关键词: 大灰象甲')">大灰象甲, 实时定量PCR')">实时定量PCR, 内参基因')">内参基因, 基因表达分析')">基因表达分析, 表达稳定性')">表达稳定性

Abstract: 【Aim】 This study aims to screen out the suitable reference genes for gene expression analysis in different tissues of adult Sympiezomias velatus. 【Methods】 The house-keeping gene sequences of S. velatus were acquired and adopted as the candidate reference genes through transcriptome sequencing technique. Their mRNA expression levels in different tissues (antenna, head, thorax, abdomen and leg) of male and female adults of S. velatus were investigated by qRT-PCR. The expression stabilities of these candidate genes were evaluated by using three software-based (geNorm, NormFinder and BestKeeper) and one web-based (RefFinder) comprehensive analysis tools. And the expression stabilities of the candidate reference genes in different adult tissues of S. velatus were further validated by using the odorant binding protein 1 gene (OBP1) as the target gene. 【Results】 Nine house-keeping genes, including beta-actin gene (ACT), glyceraldehyde-3-phosphate gene (GAPDH), 18S ribosomal RNA (18S rRNA), 60S ribosomal protein L12 gene (RPL12), 60S ribosomal protein L32 gene (RPL32), 40S ribosomal protein S20 gene (RPS20), elongation factor 2 gene (EF2), α-tubulin gene (TUA), and β-tubulin gene (TUB), were identified for the first time based on the S. velatus transcriptome data. It was revealed that RPL12 and RPS20 were the most stable reference genes based on the geNorm analysis, while TUA and TUB were the most stable reference genes based on the BestKeeper analysis and NormFinder analysis, respectively. Comprehensive analysis showed that TUB, TUA, RPS20 and RPL12 were the most stable reference genes, while three of the most widely used reference genes including 18S rRNA, ACT and GAPDH, were the least stable. Finally, the validation result of expression stability of four candidate reference genes with OBP1 as the target gene showed that the expression patterns of OBP1 indifferent tissues of adult S. velatus were basically coincident when TUB and RPL12 acted as the reference genes, while the expression pattern was a little different from that with TUB as the reference gene when RPL32 acted as the reference gene, and completely different from that with TUB as the reference gene when 18S rRNA acted as the reference gene. 【Conclusion】 TUB, TUA, RPS20 and RPL12 proved to be acceptable as reference genes for gene expression analysis in different tissues of adult S. velatus. This study lays a foundation for future studies on gene expression in S. velatus.

Key words: Sympiezomias velatus, qPCR, reference genes, gene expression analysis, expression stability