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岳华梅, 叶欢, 阮瑞, 刘志刚, 李创举. 达氏鲟gpr54基因的克隆及Kisspeptin注射对其表达的影响[J]. 水生生物学报, 2018, 42(5): 896-902. DOI: 10.7541/2018.110
引用本文: 岳华梅, 叶欢, 阮瑞, 刘志刚, 李创举. 达氏鲟gpr54基因的克隆及Kisspeptin注射对其表达的影响[J]. 水生生物学报, 2018, 42(5): 896-902. DOI: 10.7541/2018.110
YUE Hua-Mei, YE Huan, RUAN Rui, LIU Zhi-Gang, LI Chuang-Ju. MOLECULAR CLONING OF GPR54 GENES AND EFFECTS OF KISSPEPTIN INTRAPERITONEAL INJECTION ON ITS EXPRESSION IN ACIPENSER DABRYANUS[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(5): 896-902. DOI: 10.7541/2018.110
Citation: YUE Hua-Mei, YE Huan, RUAN Rui, LIU Zhi-Gang, LI Chuang-Ju. MOLECULAR CLONING OF GPR54 GENES AND EFFECTS OF KISSPEPTIN INTRAPERITONEAL INJECTION ON ITS EXPRESSION IN ACIPENSER DABRYANUS[J]. ACTA HYDROBIOLOGICA SINICA, 2018, 42(5): 896-902. DOI: 10.7541/2018.110

达氏鲟gpr54基因的克隆及Kisspeptin注射对其表达的影响

MOLECULAR CLONING OF GPR54 GENES AND EFFECTS OF KISSPEPTIN INTRAPERITONEAL INJECTION ON ITS EXPRESSION IN ACIPENSER DABRYANUS

  • 摘要: G蛋白偶联受体54(GPR54, G protein-coupled receptor 54)是kisspeptin (Kiss)的受体蛋白。Kisspeptin/GPR54系统通过调节促性腺激素释放激素(GnRH)的活性来参与鱼类生殖调控。为了研究Kisspeptin/GPR54系统对达氏鲟(Acipenser dabryanus)GnRH的调控功能, 克隆得到达氏鲟2个gpr54基因的全长cDNA序列, 命名为dsgpr54-1及dsgpr54-2, 分别编码379和368个氨基酸。氨基酸序列比对及进化树分析表明, 达氏鲟Gpr54与四足动物Gpr54序列一致性较高, 亲缘关系较近。荧光定量PCR研究发现, dsgpr54-1的转录本在精巢、卵巢、下丘脑、垂体、中脑及端脑等组织中均有表达, 且在下丘脑中转录水平最高; 而dsgpr54-2仅在脑组织中转录, 且在垂体、中脑及下丘脑中表达丰度均较高。为了研究Gpr54是否可以与其配体Kisspeptin结合调控下丘脑中gnrh基因的表达, 分别合成了达氏鲟Kiss-1和Kiss-2的核心十肽(10 nmol/L、1000 nmol/L), 腹腔注射到9月龄达氏鲟。结果表明, 不同浓度Kiss-1、Kiss-2注射均引起gpr54基因表达量升高, 并且10 nmol/L Kiss-2注射能够显著促进dsgpr54-2的表达(P<0.05)。另外, 不同浓度Kiss-1注射均造成了gnrh转录水平的下降; 而10 nmol/L Kiss-2注射使得gnrh1表达量上升, 而gnrh2的表达量下降, 1000 nmol/L Kiss-2注射则引起gnrh1表达量的下降, gnrh2的表达量没有显著变化。上述研究结果表明, 达氏鲟gpr54基因均能与其配体kiss-1、kiss-2相结合, 但表现出一定的受体-配体选择性差异。Kiss-1、Kiss-2通过激活Gpr54的活性, 调控下丘脑中gnrh基因的表达, 且其调控功能存在差异。

     

    Abstract: GPR54 is the cognate receptor of Kisspeptin, which plays a significant role in fish reproduction regulation by acting on the Gonadotropin-Releasing Hormone (GnRH). In order to study the regulation of GnRH by Kisspeptin/GPR54 system in the Dabry’s sturgeon (Acipenser dabryanus), the full-length cDNAs of two gpr54 genes were cloned, which were designated as dsgpr54-1 and dsgpr54-2, encoding 379 and 368 amino acids, respectively. Multiple amino acid sequences alignment and evolutionary tree analysis indicated that Gpr54 of Dabry’s sturgeon shared higher sequence identities and closer evolutionary distances with its counterparts in tetrapods. By quantitative real-time PCR, it showed that dsgpr54-1 were transcribed in both gonad (testis and ovary) and brain (hypothalamus, pituitary, mesencephalon and telencephalon) with the highest transcription level in the hypothalamus. Conversely, dsgpr54-2 was only found in the brain, with high expression levels in the pituitary, mesencephalon and hypothalamus. In order to investigate whether gpr54 could regulate the expression of gnrh in the hypothalamus by combined to its ligand Kisspeptin, decapeptides of both Kiss-1 and Kiss-2 (10 nmol/L and 1000 nmol/L) of Dabry’s sturgeon were synthesized and injected to the peritoneal cavities of 9 month juveniles. It showed by quantitative real-time PCR that both the two doses of Kiss-1 and Kiss-2 injection induced the increase of gpr54 expression, and 10 nmol/L Kiss-2 injection increased the transcription of dsgpr54-2 significantly (P<0.05). Besides, both two doses of Kiss-1 injection reduced thegnrh expression. However, 10 nmol/L Kiss-2 injection caused the increase of the gnrh1 expression and the decrease of gnrh2 expression, while in the 1000 nmol/L Kiss-2 injection group, gnrh1 expression decreased with no changes of the gnrh2 transcription. The above results revealed that both the two gpr54 genes could bind to its ligands kiss-1 and kiss-2, but with receptor-ligand selection discrepancy. Kiss-1 and Kiss-2 regulate the gnrh expression differently by activating their receptor GPR54 in the hypothalamus of Dabry’s sturgeon.

     

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