光裸星虫(Sipunculus nudus)不同发育时期卵细胞内参基因的筛选  

Selection of Reference Genes for qRT-PCR in Oocytes at Different Development Stages of Sipunculus nudus

作　　者：张家炜[1] 邬婧 周丹 郑哲[1] 王庆恒[1] Zhang Jiawei;Wu Jing;Zhou Dan;Zheng Zhe;Wang Qingheng(Fisheries College,Guangdong Ocean University,Zhanjiang,524088)

机构地区：[1]广东海洋大学水产学院,湛江524088

出　　处：《基因组学与应用生物学》2020年第2期575-583,共9页Genomics and Applied Biology

基　　金：广东省自然科学基金(2017A030303075);广东省科技计划(2016A020209010);广东海洋大学大学生创新创业训练计划项目(CXXL2018049);广东海洋大学研究生教育创新计划项目(201836)共同资助。

摘　　要：内参基因的选择对功能基因表达量的归一化处理尤为重要。为了筛选出光裸星虫不同发育时期卵子的最适内参基因,利用qRT-PCR测定了甘油醛-3-磷酸脱氢酶(GAPDH)、肽基脯氨酰顺反异构酶A(PPIA)、60S核糖体蛋白L10(60S-L10)、铁蛋白(Ferritin)、β-肌动蛋白(β-actin)、泛素C(UBC)、真核生物翻译起始因子(eIF)、NADH脱氢酶(NDH)、28S核糖体RNA(28S)、TATA盒结合蛋白(TBP)、18S核糖体RNA(18S)和琥珀酸脱氢酶A亚基(SDHA)共12个候选内参基因的表达水平,并通过4个程序(geNorm,NormFinder,BestKeeper以及RefFinder)综合分析了各基因的表达稳定性。结果显示:(1)12个候选内参基因均能获得特异性扩增产物,但表达情况各异;(2)对候选内参基因进行综合打分,得到候选内参基因稳定性排名为18S>GAPDH>28S>β-actin>UBC>e IF>NDH|TBP>PPIA|Ferritin>60S-L10>SDHA。18S和GAPDH稳定性较好,可作为不同发育时期卵细胞基因表达研究的单内参基因,或最优组合内参基因。The selection of reference genes for quantitative real-time PCR(qRT-PCR)plays an important role in the normalization of functional gene expression.In order to select the most suitable reference gene in oocytes at different development stages of Sipunculus nudus,qRT-PCR was used to determine the expression of twelve candidate reference genes,including GAPDH,PPIA,60 S-L10,Ferritin,β-actin,UBC,eIF,NDH,28 S,TBP,18 S and SDHA.Combining geNorm,NormFinder,BestKeeper and RefFinder software,the expression stability of twelve reference genes were analyzed.The results showed that:(1)twelve candidate reference genes all can obtain specific amplification products,but their expressions were different;(2)according to the results given by four softwares,the comprehensive ranking of stability was as follows:18 S>GAPDH>28 S>β-actin>UBC>eIF>NDH|TBP>PPIA|Ferritin>60 S-L10>SDHA.18 S and GAPDH have good stability expression.Therefore,18 S or GAPDH could be used as a suitable reference gene or the optimcal combination in qRT-PCR analysis of oocytes at different development stages.

关 键 词：光裸星虫 卵子发生 内参基因筛选 18S 

分 类 号：Q953[生物学—动物学]