徐宏超,
邢荣莲,
李源美,
丛明^,
烟台大学, 烟台 264005

作者简介: 徐宏超(1992-),男,硕士,研究方向为生态毒理学,E-mail:hongchaoxu@126.com.

通讯作者: 丛明,mcong@ytu.edu.cn

基金项目: 国家自然科学基金面上项目（41876135）；烟台大学博士启动基金（HX20B15）


中图分类号: X171.5

Screening of Reference Genes in Hepatopancreas of Clam Ruditapes philippinarum Exposed to Ammonia Nitrogen

Xu Hongchao,
Xing Ronglian,
Li Yuanmei,
Cong Ming^,
Yantai University, Yantai 264005, China

Corresponding author: Cong Ming,mcong@ytu.edu.cn

CLC number: X171.5

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摘要:利用荧光定量PCR技术对目标基因进行转录水平分析，选择合适的内参基因作为对照是其前提条件。随着氨氮胁迫菲律宾蛤仔毒性效应的深入研究，需要准确评价氨氮对蛤仔肝胰腺组织基因表达的影响。然而，不同浓度氨氮条件下的菲律宾蛤仔肝胰腺组织的内参基因的筛选尚未报道。本研究以2种不同氨氮浓度暴露菲律宾蛤仔14 d的肝胰腺组织cDNA为qRT-PCR的模板，利用geNorm、NormFinder、BestKeeper和RefFinder软件评估β-肌动蛋白（Actin）、延伸因子1-α（EF-1α）、β-微管蛋白（Tubu）、18S核糖体RNA （18S）、泛素（Ubi）和亲环蛋白A （CyPA）6个内参基因的表达稳定性。结果表明，肝胰腺组织中备选内参基因的表达稳定性依次为：Actin>18S>CyPA>Ubi>Tubu>EF-1α，即Actin为表达最稳定的内参基因。本研究为进一步分析氨氮胁迫下菲律宾蛤仔肝胰腺组织的基因表达情况，提供了可靠的内参基因。
关键词: 氨氮/
菲律宾蛤仔/
内参基因/
筛选

Abstract:It is necessary to screen a suitable reference gene to quantify the expression levels of targeted genes by fluorescence quantitative PCR technology. With the further toxic study of ammonia nitrogen on clam Ruditapes philippinarum, it is necessary to accurately evaluate the expression profiles of related genes in clam hepatopancreas. However, there's no related report about the expression stability of reference genes in hepatopancreas of R. philippinarum. In this study, clams was exposed to two different concentrations of ammonia nitrogen for 14 d, and the cDNAs of hepatopancreas at different time points were used as qRT-PCR templates. GeNorm, NormFinder, BestKeeper and RefFinder statistical softwares were used to evaluate the expression stabilities of six candidate reference genes, namely beta-Actin (Actin), elongation factor 1-alpha (EF-1α), beta-tubulin (Tubu), 18S rRNA (18S), ubiquitin (Ubi) and cyclophilin A (CyPA). Final results indicated that the expression stability order of the six candidate reference genes in the hepatopancreas were Actin>18S>CyPA>Ubi>Tubu>EF-1α, which means Actin is the most stable reference gene and provides a reliable basis for further study on expression levels of other genes in the hepatopancreas of R. philippinarum under ammonia nitrogen stress.
Key words:ammonia nitrogen/
Ruditapes philippinarum/
reference gene/
screening.

唐启升, 韩冬, 毛玉泽, 等. 中国水产养殖种类组成、不投饵率和营养级[J]. 中国水产科学, 2016, 23(4):729-758Tang Q S, Han D, Mao Y Z, et al. Species composition, non-fed rate and trophic level of Chinese aquaculture[J]. Journal of Fishery Sciences of China, 2016, 23(4):729-758(in Chinese)

Frankic A, Hershner C. Sustainable aquaculture:Developing the promise of aquaculture[J]. Aquaculture International, 2003, 11(6):517-530

中华人民共和国生态环境部. 2019中国海洋生态环境状况公报[R]. 北京:中华人民共和国生态环境部, 2019 Ministry of Ecology and Environment of the People's Republic of China. 2019 China Eco-Environment Bulletin[R]. Beijing:Ministry of Ecology and Environment of the People's Republic of China, 2019(in Chinese)

Watanabe S, Kodama M, Fukuda M. Nitrogen stable isotope ratio in the Manila clam, Ruditapes philippinarum, reflects eutrophication levels in tidal flats[J]. Marine Pollution Bulletin, 2009, 58(10):1447-1453

Liu S G, Lou S, Kuang C P, et al. Water quality assessment by pollution-index method in the coastal waters of Hebei Province in western Bohai Sea, China[J]. Marine Pollution Bulletin, 2011, 62(10):2220-2229

Fu Q, Zheng B H, Zhao X R, et al. Ammonia pollution characteristics of centralized drinking water sources in China[J]. Journal of Environmental Sciences, 2012, 24(10):1739-1743

Stürzenbaum S R, Kille P. Control genes in quantitative molecular biological techniques:The variability of invariance[J]. Comparative Biochemistry and Physiology Part B, Biochemistry & Molecular Biology, 2001, 130(3):281-289

Yoo W G, Kim T I, Li S Y, et al. Reference genes for quantitative analysis on Clonorchis sinensis gene expression by real-time PCR[J]. Parasitology Research, 2009, 104(2):321-328

李迪, 吴萍, 何美凤, 等. qRT-PCR分析鳜鱼内参基因的筛选[J]. 生命科学研究, 2016, 20(3):214-217Li D, Wu P, He M F, et al. Screening of reference genes in Siniperca chuatsi for qRT-PCR analysis[J]. Life Science Research, 2016, 20(3):214-217(in Chinese)

鲍相渤, 刘卫东, 姜冰, 等. 内参基因在虾夷扇贝定量PCR中表达稳定性的比较[J]. 水产科学, 2011, 30(10):603-608Bao X B, Liu W D, Jiang B, et al. Expression stability of reference genes for quantitative PCR in Japanese scallop Patinopecten yessoensis[J]. Fisheries Science, 2011, 30(10):603-608(in Chinese)

曹滕飞, 丛明, 李兆艳, 等. 氨态氮对菲律宾蛤仔毒理机制研究的内参基因筛选[J]. 生态毒理学报, 2017, 12(2):182-190Cao T F, Cong M, Li Z Y, et al. Selection of reference genes for toxicological mechanism research on Ruditapes philippinarum exposed to ammonia nitrogen[J]. Asian Journal of Ecotoxicology, 2017, 12(2):182-190(in Chinese)

王爱云, 曹滕飞, 吕家森, 等. 亚硝态氮胁迫下菲律宾蛤仔实时定量PCR内参基因的筛选[J]. 生态毒理学报, 2019, 14(1):153-160Wang A Y, Cao T F, Lv J S, et al. Selection of reference genes by quantitative real-time PCR for Ruditapes philippinarum exposed to nitrite[J]. Asian Journal of Ecotoxicology, 2019, 14(1):153-160(in Chinese)

Cong M, Wu H F, Cao T F, et al. Effects of ammonia nitrogen on gill mitochondria in clam Ruditapes philippinarum[J]. Environmental Toxicology and Pharmacology, 2019, 65:46-52

Cong M, Wu H F, Yang H P, et al. Gill damage and neurotoxicity of ammonia nitrogen on the clam Ruditapes philippinarum[J]. Ecotoxicology, 2017, 26(3):459-469

Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-delta delta C(T)) method[J]. Methods, 2001, 25(4):402-408

Vandesompele J, De Preter K, Pattyn F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[J]. Genome Biology, 2002, 3(7):RESEARCH0034

Andersen C L, Jensen J L, Ørntoft T F. Normalization of real-time quantitative reverse transcription-PCR data:A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[J]. Cancer Research, 2004, 64(15):5245-5250

Pfaffl M W, Tichopad A, Prgomet C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity:BestKeeper-Excel-based tool using pair-wise correlations[J]. Biotechnology Letters, 2004, 26(6):509-515

Xie F L, Xiao P, Chen D L, et al. miRDeepFinder:A miRNA analysis tool for deep sequencing of plant small RNAs[J]. Plant Molecular Biology, 2012:DOI:10.1007/s11103-012-9885-2

Radonić A, Thulke S, MacKay I M, et al. Guideline to reference gene selection for quantitative real-time PCR[J]. Biochemical and Biophysical Research Communications, 2004, 313(4):856-862

闫含笑, 史建伍, 盛军庆, 等. 池蝶蚌β-连环蛋白基因cDNA的克隆及表达特征分析[J]. 水生生物学报, 2017, 41(5):972-976Yan H X, Shi J W, Sheng J Q, et al. The structural feature and expression analysis of hsβ-catenin cDNA and protein from Hyriopsis schlegelii[J]. Acta Hydrobiologica Sinica, 2017, 41(5):972-976(in Chinese)

程雪艳, 蒋国萍, 柴雪良, 等. 泥蚶谷胱甘肽过氧化物酶基因的全长克隆与表达分析[J]. 水生生物学报, 2016, 40(6):1144-1151Cheng X Y, Jiang G P, Chai X L, et al. Full-length cDNA cloning and expression analysis of glutathione peroxidase from blood clam Tegillarca granosa[J]. Acta Hydrobiologica Sinica, 2016, 40(6):1144-1151(in Chinese)

牟政强, 闫路路, 王化敏, 等. 菲律宾蛤仔不同发育时期及成体不同组织中内参基因筛选[J]. 水产学报, 2018, 42(5):663-672Mu Z Q, Yan L L, Wang H M, et al. The selection of reference genes in different development stages and different tissues of Ruditapes philippinarum[J]. Journal of Fisheries of China, 2018, 42(5):663-672(in Chinese)

Zhang M F, Liu Q, Jia G X. Reference gene selection for gene expression studies in lily using quantitative real-time PCR[J]. Genetics and Molecular Research, 2016, 15(2):1-14

Li Y L, Ye F, Hu Y, et al. Identification of suitable reference genes for gene expression studies of human serous ovarian cancer by real-time polymerase chain reaction[J]. Analytical Biochemistry, 2009, 394(1):110-116

Penning L C, Vrieling H E, Brinkhof B, et al. A validation of 10 feline reference genes for gene expression measurements in snap-frozen tissues[J]. Veterinary Immunology and Immunopathology, 2007, 120(3-4):212-222

潘畅, 张宇宏, 庞虹. 孟氏隐唇瓢虫qRT-PCR分析中内参基因的筛选[J]. 环境昆虫学报, 2016, 38(2):261-270Pan C, Zhang Y H, Pang H. Selection of the reference genes for gene expression studies in Cryptolaemus montrouzieri Mulsant by qRT-PCR[J]. Journal of Environmental Entomology, 2016, 38(2):261-270(in Chinese)

Fernandez P, di Rienzo J A, Moschen S, et al. Comparison of predictive methods and biological validation for qPCR reference genes in sunflower leaf senescence transcript analysis[J]. Plant Cell Reports, 2011, 30(1):63-74

Kuijk E W, du Puy L, van Tol H T, et al. Validation of reference genes for quantitative RT-PCR studies in porcine oocytes and preimplantation embryos[J]. BMC Developmental Biology, 2007, 7:58