doi:

DOI: 10.3724/SP.J.1118.2019.18301

Journal of Fishery Sciences of China (中国水产科学) 2019/26:3 PP.457-464

Identification of internal reference genes for quantification of gene expression in different embryonic developmental stages of the mud crab Scylla paramamosain


Abstract:
The mud crab Scylla paramamosain is widely cultured in the coastal areas of southern China because of its short growth cycle, high nutrition value, large market demand, and high economic value. At present, most of the mud crab larvae come from the natural populations, which puts great pressure on its natural resources. Therefore, establishing industry-scale artificial reproduction and using artificial larvae will effectively reduce the harvest of wild resources and protect the natural resources of the mud crab. On the other hand, embryonic development is the basis of artificial reproduction and individual development. At present, the research on embryo development of mud crabs is mainly focused on morphological observation and measurement of physiological and biochemical indexes. Knowledge concerning the mechanisms regulating embryo development at molecular level is poor. Commonly, quantitative real time PCR (qRT-PCR) is used to accurately quantify mRNA expression levels. However, this technique is highly dependent on stably expressed housekeeping genes to avoid experimental errors and variations. Furthermore, the universal ideal reference gene suitable for all experiments has not been obtained. To identify the most stable internal reference genes for studying gene expression in different embryonic developmental stages of mud crabs, tubulin alpha 1a (Tuba1a), ribosomal protein L13 (Rpl13), ubiquitin (UBQ), ribosomal protein S6 kinase (Rps6), arginine kinase (Ak), glyceraldehyde-3-phosphate dehydrogenase (gapdh), 28S ribosomal RNA (28S), 18S ribosomal RNA (18S), and elongation factor 1A (EF-1α) were employed to quantify their expression by qRT-PCR. The qRT-PCR results were used to evaluate their expression stabilities by three algorithms-geNorm, NormFinder, and BestKeeper. The geNorm program analysis showed that the expression of EF-1α and Rpl13 had the highest stability. The results also suggested that it was better to use these two internal reference genes together to correct the quantitative results of target genes. NormFinder analysis showed that gapdh and EF-1α had the most stable expression among the nine internal candidate reference genes. BestKeeper program analysis showed that the standard deviation and coefficient of variation of gapdh were the lowest among the nine candidate genes. Combining the three internal reference gene screening softwares, EF-1α, or double internal reference genes, EF-1α and gapdh, could be used to correct the expression of target genes in different embryonic developmental stages. Furthermore, we found that the expression of many published internal reference genes was not stable enough under our conditions. In our experiment, commonly used internal reference genes such as β-actin, 18S, and 28S were not suitable for standardizing gene expression data in the different embryonic developmental stages of S. paramamosain.

Key words:Scylla paramamosain; qRT-PCR; internal reference gene; stability

ReleaseDate:2019-07-04 08:55:16



[1] Ye H H, Tao Y, Wang G Z, et al. Experimental nursery culture of the mud crab Scylla paramamosain (Estampador) in China[J]. Aquaculture International, 2011, 19(2):313-321.

[2] Jiang Q L, Bao C C, Yang Y N, et al. Transcriptome profiling of claw muscle of the mud crab (Scylla paramamosain) at different fattening stages[J]. PLoS ONE, 2017, 12(11):e0188067.

[3] Lin Q. Species composition of genus Scylla and genetic diversity of Scylla paramamosain (Estampador, 1949) population in China[D]. Xiamen:Xiamen University, 2008.[林琪. 中国青蟹属种类组成和拟穴青蟹群体遗传多样性的研究[D]. 厦门:厦门大学, 2008.]

[4] Ding H Y, Yao H F, Li G L, et al. Effect of hatching water pH on embryonic development of Scylla serrata[J]. Journal of Aquaculture, 2010, 31(9):8-11.[丁宏印, 姚海富, 李贵玲, 等. 孵化水体pH值对锯缘青蟹胚胎发育的影响[J]. 水产养殖, 2010, 31(9):8-11.]

[5] Li S J, Wang G Z. Comparative studies on the hydrolytic enzyme activities for the mud crab, Scylla serrata (Forskål) during embryonic development[J]. Journal of Xiamen University (Natural Science), 1995, 34(6):970-974.[李少菁, 王桂忠. 锯缘青蟹胚胎发育过程中几种水解酶活力的比较研究[J]. 厦门大学学报(自然科学版), 1995, 34(6):970-974.]

[6] Zeng C S. Induced out-of-season spawning of the mud crab, Scylla paramamosain (Estampador) and effects of temperature on embryo development[J]. Aquaculture Research, 2007, 38(14):1478-1485.

[7] Forlenza M, Kaiser T, Savelkoul H F J, et al. The use of real-time quantitative PCR for the analysis of cytokine mRNA levels[M]//Cytokine Protocols. Totowa:Humana Press, 2012, 820:7-23.

[8] Gadkar V J, Filion M. New developments in quantitative real-time polymerase chain reaction technology[J]. Current Issues in Molecular Biology, 2014, 16:1-6.

[9] Huang S, Chen X W, Wang J, et al. Selection of appropriate reference genes for qPCR in the Chinese mitten crab, Eriocheir sinensis (Decapoda, Varunidae)[J]. Crustaceana, 2017, 90(3):275-296.

[10] Jiang H C, Qian Z J, Lu W, et al. Identification and characterization of reference genes for normalizing expression data from red swamp crawfish Procambarus clarkii[J]. International Journal of Molecular Sciences, 2015, 16(9):21591-21605.

[11] Leelatanawit R, Klanchui A, Uawisetwathana U, et al. Validation of reference genes for real-time PCR of reproductive system in the black tiger shrimp[J]. PLoS ONE, 2012, 7(12):e52677.

[12] Frost P, Nilsen F. Validation of reference genes for transcription profiling in the salmon louse, Lepeophtheirus salmonis, by quantitative real-time PCR[J]. Veterinary Parasitology, 2003, 118(1-2):169-174.

[13] Dhar A K, Bowers R M, Licon K S, et al. Validation of reference genes for quantitative measurement of immune gene expression in shrimp[J]. Molecular Immunology, 2009, 46(8-9):1688-1695.

[14] Wu J Y, He B, Du Y J, et al. Analysis method of systematically evaluating stability of reference genes using geNorm, NormFinder and BestKeeper[J]. Modern Agricultural Science and Technology, 2017(5):278-281.[吴建阳, 何冰, 杜玉洁, 等. 利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法[J]. 现代农业科技, 2017(5):278-281.]

[15] Chen J M. Foundationl studies on the embryonic development of the mud crab, Scylla serrata (Forskål)[D]. Xiamen:Xiamen University, 2005:1-116.[陈锦民. 锯缘青蟹Scylla serrata (Forskål)胚胎发育的基础研究[D]. 厦门:厦门大学, 2005:1-116.]

[16] Fang Y B. Cloning and expression profile of two immune-related genes and studies on the applicability of eleven internal reference genes in mud crab, Scylla Paramamosain[D]. Shanghai:Shanghai Ocean University, 2013.[房娅博. 拟穴青蟹免疫相关基因的克隆及内参基因的适用性分析[D]. 上海:上海海洋大学, 2013.]

[17] Olias P, Adam I, Meyer A, et al. Reference genes for quantitative gene expression studies in multiple avian species[J]. PLoS ONE, 2014, 9(6):e99678.

[18] 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):research0034.1-0034.11.

[19] Kidd M, Nadler B, Mane S, et al. GeneChip, geNorm, and gastrointestinal tumors:novel reference genes for real-time PCR[J]. Physiological Genomics, 2007, 30(3):363-370.

[20] 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.

[21] 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.

[22] Valasek M A, Repa J J. The power of real-time PCR[J]. Advances in Physiology Education, 2005, 29(3):151-159.

[23] Dheda K, Huggett J F, Chang J S, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[J]. Analytical Biochemistry, 2005, 344(1):141-143.

[24] Zhang Y F, Zhao L J, Zeng Y L, Selection and application of reference genes for gene expression studies[J]. Plant Physiology Journal, 2014, 50(8):1119-1125.[张玉芳, 赵丽娟, 曾幼玲. 基因表达研究中内参基因的选择与应用[J]. 植物生理学报, 2014, 50(8):1119-1125.]

[25] Dheda K, Huggett J F, Chang J S, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization[J]. Analytical Biochemistry, 2005, 344(1):141-143.

[26] Kanakachari M, Solanke A U, Prabhakaran N, et al. Evaluation of suitable reference genes for normalization of qPCR gene expression studies in Brinjal (Solanum melongena L.) during fruit developmental stages[J]. Applied Biochemistry and Biotechnology, 2016, 178(3):433-450.

[27] Huang J R, Huang H Y, Ye H H, et al. Cloning and analysis of β-actin gene from Scylla paramamosain[J]. Journal of Xiamen University (Natural Science), 2012, 51(2):274-279.[黄静茹, 黄辉洋, 叶海辉, 等. 拟穴青蟹β-肌动蛋白基因的克隆与分析[J]. 厦门大学学报(自然科学版), 2012, 51(2):274-279.]

[28] Liu H F, Gao L X, Hu Y H. Reference genes discovery and selection for quantitative real-time PCR in tree peony seed and petal tissue of different development stages[J]. Journal of Agricultural Biotechnology, 2015, 23(12):1639-1648.

[29] Chen L, Zhao W, Zhan S Y, et al. The expression stability analysis of reference genes in the different tissues and skeletal muscle of different development periods in goat[J]. Acta Veterinaria et Zootechnica Sinica, 2014, 45(8):1228-1236.[陈利, 赵薇, 占思远, 等. 山羊不同组织及不同发育时期骨骼肌内参基因的表达稳定性分析[J]. 畜牧兽医学报, 2014, 45(8):1228-1236.]