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通过定量实时PCR评估水稻条纹病毒和水稻黑条矮缩病毒感染水稻中内参基因的稳定性

Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR.

作者信息

Fang Peng, Lu Rongfei, Sun Feng, Lan Ying, Shen Wenbiao, Du Linlin, Zhou Yijun, Zhou Tong

机构信息

Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing, 210014, China.

Scientific Observing and Experimental Station of Crop Pests in Nanjing, Ministry of Agriculture, China, Nanjing, 210014, China.

出版信息

Virol J. 2015 Oct 24;12:175. doi: 10.1186/s12985-015-0405-2.

DOI:10.1186/s12985-015-0405-2
PMID:26497487
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4619528/
Abstract

BACKGROUND

Stably expressed reference gene(s) normalization is important for the understanding of gene expression patterns by quantitative Real-time PCR (RT-qPCR), particularly for Rice stripe virus (RSV) and Rice black streaked dwarf virus (RBSDV) that caused seriously damage on rice plants in China and Southeast Asia.

METHODS

The expression of fourteen common used reference genes of Oryza sativa L. were evaluated by RT-qPCR in RSV and RBSDV infected rice plants. Suitable normalization reference gene(s) were identified by geNorm and NormFinder algorithms.

RESULTS

UBQ 10 + GAPDH and UBC + Actin1 were identified as suitable reference genes for RT-qPCR normalization under RSV and RBSDV infection, respectively. When using multiple reference genes, the expression patterns of OsPRIb and OsWRKY, two virus resistance genes, were approximately similar with that reported previously. Comparatively, by using single reference gene (TIP41-Like), a weaker inducible response was observed.

CONCLUSIONS

We proposed that the combination of two reference genes could obtain more accurate and reliable normalization of RT-qPCR results in RSV- and RBSDV-infected plants. This work therefore sheds light on establishing a standardized RT-qPCR procedure in RSV- and RBSDV-infected rice plants, and might serve as an important point for discovering complex regulatory networks and identifying genes relevant to biological processes or implicated in virus.

摘要

背景

通过定量实时荧光定量PCR（RT-qPCR）对稳定表达的参考基因进行标准化，对于理解基因表达模式非常重要，特别是对于在中国和东南亚对水稻植株造成严重损害的水稻条纹病毒（RSV）和水稻黑条矮缩病毒（RBSDV）而言。

方法

通过RT-qPCR评估了水稻中14个常用参考基因在感染RSV和RBSDV的水稻植株中的表达情况。使用geNorm和NormFinder算法鉴定合适的标准化参考基因。

结果

分别确定UBQ 10 + GAPDH和UBC + Actin1为RSV和RBSDV感染下RT-qPCR标准化的合适参考基因。使用多个参考基因时，两个病毒抗性基因OsPRIb和OsWRKY的表达模式与先前报道的大致相似。相比之下，使用单个参考基因（TIP41-Like）时，观察到较弱的诱导反应。

结论

我们提出，两个参考基因的组合可以在感染RSV和RBSDV的植物中获得更准确可靠的RT-qPCR结果标准化。因此，这项工作为在感染RSV和RBSDV的水稻植株中建立标准化的RT-qPCR程序提供了思路，并且可能成为发现复杂调控网络以及鉴定与生物过程相关或涉及病毒的基因的重要切入点。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/8ce8e73c7945/12985_2015_405_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/7f535d5dadc7/12985_2015_405_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/837a5109f10b/12985_2015_405_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/0e75de6c59d7/12985_2015_405_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/2559d7d056a1/12985_2015_405_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/e6c9bc748700/12985_2015_405_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/18ccc1aabdc4/12985_2015_405_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/8ce8e73c7945/12985_2015_405_Fig7_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/7f535d5dadc7/12985_2015_405_Fig1_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/837a5109f10b/12985_2015_405_Fig2_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/0e75de6c59d7/12985_2015_405_Fig3_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/2559d7d056a1/12985_2015_405_Fig4_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/e6c9bc748700/12985_2015_405_Fig5_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/18ccc1aabdc4/12985_2015_405_Fig6_HTML.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/d5cc/4619528/8ce8e73c7945/12985_2015_405_Fig7_HTML.jpg

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