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Updated: Jun 25, 2025

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在Enterobacter cancerogenus中通过qRT-PCR进行表达分析的参考基因

Yang Pan¹, Yue Zhao¹, Hua-Rui Zeng¹

¹Education Ministry Key Laboratory of Integrated Management of Crop Diseases and Pests/State & Local Joint Engineering Research Center of Green Pesticide Invention and Application, Department of Entomology, College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, China.

|May 25, 2024

概括

这项研究确定了Enterobacter cancerogenus EcHa1中定量实时PCR (qRT-PCR) 的最佳参考基因,有助于生物控制研究. 这些发现为虫病原细菌功能基因研究建立了可靠的正常化方法.

科学领域:

微生物学微生物学
分子生物学分子生物学
昆虫病理学昆虫病理学

背景情况:

肠杆菌癌原体EcHa1显示出具有生物控制类昆虫的潜力,例如Helicoverpa armigera.
使用定量实时PCR (qRT-PCR) 精确的基因表达分析需要稳定的内源参考基因 (家政基因,HKGs).
在各种条件下确定E. cancerogenus的最佳HKG对于可靠的基因表达研究至关重要.

研究的目的:

在E. cancerogenus菌株EcHa1.1中选择和评估稳定的家政基因以进行qRT-PCR正常化.
在不同的培养温度和细菌光密度 (OD) 值下确定最适合的基因基因.
为了验证选择的参考基因的实用性,在昆虫感染期间研究E. cancerogenus中的基因表达.

主要方法:

在E.cancerogenus EcHa1.1中选了13个候选的家政基因.
基因表达水平使用qRT-PCR在不同温度 (28°C,37°C) 和OD值 (0.5,1.0,2.0) 中量化.
为了评估基因稳定性,使用了五种统计算法 (Ct,BestKeeper,NormFinder,geNorm,RefFinder).

主要成果:

参考基因的稳定性随着实验条件的变化而显著变化.
最佳的基因基因对包括28°C的gyrA/gyrB,37°C的gyrA/rpoB,0.5的OD的ftsZ/gyrA,1.0的OD的rpoB/gyrB和2.0的OD的gyrA/pyk.
在所有测试条件下, gyrA 和 gyrB 的组合表现出最高的稳定性.
使用gyrA/gyrB进行正常化,观察到E.cancerogenus EcHa1侵入H.armigera组织,并在肠道,马尔皮格管道和表皮中高度表达Hcp因子.

结论:

该研究提供了经验证的基因基因基因组,用于在不同条件下在E. cancerogenus中准确的qRT-PCR.
推 gyrA 和 gyrB 作为 E. cancerogenus 功能性基因表达研究中最可靠的参考基因.
这项研究有助于更深入地研究像E. cancerogenus这样的肠病原菌的功能基因和病原性机制.

关键词:

最好的保姆是最好的保姆肠杆菌是致癌的规范寻找器 (NormFinder) 是一个寻找规范的工具.在RefFinder中找到.在GeNorm上使用.qRT-PCR 是一个很好的方法.基因基因基因基因基因基因基因基因基因基因

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