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中国农学通报

中国农学通报 ›› 2012, Vol. 28 ›› Issue (30): 201-207.doi: 10.11924/j.issn.1000-6850.2012-1707

所属专题： 生物技术

• 生物技术科学 • 上一篇    下一篇

恶疫霉实时定量RT-PCR分析中内参基因的选择

陈孝仁 张博月 邢玉平 童蕴慧 徐敬友   

  • 收稿日期:2012-05-04 修回日期:2012-07-02 出版日期:2012-10-25 发布日期:2012-10-25
  • 基金资助:

    国家自然科学基金项目“恶疫霉致病性相关RXLR 效应分子的克隆及功能分析”

Selection of Internal Control Genes for Real-time RT-PCR Data Normalization in Phytophthora cactorum

  • Received:2012-05-04 Revised:2012-07-02 Online:2012-10-25 Published:2012-10-25

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摘要：

为选择合适的内参基因用于分析植物病原卵菌恶疫霉(Phytophthora cactorum)的基因表达情况，本研究利用实时定量RT-PCR分析持家基因Ubc、Tub-b、WS21和WS41在恶疫霉生长发育阶段和侵染草莓阶段的表达差异，并利用软件geNorm、NormFinder和BestKeeper比较其表达稳定性。结果表明：基于公共数据库序列和其他疫霉菌同源序列设计的持家基因引物在恶疫霉中具有良好的扩增效率、反应有效性和特异性。通过分析，发现在恶疫霉的不同生长发育阶段（菌丝生长、游动孢子囊、游动孢子和休止孢萌发）和侵染草莓阶段（接种后3、5、7天），表现最为稳定的是Ubc基因。当使用多个内参基因时，Ubc和WS41是最适合校正恶疫霉基因表达数据的内参基因组合。本研究结果为利用实时定量RT-PCR准确分析恶疫霉基因相对表达量提供了重要的内参基因，也为其他疫霉菌的基因表达研究提供了有价值的参考。

关键词: 生态滤池, 生态滤池

Abstract:

To select proper internal control genes for gene expression analysis in mycete plant pathogen Phytophthora cactorum, the expression patterns of 4 housekeeping genes encoding ubiquitin-conjugating enzyme (Ubc), β-tubulin (Tub-b), 40S ribosomal protein S3A (WS21) and a protein of the BAR-domain family (WS41) were investigated by real-time RT-PCR during the developmental and strawberry infection stages of P. cactorum in this study. Their expression stability was also compared to each other using the softwares geNorm, NormFinder and BestKeeper. The results showed that the designed primers of 4 housekeeping genes based on the sequences deposited in the public databases and the homologous sequences in other Phytophthora species worked well with high amplification efficiency, effectivity and specificity to P. cactorum. It was found that Ubc was the most stable gene across the investigated developmental (mycelium, sporangium, zoospore and cyst germination) and strawberry infection stages (3, 5 and 7 d post-inoculation), and the combination of Ubc and WS41 could be served as internal control combination genes for normalization of real-time RT-PCR data. The results of this study provided important internal control genes for gene expression analysis of P. cactorum by real-time RT-PCR and valuable clues to the similar studies in other Phytophthora species.