水产学报2026，Vol.50Issue(6)：20-32,13.DOI:10.11964/jfc.20260315445
舒氏猪齿鱼不同组织qPCR内参基因的筛选与验证
Screening and validation of reference genes for qPCR in different tissues of blackspot tuskfish(Choerodon schoenleinii)
摘要
Abstract
Blackspot tuskfish(Choerodon schoenleinii)is an economically important coral reef fish with high aquaculture value and a unique protogynous hermaphroditic reproductive pattern.Quantitative real-time PCR(qPCR)is widely used for gene expression analysis,but its accuracy depends on the use of stable reference genes.However,suitable reference genes for C.schoenleinii have not yet been systematically evaluated.To screen for stably expressed reference genes in different tissues of C.schoenleinii,eighteen candidate reference genes(eif2s2,ube2a,actc1,rpl7l1,eif1,eif3a,ppia,rps18,rps27a,snrpd2,taf13,tor1a,tubb4b,rps27l,dera,gapdh,actg1,and hsp90ab1)were selected.Their expression levels in gonad,liver,spleen,kidney,intestine,heart,brain,muscle,skin,and gill tissues were analyzed using qPCR,and expression stability was evaluated using BestKeeper,NormFinder,geNorm,and RefFinder.Among the 18 candidate genes,all except hsp90ab1 exhibited acceptable amplification efficiencies(90%-103%)and high coefficients of determination(R2>0.98),indicating good amplification spe-cificity.CT value analysis revealed variation in expression across tissues,with eif2s2 showing the least variation and rps27a the greatest.Comprehensive analysis using the four algorithms showed generally consistent results,identifying eif3a,eif2s2,tubb4b,and rps27l as the most stable genes,while actc1 and gapdh were the least stable.geNorm analysis indicated that V4/5=0.143,suggesting that four reference genes are sufficient for normalization across multiple tissues,whereas two reference genes are adequate for single-tissue analyses.Overall,tubb4b,eif2s2,eif3a,and rps27l were stably expressed across the ten tissues and are recommended as a reference gene combination for qPCR studies in different tissues of C.schoenleinii.For studies focusing on a single tissue,a dual-reference gene strategy is recommended.This study provides reliable tools for gene expres-sion analysis and functional research in C.schoenleinii.关键词
舒氏猪齿鱼/内参基因/qPCR/不同组织/稳定性评价Key words
Choerodon schoenleinii/reference genes/qPCR/different tissues/stability assessment分类
生物科学引用本文复制引用
高佳豪,陈海茹,李晓斌,赵艳飞,张宁,廖健,董忠典..舒氏猪齿鱼不同组织qPCR内参基因的筛选与验证[J].水产学报,2026,50(6):20-32,13.基金项目
国家重点研发计划(2024YFD2401803) （2024YFD2401803）
2025 年度广东省现代化海洋牧场适养品种核心技术攻关项目(2025-MRB-00-001) （2025-MRB-00-001）
广东海洋大学水产优青项目 （）
广东海洋大学本科生创新创业项目(CXTD2023) National Key Research and Development Program of China(2024YFD2401803) （CXTD2023）
2025 Guangdong Provin-cial Project on Core Technologies for Suitable Species in Modern Marine Pastures(2025-MRB-00-001) （2025-MRB-00-001）
Guangdong Ocean University Outstanding Young Scholars Program in Fisheries （）
Guangdong Ocean University Undergraduate Innovation and Startup Project(CXTD2023) （CXTD2023）
