Validation and Evaluation of Reference Genes for Quantitative Real-Time PCR Analysis in <i>Mythimna loreyi</i> (Lepidoptera: Noctuidae)
Liuyang Wang,
Chaoxia Yang,
Qingyu Liu,
Xiaofang Zhang,
Xiangdong Mei,
Tao Zhang,
Jun Ning
Affiliations
Liuyang Wang
Plant Protection Institute, HAAFS/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, China/IPM Innovation Center of Hebei Province/International Science and Technology Joint Research Center on IPM of Hebei Province, Baoding 071000, China
Chaoxia Yang
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Qingyu Liu
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Xiaofang Zhang
Plant Protection Institute, HAAFS/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, China/IPM Innovation Center of Hebei Province/International Science and Technology Joint Research Center on IPM of Hebei Province, Baoding 071000, China
Xiangdong Mei
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Tao Zhang
Plant Protection Institute, HAAFS/Key Laboratory of IPM on Crops in Northern Region of North China, Ministry of Agriculture and Rural Affairs, China/IPM Innovation Center of Hebei Province/International Science and Technology Joint Research Center on IPM of Hebei Province, Baoding 071000, China
Jun Ning
State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China
Quantitative real-time PCR (qRT-PCR) is a widely applied technique for accurately assessing the expression of target genes. In practice, the evaluation of gene expression requires appropriate reference genes. To screen reliable reference genes for evaluating gene expression via qRT-PCR in Mythimna loreyi, a notorious migratory pest across Asia, Africa, Europe, and Australia, we assessed the expression stability of 13 candidate reference genes in M. loreyi using the ΔCt method, BestKeeper, Normfinder, GeNorm, and the web-based comprehensive platform RefFinder. These reference genes include RPL10, RPL27, RPL32, RPS3, TATA−box, GAPDH, AK, Actin, EF, α−tubulin, SOD, 18S rRNA, and FTZ−F1, which is frequently employed in Lepidoptera insects. Our findings revealed that the performance of the candidate reference gene depended on experimental conditions. Specifically, RPL27 and RPL10 were the most suitable for evaluating expression changes across developmental stages, tissues, and adult ages. The optimal reference genes were recommended in specific experiment conditions, for instance, EF and RPS3 were recommended for mating status, AK and RPL10 were recommended for temperature treatments, RPL27 and FTZ−F1 were recommended for larva diet, and EF and RPL27 were recommended for adult diet treatments. Additionally, expression profiles of pheromone-binding protein 2 (MlorPBP2) and glutathione S-transferase (MlorGST1) were used to validate the reference genes. This study provides reference genes for the accurate normalization of qRT-PCR data, laying the groundwork for studying the expression of target genes in M. loreyi.
