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近缘新对虾PCNA基因的克隆及表达分析

张艳 罗志平 李运东 杨其彬 姜松 陈创华 黄建华 杨丽诗 陈旭 江世贵 周发林

张艳, 罗志平, 李运东, 杨其彬, 姜松, 陈创华, 黄建华, 杨丽诗, 陈旭, 江世贵, 周发林. 近缘新对虾PCNA基因的克隆及表达分析[J]. 南方水产科学, 2023, 19(4): 58-67. doi: 10.12131/20220297
引用本文: 张艳, 罗志平, 李运东, 杨其彬, 姜松, 陈创华, 黄建华, 杨丽诗, 陈旭, 江世贵, 周发林. 近缘新对虾PCNA基因的克隆及表达分析[J]. 南方水产科学, 2023, 19(4): 58-67. doi: 10.12131/20220297
ZHANG Yan, LUO Zhiping, LI Yundong, YANG Qibin, JIANG Song, CHEN Chuanghua, HUANG Jianhua, YANG Lishi, CHEN Xu, JIANG Shigui, ZHOU Falin. Cloning and expression analysis of PCNA in Metapenaeus affinis[J]. South China Fisheries Science, 2023, 19(4): 58-67. doi: 10.12131/20220297
Citation: ZHANG Yan, LUO Zhiping, LI Yundong, YANG Qibin, JIANG Song, CHEN Chuanghua, HUANG Jianhua, YANG Lishi, CHEN Xu, JIANG Shigui, ZHOU Falin. Cloning and expression analysis of PCNA in Metapenaeus affinis[J]. South China Fisheries Science, 2023, 19(4): 58-67. doi: 10.12131/20220297

近缘新对虾PCNA基因的克隆及表达分析

doi: 10.12131/20220297
基金项目: 国家现代农业产业技术体系资助 (CARS-48);农业农村部财政专项 (NHYYSWZZYKZX2020);广东省现代农业产业技术体系创新团队建设专项 (2019KJ149);中国水产科学研究院南海水产研究所中央级公益性科研院基本科研业务费专项资金资助 (2021SD13)
详细信息
    作者简介:

    张艳:张 艳 (1997—),女,硕士研究生,研究方向为水产动物遗传育种与分子生物学。E-mail: zhangyan202205@126.com

    通讯作者:

    周发林 (1975—),男,研究员,博士,研究方向为水产动物遗传育种。E-mail: zhoufalin197538@163.com

  • 中图分类号: S 917.4

Cloning and expression analysis of PCNA in Metapenaeus affinis

  • 摘要: 增殖细胞核抗原 (Proliferating cell nuclear antigen, PCNA) 作为DNA聚合酶δ的辅助蛋白,在DNA复制过程中发挥重要作用。近缘新对虾 (Metapenaeus affinis) 卵巢发育过程中存在细胞增殖活动旺盛的阶段,但目前关于其卵巢发育的分子机制研究较少。利用RACE (Rapid amplification of cDNA ends) 技术获得近缘新对虾PCNA (MaPCNA) 基因的cDNA序列全长,并通过实时荧光定量PCR (qRT-PCR) 对其进行卵巢发育相关的表达分析。MaPCNA全长为1 144 bp,包含140 bp的5'非编码区,221 bp的3'非编码区,开放阅读框 (ORF) 为783 bp,编码260个氨基酸。MaPCNA蛋白的相对分子质量为28.82 kD,理论等电点为4.5。多重比对分析表明,PCNA氨基酸序列在甲壳动物中较为保守。组织表达结果显示,MaPCNA基因在检测的组织中均有表达,其中在卵巢中的表达量最为显著 (P<0.05)。在不同发育阶段的卵巢中,MaPCNA基因的表达出现变化 (P<0.05),从I期开始逐渐上升,到III期表达量最高,之后显著降低并趋于平稳。MaPCNA基因在幼体发育不同时期的表达呈现出一定的规律性趋势,在受精卵时期的表达量最高,之后呈下降趋势,从无节幼体VI期开始表达平稳。研究结果提示PCNA基因可能在近缘新对虾的卵巢发育过程中发挥重要作用。
  • 图  1  MaPCNA 基因的 cDNA 与氨基酸序列展示图

    Figure  1.  cDNA and amino acid sequence of MaPCNA gene

    图  2  MaPCNA 与其他物种的 PCNA 氨基酸序列之间的多重序列比对图

    Figure  2.  Multiple sequence alignment between MaPCNA and PCNA amino acid sequences of other species

    图  3  MaPCNA 基因的三维空间结构示意图

    Figure  3.  Three dimensional spatial structure of MaPCNA gene

    图  4  基于氨基酸序列比较的PCNA在不同物种中的系统进化树

    Figure  4.  Phylogenetic tree of PCNA in different species based on amino acid sequence

    图  5  MaPCNA 基因在不同组织中的相对表达量

    注:图中数值为“平均值±标准差”(n=3),小写字母不同表示各组之间具有显著差异 (P<0.05);后图同此。

    Figure  5.  Relative expression of MaPCNA gene in different tissues

    Note: The values are "$ {\rm{Mean}} \pm {\rm{SD}}$" (n=3). Different lowercase letters indicate significant difference among groups (P<0.05); the same case in the following figures.

    图  6  MaPCNA 基因在卵巢发育不同阶段的相对表达量

    Figure  6.  Relative expression of MaPCNA gene at different ovarian developmental stages

    图  7  MaPCNA 基因在幼体发育各阶段的相对表达量

    Figure  7.  Relative expression of MaPCNA gene at different larval developmental stages

    表  1  实验所使用的引物序列

    Table  1.   Oligonucleotide primers used in experiment

    引物
    Primer
    引物序列 (5'—3')      
    Primer sequence (5'−3')      
    用途
    Function
    MaPCNA-F GGAAGTCTGTTGAAGAAGGTGTTGGA 验证序列
    MaPCNA-R GTATTCTGCCACAAAGCCATAGTAAGC
    MaPCNA-5GSP1 TTTGGACATGCTGGTGAGGTTCATGCCC 5'RACE
    MaPCNA-5GSP2 CATGCCCATGATGAGGTTTCGGTCGC
    MaPCNA-3GSP1 AAAGCAACACCCCTTTCCCCACAGG 3'RACE
    MaPCNA-3GSP2 TCCCTGTCCATGTCTCCTGATGTACCCC
    UPM-long CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT RACE
    UPM-short CTAATACGACTCACTATAGGGC
    NUP AAGCAGTGGTATCAACGCAGAGT
    MaPCNA-qF TCATCGAGATGCAGGAGCCAGTTA qPCR
    MaPCNA-qR ATCAGGAGACATGGACAGGGAGAC
    EF-1α-qF AAGCCAGGTATGGTTGTCAACTTT 内参
    EF-1α-qR CGTGGTGCATCTCCACAGACT
    下载: 导出CSV
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  • 收稿日期:  2022-11-23
  • 修回日期:  2022-12-21
  • 录用日期:  2023-02-08
  • 网络出版日期:  2023-02-20
  • 刊出日期:  2023-08-05

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