广东农业科学2025，Vol.52Issue(2)：108-116,9.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
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 R2≥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.关键词
实时荧光定量PCR/姜荷花/内参基因/苞片颜色/MDH/基因表达模式Key words
qRT-PCR/Curcuma alismatifolia/reference gene/bract color/MDH/the expression patterns of genes分类
农业科技引用本文复制引用
谭健俊,黄李珊,周熠玮,徐晔春,叶远俊..姜荷花苞片实时荧光定量PCR内参基因筛选[J].广东农业科学,2025,52(2):108-116,9.基金项目
国家自然科学基金(32401661) （32401661）
广东省基础与应用基础研究基金(2022A1515110580) （2022A1515110580）
