| 克氏原螯虾PcCyc基因克隆及其对光周期的表达响应 |
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| 引用本文： | 谢伟,蒋琦辰,孙宾,杨颖,李鹏,严洁,周开亚.克氏原螯虾PcCyc基因克隆及其对光周期的表达响应[J].水产科学,2021(1):72-80. |
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| 作者姓名： | 谢伟 蒋琦辰 孙宾 杨颖 李鹏 严洁 周开亚 |
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| 作者单位： | 南京师范大学生命科学学院;江苏省淡水水产研究所 |
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| 基金项目： | 国家虾蟹产业技术体系克氏原螯虾种质资源与遗传育种岗位项目(CARS-48);江苏省高等学校自然科学研究重大项目(19KJA330001);江苏省水生生物资源重大专项(ZYHB16-3);江苏省海洋渔业资源环保项目;江苏省现代农业(克氏原螯虾)产业技术体系项目(JATS[2020]365);江苏省农业重大新品种创制项目(PZCZ201746);江苏省高等学校自然科学研究面上项目(17KJD240001). |
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| 摘 要： | 利用cDNA末端快速扩增技术克隆克氏原螯虾生物钟核心基因之一的周期循环蛋白(CYC)基因(命名为PcCyc基因)的部分cDNA序列。取雄性克氏原螯虾(体长9~11 cm)在全光谱日光灯不同光周期下(12L∶12D、0L∶24D和0L∶72D)培养结束后,于7:00、11:00、15:00、19:00、23:00、翌日03:00、7:00取样,采用实时荧光定量PCR检测PcCyc基因在脑、眼柄和肝胰腺中的mRNA表达水平。试验结果显示,PcCyc基因完整的开放阅读框共2010个碱基,可编码669个氨基酸。氨基酸序列与红螯螯虾CYC蛋白相似性为94%;蛋白分子量约73.963 ku,理论等电点6.56,为疏水性非分泌蛋白,无信号肽;结构域分析结果显示,PcCYC蛋白含有BHLH/PAS结构域,包括1个HLH结构域和2个PAS结构域;二级结构包含11个α-螺旋、12个β-折叠和24个无规则卷曲。不论光照如何改变,PcCyc基因在眼柄和肝胰腺中都基本遵循着24 h的节律振荡,而在大脑中的节律振荡随光照周期改变被打破。试验结果将为进一步探究克氏原螯虾Cyc基因在其昼夜生物钟节律中发挥的作用及机制奠定基础。
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| 关 键 词： | 克氏原螯虾 Cyc基因 克隆 MRNA表达 生物信息学 |
Cloning and Expression Response of PcCyc Gene to Photoperiod in Red Swamp Crayfish Procambarus clarkii |
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| XIE Wei,JIANG Qichen,SUN Bin,YANG Ying,LI Peng,YAN Jie,ZHOU Kaiya.Cloning and Expression Response of PcCyc Gene to Photoperiod in Red Swamp Crayfish Procambarus clarkii[J].Fisheries Science,2021(1):72-80. |
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| Authors: | XIE Wei JIANG Qichen SUN Bin YANG Ying LI Peng YAN Jie ZHOU Kaiya |
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| Institution: | (Jiangsu Key Laboratory for Biodiversity&Biotechnology,College of Life Sciences,Nanjing Normal University,Nanjing 210023,China;Jiangsu Province Fisheries Research Institute,Nanjing 210017,China) |
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| Abstract: | In this study,a partial cDNA sequence of cycle gene(PcCyc),one of the core genes of the circadian clock,was cloned from red swamp crayfish Procambarus clarkii with body length of 9—11 cm by rapid amplification of cDNA ends(RACE)technology,and then the mRNA expression levels of PcCyc gene were detected in brain,eye stalk and hepatopancreas of the red swamp crayfish exposed to various photoperiods of 12 L∶12 D,0 L∶24 D,and 0 L∶72 D under full-spectrum fluorescent lamps at 7:00,11:00,15:00,19:00,23:00,03:00 and 7:00 the next day by real-time quantitative PCR.The results showed that the mRNA of PcCyc gene had a complete open reading frame(ORF)of 2010 base pairs encoding 669 amino acids,and the amino acid sequence of PcCYC had the maximal similarity(94%)to that of the red claw crayfish Cherax quadricarinatus,with the protein molecular weight of about 73.963 ku,the theoretical isoelectric point of 6.56,which is a hydrophobic non-secreted protein with no signal peptide.The structural domain analysis revealed that there were a BHLH/PAS domain,including one HLH domain and two PAS domains in the PcCYC protein,with secondary structure of 11 alpha-helices,12 beta-sheets,and 24 random coils.The PcCyc gene was shown to be 24-hour rhythm fluctuation in both eyestalk and hepatopancreas,no matter what photoperiod was.However,the rhythmic fluctuation was broken in the brain as the photoperiod changed.The findings provide the foundation for further exploration of the role and mechanism of the Cyc gene in circadian clock of the red swamp crayfish. |
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| Keywords: | Procambarus clarkii cycle gene cloning mRNA expression bioinformation |
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