原创力文档validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr验证参考基因的基因表达研究病毒感染烟草benthamiana使用定量实时pcr.pdfVIP
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validation of reference genes for gene expression studies in virus-infected nicotiana benthamiana using quantitative real-time pcr验证参考基因的基因表达研究病毒感染烟草benthamiana使用定量实时pcr
Validation of Reference Genes for Gene Expression
Studies in Virus-Infected Nicotiana benthamiana Using
Quantitative Real-Time PCR
. .
Deshui Liu , Lindan Shi , Chenggui Han, Jialin Yu, Dawei Li, Yongliang Zhang*
State Key Laboratory of Agro-Biotechnology, College of Biological Sciences, China Agricultural University, Beijing, China
Abstract
Nicotiana benthamiana is the most widely-used experimental host in plant virology. The recent release of the draft genome
sequence for N. benthamiana consolidates its role as a model for plant–pathogen interactions. Quantitative real-time PCR
(qPCR) is commonly employed for quantitative gene expression analysis. For valid qPCR analysis, accurate normalisation of
gene expression against an appropriate internal control is required. Yet there has been little systematic investigation of
reference gene stability in N. benthamiana under conditions of viral infections. In this study, the expression profiles of 16
commonly used housekeeping genes (GAPDH, 18S, EF1a, SAMD, L23, UK, PP2A, APR, UBI3, SAND, ACT, TUB, GBP, F-BOX, PPR
and TIP41) were determined in N. benthamiana and those with acceptable expression levels were further selected for
transcript stability analysis by qPCR of complementary DNA prepared from N. benthamiana leaf tissue infected with one of
five RNA plant viruses (Tobacco necrosis virus A, Beet black scorch virus, Beet necrotic yellow vein virus, Barley stripe mosaic
virus and Potato virus X). Gene stability was analysed in parallel by three commonly-used dedicated algorithms: geNorm,
NormFinder and BestKeeper. Statistical analysis revealed that the PP2A, F-BOX and L23 genes were the most stable overall,
and that the combination of these three genes was sufficient for accurate normalisation. In addition, the suitability of PP2A,
F-BOX and L23 as refe
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