;o. 视囊形成期;p. 肌节出现期;q. 晶体形成期;r. 尾芽期;s. 尾芽游离期;t. 肌肉效应期;u. 心跳期;v. 将孵期;w.破膜期

    Fig.  4  Embryonic development of A.latus

    a. 2-cell stage; b. 4-cell stage; c. 8-cell stage; d. 16-cell stage; e. 32-cell stage; f. 64-cell stage; g. multicellular stage; h. morula; i. high blastula; j. low blastula; k. early-gastrula stage; l. mid-gastrula stage; m. late-gastrula stage; n. closure of blastopore; o. eye vesicle formation stage; p. muscle burl stage; q. crystal stage of eyes; r. tail-bud stage; s. tail-bud free stage; t. muscular effect stage; u. heart-beating stage; v. pre-hatching stage; w. hatching stage

    图  5  黄鳍棘鲷胚胎发育过程中IGF-1/IGF-2 mRNA的相对表达量

    Fig.  5  The relative expression of IGF-1/IGF-2 mRNA in different embryo development stages of A.latus

    表  1  本实验所用引物

    Tab.  1  The primers used in the experiment

    引物名称序列(5′-3′)用途
    IGF-2-FTCATCTCAGCCGCACCAACTCDS序列克隆
    IGF-2-RAAAAGGTGCTGGAACAGGAATC
    5-GSP1GGCATCACGGGTAAGACCTGTA5'末端序列克隆
    5-GSP2GTGGCAAAGTGAGTGGCGTC
    3-GSP1TCTGAACTCTTTCGCTCCCTCT3'末端序列克隆
    3-GSP2ATTAGATTCCTGTTCCAGCACCTT
    M13F-47CGCCACCCTTTTCCCAGTCACGAC菌液检测
    M13R-48AGCGGATAACAATTTCACACAGGA
    IGF-1-F1TAGCCACACCCTCTCACTACTG荧光定量
    IGF-1-R1AAGCCTCTCTCTCCACACACAA
    IGF-2-F1CCGTAGCTGTGACCTCAACC荧光定量
    IGF-2-R1TCCTCTGCCACACCTCGTAT
    β-actin-FACCCAGATCATGTTCGAGACC内参基因
    β-actin-RATGAGGTAGTCTGTGAGGTCG
    下载: 导出CSV

    表  2  海水鱼类胚胎发育特征的比较分析

    Tab.  2  Comparison of embryonic developmental characteristics of marine fish

    物种受精卵直径/
    mm
    油球直径/
    mm
    孵化水温/
    °C
    胚胎发育
    时间
    黄鳍棘鲷
    A. latus
    0.900±0.0500.250±0.02026.5±0.525 h30 min
    条石鲷
    O. fasciatus[28]
    0.910±0.0380.210±0.02823.9±0.430 h32 min
    黑棘鲷
    A.schlegelii[29]
    0.895±0.05521.0±0.532 h
    大西洋鲷
    S.aurata[30]
    0.870±0.0500.205±0.02519±0.544 h48 min
    军曹鱼
    R. canadum[31]
    1.245±0.0650.325±0.02727.0±0.526 h30 min
    鞍带石斑鱼
    E. lanceolatus[32]
    0.820±0.0300.230±0.03029.0±0.518 h30 min
    多纹钱蝶鱼
    (S. multifasciata)[21]
    0.605±0.0050.237±0. 01327.0±1.018 h30 min
    黄姑鱼
    N.albiflora[33]
    0.830±0.0180.240±0.01223.2±0.1522 h59 min
    下载: 导出CSV
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  • 收稿日期:  2022-02-15
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    /

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    返回