姜荷花苞片实时荧光定量PCR内参基因筛选

谭健俊; 黄李珊; 周熠玮; 徐晔春; 叶远俊

doi:10.16768/j.issn.1004-874X.2025.02.009

姜荷花苞片实时荧光定量PCR内参基因筛选

Screening of Reference Genes for Quantitative Real-time PCR in Curcuma alismatifolia Bracts

摘要

摘要:
目的筛选适用于姜荷花苞片组织qRT-PCR分析的内参基因，为苞片颜色相关基因功能研究奠定理论基础。
方法以9个不同颜色的姜荷花品种为试材，根据姜荷花基因组数据选择常用的内参基因，包括甘油醛-3-磷酸脱氢酶（GAPDH）、18S核糖体RNA（18S rRNA）、蛋白磷酸酶2A（PP2A）、泛素蛋白（UBQ）、苹果酸脱氢酶（MDH）、肌动蛋白（Actin）、微管蛋白（TUB）和苯丙氨酸解氨酶（PAL）8个看家基因，使用ΔCt、geNorm、NormFinder和BestKeeper 4种分析方法评估基因表达的稳定性，通过RefFinder程序综合评价以筛选出姜荷花苞片组织的最佳内参基因。
结果 8个内参基因均扩增出单一主峰，引物标准曲线相关系数R2 ≥ 0.98，扩增效率为99.5%~125.0%，PCR产物经1% 琼脂糖电泳检测显示条带清晰单一，表明引物设计特异性好。4种不同的算法分析获得的结果排名有差异，其中MDH在ΔCt、geNorm和NormFinder分析中为表达最稳定的内参基因，18S rRNA在BestKeeper分析中为表达最稳定的内参基因。利用RefFinder程序综合评价得出候选内参基因稳定性综合排名，排序依次为MDH＞Actin＞TUB＞18S rRNA＞PP2A＞GAPDH＞UBQ＞PAL，表明不同品种姜荷花苞片最佳内参基因是MDH，其次为Actin和TUB，PAL稳定性最差、不适合作为姜荷花苞片组织的内参基因。
结论 MDH可作为不同品种姜荷花苞片组织的稳定内参基因，为后续深入开展姜荷花苞片颜色合成相关基因的表达模式分析提供参考。

Abstract:
Objective To screen suitable reference genes for Quantitative Real-time PCR(qRT-PCR) analysis in Curcuma alismatifolia bract, establishing the theoretical foundation for investigating the functional genes associated with bract color.
Method Nine C. alismatifolia cultivars with different colors were employed in this experiment. Based on the genomic information of C. alismatifolia, eight housekeeping genes including glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 18S rRNA, protein phosphatase 2A (PP2A), ubiquitin (UBQ), malic dehydrogenase (MDH), actin 1 (Actin), β-tubulin (TUB), and phenylalanine ammonia lyase (PAL) were selected. ΔCt, geNorm, NormFinder and BestKeeper were used to evaluate the stability of genes expression. The RefFinder program was utilized to comprehensively assess the optimal reference genes for C. alismatifolia bract.
Result Results indicated that all the eight reference genes amplified a single peak, and the standard curve with R ≥ 0.98 and amplification efficiency value of 99.5%~125.0%. The PCR products were analyzed using 1% agarose gel electrophoresis and exhibited distinct and single bands, which suggested that the primers were highly specific. The ranking of results obtained from the four different algorithms varied. Specifically, MDH was identified as the most stable reference gene in the ΔCt, geNorm, and NormFinder methodologies analysis, whereas 18S rRNA was the most stable reference gene in the BestKeeper analysis. The comprehensive ranking of candidate reference genes stability was obtained using the RefFinder program, with the order from high to low being MDH > Actin > TUB > 18S rRNA > PP2A > GAPDH > UBQ > PAL. The most suitable reference gene for different cultivars of C. alismatifolia bract is MDH, followed by Actin and TUB, while PAL is the least stable and unsuitable as a reference gene for C. alismatifolia bract..
Conclusion MDH can be used as a stable reference gene for different cultivars of C. alismatifolia bract, providing a reference for further analysis of the expression patterns of genes related to color synthesis in C. alismatifolia bracts.

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