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大黄鱼slitrk3基因启动子克隆及转录调控分析

周锐涛 岳珠峰 纪焦军 温靖 姜丹 王志勇 方铭

周锐涛, 岳珠峰, 纪焦军, 温靖, 姜丹, 王志勇, 方铭. 大黄鱼slitrk3基因启动子克隆及转录调控分析[J]. 南方水产科学. doi: 10.12131/20220309
引用本文: 周锐涛, 岳珠峰, 纪焦军, 温靖, 姜丹, 王志勇, 方铭. 大黄鱼slitrk3基因启动子克隆及转录调控分析[J]. 南方水产科学. doi: 10.12131/20220309
ZHOU Ruitao, YUE Zhufeng, JI Jiaojun, WEN Jing, JIANG Dan, WANG Zhiyong, FANG Ming. Cloning and transcriptional regulation of slitrk3 gene promoter in large yellow croaker (Larimichthys crocea)[J]. South China Fisheries Science. doi: 10.12131/20220309
Citation: ZHOU Ruitao, YUE Zhufeng, JI Jiaojun, WEN Jing, JIANG Dan, WANG Zhiyong, FANG Ming. Cloning and transcriptional regulation of slitrk3 gene promoter in large yellow croaker (Larimichthys crocea)[J]. South China Fisheries Science. doi: 10.12131/20220309

大黄鱼slitrk3基因启动子克隆及转录调控分析

doi: 10.12131/20220309
基金项目: 国家重点研发计划项目 (2018YFD0901201)
详细信息
    作者简介:

    周锐涛 (1998—),男,硕士研究生,研究方向为水产动物遗传与育种。E-mail: zhourt0710@163.com

    通讯作者:

    方 铭 (1979—),男,教授,博士,研究方向为水产动物遗传与育种。E-mail: fangming618@126.com

  • 中图分类号: S 917.4

Cloning and transcriptional regulation of slitrk3 gene promoter in large yellow croaker (Larimichthys crocea)

  • 摘要: 神经突触相关蛋白Slitrk3具有调节抑制性突触发育的作用。研究大黄鱼 (Larimichthys crocea) 神经突触黏附分子slitrk3基因的转录调控机制,可为解决大黄鱼养殖面临的生长、应激及抗逆等问题提供新思路。通过生物信息学方法对大黄鱼及其他物种的Slitrk3氨基酸进行多重序列比对和系统进化树分析,并预测大黄鱼slitrk3基因启动子区域相关的调控元件,采用双荧光素酶报告基因系统检测大黄鱼slitrk3基因启动子区域的转录活性。生物信息学分析结果显示,Slitrk3氨基酸序列在鱼类中具有较高的保守性;大黄鱼slitrk3基因启动子区域预测存在2个潜在的转录起始位点、2个CpG岛以及Sp1、GR、C/EBPα和C/EBPβ等多种转录因子结合位点。双荧光素酶报告基因系统结果显示,大黄鱼slitrk3基因启动子区域的−1970—−1614 bp、−1210—−667 bp存在正调控元件,−1614—−1210 bp、−667—−376 bp、−376—−147 bp存在负调控元件,推测−147—+16 bp为核心启动子。
  • 图  1  大黄鱼及其他物种的Slitrk3氨基酸序列多重序列比对

    Figure  1.  Multiple alignments of amino acid sequences of Slitrk3 in L. crocea and other species

    图  2  大黄鱼及其他物种的Slitrk3氨基酸序列系统进化树

    Figure  2.  Phylogenetic tree of amino acid sequences of Slitrk3 in L. crocea and other species

    图  3  大黄鱼slitrk3基因启动子CpG岛的预测

    Figure  3.  Prediction of CpG islands of slitrk3 gene promoter in L. crocea

    图  4  大黄鱼slitrk3基因启动子不同长度片段的PCR产物电泳

    注:M. DL2000 DNA Marker;1. slitrk3-P1片段;2. slitrk3-P2片段;3. slitrk3-P3片段;4. slitrk3-P4片段;5. slitrk3-P5片段;6. slitrk3-P6片段。

    Figure  4.  Electrophoresis of PCR products of slitrk3 gene promoter different length fragments in L. crocea

    Note: M. DL2000 DNA Marker; 1. slitrk3-P1 fragment; 2. slitrk3-P2 fragment; 3. slitrk3-P3 fragment; 4. slitrk3-P4 fragment; 5. slitrk3-P5 fragment; 6. slitrk3-P6 fragment.

    图  5  大黄鱼slitrk3基因启动子重组质粒酶切电泳

    注:M. DL5000 DNA Marker;0. pGL3-Basic质粒;1. pGL3-slitrk3-P1质粒;2. pGL3-slitrk3-P2质粒;3. pGL3-slitrk3-P3质粒;4. pGL3-slitrk3-P4质粒;5. pGL3-slitrk3-P5质粒;6. pGL3-slitrk3-P6质粒。

    Figure  5.  Electrophoresis of restriction enzymes digestion of slitrk3 gene promoter recombinant plasmids in L. crocea

    Note: M. DL5000 DNA Marker; 0. pGL3-Basic plasmid; 1. pGL3-slitrk3-P1 plasmid; 2. pGL3-slitrk3-P2 plasmid; 3. pGL3-slitrk3-P3 plasmid; 4. pGL3-slitrk3-P4 plasmid; 5. pGL3-slitrk3-P5 plasmid; 6. pGL3-slitrk3-P6 plasmid.

    图  6  大黄鱼slitrk3基因启动子不同长度片段活性分析

    注:数据均以平均值±标准误表示 (每个样品3次重复);不同字母表示差异极显著 (P<0.01);*. 差异显著 (P<0.05)。

    Figure  6.  Activity analysis of different length fragments of slitrk3 gene promoter in L. crocea

    Note: Data are described as $ \overline { X}\pm { \rm {SD}}$1 (N=3); different letters above the bars indicate extremely significant differences at P<0.01; *. Significant differences at P<0.05.

    表  1  大黄鱼及其他物种的Slitrk3氨基酸序列的GeneBank登录号

    Table  1.   GeneBank ID of amino acid sequences of Slitrk3 in L. crocea and other species

    物种
    Species
    GeneBank登录号
    GeneBank ID
    大黄鱼 Larimichthys crocea XP_010750353.1
    棘头梅童鱼 Collichthys lucidus TKS81208.1
    金钱鱼 Scatophagus argus XP_046245335.1
    大西洋鲷 Sparus aurata XP_030298892.1
    黃鲈 Perca flavescens XP_028430026.1
    大鳍弹涂鱼 Periophthalmus magnuspinnatus XP_033832314.1
    斑马鱼 Danio rerio XP_021323398.1
    大鼠 Rattus norvegicus NP_001101153.1
    小鼠 Mus musculus NP_001344780.1
    智人 Homo sapiens NP_001305739.1
    下载: 导出CSV

    表  2  大黄鱼slitrk3基因启动子不同长度片段的扩增引物

    Table  2.   Primers used for fragments amplification primers of L. crocea slitrk3 gene

    引物
    Primer
    序列 (5'—3')
    Sequence (5'−3')
    扩增区域
    Amplification region/bp
    产物大小
    Amplification size/bp
    slitrk3-P1 F: ctatcgataggtaccGAGCTCATCCGTAGCTCATTCACATGC −1970—+16 2029
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    slitrk3-P2 F: ctatcgataggtaccGAGCTCGCGGAATCAATCATTTCTGGA −1614—+16 1673
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    slitrk3-P3 F: ctatcgataggtaccGAGCTCGAATCCTCGATCAGCCGATGT −1210—+16 1269
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    slitrk3-P4 F: ctatcgataggtaccGAGCTCCAAACTGACACCTATTTTTCC −667—+16 726
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    slitrk3-P5 F: ctatcgataggtaccGAGCTCTGTCTGAGTGGAGAGATTTGT −376—+16 435
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    slitrk3-P6 F: ctatcgataggtaccGAGCTCCTTTGTGAGGGTGTGTGTATC −147—+16 206
    R: cagtaccggaatgccAAGCTTAGGTAACCCACAGCATCCTCG
    注:单下划线为Sac I酶切位点,下划线为Hind III酶切位点,小写字母为质粒同源序列。 Note: The single underline are the Sac I restriction enzyme digestion sites; the underline is the Hind III restriction enzyme digestion site; and the lowercase letters are the plasmid homologous sequences.
    下载: 导出CSV

    表  3  大黄鱼slitrk3基因启动子转录因子结合位点的预测

    Table  3.   Prediction of transcription factor binding sites of slitrk3 gene promoter in L. crocea

    转录因子
    Transcription factor
    起始
    Star/bp
    终止
    End/bp
    序列 (5'—3')
    Sequence (5'−3')
    Sp1 −1 901 −1 889 gtgcccccccctc
    −1 422 −1 413 gctcaggcca
    −1 037 −1 028 cgcccactgc
    −646 −637 ccgtcctctt
    −520 −511 gtctcctcac
    −504 −495 cctcacccac
    −145 −136 tgtgagggtg
    −107 −98 tccctccttt
    −84 −75 cccatcccct
    GR −1 893 −1 884 ccctctgttc
    −1 801 −1 792 aacagaacac
    −78 −69 ccctctgttc
    C/EBPα −1 697 −1 688 ttattttttt
    −551 −542 tgttatgtaa
    −361 −352 tttgtttgca
    −312 −303 tatattttgt
    −67 −58 gttttgcagt
    C/EBPβ −979 −970 attggaaaat
    GATA-1 −1 735 −1 726 gacatgataa
    TBP −1 691 −1 682 tttttagatt
    TEC1 −1 577 −1 568 cggaatgaaa
    RAP1 −1 327 −1 318 gtgtttgtgt
    NF-1 −757 −748 tggcacccat
    MEB-1 −698 −689 ttatttttaa
    GLO −698 −689 ttatttttaa
    E1 −498 −489 ccacctgctg
    Myf-3 −498 −489 ccacctgctg
    Oct-1 −339 −330 aaattatcca
    IRF-1 −72 −63 gttctgtttt
    下载: 导出CSV
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  • 收稿日期:  2022-12-04
  • 修回日期:  2023-02-27
  • 录用日期:  2023-03-13
  • 网络出版日期:  2023-03-23

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