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小苍兰实时荧光定量PCR中的内参基因筛选
Screening on reference genes for real⁃time fluorescent quantitative PCR of Freesiahybrida
筛选适用于小苍兰实时荧光定量PCR的内参基因，为准确研究小苍兰基因表达，尤其是种球发育相关基因的表达分析提供参考。
以小苍兰‘上农金皇后’的花瓣、雌雄蕊、花葶、叶片、球茎、根等6个不同组织器官，5个不同发育时期的球茎以及不同外源激素处理后的球茎为材料，通过实时荧光定量PCR(qRT-PCR)方法分析了小苍兰中8个常用持家基因的表达情况，进而利用geNorm、NormFinder以及BestKeeper等3个常用软件综合分析各基因表达的稳定性。
在小苍兰‘上农金皇后’6个不同的组织器官中，所选8个持家基因均有表达，但稳定性存在差异。综合分析表明，Actin和18S rRNA是最合适的内参基因，而EF-1β经3个软件分析均表现为最不稳定，不适合后续应用于小苍兰的基因表达分析。在5个不同发育时期的球茎组织中，表达最为稳定的是QCR、Actin和TUB，适宜今后研究球茎发育相关的基因表达特征时作为内参基因，而表达最不稳定的CYP和18S rRNA则不宜使用。在3种不同外源激素诱导下的小苍兰种球中，18S rRNA和CYP的表达水平相对一致且稳定性较好，可以用来作为内参基因进行相关目的基因的定量PCR研究，而EF-1β表达最不稳定，不推荐使用。
综合来看，小苍兰基因表达研究中最适内参基因应根据不同情况而定，Actin和18S rRNA是分析不同组织器官中基因表达时的首选内参基因；QCR、Actin和TUB是研究不同发育时期球茎中基因表达的适宜内参基因；18S rRNA和CYP是研究外源激素诱导下基因表达的首选内参基因。
Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) has become the most common method for studying gene expression. The appropriate application of qRT-PCR in such studies requires the use of reference gene(s) as an internal control in order to normalize the mRNA levels between different samples for an exact comparison of gene expression levels. The aim of the present study was to screen optimized reference genes for qRT-PCR in Freesiahybrida for future studies on the gene expression of Freesia, especially the genes relative to corm development.
Freesia hybrida cultivar ‘Shangnong Golden Queen’ was used as the plant material in the study. Six different tissues or organs including tepal, pistil and stamen (mixed samples), flower scape, leaf, corm and root, corms at five developmental stages and corms under treatments with three exogenous hormones, were collected, respectively. qRT-PCR was used to analyze the gene expression level of selected eight commonly used housekeeping genes, including Actin, Glyceraldehyde-3-phosphate (GAPDH), β-tubulin (TUB), 18S rRNA, elongation factor 1 beta (EF-1β), ubiquinol-cytochrome C reductase (QCR), cyclophilin (CYP) and S-adenosylmethionine decarboxylase (SAMDC). Three widely-used software, including geNorm, NormFinder and BestKeeper, were used to make a comprehensive analysis on the stability of gene expression.
The eight candidate housekeeping genes could be expressed in diffe-rent tissues and organs of Freesia hybrida ‘Shangnong Golden Queen’ with individual expression richness. Among those, two genes, 18S rRNA and Actin were the most stable, while EF???1β was the most unstable gene based on the analysis with the three software. In the corms at five developmental stages, three candidate genes, QCR, Actin and TUB presented a stable expression level, which can be selected as reference genes in future studies on the gene expression involving corm development. While CYP and 18S rRNAwere not stable, these two genes were not introduced to analyze gene expression in corms at different developmental stages. In corms under treatments with exogenous hormones, the expression of CYP and 18S rRNA was quite stable, while EF?1β was the most unstable gene in this experimental condition.
This is the first time reference genes were screened for a given set of experimental conditions in Freesia hybrida. Taken together, 18S rRNAand Actin were the preferred internal reference genes for gene expression analysis in different tissues of Freesia. QCR，Actin and TUB were the optimized genes in corm development, while for the gene expression analysis under hormone induction, the preferred reference genes were 18S rRNA and CYP. Our findings provide a guideline for any future work on gene expression in Freesia by using qRT-PCR.
小苍兰 / 内参基因 / 实时荧光定量PCR / 球茎 / 香雪兰属
Freesia hybrida / reference genes / real-time fluorescence quantitative PCR(qRT-PCR) / corm / Freesia
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