Hou Lijuan, Zhu Haixia, Xian Wenrong, Ma Yongqiang
Academy of Agriculture and Forestry Sciences, Qinghai University, No. 251, Ningda Road, Xining, 810016, Qinghai, People's Republic of China.
Key Laboratory of Agricultural Integrated Pest Management in Qinghai, Xining, 810016, China.
Sci Rep. 2025 Apr 23;15(1):14205. doi: 10.1038/s41598-025-97542-x.
Potato blackleg disease is caused by Pectobacterium atrosepticum, can seriously destroy potatoes' growth and development. To accurately evaluate the expression levels of genes involved in potato (Solanum tuberosum) responses to P. atrosepticum infection, seven candidate reference genes (EF1α, eIF5A3, Tubulin, Ubiquitin, GAPDH, Actin, and CYP3) were systematically assessed for their expression stability at 1, 3, and 5 days after inoculation (dai) using the geNorm, NormFinder, BestKeeper, ∆Ct, and RefFinder algorithms. The results demonstrated that EF1α exhibited the highest stability among all experimental conditions, followed by eIF5A3 and Tubulin, whereas Ubiquitin displayed the least stability. To validate the screening outcomes, the expression patterns of four disease resistance-related genes (RBOHC, WRKY24, MPK3, and CPK32) were analyzed in both resistant and susceptible potato cultivars using the EF1α as the most stable and Ubiquitin as the least stable. Validation experiments revealed that the expression levels of disease resistance-related genes were stable and consistent with the RNA-Seq data when EF1α was used as a reference gene. In contrast, using Ubiquitin as a reference gene led to significant variability. Therefore, EF1α can be employed as the reference gene when studying the interaction between the potato and P. atrosepticum, providing a standardized reference for the subsequent studies on screening of disease resistance genes and exploring of disease resistance mechanism in potato.
马铃薯黑胫病由黑腐果胶杆菌引起，会严重破坏马铃薯的生长发育。为了准确评估参与马铃薯（Solanum tuberosum）对黑腐果胶杆菌感染反应的基因的表达水平，使用geNorm、NormFinder、BestKeeper、∆Ct和RefFinder算法，系统评估了七个候选内参基因（EF1α、eIF5A3、微管蛋白、泛素、甘油醛-3-磷酸脱氢酶、肌动蛋白和CYP3）在接种后1、3和5天（dai）的表达稳定性。结果表明，EF1α在所有实验条件下表现出最高的稳定性，其次是eIF5A3和微管蛋白，而泛素的稳定性最低。为了验证筛选结果，以最稳定的EF1α和最不稳定的泛素为内参基因，分析了四个抗病相关基因（RBOHC、WRKY24、MPK3和CPK32）在抗病和感病马铃薯品种中的表达模式。验证实验表明，以EF1α为内参基因时，抗病相关基因的表达水平稳定且与RNA测序数据一致。相比之下，以泛素为内参基因则导致显著的变异性。因此，在研究马铃薯与黑腐果胶杆菌的相互作用时，EF1α可作为内参基因，为后续马铃薯抗病基因筛选和抗病机制探索的研究提供标准化参考。
