布氏鲳鲹microRNA-novel_176组织表达及靶基因验证  

Tissue expression and target gene verification of microRNA-novel_176 in Trachinotus blochii

作　　者：宋飞彪 施立萍 郑达 梁欢 纪馨馨 佟金凤 卜邵洋 孙俊龙 骆剑[1] SONG Feibiao;SHI Liping;ZHENG Da;LIANG Huan;JI Xinxin;TONG Jinfeng;BU Shaoyang;SUN Junlong;LUO Jian(Hainan Aquaculture Breeding Engineering Research Center,Sanya Nanfan Research Institute of Hainan University,School of Marine Biology and Fisheries,Hainan University,Haikou 570228,China)

机构地区：[1]海南大学海洋生物与水产学院,海南大学三亚南繁研究院,海南省水产种业工程研究中心,海口570228

出　　处：《水产学报》2025年第4期40-48,共9页Journal of Fisheries of China

基　　金：海南省自然科学基金(322QN236);国家自然科学基金(32160862);国家重点研发计划(2022YFD2400504)。

摘　　要：【目的】研究microRNA在布氏鲳鲹性腺发育中的作用。【方法】本研究以前期发现的性腺发育关键miR-novel_176为对象,采用荧光定量PCR、荧光原位杂交、双荧光素酶报告基因、RNA干扰和过表达等实验手段,系统验证其与靶基因的调控关系及潜在的生物学功能。【结果】miR-novel_176在布氏鲳鲹各组织中均表达,在卵巢中的表达量高于精巢,但差异不显著;miR-novel_176主要定位于卵巢细胞质,阳性信号主要位于卵母细胞和颗粒细胞中,在精巢中的支持细胞和精原细胞也显示出微弱杂交信号;miR-novel_176与esr2和foxo3基因的3'-UTR互补,显著抑制荧光素酶的活性;细胞转染novel_176-antagomir和novel_176-agomir后,miR-novel_176的表达水平显著下调和上调,靶基因foxo3和esr2的表达水平发生显著相反的表达变化。【结论】miR-novel_176的靶基因为esr2和foxo3,miR-novel_176能够特异互补3'UTR区域进而调控其表达水平。本研究为进一步揭示miRNA在鱼类性腺发育中的功能提供了新证据,同时为布氏鲳鲹性腺发育研究提供了新的候选miRNA。MicroRNAs,as non-coding RNAs,play important roles in many key physiological processes in organisms and can significantly impact gonadal development in fish through the regulation of target gene transcription.Therefore,this study investigated the previously identified key miRNA-novel_176 involved in gonadal development.Various experimental techniques including fluorescence quantitative PCR,fluorescence in situ hybridization,dual-luciferase reporter gene assay,RNA interference,and overexpression were employed to systematically validate its regulatory relationship with target genes and explore its potential biological functions.The results showed that miRnovel_176 was expressed in various tissues of Trachinotus blochii,with higher expression levels observed in the ovary than in the testis,although the difference was insignificant.miRnovel_176 was primarily localized in the cytoplasm of ovarian cells,with positive signals mainly detected in oocytes and granulosa cells.Weak hybridization signals were also observed in the supporting cells and spermatogonia of the testis.miRnovel_176 was found to target the 3'-UTRs of the esr2 and foxo3 genes,significantly inhibiting luciferase activity.Following transfection with novel_l76-antagomir and novel_l76-agomir,the expression levels of miRnovel_176 were significantly downregulated and upregulated,respectively,leading to significant opposite expression changes in the target genes foxo3 and esr2.This study indicates that the target genes of miRnovel_176 are esr2 and foxo3,and miRnovel_176 can regulate their expression levels by targeting their 3'UTR regions.This study provides new evidence for further revealing the function of miRNAs in the gonadal development of fish and also provides new candidate miRNAs for the study of gonadal development of T.blochii.

关 键 词：布氏鲳鲹 miRNA 性腺发育 细胞定位 RNA干扰 基因过表达 

分 类 号：Q786[生物学—分子生物学] S968.1[农业科学—水产养殖]