曹滕飞¹,
丛明²,
李兆艳³,
赵建民²,
吕家森³,
李成华¹
1. 宁波大学海洋学院, 宁波 315211;
2. 中国科学院烟台海岸带研究所, 烟台 264003;
3. 烟台大学生命学院, 烟台 264005

作者简介: 曹滕飞(1992-),男,硕士,研究方向为生态毒理和分子生物学,E-mail:caotengfei2012@163.com.

基金项目: 国家自然科学基金(41406132)


中图分类号: X171.5

Selection of Reference Genes for Toxicological Mechanism Research on Ruditapes philippinarum Exposed to Ammonia Nitrogen

Cao Tengfei¹,
Cong Ming²,
Li Zhaoyan³,
Zhao Jianmin²,
Lv Jiasen³,
Li Chenghua¹
1. School of Marine Sciences, Ningbo University, Ningbo 315211, China;
2. Yantai Institute of Coastal Zone Research, Chinese Academy of Sciences, Yantai 264003, China;
3. College of Life Science, Yantai University, Yantai 264005, China

CLC number: X171.5

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摘要:为了准确和系统地分析NH₃-N对菲律宾蛤仔(Ruditapes philippinarum)毒性的分子机制，并对相关基因的转录组学数据进行验证，利用geNorm和Normfinder这2种软件，以暴露30 d中不同时间点的鳃组织cDNA为模板，从18S rRNA (18S)、beta-actin (Actin)、beta-tubulin (Tubu)、elongation factor 1-alpha (EF1α)、cyclophilin A (CyPA)、Ubiquitin (Ub)等6个备选管家基因中，筛选可以稳定表达的内参基因，作为转录组学分析的内参基因。以稀释50倍的鳃组织cDNA模板作为检测模板，geNorm软件分析获得6对备选内参基因的表达稳定性依次为：EF1α>CyPA>Actin>Tubu>Ub>18S，较优内参为EF1α和CyPA；NormFinder软件分析获得6对备选内参基因的表达稳定性为：EF1α>Actin>Tubu>CyPA>Ub>18S，最优内参基因为EF1α。为了验证上述筛选结果，分别以EF1α和CyPA作为内参基因研究unigene1的表达趋势，发现以EF1α为内参基因时，unigene1的qRT-PCR的表达趋势与其转录组表达倍数的变化趋势一致。因此确定NH3-N暴露菲律宾蛤仔后，鳃组织中表达最为稳定的内参基因为EF1α。
关键词: 氨氮/
菲律宾蛤仔/
内参基因

Abstract:In order to analyze the transcriptome data accurately and systemically, a suitable reference gene is necessary. In the present study, geNorm and Normfinder softwares were used to analyze the expression stabilities of six candidate reference genes of Ruditapes philippinarum after ammonia nitrogen exposure for 30 days. The reference genes included 18S rRNA (18S), beta-actin (Actin), beta-tubulin (Tubu), elongation factor 1-alpha (EF1α), cyclophilin A (CyPA) and Ubiquitin (Ub). Based on a 50-times diluted cDNA in gills as the quantitative real-time PCR template, the geNorm software analysis demonstrated that the expression stabilities of the six candidate reference gene after NH₃-N exposure were as follows: EF1α>CyPA>Actin>Tubu>Ub>18S, and EF1α and CyPA were the best two ones among the six reference genes. A little different order was detected by the NormFinder software as follows: EF1α>Actin>Tubu>CyPA>Ub>18S. And EF1α was recommended as the optimal reference gene. As an assay, EF1α and CyPA were employed to examine one target unigene by qRT-PCR respectively. Final results showed that the variation trend detected by EF1α was more consistent with the foldchange value of the transcriptome. So EF1α was selected as the housekeeping gene to analyze the transcriptome data of gills in Ruditapes philippinarum after ammonia nitrogen exposure.
Key words:ammonia nitrogen/
Ruditapes philippinarum/
reference gene.