亚硝态氮胁迫下菲律宾蛤仔实时定量PCR内参基因的筛选  

Selection of Reference Genes by Quantitative Real-Time PCR for Ruditapes philippinarum Exposed to Nitrite

作　　者：王爱云[1] 曹滕飞 吕家森[2] 丛明[3] Wang Aiyun;Cao Tengfei;Lv Jiasen;Cong Ming(School of Life Sciences,Ludong University,Yantai 264025,China;College of Life Science,Yantai University,Yantai 264003,China;Yantai Institute of Coastal Zone Research,Chinese Academy of Sciences,Yantai 264003,China)

机构地区：[1]鲁东大学生命科学学院,烟台264025 [2]烟台大学生命学院,烟台264003 [3]中国科学院烟台海岸带研究所,烟台264003

出　　处：《生态毒理学报》2019年第1期153-160,共8页Asian Journal of Ecotoxicology

基　　金：国家自然基金面上项目(41876135);鲁东大学高端人才团队建设费(108-10000318)

摘　　要：为了更好地从基因角度研究亚硝态氮对贝类的生态毒性,需要筛选一个合适的内参基因作为参考,对其毒性相关基因进行定量表达分析。本研究以菲律宾蛤仔(Ruditapes philippinarum)作为研究对象,以亚硝态氮胁迫的鳃组织cDNA作为模板,利用geNorm软件和NormFinder软件对6个候选内参基因进行表达稳定性分析,并对2个结果做了综合赋权分析。结果显示,geNorm分析的基因表达稳定性结果为Actin>CyPA>EF1α>Ubi>18S>Tubu,NormFinder软件分析的结果为Actin>Ubi>CyPA>EF1α>18S>Tubu,最后经加权赋值法综合分析获得6个候选基因的表达稳定性为Actin>CyPA>Ubi>EF1α>18S>Tubu,即Actin为表达最稳定的基因。因此,Actin可以作为衡量亚硝态氮胁迫后菲律宾蛤仔鳃组织中基因表达的内参基因。In order to well investigate the eco-toxicity of nitrite on molluscs,a suitable housekeeper gene was needed for further analysis to qualify the toxicity-related genes.In the present study,the clam Ruditapes philippinarum was used as the target mollusc and cDNAs templates were got from gill tissue RNA of R.philippinarum after nitrite exposure.Six candidate genes were assayed in terms of their expression stabilities by geNorm and NormFinder softwares,followed by a comprehensive weighting comparison.GeNorm analysis indicated that the stability order for the six genes were Actin > CyPA > EF1 α> Ubi > 18S > Tubu.However,a little different order was suggested by the Normfinder software,Actin > Ubi > CyPA > EF1α> 18S > Tubu.After a comprehensive weighting comparison,the final stability order of the six genes was Actin > CyPA > Ubi > EF1α> 18S > Tubu.So,the Actin gene was the most suitable reference gene to qualify target genes for their expression levels in the clam R.philippinarum after nitrite exposure.

关 键 词：亚硝态氮 菲律宾蛤仔 内参基因 转录组学 实时荧光定量PCR 

分 类 号：X171.5[环境科学与工程—环境科学]