Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes
Selection of endogenous reference genes for qRT-PCR analysis in Camelina sativa and identification of FLOWERING LOCUS C allele-specific markers to differentiate summer- and winter-biotypes
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DOI：
10.1016/j.indcrop.2018.12.017
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发表时间：
2019-03-01
影响因子：
5.9
通讯作者：
Anderson, James V.
中科院分区：
文献类型：
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作者：
Chao, Wun S.;Wang, Hongxia;Anderson, James V.
定量实时聚合酶链反应（qRT-PCR）分析极大地依赖于使用稳定表达的参考基因的转录物标准化。本研究在亚麻荠不同器官和春化组织中鉴定了用于qRT-PCR分析的参考基因。通过对夏季-（CO 46）和冬季-（Joelle）生物型的春化前和春化后组织的RNAseq分析，从22，157个基因中鉴定出19个稳定表达的候选参考基因。另外两个候选参考基因也选自编码肌动蛋白2（ACT 2）和非由核盘菌感染诱导（NIS）的拟南芥的直系同源物。我们还评估了三种亚麻荠植物基因的转录水平，CO 1过表达的支持基因（SOC 1）、FLOWERLOCUS C（FLC）和MADS影响FLOWERLOCUS 2（MAF 2），这些基因已知在响应低温时受到差异调节。使用Normandy、geNorm、BestKeeper和Comparative Delta C-T软件对转录物丰度的稳定性进行排序。外孢囊复合物组分SEC 3A（SEC 3A）、泛醌氧化还原酶（UbOxRed）和含RING-U-BOX结构域蛋白（RUB）是用于亚麻荠属基因表达标准化的最合适的参考基因。在这项研究中，SEC 3A被用来标准化qRT-PCR表达数据，获得使用两套等位基因特异性FLC标记，以区分夏季和冬季生物型在30个加入的亚麻荠。
Quantitative real-time polymerase chain reaction (qRT-PCR) analysis greatly relies on transcript normalization using stably expressed reference genes. This study identifies reference genes for qRT-PCR analysis in different organs and vernalized tissues of camelina (Camelina sativa). Nineteen stably expressed candidate reference genes from 22,157 genes were identified by RNAseq analysis of pre- and post-vernalized tissues of a summer- (CO46) and winter- (Joelle) biotype. Two additional candidate reference genes were also selected from orthologs of arabidopsis encoding ACTIN2 (ACT2) and NOT INDUCED BY SCLEROTINIA INFECTION (NIS). We also evaluated the transcript levels of three camelina plant genes, SUPPRESSOR OF OVEREXPRESSION OF CO 1 (SOC1), FLOWERING LOCUS C (FLC), and MADS AFFECTING FLOWERING 2 (MAF2), which are known to be differentially-regulated in response to cold temperatures. The stability of transcript abundance was ranked using NormFinder, geNorm, BestKeeper, and Comparative Delta C-T software. EXOCYST COMPLEX COMPONENT SEC3A (SEC3A), UBIQUINONE OXIDOREDUCTASE (UbOxRed), and RING-U-BOX domains-containing protein (RUB) were the most suitable reference genes for normalization of gene expression in camelina. In this study, SEC3A was used to normalize qRT-PCR expression data obtained using two sets of allele-specific FLC markers to differentiate summer- and winter-biotypes across 30 accessions of camelina.