绿太阳鱼不同组织qRT-PCR内参基因的筛选

doi:10.16378/j.cnki.1003-1111.25172

Abstract
Figure/Table
References (0)
Related Citation (7)

Download: PDF (8615 KB) HTML (1 KB)
Export: BibTeX | EndNote (RIS)

Abstract In order to screen out the reference genes suitable for different tissues of green sunfish Lepomis cyanellus, the expression levels of six candidate reference genes, ubiquitin folding modifier gene (ufm1), α-tubulin gene (TUBA), cytochrome b gene (CYTB), precursor protein transporter gene (SEC62), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH) and β-actin gene (ACTB), were detected in intestine, liver, muscle, brain, gill, gonad and other tissues in 9 samples of green sunfish by real-time fluorescence quantitative PCR (qRT-PCR). Stability of the candidate reference genes were evaluated by Norm Finder and BestKeeper software, and these algorithms were summarize by ReFinder to screen out the best reference genes. The results showed that the specific amplification products were found by quantitative PCR primers of the six candidate reference genes. The descending order of the expression levels of the six candidate reference genes in different tissues were ranked as CYTB>TUBA>ufm1>SEC62>ACTB>GAPDH, with the most stable reference gene of CYTB in the intestine, ufm1 in liver, muscle, gill and gonad, and TUBA in brain screened by ReFinder. The paired difference analysis by geNorm software revealed that at least two optimal candidate reference genes should be used. There was similar expression stability of ufm1 and TUBA in different tissues, with the overall best stability. The findings can be used for qRT-PCR analysis of gene expression differences in different tissues of green sunfish, and provide reference genes and theoretical basis for future gene expression analysis of green sunfish.

Key words： Lepomis cyanellus reference gene qRT-PCR expression stability

Received: 10 July 2025

PACS:

S961

	Service

	E-mail this article
	Add to my bookshelf
	Add to citation manager
	E-mail Alert
	RSS
	Articles by authors
	QIN Lipeng
	CAO Lu
	ZHANG Huiman
	LIU Qing
	LIU Shaozhen
	SONG Jing

Cite this article:

QIN Lipeng,CAO Lu,ZHANG Huiman, et al. Screening of qRT-PCR Reference Genes in Different Tissues of Green Sunfish Lepomis cyanellus[J]. Fisheries Science, 2026, 45(2): 259-268.

URL:

https://www.shchkx.com/EN/10.16378/j.cnki.1003-1111.25172 OR https://www.shchkx.com/EN/Y2026/V45/I2/259

[1] FULLER P, CANNISTER M, NEILSON M. Lepomis cyanellus Rafinesque, 1819[J]. Geological Survey. 2021, 88:417-429.
[2] YUN C. Gonadal development in green sunfish, Lepomis cyanellus: a histological study[J]. Chinese Journal of Zoology,2008,43(1):88-95.
[3] DUDLEY R K, MATTER W J. Effects of small green sunfish (Lepomis cyanellus) on recruitment of Gila chub (Gila intermedia) in Sabino Creek, Arizona[J]. Southwestern Naturalist,2000,45(1):24-29.
[4] TEAL C N, SCHILL D J, FOGELSON S B, et al. The effects of estradiol-17β on the sex reversal, survival, and growth of green sunfish Lepomis cyanellus[J]. Aquaculture,2023,562:738853.
[5] MORRIS J E, MISCHKE C C, LANE R L. Culture and propagation of sunfish and their hybrids[C]// Resource Ecology and Management Conference Papers, Posters and Presentations. Rockville:American Fisheries Society, 2005:26.
[6] TEAL C N, SCHILL D J, FOGELSON S B, et al. Development of aquaculture protocols and gonadal differentiation of green sunfish (Lepomis cyanellus) [J]. Aquaculture,2022,547:737515.
[7] WERNER E E, HALL D J. Competition and habitat shift in two sunfishes (Centrarchidae)[J]. Ecology,1977,58(4):869-876.
[8] 万安,张晓可,刘志刚,等.大别山区黑沟绿太阳鱼种群的形态特征研究[J].水生生物学报,2017,41(1):194-200.
[9] 赵仲孟,周剑,李强,等.四川嘉陵江乌木滩水库发现外来物种蓝太阳鱼[J].水生生物学报,2020,44(1):103.
[10] VALASEK M A, REPA J J. The power of real-time PCR[J]. Advances in Physiology Education,2005,29(3):151-159.
[11] MACKAY I M. Real-time PCR in the microbiology laboratory[J]. Clinical Microbiology and Infection,2004,10(3):190-212.
[12] BUSTIN S A. Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[J]. Journal of Molecular Endocrinology,2000,25(2):169-193.
[13] GUTIERREZ L, MAURIAT M, GUÉNIN, S, et al. The lack of a systematic validation of reference genes: a serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants[J]. Plant Biotechnology Journal,2008,6(6):609-618.
[14] LEE S, JO M, LEE J, et al. Identification of novel universal housekeeping genes by statistical analysis of microarray data[J]. Journal of Biochemistry and Molecular Biology,2007,40(2):226-231.
[15] CZECHOWSKI T, STITT M, ALTMANN T, et al. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis[J]. Plant Physiology,2005,139(1):5-17.
[16] 唐永凯,俞菊华,徐跑,等.建鲤内参基因EF-1α的实时荧光定量 PCR方法的建立[J].江西农业大学学报,2012,34(3):580-584.
[17] 潘沫晗,陆添权,田波.滇牡丹种子实时荧光定量PCR分析中内参基因的筛选与验证[J].生物技术通报,2020,36(9):218-226.
[18] HELLEMANS J, VANDESOMPELE J. Selection of reliable reference genes for RT-qPCR analysis[J]. Methods in Molecular Biology,2014,1160:19-26.
[19] DE SPIEGELAERE W, DERN-WIELOCH J, WEIGEL R, et al. Reference gene validation for RT-qPCR, a note on different available software packages[J].PLoS One,2015,10(3):e0122515.
[20] ROYER C, BÉGIN A G, PLAWINSKI L, et al. Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells[J]. Experimental Cell Research,2018,370(2):389-398.
[21] HADADI E, DE SOUZA L E B, BENNACEUR-GRISCELLI A,et al.Identification of valid reference genes for circadian gene-expression studies in human mammary epithelial cells[J]. Chronobiology International,2018,35(12):1689-1701.
[22] WEAVER R F. Molecular Biology[M]. Boston: McGraw-Hill,2005.
[23] 刘思嘉,祁得林,祁洪芳,等.青海湖裸鲤实时荧光定量PCR内参基因筛选与验证[J].水产科学,2023,42(3):413-423.
[24] INGERSLEV H C, PETTERSEN E F, JAKOBSEN R A, et al. Expression profiling and validation of reference gene candidates in immune relevant tissues and cells from Atlantic salmon (Salmo salar L.) [J]. Molecular Immunology,2006,43(8):1194-1201.
[25] MCCURLEY A T, CALLARD G V. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment[J]. BMC Molecular Biology,2008,9:102.
[26] ZHENG W J, SUN L. Evaluation of housekeeping genes as references for quantitative real time RT-PCR analysis of gene expression in Japanese flounder (Paralichthys olivaceus) [J]. Fish & Shellfish Immunology,2011,30(2):638-645.
[27] ØVERGÅRD A C, NERLAND A H, PATEL S. Evaluation of potential reference genes for real time RT-PCR studies in Atlantic halibut (Hippoglossus hippoglossus L.); during development, in tissues of healthy and NNV-injected fish, and in anterior kidney leucocytes[J]. BMC Molecular Biology,2010,11(1):36.
[28] 彭冰冰,宾东超,周于娜,等.方格星虫实时荧光定量PCR内参基因的选择与验证[J].水产科学,2022,41(2):304-310.
[29] 张宁,杜文文,王中铎,等.多鳞鱚不同组织荧光定量PCR内参基因筛选[J].广东海洋大学学报,2018,38(5):8-14.
[30] 胡秀芹,王婧华,宋晗,等.栝楼性别分化期花芽及不同器官内参基因的筛选[J/OL].分子植物育种,2023:1-10 (2023-08-31) [2025-07-05]. https://link.cnki.net/urlid/46.1068.S.20230831.1134.002.
[31] JORGENSEN S M, KLEVELAND E J, GRIMHOLT U, et al. Validation of reference genes for real-time polymerase chain reaction studies in Atlantic salmon [J]. Marine Biotechnology,2006,8(4):398-408.
[32] TANG R Y, DODD A, LAI D, et al. Validation of zebrafish (Danio rerio) reference genes for quantitative real-time RT-PCR normalization[J]. Acta Biochimica et Biophysica Sinica,2007,39(5):384-390.
[33] TANG Y K, YU J H, XU P, et al. Identification of housekeeping genes suitable for gene expression analysis in Jian carp (Cyprinus carpio var. jian) [J]. Fish & Shellfish Immunology,2012,33(4):775-779.
[34] MA L M, WANG W J, LIU C H, et al. Selection of reference genes for reverse transcription quantitative real-time PCR normalization in black rockfish (Sebastes schlegeli) [J]. Marine Genomics,2013,11:67-73.
[35] YANG C G, WANG X L, TIAN J, et al. Evaluation of reference genes for quantitative real-time RT-PCR analysis of gene expression in Nile tilapia (Oreochromis niloticus) [J]. Gene,2013,527(1):183-192.
[36] AURSNES I A, RISHOVD A L, KARLSEN H E, et al. Validation of reference genes for quantitative RT-qPCR studies of gene expression in Atlantic cod (Gadus morhua L.) during temperature stress[J]. BMC Research Notes,2011,4:104.
[37] MO F, ZHAO J, LIU N, et al. Validation of reference genes for RT-qPCR analysis of CYP4T expression in crucian carp[J]. Genetics and Molecular Biology,2014,37(3):500-507.
[38] ZHANG J, HU Y H, SUN B G, et al. Selection of normalization factors for quantitative real time RT-PCR studies in Japanese flounder (Paralichthys olivaceus) and turbot (Scophthalmus maximus) under conditions of viral infection[J]. Veterinary Immunology and Immunopathology,2013,152(3/4):303-316.
[39] SILVEIRA T L R, DOMINGUES W B, REMIĀO M H, et al. Evaluation of reference genes to analyze gene expression in silverside Odontesthes humensis under different environmental conditions[J]. Frontiers in Genetics,2018,9:75.