Evaluation of Reference Genes in Glenea cantor (Fabricius) by Using qRT-PCR

Kapok is the main host of Glenea cantor (Fabricius), which causes serious damage and is difficult to control. In severe cases, it often causes the kapok trees to die continuously, which seriously affects the results of urban landscaping. To provide reference for the functional research on related genes in G. cantor, we screened the stable expression of candidate reference genes at different developmental stages (i.e., eggs, larvae, pupae, and adults), in various adult tissues (i.e., head, thorax, abdomen, feet, antennae, and wings), and sexes (i.e., male pupae, female pupae, male adults, and female adults). In this study, 12 candidate reference genes (i.e., ACTINLIKE, ACTININ, TUB, RPL36, RPL32, RPS20, TBP, GAPDH, 18S rRNA, EF1A1, EF1A2, and UBQ) were evaluated using different adult tissues, developmental stages, and sexes. RefFinder, geNorm, NormFinder, and BestKeeper were used to evaluate and comprehensively analyze the stability of the expression of the candidate reference genes. The results show that RPL32 and EF1A1 were the most suitable reference genes in the different adult tissues, and RPL36 and EF1A1 were best at the different developmental stages. RPL36 and EF1A2 were the best fit for the qRT-PCR reference genes in the different sexes, while RPL36 and EF1A1 were the most appropriate qRT-PCR reference genes in all samples. Results from geNorm showed that the optimal number of reference genes was two. We also surveyed the expression of cellulase at the different developmental stages and in the different adult tissues. Results further verified the reliability of the reference genes, and confirmed the best reference genes under the different experimental conditions. This study provides a useful tool for molecular biological studies on G. cantor.

Automated summary

The study screened and evaluated stable reference genes for Glenea cantor in different adult tissues, developmental stages, and sexes, with RPL32 and EF1A1 identified as the most suitable in different adult tissues, RPL36 and EF1A1 as the best in different developmental stages, and RPL36 and EF1A2 as the optimal for different sexes. GeNorm results indicated that the optimal number of reference genes was two, and the study provided a useful tool for molecular biological studies on G. cantor.