Department of Arid Land Agriculture, College of Agricultural and Food Sciences, King Faisal University, P.O. Box 420, 31982, Al-Hofuf, Saudi Arabia.
Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), Giza, 12619, Egypt.
Mol Biol Rep. 2024 Sep 30;51(1):1028. doi: 10.1007/s11033-024-09975-8.
Normalization with respect to stable housekeeping genes is important to facilitate gene transcription regulation research and acquire more accurate quantitative polymerase chain reaction (qPCR) data. In the current study, five candidates housekeeping genes of the cotton leafworm, Spodoptera littoralis encoding for Actin (Actin), elongation factor 1-alpha (EF1α), ribosomal protein S3 (RPS3), ribosomal protein 49 (RP49), and Ubiquitin (Ubi), were evaluated as normalization housekeeping genes under Spodoptera littoralis nucleopolyhedrovirus (SpliNPV) viral infection.
The qPCR results confirmed the expression of all five housekeeping genes in S. littoralis viral infected larvae. The expression profiles of the housekeeping genes showed that the EF1α, Actin, and RP49 had the minimum average Ct values of 18.41 ± 0.66, 18.84 ± 0.90 and 19.01 ± 0.87 in all infected samples, respectively. While RPS3 and Ubi showed the maximum average Ct of 21.61 ± 0.51 and 21.11 ± 0.82, respectively. According to the results of ΔCt and geNorm analysis, EF1α was ranked as the most stable housekeeping gene during infection time-course. While by using BestKeeper, geNorm and NormFinder, the Ubi, RP49, and RPS3 showed the most genes transcription stability. The obtained results were also validated using the Cytochrome c oxidase (COX) gene transcripts in response to SpliNPV infection.
The results revealed that EF1α and Ubi were the most stable housekeeping genes to be used for normalizing S. littoralis gene transcription regulation under SpliNPV infection. These findings, provide a significant addition for gene transcription regulation studies of S. littoralis upon infection using SpliNPV as a bio-agent.
相对于稳定的管家基因进行归一化对于促进基因转录调控研究和获得更准确的定量聚合酶链反应（qPCR）数据非常重要。在本研究中，评估了编码肌动蛋白（Actin）、延伸因子 1-α（EF1α）、核糖体蛋白 S3（RPS3）、核糖体蛋白 49（RP49）和泛素（Ubi）的 5 种棉铃虫候选管家基因，作为棉铃虫核多角体病毒（SpliNPV）病毒感染下的归一化管家基因。
qPCR 结果证实了所有 5 种管家基因在 S. littoralis 病毒感染幼虫中的表达。管家基因的表达谱显示，EF1α、Actin 和 RP49 在所有感染样本中的平均 Ct 值最小，分别为 18.41±0.66、18.84±0.90 和 19.01±0.87。而 RPS3 和 Ubi 的平均 Ct 值最大，分别为 21.61±0.51 和 21.11±0.82。根据ΔCt 和 geNorm 分析的结果，EF1α在感染时间过程中被评为最稳定的管家基因。而通过使用 BestKeeper、geNorm 和 NormFinder，Ubi、RP49 和 RPS3 显示出最稳定的基因转录稳定性。使用 SpliNPV 感染响应的细胞色素 c 氧化酶（COX）基因转录物也验证了这些结果。
结果表明，EF1α和 Ubi 是最稳定的管家基因，可用于归一化 SpliNPV 感染下棉铃虫基因转录调控。这些发现为使用 SpliNPV 作为生物制剂感染 S. littoralis 时的基因转录调控研究提供了重要补充。
