中华鳖Dkkl1基因的分子特征及对外源性激素处理的响应  

Molecular characterization of Dkkl1 gene and its response to exogenous hormone treatment in Pelodiscus sinensis

作　　者：汪泳昌 祝骏贤 李建松 陈辰 纪利芹 洪孝友[2] 刘晓莉 王亚坤[2] 吴聪聪 余汶君 罗来福 陈海港 魏成清[2] 朱新平[2] 张俊杰[1] 李伟[2] WANG Yongchang;ZHU Junxian;LI Jiansong;CHEN Chen;JI Liqin;HONG Xiaoyou;LIU Xiaoli;WANG Yakun;WU Congcong;YU Wenjun;LUO Laifu;CHEN Haigang;WEI Chengqing;ZHU Xinping;ZHANG Junjie;LI Wei(College of Life Sciences,Xinjiang Agricultural University,Urumqi 830052,China;Pearl River Fisheries Research Institute,Chinese Academy of Fishery Sciences/Key Laboratory of Tropical&Subtropical Fishery Resource Application&Cultivation of Ministry of Agriculture and Rural Affairs,Guangzhou 510380,China;Huizhou Wealth Xing Industrial Co.,Ltd.,Huizhou 516000,China)

机构地区：[1]新疆农业大学生命科学学院,新疆乌鲁木齐830052 [2]中国水产科学研究院珠江水产研究所/农业农村部热带亚热带水产资源利用与养殖重点实验室,广东广州510380 [3]惠州市财兴实业有限公司,广东惠州516000

出　　处：《南方水产科学》2023年第6期166-172,共7页South China Fisheries Science

基　　金：国家自然科学基金青年科学基金项目(32102792);广东省农业农村厅种业振兴项目(2022-SJS-00-00,2022-SPY-00-015);中国水产科学研究院中央级公益性科研院所基本科研业务费专项资金资助(2023SJHX1);广东省现代农业产业技术体系创新团队建设项目(2019KJ150);中国-东盟海洋合作基金(CAMC-2018F);广东省淡水水产种质资源库运行项目(2022-SBH-00-001);国家淡水水产种质资源库珠江分库(FGRC18537)。

摘　　要：Dkkl1基因在哺乳动物睾丸发育和精子发生过程中扮演着重要角色,然而其在龟鳖类动物中的研究十分有限。为探索龟鳖类Dkkl1基因的潜在功能和作用机制,克隆了中华鳖(Pelodiscus sinensis)Dkkl1基因的cDNA片段,分析其序列特征、表达模式以及对外源性激素处理的响应。克隆获得的Dkkl 1基因cDN A序列长度为823 bp,其中3'-非编码区(UTR)为67 bp,5'UTR为90 bp,开放阅读框为666 bp,共编码222个氨基酸。中华鳖Dkkl1蛋白是一种稳定性较差、亲水性较强的碱性蛋白,其二级结构和三级结构主要以α-螺旋和无规则卷曲为主。氨基酸序列同源性比对结果显示其与中华草龟(Chinemys reevesii)Dkkl1蛋白的相似性较高(81%),与棱皮龟(Dermochelys coriacea)Dkkl1蛋白的同源性较低(70%)。通过RT-PCR和RT-qPCR分析发现,中华鳖Dkkl1 mRNA在3冬龄成体精巢中极显著性高表达(P<0.001),而在其余体组织中几乎不表达。并且随着年龄的增长,中华鳖精巢中Dkkl1基因的表达量逐渐上升,并在3冬龄时达到顶峰。此外,17β-雌二醇(E2)和17α-甲基睾酮(17α-MT)处理均可显著抑制成体中华鳖精巢中Dkkl1基因的表达(P<0.05)。研究表明,Dkkl1基因可能在中华鳖睾丸发育和精子发生过程中起着重要作用。Dkkl1 gene plays an important role in mammalian testicular development and spermatogenesis,but the research of Dkkl1 gene in turtles is still limited.Therefore,in order to explore its potential function and mechanism of action in turtles,we cloned a cDNA fragment of Dkkl1 gene from Chinese soft-shelled turtle(Pelodiscus sinensis),and analyzed its sequence characteristics,expression pattern and response to exogenous hormone treatment.The cDNA of Dkkl1 gene was 823 bp in length,with 3'UTR of 67 bp,5'UTR of 90 bp,open reading frame of 666 bp,and encoded 222 amino acids in total.Dkkl1 protein is a kind of alkaline protein with poor stability and high hydrophilicity.The secondary and tertiary structures of Dkkl1 protein are mainly dominated byα-helix and irregular coil.Amino acid sequence homology comparison showed high similarity with Chinemys reevesii Dkkl1 protein(81%),but low homology with Dermochelys coriacea Dkkl1 protein(70%).RT-PCR and RT-qPCR analyses reveal that Dkkl1 mRNA was significantly highly expressed in spermathecae of 3-winter-age P.sinensis adults(P<0.001),while it was hardly expressed in the rest of somatic tissues.Moreover,the expression of Dkkl1 gene in P.sinensis spermathecae gradually increased with age and peaked at 3-winter-age.In addition,both 17β-estradiol and 17α-methyltestosterone treatments significantly inhibited the expression of Dkkl1 gene in adult P.sinensis spermathecae(P<0.05).The results suggest that the Dkkl1 gene might play an important role in the process of testicular development and spermatogenesis in P.sinensis.

关 键 词：中华鳖 Dkkl1基因 睾丸发育 精子发生 生物信息学分析 外源性激素 

分 类 号：S917.4[农业科学—水产科学]