Screening and stable expression analysis of internal reference genes in different tissues of Tulipa gesneriana L.
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摘要: 【目的】筛选郁金香在盐胁迫条件下稳定表达的内参基因，为开展郁金香耐盐相关基因表达调控相关研究提供理论参考。【方法】以郁金香栽培品种红色印象为材料，于苗期对其进行盐胁迫处理，不同时间（0、2、4、6、8、10、12、24和48 h）采集根、鳞茎和叶，通过实时荧光定量PCR检测候选内参基因在郁金香不同组织中的表达情况，再利用Delta CT计算每个样本的平均Ct值，再结合3个常用内参基因稳定性评估软件（geNorm、NormFinder、BestKeeper）的分析结果，利用RefFinder在线软件综合评价候选内参基因的表达稳定性，并通过目的基因质膜Na+/H+逆向转运蛋白基因（SOS1）和液泡膜Na+/H+逆向转运蛋白基因（NHX1）的表达分析验证内参基因的可靠性。【结果】在15个候选内参基因中筛选到9个可用于郁金香各组织实时荧光定量PCR的内参基因。候选内参基因的Ct值分析结果显示，25S rRNA在不同组织中的平均Ct值最低，表达丰度最高，Ct值分布范围相对集中，是根、鳞茎和叶均能稳定表达的内参基因。geNorm和NormFinder分析结果均显示，18S rRNA均能在根、鳞茎和叶中稳定表达，25S rRNA在根和叶中表现出较好的基因稳定性，NADP基因在鳞茎中基因稳定性较好。BestKeeper分析结果显示，根、鳞茎和叶组织中最佳的内参基因为25S rRNA，同时18S rRNA在根和叶中稳定性较好。结合上述4种分析结果通过RefFinder软件得到各候选基因在郁金香不同组织中的表达稳定性综合排名，其中根组织：25S rRNA>18S rRNA>TUB-6>EF1α>NADP>EF-1α>β-actin；鳞茎组织：NADP>25S rRNA>18S rRNA>EF-1α>EF1α>TUB-6；叶组织：25S rRNA>18S rRNA>EF-1α>CYP>UBC。【结论】9个候选内参基因在郁金香不同组织中的表达情况存在差异，25S rRNA和18S rRNA是在郁金香各组织的不同盐胁迫处理中稳定表达的内参基因，同时使用2个内参基因更有利于得到准确的基因表达定量结果。Abstract: 【Objective】The purpose of the study was to screen the internal reference genes stably expressed in Tulipa gesneriana L. under salt stress conditions,and to provide theoretical references for carrying out the studies related to the regulation of the expression of salt tolerance related genes in T. gesneriana.【Method】T. gesneriana cultivar Red Impression was used as the materials,and it was treated with salt stress at the seedling stage. Roots,bulbs and leaves were collected at different times(0,2,4,6,8,10,12,24 and 48 h),and the expression of candidate internal reference genes in different tissues of T. gesneriana was detected by real-time fluorescence quantitative PCR. The average Ct value for each sample was calculated by Delta CT. Combined with the analysis results of three commonly used software for evaluating the stability of internal reference genes(geNorm,NormFinder and BestKeeper),RelFinder online software was used to comprehensively evaluate the expression stability of candidate internal reference genes and to verify the reliability of the internal reference genes through the expression analysis of the target gene plasma membrane Na+/H+ reverse transporter protein gene(SOS1)and vesicular membrane Na+/H+ reverse transporter protein gene(NHX1).【Result】Among the 15 candidate internal reference genes,9 internal reference genes that could be used for real-time fluorescent quantitative PCR in various tissues of T. gesneriana were screened. The results of Ct value analysis of the candidate internal reference genes showed that the average Ct value of 25S rRNA in different tissues was the lowest,the expression abundance was the highest,and the distribution range of Ct value was relatively concentrated,which was an internal reference gene that could be stably expressed in root,bulb and leaf. The results of geNorm and NormFinder analysis showed that 18S rRNA was stably expressed in roots,bulbs and leaves. The 25S rRNA showed good gene stability in roots and leaves,and NADP gene had good gene stability in bulbs. The results of BestKeeper analysis showed that the most suitable internal reference gene in root,bulb and leaf tissues was 25S rRNA,and 18S rRNA had good stability in root and leaf. Combined with the results of the above four analysis,the comprehensive ranking of the expression stability of each candidate gene in different tissues of T. gesneriana was obtained through RefFinder software. Among them,root tissue:25S rRNA>18S rRNA>TUB-6>EF1α>NADP>EF-1α>β-actin;bulb tissue:NADP>25S rRNA>18S rRNA>EF-1α>EF1α>TUB-6;leaf tissue:25S rRNA>18S rRNA>EF-1α>CYP>UBC.【Conclusion】There are differences in the expression of 9 candidate internal reference genes in different tissues of T. gesneriana. 25S rRNA and 18S rRNA are internal reference genes stably expressed in different salt stress treatments in various tissues of T. gesneriana. The use of two internal reference genes at the same time is more conducive to obtaining accurate quantitative results of gene expression.
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