龙须菜5种生活史阶段藻体间内参基因的筛选

Selection of Internal Reference Genes in Gracilariopsis lemaneiformis at 5 Life-History Stages

为筛选龙须菜5个枝叶状生活史阶段藻体中稳定表达的内参基因,本研究对9个候选内参基因,包括18S rRNA、28S rRNA、GAPDH、ACT、EF1、APC、PEB、rbcL和COX在具有世代交替生活史的产琼胶红藻龙须菜(Gracilariopsis lemaneiformis)不同生活史阶段中的表达稳定性进行评价,拷贝数分析结果表明,这些候选管家基因在不同倍性的藻体间表达差异最大,其次是雌雄配子体间的表达差异。geNorm和Normfinder软件对全部样品进行分析,发现结果与使用拷贝数的分析结果总体一致。结果表明,18S rRNA和28S rRNA比较适合作为二倍四分孢子体研究的内参基因;EF1和28S rRNA适合作为单倍体雌、雄配子体研究的内参基因;而针对5种生活史阶段藻体间的研究时,推荐28S rRNA和ACT为最适内参基因。

Selection of an appropriate internal reference gene is very important for obtaining accurate results in gene expression research.In this study,9 candidate internal reference genes in different life-history stages of agar-producing red algae Gracilariopsis lemaneiformis was comprehensively evaluated,including 18S rRNA,28S rRNA,ACT,GAPDH,EF1,APC,PEB,Rbc-L and COX.Copy number calculation results indicated that these candidate internal reference genes showed the biggest expression difference among algae with different ploidy,and then followed by the difference between male and female gametophytes.All samples were analyzed by geNorm and Normfinder software,and the results were found to be consistent with the analysis results using copy number.According to the comparison results,we suggest that 18S rRNA and 28S rRNA are more suitable as internal reference genes for the study of diploid tetrasporophyte.EF1 and 28 S rRNA are suitable as internal reference genes for haploid female and male gametophytes research.28S rRNA and ACT are recommended as the most suitable internal reference genes for the study of algae in five life cycle stages.