Screening of reference genes for gene expression study in different tissues from the transcriptome data of the vector...

Selecting appropriate reference genes is crucial for ensuring the accuracy and reliability of gene expression study using reverse transcription-quantitative PCR (RT-qPCR). To screen the optimal reference genes for analyzing gene expression in different tissues of the vector leafhopper Psammotettix striatus which causes extensive damage to a wide range of crops by vectoring multiple plant pathogenic microorganisms, the transcriptome data from Malpighian tubules (MTs) of P. striatus were mined. Twenty alternative candidate reference genes were initially selected for screening, among which seven genes with diverse Gene Ontology (GO) annotations were choosed as candidate reference genes, i.e., ribosomal protein L7A (RPL7A), ribosomal protein S28 (RPS28), ribosomal protein L22 (RPL22), ribosomal protein LP2 (RPLP2), H3 histone family 3A (H3F3A), elongation factor 1γ (EF-1γ), and elongation factor 1α (EF-1α). Gene expression levels in different tissues of P. striatus adults were examined using RT-qPCR, and their expression stability was analyzed using multiple reference gene screening software. This study revealed EF-1α as the most abundantly expressed gene, while RPL22 exhibited the lowest expression levels. EF-1α showed the most stable expression, whereas RPS28 showed the least stability. Various software tools confirmed EF-1α as the most stable single reference gene, and EF-1α and RPLP2 an optimal combination. This study provides a foundation for future investigation of the transmission of pathogenic microorganisms mediated by the vector leafhoppers, the function of the MTs, the biosynthesis of brochosomes, the coevolutionary processes and nutritional interactions of symbionts and host insects, and the gene expression study of other sap-sucking insects.