Abstract
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.
Highlights
Glenea cantor (Fabricius) (Coleoptera: Cerambycidae: Lamiinae) is one of the dominant trunk borers of kapok trees, Bombax ceiba [1]
The amplification efficiency of the primers in G. cantor was 91.0% (RPS20)–109.4% (18S), which was within the required range of 80.0–120.0%
The results showed that ribosomal protein L32 (RPL32) and elongation factor 1-alpha1 (EF1A1) were the most stable reference genes in the different adult tissues, followed by ribosomal protein L36 (RPL36), and the least stable gene was 18s ribosome RNA (18S)
Summary
Glenea cantor (Fabricius) (Coleoptera: Cerambycidae: Lamiinae) is one of the dominant trunk borers of kapok trees, Bombax ceiba [1]. In 2005, this pest killed over 10% of the trees in a B. ceiba plantation in Nanning, Guangxi Province, China [1]. In 2018, B. ceiba was discovered as a new important host plant for long-horned beetles in Nanning, Guangxi Province [3]. G. cantor lives for four generations a year, with a period of 70 days per generation in Nanning, Guangxi Province in China [4,5]. The peak period for G. cantor is from March to July, and the larvae mainly hibernate in the affected branches during their fourth instar [4,5]. G. cantor is a good reproductive behavior model in the regulatory mechanism, as a polygamous long-horned beetle and R-strategist. Transcriptome analysis was performed (unpublished data), in which the validation of the transcript expression and gene expression studies required quantitative real-time PCR (qRT-PCR) by using the validated reference genes
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