Identification and Validation of Reference Genes for Quantitative Real-time PCR in Stipa breviflora under Conditions of Simulated Warming

doi:10.11733/j.issn.1007-0435.2017.05.016

Acta Agrestia Sinica ›› 2017, Vol. 25 ›› Issue (5): 1020-1028.DOI: 10.11733/j.issn.1007-0435.2017.05.016

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Identification and Validation of Reference Genes for Quantitative Real-time PCR in Stipa breviflora under Conditions of Simulated Warming

GAO Jin-yu¹, GUO Hui-qin¹, CAO Lu¹, HAN Bing^1,2

1. College of Life Science, Inner Mongolia Agricultural University, Hohhot, Inner Mongolia 010018, China;
2. Institute of Grassland Research of CAAS, Hohhot, Inner Mongolia 010018, China

Received:2015-10-12 Revised:2017-09-06 Online:2017-10-15 Published:2018-01-25

模拟增温下短花针茅实时荧光定量内参基因的筛选及验证

高金玉¹, 郭慧琴¹, 曹路¹, 韩冰^1,2

1. 内蒙古农业大学生命科学学院, 内蒙古呼和浩特 010018;
2. 中国农业科学院草原研究所, 内蒙古呼和浩特 010018

通讯作者: 韩冰,E-mail:hb_nmg@163.com
作者简介:高金玉(1991-),女,河北衡水人,硕士研究生,研究方向为植物细胞生物学,E-mail:gaojy0423@163.com
基金资助:
内蒙古自然科学基金（2015MS0305）；中国农业学院草原研究所科技创新工程项目资助

Abstract

Abstract:

Under simulation of warming codition, we used Stipa breviflora as material tested the expression stability of nine candidate reference genes by qRT-PCR technology. Delta-Ct method, geNorm, normFinder, Bestkeeper and RefFinder were used to analyze the data. In S. breviflora, the ranking order of six reference genes from better to average was as following:TLF, 18s rRNA, APRT, TUBα, EF-1α, Actin2, after removal of three internal genes by common PCR. Five genes were selected from the transcriptome database of S. breviflora under the conditions of simulated warming. The results showed that there was a change in the relative expression level of the five genes between the warming and the control using TLF as the internal reference gene. The results in quantitative real-time PCR were consistent with the results obtained in the transcriptome sequencing of S. breviflora. The optimal internal reference gene of Stipa brevifolia was TLF under simulated warming.

Key words: Simulated warming, Stipa breviflora, Quantitative real-time PCR, Reference genes

摘要：

本研究以荒漠草原植物短花针茅（Stipa breviflora）为材料，在模拟增温处理下，通过qRT-PCR试验分析9个候选内参基因的表达稳定性，运用Delta-CT法，geNorm，normFinder，Bestkeeper软件及RefFinder在线工具进行候选内参基因的稳定性分析。经普通PCR剔除3个内参基因后，6个候选内参基因表达稳定性由高到低排序为：TLF，18s rRNA，APRT，TUBα，EF-1α，Actin2。以TLF作为内参基因，在模拟增温下短花针茅转录组数据库中随机选择5个基因进行验证。qRT-PCR结果表明，5个验证基因在增温与对照之间的相对表达量存在倍率变化，且与转录组测序中的变化趋势一致，从而确定TLF为短花针茅增温处理下的最佳内参基因。

关键词: 模拟增温, 短花针茅, 实时荧光定量PCR, 内参基因

CLC Number:

Q503

GAO Jin-yu, GUO Hui-qin, CAO Lu, HAN Bing. Identification and Validation of Reference Genes for Quantitative Real-time PCR in Stipa breviflora under Conditions of Simulated Warming[J]. Acta Agrestia Sinica, 2017, 25(5): 1020-1028.

高金玉, 郭慧琴, 曹路, 韩冰. 模拟增温下短花针茅实时荧光定量内参基因的筛选及验证[J]. 草地学报, 2017, 25(5): 1020-1028.

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Identification and Validation of Reference Genes for Quantitative Real-time PCR in Stipa breviflora under Conditions of Simulated Warming

模拟增温下短花针茅实时荧光定量内参基因的筛选及验证

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