文章摘要
Ccr-lncRNA172145靶向miR-206在锦鲤体色调控中的作用初探
Preliminary study on the role of Ccr-lncRNA172145 in targeting miR-206 in the regulation of skin color of Koi carp (Cyprinus carpio var. koi)
投稿时间:2020-11-19  修订日期:2021-01-02
DOI:
中文关键词: 锦鲤  ccr-lncRNA172145  miR-206  体色
英文关键词: Cyprinus carpio var. koi  Ccr-lncRNA172145  miR-206  skin color
基金项目:中央级公益性科研院所基本科研业务费专项资金(2019ZY22);农业农村部现代农业人才支撑计划项目(2016142);江苏省研究生科研与实践创新计划(KYCX18_0738)
作者单位邮编
罗明坤 南京农业大学无锡渔业学院 214081
王兰梅 中国水产科学研究院淡水渔业研究中心 
朱文彬 中国水产科学研究院淡水渔业研究中心 
傅建军 中国水产科学研究院淡水渔业研究中心 
董在杰 南京农业大学无锡渔业学院 214081
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中文摘要:
      基于前期对锦鲤皮肤组织的转录组测序数据,筛选到4条在3种皮肤(黑色、红色、白色)中显著差异表达的lncRNA;通过RNAhybrid和TargetScan靶基因软件,发现lncRNA172145与黑色素合成通路中miR-206之间存在靶向结合位点。基于CPC、CPAT及CNIT软件,对lncRNA172145进行编码能力分析,证实该序列为lncRNA,不具备编码蛋白能力。然后,利用qRT-PCR技术对该序列时空表达水平进行了检测,发现在眼睛、黑色皮肤、鳍条及血液中的表达量显著高于其他组织;自原肠胚时期表达量开始显著性上升,高水平趋势一直持续到孵化后20 d。借助双荧光素酶报告实验,进一步证实lncRNA172145与miR-206之间存在靶向调控关系。最后,通过合成miR-206拮抗剂,对miR-206进行了体内沉默,发现与注射阴性对照拮抗剂组和PBS组相比,miR-206拮抗剂组的lncRNA172145表达水平显著性升高。研究结果说明lncRNA172145可能通过靶向到miR-206,参与到黑色素合成通路的调控,这为后续深入挖掘二者在黑色素合成通路中具体的分子作用机制提供了基础资料。
英文摘要:
      Based on the transcriptome sequencing data of Cyprinus carpio var. koi skin tissues in the previous research, four lncRNAs were screened that were significantly differentially expressed in the three skin types (black, red, and white); and target binding sites between lncRNA172145 and miR-206 in the melanogenesis pathway were identified through RNAhybrid and TargetScan target gene tools. Then, the coding ability of lncRNA172145 was detected by CPC, CPAT, and CNIT software, and the results confirmed that this sequence is lncRNA with no protein coding ability. The temporal and spatial expression levels of this sequence were further analyzed by qRT-PCR, and it was demonstrated that the levels were significantly higher in the eyes, black skin, fins and blood than that in other tissues; and the expression levels started to increase significantly from the gastrulation stage, and the high-level trend continued until 20 days after hatching. Additionally, with the help of dual luciferase reporter assay, the existence of a targeted regulatory relationship between lncRNA172145 and miR-206 was further verified. Finally, miR-206 was silenced in vivo by synthesizing miR-206 antagonist, and the results showed that the expression level of lncRNA172145 in the miR-206 antagonist group, was significantly higher compared to the negative control antagonist group and the PBS group. This study suggests that lncRNA172145 may be involved in the regulation of melanogenesis pathway by targeting to miR-206, which provides fundamental information for the subsequent in-depth investigation of the specific molecular mechanisms of them in the regulation of skin color.
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