文章摘要
黄带拟鲹qPCR内参基因筛选及验证
Screening of Reference Genes for Quantitative Real-time PCR in striped jack (Pseudocaranx dentex)
投稿时间:2022-06-22  修订日期:2022-07-20
DOI:
中文关键词: 黄带拟鲹  qPCR  内参基因  稳定性
英文关键词: Pseudocaranx dentex  qPCR  reference gene  stability
基金项目:国家自然科学基金项目(42076132, 42106129和32102768);财政部和农业农村部:国家现代农业产业技术体系资助(CARS-47)[This work was supported by the National Natural Science Foundation of China (42076132, 42106129 and 32102768), and the China Agriculture Research System of MOF and MARA (CARS-47)]
作者单位邮编
李杰锋 上海海洋大学 水产与生命学院 上海中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛 201306;266071
王焕 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛 
李步苏 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛青岛海洋科学与技术试点国家实验室 海洋渔业科学与食物产出过程功能实验室 山东 青岛 
曾祥辉 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛大连海洋大学 水产与生命学院 辽宁 大连 
柳淑芳 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛青岛海洋科学与技术试点国家实验室 海洋渔业科学与食物产出过程功能实验室 山东 青岛 266071
庄志猛 中国水产科学研究院黄海水产研究所 农业农村部海洋渔业可持续发展重点实验室 山东 青岛 
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中文摘要:
      实时荧光定量PCR(qPCR)是研究基因表达的一种广泛使用且有效的方法,选用黄带拟鲹(Pseudocaranx dentex)合适的内参基因,是qPCR技术获得该物种基因表达可靠结果的关键。本研究首先测定了9个常用内参基因(ACTIN、RPL13、EF-1α、GAPDH、HPRT、PPIA、β2M、TUB和PP2A)在黄带拟鲹成鱼组织中的表达丰度;同时,利用BestKeeper、NormFinder、geNorm和RefFinder 4种模型预测了内参基因的表达稳定性,发现其表达稳定性排序为:RPL13>EF-1α>PP2A>HPRT>PPIA>TUB>β2M>ACTIN>GAPDH。进一步通过定量检测目标基因myod1在不同组织的表达情况,验证上述预测结果的准确性。研究发现,RPL13和EF-1α单独或联合作为黄带拟鲹qPCR内参基因,可显著提高目标基因表达量检测结果的稳定性与可靠性。结果表明,RPL13和EF-1α可作为黄带拟鲹不同组织qPCR分析的内参基因。同时,也证实了并不是管家基因的表达在所有物种中具有良好的稳定性,需要根据实际情况筛选适宜的内参基因。本研究结果可为后续黄带拟鲹功能基因表达特征的研究提供技术支撑,有望适用于其他鲹科鱼类。
英文摘要:
      Background: Quantitative real-time polymerase chain reaction (qPCR) is one of the most widely-used molecular technique for detecting and measuring gene expression because of its high sensitivity, sharp specificity, and good reproducibility. To obtain the reliable and comparable relative quantitative results using qPCR, appropriate reference genes are required for eliminating the non-biological variations caused by initial RNA templates, efficiency of cDNA synthesis, and laboratory procedures. However, previous studies have reported that the stability of reference genes may be variable across different species, tissue types, cell lines, developmental stages, and experimental treatments, yielding inaccurate or even wrong gene expression results. Therefore, selection and validation of stable reference genes for different tissues of a specific species is especially important to obtain accurate target gene expression results. The striped jack Pseudocaranx dentex belonging to the order Perciformes and the family Carangidae, as a pelagic migratory fish with high nutritional value, has already received extensive attention in global aquaculture production and is regarded as a candidate species for far-reaching marine aquaculture in China. At present, the molecular biology and genetics researches of P. dentex are being carried out extensively, and there is an increasing demand for quantitative gene expression analysis by qPCR for studying gene function. However, little study has evaluated reference genes in this species. The objective of this research is to identify suitable reference genes in P. dentex among different tissues, which will provide the basis for subsequent gene expression patterns analysis. Materials and Methods: Here, nine common-used reference genes, also known as housekeeping genes, including beta actin (β-actin), ribosomal protein L13 (RPL13), elongation factor 1 alpha (EF-1α), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl transferase (HPRT), peptidylprolyl isomerase A (PPIA), beta 2-microglobulin (β2M), beta tubulin (TUB), and serine/threonine-protein phosphatase2A catalytic subunit (PP2A) were selected as candidate reference genes for qPCR analysis among different tissues of P. dentex. The expression stability in ten tissues (brain, gill, heart, intestine, kidney, liver, spleen, stomach, slow-twitch muscle and fast-twitch muscle) from three adult individuals of P. dentex were evaluated using four independent methods: BestKeeper, NormFinder, geNorm, and RefFinder. To validate the results obtained from above analysis, the expression profile of a target gene myoblast determination protein 1 (myod1) was investigated in muscle tissues (slow-twitch muscle and fast-twitch muscle) and non-muscle tissues (kidney and gills) using different reference genes or combinations recommended. Results: Expression levels analysis showed that RPL13 was the most highly expressed gene, followed by EF-1α, β2M, ACTIN, PPIA, HPRT, TUB, and PP2A, while GAPDH was the most weakly expressed in ten tissues of P. dentex. In addition, nine candidate reference genes exhibited relatively different variation in Ct values among ten tissues. The stability result in BestKeeper, evaluated by standard deviation (SD) and coefficient of variation (CV), was shown in order of the most stable to least stable EF-1α > RPL13 > PP2A > PPIA >HPRT > β2M > TUB > GAPDH > ACTIN. According to the stability value (SV) of each candidate gene analyzed by NormFinder, the ranking of most to least stable reference genes was as follows: RPL13 > EF-1α> HPRT > TUB > PP2A > PPIA > β2M > GAPDH > ACTIN. According to the principle that the lower the expression stability value (M), the better stability of the gene expression, the expression stability by geNorm is classified in descending order: RPL13 = EF-1α > ACTIN > GAPDH > PP2A > TUB > PPIA > HPRT > β2M. Furthermore, the results of RefFinder analysis showed that the comprehensive stability ranking of each gene from high to low: RPL13 > EF-1α > PP2A > HPRT > PPIA > TUB > β2M > ACTIN > GAPDH. What is more, all the paired coefficients of variation Vn/(n+1) by geNorm were lower than the critical value 0.15, indicating that the inclusion of additional reference genes have little effect on the V value. The combination of the two best reference genes is a valid normalization strategy, and the results could meet the requirement of correcting the expression level of target genes. Obviously, RPL13 and EF-1α were the two best reference genes in adult tissues combining the results of four methods. Finally, the relative expression levels of myod1 in slow-twitch muscle, fast-twitch muscle, kidney and gills showed that there are no significant differences when using RPL13, EF-1α and their combination as reference genes, while significant differences were existed when using three least stable genes β2M, ACTIN and GAPDH. This result confirmed that the stability evaluation by four methods was necessary and effective. Conclusions: In conclusion, we recommend RPL13 and EF-1α and their combination were the ideal reference genes for all the tested adult tissue types in P. dentex verified by four individual algorithms. The results of this study provide the basis for the standardized and quantitative detection of functional genes, and provide a technical support for the comprehensive and systematic molecular biology and genetic research of P. dentex in the later stage.
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