鹿茸组织中内参基因的筛选和验证

为筛选在不同生长时期鹿茸组织中稳定表达的内参基因,试验以不同生长时期(分别为脱盘后10、20、40和60 d)的鹿茸组织为材料,采用实时荧光定量PCR(qRT-PCR)方法分析甘油醛-3-磷酸脱氢酶(GAPDH)、β2-微球蛋白(B2M)、还原型辅酶Ⅰ(NADH)、60S 核糖体蛋白L40(RPL40)、谷胱甘肽还原酶7(GPx)和β肌动蛋白(ACTB)6个看家基因的表达情况,并运用 geNorm和NormFinder 两个程序综合分析6个看家基因的表达稳定性.结果显示,GAPDH、ACTB、RPL40表达稳定性较好,可用作鹿茸基因表达研究的内参基因,而NADH和GPx的稳定性最差,不适合作内参基因.通过对鹿茸生长相关基因(ANXA5、HSP27、PRD2、CRABP1、LGALS1)表达分析,进一步验证了上述结果,并且发现这5种基因均在脱盘后10 d的鹿茸组织中高表达.该研究结果为鹿茸快速生长及骨化相关基因的研究奠定了一定基础.

Selection and Validation of Reference Genes in Velvet Antlers Tissues

The aim of this study was to identify the most stable gene to be used as reference genes for qRT-PCR analysis in Sika deer antler tissues. The expression patterns of 6 housekeeping genes were analyzed, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), beta-2-microglobulin (B2M), NADH dehydrogenase (NADH), 60S ribosomal protein L40 (RPL40), glutathione peroxidase 7 (GPx) and beta actin (ACTB), in Sika deer antler at different stages (10, 20, 40 and 60 d). The stability of housekeeping gene expression was analyzed with use of 2 software packages, including geNorm and NormFinder. The results showed that GAPDH, ACTB, RPL40 were suitable reference genes for efficient normalization of qRT-PCR data, whereas NADH and GPx were not suitable for analysis of Sika deer antler. These findings were confirmed by comparative profiling of 5 antler genes associated with antler rapid growth (ANXA5, HSP27, PRD2, CRABP1 and LGALS1), while it was observed that the 5 genes were significanly expressed in antler aged 10 d. These results will provide a necessary basis for the further research on rapid growth of antler and ossification genes.