天府肉鹅母系不同阶段颗粒细胞内参基因的选择

doi:10.3785/j.issn.1008-9209.2018.03.132

浙江大学学报（农业与生命科学版）

2019, Vol. 45

Issue (3): 376-384 DOI: 10.3785/j.issn.1008-9209.2018.03.132

动物科学与动物医学

天府肉鹅母系不同阶段颗粒细胞内参基因的选择

莫远亮(

),王郁石,王继文(

)

四川农业大学动物科技学院/畜禽遗传资源发掘与创新利用四川省重点实验室，成都 611130

Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese.

Yuanliang MO(

),Yushi WANG,Jiwen WANG(

)

College of Animal Science and Technology, Sichuan Agricultural University/Farm Animal Genetic Resources Exploration and Innovation Key Laboratory of Sichuan Province, Chengdu 611130, China

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摘要：

为筛选出鹅卵泡不同阶段颗粒细胞稳定表达的内参基因，以产蛋期天府肉鹅母系卵泡颗粒层细胞为材料，利用实时荧光定量聚合酶链式反应（real-time fluorescence quantitative polymerase chain reaction, RT-qPCR）技术分别对GAPDH、ACTB、TUB、UBC、HMBS、SDH、18S、28S、TBP和HPRT1等10个候选内参基因的相对表达量进行测定，采用ΔC _T法、qbase⁺、NormFinder、BestKeeper等4种评价方法对9个不同阶段卵泡颗粒细胞的表达稳定性进行评定。RT-qPCR熔解曲线和PCR扩增显示，10个候选内参基因的引物特异性良好；通过构建标准曲线，表明各内参基因在系列稀释的浓度梯度内具有良好的线性关系；综合4种方法的评价结果，发现在不同阶段颗粒细胞中，最稳定的3个内参基因依次为SDH、HMBS和18S，稳定性最差的3个内参基因依次为UBC、GAPDH和TUB。在不同阶段颗粒细胞中最稳定的内参基因为SDH和HMBS，以最稳定的2个内参基因的几何平均数作为标准化校正因子可得到更加准确的结果。

关键词： 鹅; 颗粒细胞; 内参基因; 稳定性; 选择

Abstract:

In order to screen out the most stable reference genes in different periods of granulosa cells in goose, we selected 10 candidate reference genes (GAPDH, ACTB, TUB, UBC, HMBS, SDH, 18S, 28S, TBP, HPRT1) to determine the relative expression levels by the real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). The expression stabilities of 10 candidate reference genes in nine different stages of granulosa cells were systematically analyzed by delta-C _T, qbase⁺, NormFinder and BestKeeper, respectively. The results of RT-qPCR melting curve and PCR amplification showed that the 10 candidate reference genes were specifically amplified. By constructing a standard curve, between C _q value and the logarithm of relative copy number exhibited a good linear relationship in the serial dilution concentration gradient. Based on the evaluation results of four different algorithms, SDH, HMBS and 18S were found to be three of the most stable reference genes, but UBC, GAPDH and TUB were three of the least stable reference genes in the different periods of granulosa cells. Therefore, the most stable internal reference genes were SDH and HMBS in granulosa cells at different developmental stages, and it could get more accurate normalization of RT-qPCR data by geometric averaging of the most stable reference genes.

Key words: geese granulosa cell reference gene stability selection

收稿日期: 2018-03-13 出版日期: 2019-06-25

CLC:

S 835

基金资助: 国家自然科学基金(31672424);国家现代水禽产业技术体系(CARS-42-4);国家科技支撑计划(2015BAD03B06)

通讯作者: 王继文 E-mail: evianmoyl@foxmail.com;wjw2886166@163.com

作者简介: 莫远亮（https://orcid.org/0000-0002-4945-5028），E-mail：evianmoyl@foxmail.com

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引用本文:

莫远亮,王郁石,王继文. 天府肉鹅母系不同阶段颗粒细胞内参基因的选择[J]. 浙江大学学报（农业与生命科学版）, 2019, 45(3): 376-384.

Yuanliang MO,Yushi WANG,Jiwen WANG. Identification for internal reference genes in different periods of granulosa cells of Tianfu meat geese.. Journal of Zhejiang University (Agriculture and Life Sciences), 2019, 45(3): 376-384.

链接本文:

http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2018.03.132 或 http://www.zjujournals.com/agr/CN/Y2019/V45/I3/376

表1 候选内参基因引物信息

图 1 候选内参基因的RT-qPCR熔解曲线

图 2 内参基因的PCR扩增

表2 候选内参基因的标准曲线

图3 候选内参基因在颗粒细胞中的 C q 值

图4 基于不同方法的内参基因稳定性分析结果

表3 内参基因表达稳定性综合排序

图5 最佳内参基因数目的确定

图6 FSHR 在不同阶段颗粒细胞中的表达分析

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