黑线姬鼠睾丸实时荧光定量PCR内参基因的筛选及验证  

Screeningand Verification of Reference Genes for Real-time Quantitative PCR Analysis in Apodemus agrarius Testicles

作　　者：金志民[1] 李强 王迎新 李璐 任占辰 贾修歧 JIN Zhimin;LI Qiang;WANG Yingxin;LI Lu;REN Zhanchen;JIA Xiuqi(College of life science and technology Mudanjiang Normal University,Mudanjiang,Heilongjiang 157011,China)

机构地区：[1]牡丹江师范学院生命科学与技术学院,黑龙江牡丹江157011

出　　处：《天津农业科学》2024年第7期54-59,66,共7页Tianjin Agricultural Sciences

基　　金：国家重点研发计划子课题(2023YFF0724603-4);黑龙江省2023年度省属高校基本科研业务费科研项目(1453QN029);牡丹江师范学院青年学术骨干项目(MNUQN202305);生物学学科卓越研究生培养模式改革创新项目(MSYZYXM2022004)。

摘　　要：为筛选出不同季节黑线姬鼠睾丸的最适内参基因,基于黑线姬鼠睾丸转录组数据,初步筛选出12个候选内参基因,即ALDOA、GLUL、RABAC1、TEGT、EID3、SYF2、MRPL20、GAPDH、HPRT、18s、B2M、NWD1。以不同季节(春季、夏季、秋季)成年黑线姬鼠的睾丸作为试验材料,进行RNA的提取并反转录成cDNA,再通过实时荧光定量PCR技术(Real-time Quantitative PCR,RT-qPCR)检测上述候选内参基因的表达水平。将所得到的循环阈值(Cyclethreshold,Ct)通过GeNorm、NormFinder、BestKeeper和RefFinder等分析软件进行基因稳定性的综合分析。结果表明,不同季节黑线姬鼠睾丸中12个候选内参基因表达稳定性由高到低排序为STF2>B2M>GAPDH>MRPL20>18s>HPRT>ALDOA>EID3>RABAC1>TEGT>NWD1>GLUL,其中SYF2是最稳定的内参基因,GLUL是最不稳定的基因。综上,本研究确定了不同季节下黑线姬鼠睾丸的内参基因,为黑线姬鼠功能基因的开发及基因表达的研究提供基础,并为后续黑线姬鼠基因组学的研究提供参考。In order to screen out the optimal internal reference genes of Apodemusagrarius testicles in different seasons,this study was based on the transcriptome data of Apodemus agrarius testicles,twelve candidate reference genes were initially selected,namely ALDOA,GLUL,RABAC1,TEGT,EID3,SYF2,MRPL20,GAPDH,HPRT,18s,B2M and NWD1.The testicles of different seasons(spring,summer and autumn)were extracted from RNA and reverse transcribed into cDNA.The expression levels of the above candidate reference genes were then measured by Real-time Quantitative PCR.The Cyclethreshold was analyzed by the analysis software GeNorm,NormFinder,BestKeeper and RefFinder.The results showed that the expression stability of 12 candidate reference genes in the testis of Apodemus agrus in different seasons was ranked from high to low as STF2>B2M>GAPDH>MRPL20>18s>HPRT>ALDOA>EID3>RABAC1>TEGT>NWD1>GLUL,among which SYF2 was the most stable reference gene,GLUL was the most unstable gene.Among them,SYF2 was the most stable reference gene,GLUL was the least unstable gene.This study provides a basis for functional gene development and gene expression research of Apodemus agrarius,and provides a reference for subsequent genomics research of Apodemus agrarius.

关 键 词：黑线姬鼠 内参基因 表达稳定性 不同季节 QRT-PCR 

分 类 号：Q493.5[生物学—生理学]