摘要
[目的]筛选出木香薷花发育相关稳定表达的内参基因,为后续开展木香薷花发育相关的基因功能分析及调控机理研究奠定基础。[方法]从木香薷花瓣转录组数据库中选择了9个内参基因(GAPDH、ACT、UBQ、UBC、TUA、TUB、EF1-α、eIF3K和SEP),采用实时定量PCR技术检测其在木香薷花不同部位及不同发育阶段的小花中的特异性及表达情况,并利用ΔCT程序、GeNorm、NormFinder、Bestkeeper及Refinder网站对其表达稳定性进行综合评估,最后通过木香薷花青素合成过程中关键基因ElstANS对筛选出的内参基因的可靠性进行验证。[结果]在木香薷花不同部位,内参基因UBQ和eIF3K表达最稳定;在小花的各个发育阶段,内参基因UBQ和TUA表达最为稳定;在所有样品中,内参基因ACT和UBQ表达为最稳定,最不稳定的是UBC基因。ElstANS验证发现无论ACT、UBQ单独使用还是组合使用,ElstANS在木香薷花不同部位或不同发育阶段均现出相同的表达趋势,而稳定性最差的UBC无法准确校正qRT-PCR结果。[结论]在进行木香薷花发育调控相关基因的表达分析时,推荐单独使用UBQ、ACT或组合使用UBQ+ACT作为内参基因。
[Objective]This study aimed to identify stably expressed reference genes associated with flower development in Elsholtzia stauntonii in order to provide the basis for subsequent study on gene function and regulation mechanism in E.stauntonii.[Method]Based on the transcriptome data of E.stauntonii petals,nine candidate reference genes(GAPDH,ACT,UBQ,UBC,TUA,TUB,EF1-α,eIF3K and SEP)were detected using qRT-PCR in different parts and different developmental stages of flower.Their expression stability was analyzed using four statistical algorithms namely theΔCT method,geNorm,NormFinder,BestKeeper and Refinder program.To validate the reliability of the selected reference genes,the expression pattern of ElstANS,a key gene involved in anthocyanin synthesis pathway,was analyzed.[Result]UBQ and eIF3K were the most stably expressed reference genes among different flower organs.TUA and UBQ exhibited the highest stability across different developmental stages.In all samples,UBQ and ACT were the most stable reference genes,while UBC showed the least stable expression.Validation using ElstANS demonstrated that whether UBQ or ACT was used individually or in combination,consistent expression patterns were observed across samples,whereas normalization with the unstable UBC gene failed to yield accurate qRT-PCR results.[Conclusion]UBQ and ACT alone or in combination can be used as suitable reference genes to normalize qRT-PCR data in different parts and developmental stages of flower of E.stauntonii.
作者
刘苗苗
杨瑾
陈山
周秀梅
LIU Miao-miao;YANG Jin;CHEN Shan;ZHOU Xiu-mei(Henan Engineering Research Center of the Development and Utilization of Characteristic Horticultural Plant,School of Horticulture and Landscape architecture,Henan Institute of science and technology,Xinxiang 453003,Henan,China)
出处
《林业科学研究》
北大核心
2025年第4期165-174,共10页
Forest Research
基金
河南省省重点研发与推广专项项目(232102111081)省市级。
关键词
木香薷
内参基因
QRT-PCR
基因表达
花发育
Elsholtzia stauntonii
reference gene
quantitative Real-time PCR(qRT-PCR)
gene expression
flower development
