Yang Liang, Chen Juanni, Liu Ying, Zhang Shuting, Li Shili, Ding Wei
Laboratory of Natural Products Pesticides, College of Plant Protection, Southwest University, Chongqing 400715, China.
Laboratory of Natural Products Pesticides, College of Plant Protection, Southwest University, Chongqing 400715, China.
J Microbiol Methods. 2018 May;148:104-109. doi: 10.1016/j.mimet.2018.04.004. Epub 2018 Apr 11.
Quantitative real-time reverse transcriptase PCR (qRT-PCR) has become the method choice for quantification of gene expression changes, however, the accuracy of the method depends on the stability of reference genes. Ralstonia pseudosolanacearum (R. pseudosolanacearum) is an important plant pathogen, infecting >450 plant species and causing bacterial wilt. In order to identify stable reference genes in R. pseudosolanacearum CQPS-1 under different environment stresses. We used five tools (△Ct method, GeNorm, NormFinder, BestKeeper, and RefFinder) to evaluate the stability of seven candidate reference genes including phosphoglycerate kinase (PGK), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 16S ribosomal RNA (16S), cell division protein ftsZ (ftsZ), DNA gyrase subunit A (gyrA), Ribosomal protein L13 (rplM), and phosphoserine aminotransferase (serC) under biotic (growth phases) and abiotic stress (temperature, hydroxycoumarins, nutrition). Overall, gyrA and serC were the most stable genes under different growth phases, while serC, gyrA and ftsZ during temperature stress, gyrA, ftsZ and 16S under hydroxycoumarins stress, and serC and 16S under nutrition stress conditions. This study provides useful resources for normalizing expression changes of target genes in R. pseudosolanacearum subjected to environment stress.
定量实时逆转录聚合酶链反应（qRT-PCR）已成为定量基因表达变化的方法选择，然而，该方法的准确性取决于内参基因的稳定性。青枯雷尔氏菌是一种重要的植物病原菌，可感染450多种植物并引起青枯病。为了鉴定青枯雷尔氏菌CQPS-1在不同环境胁迫下的稳定内参基因。我们使用了五种工具（△Ct法、GeNorm、NormFinder、BestKeeper和RefFinder）来评估七个候选内参基因的稳定性，这些基因包括磷酸甘油酸激酶（PGK）﹑甘油醛-3-磷酸脱氢酶（GAPDH）、16S核糖体RNA（16S）、细胞分裂蛋白ftsZ（ftsZ）、DNA促旋酶亚基A（gyrA）、核糖体蛋白L13（rplM）和磷酸丝氨酸转氨酶（serC）在生物胁迫（生长阶段）和非生物胁迫（温度、羟基香豆素、营养）下的稳定性。总体而言，gyrA和serC是不同生长阶段最稳定的基因，而serC、gyrA和ftsZ在温度胁迫期间最稳定，gyrA、ftsZ和16S在羟基香豆素胁迫下最稳定，serC和16S在营养胁迫条件下最稳定。本研究为在环境胁迫下青枯雷尔氏菌中靶基因表达变化的标准化提供了有用的资源。
