| 摘要: |
| 密斑刺鲀（Diodon hystrix）不仅具有食用价值，同时也是传统的中药及保健食品，但目前关于密斑刺鲀的生殖内分泌调控研究较少，限制了密斑刺鲀的人工繁育基础理论的发展。总所周知，促性腺激素释放激素（gonadotropin-releasing hormone，GnRH）是生殖内分泌调控的关键因子之一。利用转录组分析及分子克隆技术，获得密斑刺鲀促性腺激素释放激素（gonadotropin-releasing hormone，GnRH）gnrh2和gnrh3基因的cDNA开放阅读框（Open Reading Frame，ORF）序列，并利用生物信息学的方法，获得密斑刺鲀gnrh2 cDNA开放阅读框共279 bp，可编码92个氨基酸残基；密斑刺鲀gnrh3 cDNA开放阅读框共270 bp，可编码89个氨基酸残基。通过系统进化分析表明，密斑刺鲀GnRH与鲀科类的鱼具有较近的亲缘关系。通过聚合酶链式反应（Polymerase Chain Reaction，PCR）半定量的方法检测gnrh2和gnrh3基因在密斑刺鲀各组织中的分布，结果发现gnrh2和gnrh3基因在脾、鳃和垂体中的表达量较高，而在心脏中的表达量最低。此外，利用实时荧光定量的方法检测了密斑刺鲀gnrh2和gnrh3基因在性腺发育不同时期的表达模式，结果显示密斑刺鲀gnrh2基因在不同的性腺发育时期中没有显著性的变化，而gnrh3基因随着性腺发育成熟而不断升高。研究主要获得两个创新性的重要结果：首先是证实了密斑刺鲀存在两种gnrh基因（gnrh2和gnrh3），其次是提示了gnrh3基因可能是密斑刺鲀生殖调控的主要gnrh类型。研究结果可为深入研究密斑刺鲀生殖内分泌调控机理提供研究方向和基础。 |
| 关键词: 密斑刺鲀(Diodon hystrix) GnRH 分子克隆 组织分布 |
| DOI：10.11693/hyhz20210100022 |
| 分类号:S965.3 |
| 基金项目:海南省重点研发计划项目，ZDYF2018225号；热带海洋生物资源利用与保护教育部重点实验室2020年开放课题，UCTMB20201号；海南省重大科技计划项目，ZDKJ2016009号；海南热带海洋学院2016年学科带头人和博士研究生科研启动项目，RHDXB201612号。 |
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| CLONING AND EXPRESSION OF gnrh GENE IN DIODON HYSTRIX |
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CHEN Hua-Pu1, HUANG Chun-Ren2, HE Rui-Qi1, DAI Ming-Shu1, ZHANG Ming-Zhen1, LI Zhi-Yuan1, HUANG Hai3, LI Guang-Li1
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1.Guangdong Research Center on Reproductive Control and Breeding Technology of Indigenous Valuable Fish Species, Guangdong Provincial Engineering Laboratory for Mariculture Organism Breeding, Fisheries College, Guangdong Ocean University, Zhanjiang 524088, China;2.Hainan Chenhai Aquatic Products Co., Ltd, Sanya 572000, China;3.Key Laboratory of Utilization and Conservation for Tropical Marine Bioresources, Ministry of Education, Hainan Key Laboratory for Conservation and Utilization of Tropical Marine Fishery Resources, Hainan Tropical Ocean University, Sanya 572022, China
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| Abstract: |
| Diodon hystrix is an edible fish with commercial value and additional value for traditional Chinese medicine and health care. However, studies on its endocrinology and artificial breeding are few, which hinders the practice of artificial breeding. It is known that gonadotropin-releasing hormone (GnRH) is a key regulation factor of reproduction. In the present study, two subtypes of gnrh genes, named as gnrh2 and gnrh3, were cloned form the brain of D. hystrix by transcriptome analysis and molecular cloning. Results show that the open reading frame (ORF) of gnrh2 gene cDNA was 279 bp, encoding 92 amino acids, and the ORF of gnrh3 gene cDNA was 270 bp, encoding 89 amino acids. The phylogenetic analysis showed that D. hystrix GnRHs are closely related to the GnRHs of pufferfish family. D. hystrix gnrh2 and gnrh3 genes were expressed under different levels in all the detected tissues with semi-quantitative polymerase chain reaction (PCR) method, showing the lowest expressions in heart and the rather high expressions in spleen, gill, and pituitary. Furthermore, real-time quantitative PCR was used to detected the expression profiles of gnrh2 and gnrh3 genes during the ovarian and testicular developments, showing no significant change in the expressions of gnrh2 in both of ovarian and testicular developments, while the expression of gnrh3 showed gradual significant increase with the maturation processes of ovarian and testicular. Two important facts were revealed in this study. First, two gnrh genes (gnrh2 and gnrh3) were confirmed in D. hystrix; and secondly, the gnrh3 gene was the main gnrh type of reproductive regulation in D. hystrix. The present study provides a reference for future study on the mechanism of the reproductive endocrine regulation in D. hystrix. |
| Key words: Diodon hystrix GnRH molecular cloning tissue distribution |
