Systematic selection of suitable reference genes for quantitative real-time PCR normalization studies of gene expression in Lutjanus erythropterus.

Quantitative real-time PCR (qRT-PCR) has been widely employed for the study of gene expression in fish, and accurate normalization is crucial. In this study, we aimed to identify the most stably expressed genes in various tissues, different developmental stages, and within astaxanthin treatment groups in Lutjanus erythropterus. Twelve candidate genes (EEF1A, CYB5R3, DLD, IDH3A, MRPL17, MRPL43, NDUFS7, PABPC1, PAGR1, PFDN2, PSMC3, and RAB10) were examined via qRT-PCR. We employed geNorm and NormFinder to assess their stability. The results revealed that RAB10 and PFDN2 exhibited relatively stable expression patterns across different tissue and astaxanthin treatment groups, while NDUFS7 and MRPL17 proved to be the most reliable reference gene combinations across various developmental stages. The stability of these selected genes was further validated by assessing the expression of two target genes, CRADD and CAPNS1, across developmental stages, reinforcing the reliability of NDUFS7 as it closely aligned with transcriptome-wide expression patterns at these stages. The present results will help researchers to obtain more accurate results in future qRT-PCR analysis in L. erythropterus.

Analysis of opsin gene family of Crimson snapper (Lutjanus erythropterus).

Opsin is a fellow of the G protein-coupled receptors (GPCRs) superfamily. It can be divided into visual and non-visual opsin according to whether it is directly involved in visual imaging. Opsin plays an important role in visual image formation and the regulation of non-image forming functions such as circadian entrainment in the growth, development and evolution of fish. Crimson snapper belongs to Perciforme mainly found in the Indo-West Pacific and the South China Sea. It is one of the most influential economic fishes in the South China Sea. In order to study the existence and expression of opsin gene in Crimson snapper, we sequenced the genome and tissue sample transcriptome of Crimson snapper. In this study, 32 opsin genes were identified from the genome of Crimson snapper. The length of these genes ranged from 1061 bp to 86203 bp and were distributed on 15 different chromosomes. The analysis of opsin gene family of Crimson snapper showed that the sws2 had two extra copies as compared with that of Zebrafish. Domain and motif analysis revealed that all the 32 opsin genes have seven-(pass)-transmembrane domain receptors (7TM receptors) each, and the opsin family contained 10 common motifs. The expression level of opsin gene, confirmed by RT-qPCR, was analyzed by using nine tissues transcriptome databases of Crimson snapper. The results showed that almost all opsin genes were highly expressed in the retina and brain, except opn7a and opn7b which were expressed in intestine and red skin, and almost no expression in other tissues. Our results provide a comprehensive basic knowledge for the opsin gene family of Crimson snapper, which has significance for the study of the function of opsin in Lutjanidaes.