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

Abstract

PDF HTML阅读 XML下载 导出引用 引用提醒 拟穴青蟹不同发育时期胚胎基因表达的内参基因的筛选 DOI: 作者: 作者单位: 1. 集美大学水产学院, 农业部东海海水健康养殖重点实验室, 福建 厦门 361021;2. 福建农林大学动物科学学院, 福建 福州 350002 作者简介: 吕梁(1993-),男,硕士研究生,主要从事甲壳动物生殖和发育生物学研究.E-mail:1121098527@qq.com 通讯作者: 中图分类号: 917 基金项目: 国家自然科学基金项目（41676161；31672681；31472266）. Identification of internal reference genes for quantification of gene expression in different embryonic developmental stages of the mud crab Scylla paramamosain Author: Affiliation: 1. Fisheries College, Jimei University;Key Laboratory of Healthy Mariculture for the East China Sea, Ministry of Agriculture, Xiamen 361021, China;2. College of Animal Sciences, Fujian Agriculture and Forestry University, Fuzhou 350002, China Fund Project: 摘要 | 图/表 | 访问统计 | 参考文献 | 相似文献 | 引证文献 | 资源附件 | 文章评论 摘要:为获得适用于研究拟穴青蟹（）不同发育时期胚胎基因表达的内参基因，本研究采用3个内参基因筛选软件geNorm、NormFinder及BestKeeper对微管蛋白Ak）、3-磷酸甘油脱氢酶（）等9个常用候选内参基因在拟穴青蟹不同发育时期胚胎的表达稳定性进行分析。geNorm分析表明，的表达稳定性最高，并且采用2个内参基因同时校正目标基因的定量结果可以达到最优效果；NormFinder分析表明，的标准偏差和变异系数是9个候选基因中最低的。综合3个内参基因筛选软件，可以选用双内参同时校正拟穴青蟹不同胚胎发育时期各基因的表达情况。 Abstract:The mud crab 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 (), ribosomal protein S6 kinase (), glyceraldehyde-3-phosphate dehydrogenase (), 28S ribosomal RNA (28S), 18S ribosomal RNA (18S), and elongation factor 1A () 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 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 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 were the lowest among the nine candidate genes. Combining the three internal reference gene screening softwares, , 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 -actin, 18S, and 28S were not suitable for standardizing gene expression data in the different embryonic developmental stages of . 参考文献 相似文献 引证文献