菲律宾蛤仔不同发育时期及成体不同组织中内参基因筛选

摘要

为筛选出菲律宾蛤仔17个发育时期及成体7个组织中的最适内参基因,实验采用3个内参基因筛选方法geNorm、NormFinder及ΔCt对菲律宾蛤仔不同发育时期和成体不同组织中的12个候选内参基因延伸因子1α基因(EF1A)、TATA盒结合蛋白基因(TBP)、组蛋白H3基因(HIS)、细胞色素b5基因(CYTB5)、泛素缀合酶基因(UCE)、核糖体蛋白L8基因(RPL8)、核糖体蛋白S23基因(RPS23)、核糖体蛋白L2基因(RPL2)、细胞色素C基因(CYTC)、生长因子受体结合蛋白2基因(GFRP2)、肌动蛋白基因(ACT)和微管蛋白基因(TUB)进行表达稳定性分析.结果显示,菲律宾蛤仔不同发育时期qRT-PCR分析需要3个内参基因,分别为CYTC、CYTB5和RPS23;菲律宾蛤仔成体不同组织qRT-PCR分析需要2个内参基因,分别为CYTB5和GFRP2.ACT在菲律宾蛤仔不同发育阶段和不同组织中表达最不稳定.%To select the reference genes applicable for 17 development stages and 7 tissues of Ruditapes philip-pinarum,expression stabilities of elongation factor 1 alpha gene(EF1A),TATA-box binding protein gene(TBP), histone gene(HIS),cytochrome b5 gene(CYTB5),ubiquitin-conjugating enzyme gene(UCE),ribosomal protein L8 gene(RPL8),ribosomal protein S23 gene(RPS 23),ribosomal protein L2 gene(RPL 2),cytochrome C gene (CYTC),growth factor receptor-bound protein 2 gene(GFRP2),β-actin gene(ACT)and tubulin gene(TUB)were then evaluated with three algorithms—geNorm, NormFinder and ΔCt methods, respectively. The results suggest that CYTC,CYTB5 and RPS23 were the optimal reference gene combination for different development stages;CYTB5 and GFRP2 were recommended for qRT-PCR normalization of different tissues.The ACT was proved to be the most unstable in different development stages and different tissues of R.philippinarum.