新西兰兔下丘脑qPCR内参基因的筛选和验证  

Selection and validation of qPCR reference genes in hypothalamus of New Zealand rabbits

作　　者：姜幸慧 陈晶 张洺瑞 邱水玲 甘乾福[1] JIANG Xinghui;CHEN Jing;ZHANG Mingrui;QIU Shuiling;GAN Qianfu(College of Animal Science,Fujian Agriculture and Forestry University,Fuzhou 350002,China)

机构地区：[1]福建农林大学动物科学学院,福建福州350002

出　　处：《畜牧与兽医》2024年第9期48-55,共8页Animal Husbandry & Veterinary Medicine

基　　金：福建省中青年教师教育科研项目(JAT220070);福建省科技计划项目[高校产学合作项目(2023N5004),星火项目(2023S0007,2023S0015,2023S0054),对外合作项目(2023I1009)];福建农林大学农村振兴服务团队-草食性动物产业服务团队项目(11899170139)。

摘　　要：为筛选适合新西兰白兔下丘脑的最佳内参基因,试验选定甘油醛-3-磷酸脱氢酶(GAPDH)、肌动蛋白β(ACTB)、β-2-微球蛋白(B2M)、酪氨酸3-单加氧酶(YWHAZ)、次黄嘌呤磷酸核糖转移酶1(HPRT1)、核糖体蛋白L4(RPL4)和TATA-box结合蛋白(TBP)共7个候选内参基因,利用实时荧光定量PCR(qPCR)技术对不同年龄、性别及发情状态的新西兰白兔下丘脑组织的基因表达水平进行检测;通过RefFinder在线软件综合ΔCt、GeNorm、NormFinder和BestKeper等4种算法的分析结果来评估内参基因表达的稳定性,并选取在下丘脑表达的功能基因雌激素受体(ER1)、孕激素受体(PGR)和RFamide神经肽VF(NPVF)对候选内参基因稳定性和评价结果的可靠性进行验证。结果:不同年龄的雌性新西兰白兔下丘脑稳定表达的内参基因是YWHAZ和GAPDH;雄性新西兰白兔下丘脑稳定性表达的内参基因YWHAZ和TBP,而不同发情期新西兰白兔适合使用HPRT1和YWHAZ作为内参基因。当使用不同内参基因进行标准化时,目的基因表达水平受到样本不同生理状态而出现明显变化,强调了先行验证内参基因对于qPCR研究基因表达水平的重要性。We selected seven reference genes as candidates,including GAPDH,ACTB,B2M,YWHAZ,HPRT1,RPL4,and TBP,to find a suitable gene for the hypothalamus of New Zealand white rabbits.The gene expression levels in the hypothalamus of New Zealand white rabbits of different ages,genders,and estrus states were detected by qPCR.Then,RefFinder was used to evaluate the stability of the reference gene expression by combining the analysis results ofΔCt,GeNorm,NormFinder and BestKeper.Next,ER1,PGR and NPVF were used to verify the stability of the candidate reference genes and the reliability of the analysis results.The results showed that YWHAZ and GAPDH were the most stable hypothalamic reference genes for the female rabbits,while YWHAZ and TBP were recommended for the male rabbits;and HPRT1 and YWHAZ were good for the rabbits in different estrus cycles.When different reference genes were used for normalization,the expression level of the target gene would change significantly depending on the physiological state of the sample,underlining the importance of a priori validation of reference genes for qPCR studies.

关 键 词：新西兰白兔 下丘脑 内参基因 稳定性评价和验证 

分 类 号：S829.1[农业科学—畜牧学]