半滑舌鳎生物钟相关基因克隆及在卵巢发育成熟过程中表达特征分析  

Cloning and Expression Analysis of Clock Genes During Ovarian Development and Maturation of Tongue Sole(Cynoglossus semilaevis)

作　　者：晏科文 马晓东 史宝 程汉良[1] 王重女 赵新宇 YAN Kewen;MA Xiaodong;SHI Bao;CHENG Hanliang;WANG Chongnü;ZHAO Xinyu(College of Marine Science and Fisheries,Jiangsu Ocean University,Lianyungang 222005,China;Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,State Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao 266071,China)

机构地区：[1]江苏海洋大学海洋科学与水产学院,江苏连云港222005 [2]中国水产科学研究院黄海水产研究所、海水养殖生物育种与可持续产出全国重点实验室、青岛海洋科技中心海洋渔业科学与食物产出过程功能实验室,山东青岛266071

出　　处：《渔业科学进展》2024年第3期101-116,共16页Progress in Fishery Sciences

基　　金：国家重点研发计划项目(2022YFD2400401);国家自然科学基金项目(31772829);山东省重点研发计划项目(2021LZGC028);中国水产科学研究院黄海水产研究所基本科研业务费(20603022023023;2020TD47);国家现代农业产业技术体系(CARS-47)共同资助。

摘　　要：为阐明生物钟相关基因在半滑舌鳎(Cynoglossus semilaevis)卵巢成熟过程中的作用及机制,采集性成熟半滑舌鳎不同发育期的卵巢组织提取总RNA,通过反转录聚合酶链式反应(RT-PCR)技术对5个核心生物钟基因Clock1a、Bmal1a、Cry1a、Cry2和Per2的编码区(CDS)序列进行克隆和序列分析。结果显示,Clock1a的CDS序列编码712个氨基酸,序列中有功能性结构域PASD1和PAS11。Bmal1a的CDS序列编码626个氨基酸,序列中有功能性结构域PASD3和PAS11。Cry1a和Cry2的CDS序列分别编码631个、669个氨基酸,序列中均有功能性结构域FAD7。Per2的CDS序列编码1415个氨基酸,序列中有功能性结构域PeriodC和PAS11。通过实时荧光定量PCR(qRT-PCR)分析5个生物钟基因mRNA在性成熟半滑舌鳎卵巢不同发育期的表达特性,发现5个生物钟基因存在性腺发育周年表达规律,且在卵巢Ⅱ期和Ⅲ期相对高表达(P<0.05),在卵巢Ⅳ、Ⅴ和Ⅵ期相对低表达,这说明生物钟基因对半滑舌鳎的卵巢发育成熟起到重要的调节作用。研究结果为进一步探究生物钟基因调控半滑舌鳎卵巢发育成熟的机制提供了重要参考,为提高半滑舌鳎的繁育效率提供了理论基础。Clock genes play a pivotal role in the rhythm maturation of bony fish ovaries.To explore the role of clock genes in the rhythmic ovarian development and maturation of tongue sole(Cynoglossus semilaevis),we used real-time fluorescent quantitative PCR technique to analyze the expression profiles of circadian locomotor output cycles Kaput 1a(Clock1a),brain and muscle Arntlike 1a(Bmal1a),Cryptochrome 1a(Cry1a),Cryptochrome 2(Cry2),and Period 2(Per2)at the ovarian stagesⅡ,Ⅲ,Ⅳ,Ⅴ,andⅥ.The coding DNA sequence(CDS)of five clock genes were cloned and phylogenetically analyzed.We found that the CDS sequence length of Clock1a was 1620 bp and encoded 539 amino acids,with the encoded amino acid sequence of Clock1a having a predicted molecular weight of 81.9 kDa.Clock1a has the functional domain PASD1(consisting of 64 amino acids)and PAS11(consisting of 103 amino acids).The CDS sequence length of Bmal1a was 1881 bp and encoded 626 amino acids,with the encoded amino acid sequence of Bmal1a having a predicted molecular weight of 68.9 kDa.In the Bmal1a sequence,the functional domain PASD3 is composed of 63 amino acids,and the functional domain PAS11 is composed of 103 amino acids.The CDS sequence length of Cry1a was 1896 bp and encoded 631 amino acids,with the encoded amino acid sequence of Cry1a having a predicted molecular weight of 71.4 kDa.Its functional domain FAD7 was composed of 199 amino acids.The CDS sequence length of Cry2 was 2007 bp and encoded 669 amino acids,with the encoded amino acid sequence of Cry2 having a predicted molecular weight of 76.0 kDa.The Cry2 sequence contains a functional domain FAD7 consisting of 199 amino acids.The CDS sequence length of Per2 was 4248 bp,encoding 1415 amino acids,and the encoded amino acid sequence of Per2 has a predicted molecular weight of 154.0 kDa.In the Per2 sequence,the functional domain PeriodC consists of 295 amino acids and a functional domain PAS11 consists of 102 amino acids.The neighbor-joining method was used to analyze the Clock1a,Bmal1a,Cry1a,Cry2,and Per2

关 键 词：生物钟基因 半滑舌鳎 卵巢 周年表达规律 

分 类 号：S917.4[农业科学—水产科学]