杜娜^1,2,3, 张敏⁴, 吴志昊^1,2, 焦爽^1,2, 尤锋^1,2, 谭训刚^1,2
1.中国科学院海洋研究所 中国科学院海洋大科学研究中心, 中国科学院 实验海洋生物学重点实验室 山东省实验海洋生物学重点实验室, 山东 青岛 266071;2.青岛海洋科学与技术试点国家实验室 海洋生物与生物技术实验室, 山东 青岛 266237;3.中国科学院大学, 北京 100049;4.青岛科技大学, 山东 青岛 266061

摘要:
多倍体诱导会造成物种中不同基因的表达水平变化不同，以内参基因作为对照的相对实时定量PCR是检测基因表达水平变化的一种高效方法，这就需要对多倍体中的内参基因进行筛选以获得较适宜的参比基因。本研究以二倍体和三倍体牙鲆肌肉和脑组织为对象，以绝对定量及2^–ΔCt等方法分析管家基因18S rRNA、β-肌动蛋白基因（β-actin），β-2-微球蛋白基因（b2m），3-磷酸甘油醛脱氢酶基因（gapdh），核糖体蛋白L17基因（rpl17），α-微管蛋白基因（α-tub），延伸因子-1-α基因（ef1-α）和泛素结合酶基因（ubc-e）的表达水平稳定性。绝对定量分析发现18S rRNA和α-tub的表达在牙鲆二、三倍体肌肉之间有显著性差异，其他基因的表达没有显著性差异，利用2^–ΔCt分析方法分析发现只有α-tub的表达在二、三倍体牙鲆肌肉之间有显著性差异，其他基因的表达没有显著性差异；在牙鲆三倍体的脑和二倍体的脑之间，这些基因的表达均没有显著性差异。综合基因表达稳定性和Normfinder分析的结果发现，ef1-α是定量分析牙鲆三倍体的肌肉和二倍体的肌肉之间基因表达差异的较适合的内参基因，α-tub、ubc-e、rpl17、β-actin只在其中一种分析方法中符合要求，而其他基因不符合任何一种分析方法的要求；ef1-α、rpl17是定量分析牙鲆二、三倍体脑组织之间基因表达差异的比较适合的内参基因，β-actin只在基因表达稳定性上符合要求，其他基因不符合任何一种分析方法的要求。本研究为分析牙鲆二倍体和三倍体同一组织之间基因表达的差异奠定了基础。
关键词:二倍体牙鲆三倍体牙鲆内参基因肌肉脑
DOI：10.11759/hykx20210408001
分类号:Q78;Q17
基金项目:国家自然科学基金项目（31672636）

Selection and identification of potential reference genes for qPCR in muscle and brain of diploid and triploid olive flounder
DU Na^1,2,3, ZHANG Min⁴, WU Zhi-hao^1,2, JIAO Shuang^1,2, YOU Feng^1,2, TAN Xun-gang^1,2
1.CAS and Shandong Province Key Laboratory of Experimental Marine Biology, Center for Ocean Mega-Science, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, China;2.Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266237, China;3.University of Chinese Academy of Sciences, Beijing 100049, China;4.Qingdao University of Science and Technology, Qingdao 266061, China
Abstract:
Polyploidy induction is a breeding method to produce new genotypes, with the double of chromosomes, the expression level of its genes changes. Relative real-time quantitative PCR is an efficient method to detect the changes of gene expression levels. In order to screen the internal reference genes suitable for diploid and triploid tissues, this study analyzed the expression and stability of 18s rRNA, β-actin (β-actin), β-2-microglobulin (b2m), 3-Glyceraldehyde phosphate dehydrogenase (gapdh), ribosomal protein L17 (rpl17), α-tubulin (α-tub), elongation factor-1-α (ef1-α), ubiquitin conjugating enzyme (ubc-e) in muscle and brain of diploid and triploid olive flounder. The results of absolute quantitative PCR found that the expression of 18S rRNA and α-tub were significantly different in muscle between the triploid and diploid flounder, while only the expression of α-tub was significantly different when 2^-ΔCt method was used and the expression of the rest genes were no significantly different. The expression of most genes in brain were not significantly different between the triploid and diploid flounder. So, there was a dose compensation effect. The comprehensive results of
gene stability analysis and Normfinder analysis showed that ef1-α might be suitable internal reference genes in the muscle of olive flounder during qPCR analysis, α-tub、ubc-e、rpl17、β-actin met one requirement of them, while the rest genes were not suitable. In the brain, ef1-α, rpl17 might be suitable reference genes, β-actin only met the requirement of gene stability, while the others were not suitable. This study laid a foundation for the analysis of the differential gene expression between diploid and triploid olive flounder.
Key words:diploid olive floundertriploid olive flounderreference genesmusclebrain





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