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缢蛏生物钟基因Tim的鉴定、昼夜表达模式及对性激素分泌的影响

郦琪钧 郑向阳 柳燕子 严玉箫 陈妍蕾 姚韩韩 董迎辉

郦琪钧,郑向阳,柳燕子,等. 缢蛏生物钟基因Tim的鉴定、昼夜表达模式及对性激素分泌的影响[J]. 海洋学报,2023,45(12):101–111 doi: 10.12284/hyxb2023181
引用本文: 郦琪钧,郑向阳,柳燕子,等. 缢蛏生物钟基因Tim的鉴定、昼夜表达模式及对性激素分泌的影响[J]. 海洋学报,2023,45(12):101–111 doi: 10.12284/hyxb2023181
Li Qijun,Zheng Xiangyang,Liu Yanzi, et al. Identification and circadian expression patterns of circadian clock gene Tim and its effects on sex hormone secretion in Sinonovacula constricta[J]. Haiyang Xuebao,2023, 45(12):101–111 doi: 10.12284/hyxb2023181
Citation: Li Qijun,Zheng Xiangyang,Liu Yanzi, et al. Identification and circadian expression patterns of circadian clock gene Tim and its effects on sex hormone secretion in Sinonovacula constricta[J]. Haiyang Xuebao,2023, 45(12):101–111 doi: 10.12284/hyxb2023181

缢蛏生物钟基因Tim的鉴定、昼夜表达模式及对性激素分泌的影响

doi: 10.12284/hyxb2023181
基金项目: 国家自然科学青年基金项目(31902393);宁波市重大科技攻关暨“揭榜挂帅”项目(2021Z114);宁波市公益性科技计划项目(2022S168);国家海洋水产种质资源库项目(2023);浙江省一流学科“生物工程”学生创新项目(CX2023007)。
详细信息
    作者简介:

    郦琪钧(1999—),男,浙江省绍兴市人,主要从事贝类分子遗传学研究。E-mail:1063999318@qq.com

    通讯作者:

    姚韩韩(1984—),女,高级实验师,主要从事贝类分子遗传学研究。E-mail:yaohanhan1020@126.com

    董迎辉(1980—),男,教授,主要从事贝类分子育种学研究。E-mail:dongyinghui118@126.com

  • 中图分类号: P735

Identification and circadian expression patterns of circadian clock gene Tim and its effects on sex hormone secretion in Sinonovacula constricta

  • 摘要: TimTimeless)是继Per基因发现后的第2个核心生物钟基因,在动物繁殖调控中发挥重要作用。为探讨生物钟基因Tim对缢蛏夜间产卵的调控作用,本研究克隆了缢蛏Tim基因的cDNA全长序列并进行序列分析,利用qRT-PCR和免疫荧光技术研究了Tim基因/蛋白在不同组织中的昼夜表达模式,并用siRNA干扰技术初步研究了Tim基因对性激素分泌的影响。结果显示,缢蛏Tim基因开放阅读框序列为3 090 bp,编码1 029个氨基酸,具备3个保守结构域(TIMELESS superfamily、TIMELESS_C和FIil);Tim基因在缢蛏各个组织中均有表达,其中在肝胰腺、水管、卵巢、精巢中的表达水平较高;Tim基因在卵巢和精巢中呈现昼夜规律性表达,均在白天12:00–18:00较高,夜间00:00–06:00较低;Tim蛋白主要定位在肝胰腺的肝细胞、水管末端的上皮细胞、卵巢的成熟卵母细胞及精巢的精子细胞中,且在卵巢和精巢中的阳性信号均是白天12:00稍强于夜间00:00;siRNA干扰后,Tim基因在精巢、卵巢中的表达量均在干扰后第3~7 天显著下降(P < 0.05),而精巢中睾酮和卵巢中雌二醇含量均在干扰后第1~9 天极显著下降(P < 0.01)。由此可见,Tim基因/蛋白在精巢和卵巢中的表达均呈昼夜规律性变化,并且其被干扰后性激素含量显著降低,推测Tim基因可能通过介导性激素的分泌进而调控缢蛏夜间产卵过程。本研究初步探讨了缢蛏生物钟基因Tim的昼夜表达模式及其对性激素分泌的影响,将对深入解析贝类产卵机制和优化工厂化育苗技术具有重要应用价值。
  • 图  1  Tim蛋白的系统进化树(a)、motif(b)及结构域(c)分析

    Fig.  1  Phylogenetic tree (a), motif (b) and domain (c) analysis of the Tim proteins

    图  2  Tim蛋白的系统进化树(最大似然法,Bootstrap值 = 1 000)

    Fig.  2  Phylogenetic tree of the Tim proteins (maximum likelihood method, Bootstrap value = 1 000)

    图  3  缢蛏Tim基因在不同组织中的表达分析(n = 6,不同小写字母代表差异显著P < 0.05)

    Fig.  3  Relative expression of Tim gene in different adult tissues of Sinonovacula constricta (n = 6, superscript letters represent statistically significant differences at P < 0.05)

    图  4  缢蛏Tim基因在4个组织中的昼夜表达分析(n = 6)

    Fig.  4  Diurnal expression analysis of Tim gene in four tissues of Sinonovacula constricta (n = 6)

    图  5  缢蛏Tim蛋白抗体的特异性检测

    1: 肝胰腺;2:水管;3:卵巢;4:精巢;5,6,7,8:分别代表肝胰腺、水管、卵巢、精巢未加一抗的空白对照

    Fig.  5  Specificity of Tim antibody in Sinonovacula constricta

    1: Hepatopancreas; 2: siphon; 3: ovary; 4: testis; 5, 6, 7, 8: lacking the primary antibody in hepatopancreas, siphon, ovary and testis, respectively

    图  6  缢蛏Tim蛋白在4个组织中的昼夜表达分析

    HC:肝细胞,EC:上皮细胞,MO:成熟卵母细胞,ST:精子细胞;比例尺为100 μm

    Fig.  6  Diurnal expression analysis of Tim protein in four tissues of Sinonovacula constricta

    HC: Hepatocytes, EC: epithelial cell,MO: mature oocytes, ST: spermatid;scale bars were 100 μm

    图  7  siRNA注射后Tim基因表达及睾酮和雌二醇含量变化(n = 6)

    a1,a2:siRNA干扰后Tim基因在精巢和卵巢中的表达变化;b1,b2:siRNA干扰后精巢中睾酮和卵巢中雌二醇的含量变化;*代表P < 0.05;**代表P < 0.01

    Fig.  7  Gene expression of Tim and testosterone and estradiol contents after siRNA injection (n = 6)

    a1, a2: Relative expressions of Tim in testis and ovary among groups after siRNA injection; b1, b2: the testosterone (testis) and estradiol (ovary) content changes among groups after siRNA injection; * represents P < 0.05, and ** represents P < 0.01

    表  1  实验所用引物序列信息

    Tab.  1  Information of primers used in the experiments

    引物名称 引物序列(5′-3′) 用途
    RACE- Tim-5 CCATAGACCTTTGTCGGCGAGTTGCTGT 基因克隆
    RACE- Tim-3 TGGCGACACCTACTTCAGGGCTTTTATT 基因克隆
    Tim-F1 GTAATGAACGGAATGGGGCT 序列验证
    Tim-R1 ATGGGGCTAAATGGAAGTCTCA 序列验证
    Tim-F2 GGAACACCTACTGGATGACGAC 序列验证
    Tim-R2 TGTTATGGCTGGTGGCAATG 序列验证
    Tim-F3 AATCAACTTTTCAAGGGGACG 序列验证
    Tim-R3 GTCTTCTCTTTGATTGATGTCGTG 序列验证
    Real-Tim-F CGGAATGGGGCTAAATGG 荧光定量
    Real-Tim-R CGTCATCCAGTAGGTGTTCCA 荧光定量
    18S rRNA-F TCGGTTCTATTGCGTTGGTTTT 荧光定量
    18S rRNA-R CAGTTGGCATCGTTTATGGTCA 荧光定量
    siRNA-Tim-F GGAAGGAAAUAGAGUUUAATT siRNA干扰
    siRNA-Tim-R UUAAACUCUAUUUCCUUCCTT siRNA干扰
    NC-F UUCUCCGAACGUGUCACGUTT siRNA干扰
    NC-R ACGUGACACGUUCGGAGAATT siRNA干扰
    下载: 导出CSV
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  • 收稿日期:  2023-07-14
  • 修回日期:  2023-11-27
  • 网络出版日期:  2024-01-12
  • 刊出日期:  2023-12-01

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