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东亚飞蝗(直翅目:蝗科)定量PCR标准化中内参基因的筛选与评估

Selection and assessment of reference genes for quantitative PCR normalization in migratory locust Locusta migratoria (Orthoptera: Acrididae).

作者信息

Yang Qingpo, Li Zhen, Cao Jinjun, Zhang Songdou, Zhang Huaijiang, Wu Xiaoyun, Zhang Qingwen, Liu Xiaoxia

机构信息

Department of Entomology, China Agricultural University, Beijing, China.

Department of Entomology, China Agricultural University, Beijing, China; Department of Horticulture, Beijing Vocational College of Agriculture, Beijing, China.

出版信息

PLoS One. 2014 Jun 2;9(6):e98164. doi: 10.1371/journal.pone.0098164. eCollection 2014.

Abstract

Locusta migratoria is a classic hemimetamorphosis insect and has caused widespread economic damage to crops as a migratory pest. Researches on the expression pattern of functional genes in L. migratoria have drawn focus in recent years, especially with the release of genome information. Real-time quantitative PCR is the most reproducible and sensitive approach for detecting transcript expression levels of target genes, but optimal internal standards are key factors for its accuracy and reliability. Therefore, it's necessary to provide a systematic stability assessment of internal control for well-performed tests of target gene expression profile. In this study, twelve candidate genes (Ach, Act, Cht2, EF1α, RPL32, Hsp70, Tub, RP49, SDH, GAPDH, 18S, and His) were analyzed with four statistical methods: the delta Ct approach, geNorm, Bestkeeper and NormFinder. The results from these analyses aimed to choose the best suitable reference gene across different experimental situations for gene profile study in L. migratoria. The result demonstrated that for different developmental stages, EF1α, Hsp70 and RPL32 exhibited the most stable expression status for all samples; EF1α and RPL32 were selected as the best reference genes for studies involving embryo and larvae stages, while SDH and RP49 were identified for adult stage. The best-ranked reference genes across different tissues are RPL32, Hsp70 and RP49. For abiotic treatments, the most appropriate genes we identified were as follows: Act and SDH for larvae subjected to different insecticides; RPL32 and Ach for larvae exposed to different temperature treatments; and Act and Ach for larvae suffering from starvation. The present report should facilitate future researches on gene expression in L. migratoria with accessibly optimal reference genes under different experimental contexts.

摘要

东亚飞蝗是一种典型的渐变态昆虫,作为迁飞性害虫,它给农作物造成了广泛的经济损失。近年来,随着基因组信息的发布,对东亚飞蝗功能基因表达模式的研究受到了关注。实时定量PCR是检测靶基因转录表达水平最可重复且最灵敏的方法,但最佳内参基因是其准确性和可靠性的关键因素。因此,有必要对内部控制进行系统的稳定性评估,以便对靶基因表达谱进行良好的检测。在本研究中,使用四种统计方法(ΔCt法、geNorm、Bestkeeper和NormFinder)对12个候选基因(Ach、Act、Cht2、EF1α、RPL32、Hsp70、Tub、RP49、SDH、GAPDH、18S和His)进行了分析。这些分析结果旨在为东亚飞蝗基因谱研究选择在不同实验条件下最合适的参考基因。结果表明,对于不同发育阶段,EF1α、Hsp70和RPL32在所有样本中表现出最稳定的表达状态;EF1α和RPL32被选为涉及胚胎和幼虫阶段研究的最佳参考基因,而SDH和RP49被确定为成虫阶段的最佳参考基因。在不同组织中排名最佳的参考基因是RPL32、Hsp70和RP49。对于非生物处理,我们确定的最合适基因如下:不同杀虫剂处理的幼虫中为Act和SDH;不同温度处理的幼虫中为RPL32和Ach;饥饿处理的幼虫中为Act和Ach。本报告应有助于在不同实验背景下,通过可获取的最佳参考基因,推动未来对东亚飞蝗基因表达的研究。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/2c65/4041718/c31850452801/pone.0098164.g001.jpg

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