东方蜜蜂微孢子虫实时荧光定量PCR内参基因筛选OA

[Purpose]This study aims to screen stably expressed reference genes in Nosema ceranae within 1 to 10 days post-infec-tion in Apis mellifera,providing a reliable basis for gene expression analysis.[Method]Through literature retrieval and BLAST a-lignment,seven homologous genes—GAPDH,TUA,TUBB,ACTB,TBP,SYB,and RPL32—of N.ceranae were selected as candi-date reference genes.Real-time quantitative PCR was performed to determine the expression levels of these seven candidate reference genes within 1 to 10 days post-infection.EΔCq,GeNorm,NormFinder,and BestKeeper were used to evaluate the expression stabili-ty,followed by comprehensive ranking via RefFinder.[Result]EΔCq and BestKeeper had consistent results,both showing the stabil-ity order:TUA>GAPDH>TBP>SYB>ACTB>TUBB>RPL32.GeNorm analysis showed that TBP and TUA were the most stable genes,with an optimal number of reference genes set to two.NormFinder identified TBP as the most stable gene.All the four algorithms consistently identified RPL32 as the least stable gene.The comprehensive ranking by RefFinder showed the stability order of the sev-en candidate reference genes as:TUA>TBP>GAPDH>SYB>ACTB>TUBB>RPL32.[Conclusion]TUA and TBP can be used as ref-erence genes for quantifying the relative expression of target genes in N.ceranae.The two genes can be selected for calibration in sub-sequent studies of gene expression to ensure the result accuracy.