Institute of Plant Protection, Hunan Academy of Agricultural Science, Changsha, 410125, China.
Key Laboratory of Integrated Management of the Pests and Diseases on Horticultural Crops in Hunan Province, Changsha, 410125, China.
Mol Biol Rep. 2019 Dec;46(6):5767-5776. doi: 10.1007/s11033-019-05010-3. Epub 2019 Aug 5.
Quantitative real time PCR (qRT-PCR) is a nucleic acid quantitative technique and is also considered as a validation tool. The Cry1Ia36 protein isolated from Bacillus thuringiensis (Bt) strain YC-10 has high nematicidal activity against nematodes. Caenorhabditis elegans is one of the major model organisms and a readily accessible source of biological material for gene expression studies. To evaluate the expression stability of 12 candidate reference genes of C. elegans for exposing to different concentrations of Cry1Ia36 protein and different treat time, five statistical approaches (the comparative delta-Ct method, BestKeeper, NormFinder, Genorm and RefFinder) were used to evaluate each individual candidate reference gene. The results indicated that cdc-42 and F35G12.2 were the best reference genes for performing reliable gene expression normalization in the impact of Cry1Ia36 protein. In addition, when C. elegans was exposed to Cry1Ia36 protein and other nematicides, avermectin and 5-aminolevulinic acid, cdc-42 was recommended as the most reliable reference genes. Y45F10D.4 was the least stable reference genes in our experimental settings. Therefore, cdc-42 was reliable reference gene for gene expression studies in C. elegans exposed to Cry1Ia36 protein and other nematicides.
实时荧光定量 PCR（qRT-PCR）是一种核酸定量技术，也被认为是一种验证工具。从苏云金芽孢杆菌（Bt）YC-10 菌株中分离出的 Cry1Ia36 蛋白对线虫具有很高的杀线虫活性。秀丽隐杆线虫是主要的模式生物之一，也是用于基因表达研究的生物材料的易得来源。为了评估 12 个候选参考基因在暴露于不同浓度的 Cry1Ia36 蛋白和不同处理时间下的表达稳定性，使用了五种统计方法（比较 delta-Ct 法、BestKeeper、NormFinder、Genorm 和 RefFinder）来评估每个候选参考基因。结果表明，在 Cry1Ia36 蛋白的影响下，cdc-42 和 F35G12.2 是进行可靠基因表达归一化的最佳参考基因。此外，当秀丽隐杆线虫暴露于 Cry1Ia36 蛋白和其他杀线虫剂，阿维菌素和 5-氨基乙酰丙酸时，cdc-42 被推荐为最可靠的参考基因。在我们的实验设置中，Y45F10D.4 是最不稳定的参考基因。因此，cdc-42 是秀丽隐杆线虫暴露于 Cry1Ia36 蛋白和其他杀线虫剂时进行基因表达研究的可靠参考基因。
