摘要
【目的】筛选芍药切花开放至衰老过程中和盛开期不同组织中稳定表达的最适内参基因,深入解析ACS和ACO基因在乙烯介导的芍药切花开放至衰老过程中的功能。【方法】(1)以芍药‘桃花飞雪’为材料,基于qRT-PCR技术分析13个候选内参基因在芍药切花开放至衰老不同时期及盛开期不同组织中表达水平的变化,应用geNorm、NormFinder、BestKeeper等软件和ΔCt法对其表达稳定性进行了评价;通过在线RefFinder网站分析获得候选内参基因表达稳定性的综合排名。(2)利用筛选出的双内参基因,对乙烯生物合成关键基因ACS和ACO在芍药切花开放至衰老不同时期及盛开期不同组织中的表达水平进行定量分析。【结果】(1)在芍药切花开放至衰老的不同时期,geNorm分析表明GAPDH和RAN3表达稳定性最好;NormFinder分析显示RAN3表达最稳定;BestKeeper分析表明18S rRNA表达稳定性最好;RefFinder综合分析表明RAN3和GAPDH为最适内参基因。(2)在盛开期芍药切花花器官不同组织中,geNorm分析表明ARF和PP2A表达稳定性最好;NormFinder分析显示UBQ表达最稳定;BestKeeper分析表明GAPDH表达稳定性最好;RefFinder综合分析表明UBQ和ARF为最适内参基因。(3)在芍药切花开放至衰老过程中,ACS基因表达量显著低于ACO基因表达量,是调控乙烯合成的开关;ACO主要在切花开放前期(瓶插0~12 h)发挥作用;ACS表达量在绽口期和衰败期(瓶插6 h和144 h)均发挥重要作用,并在盛开期对乙烯合成起到限制作用;(4)在芍药切花盛开期不同组织中,ACS基因表达量显著低于ACO,ACS和ACO基因的表达存在组织差异性,且均在茎和花萼中表达较高。【结论】本研究筛选出芍药切花开放至衰老不同时期(RAN3和GAPDH)以及盛开期不同组织中(UBQ和ARF)qPCR定量表达分析的双内参基因组合;乙烯生物合成关键基因ACO主要在芍药切花开放过程发挥作用,ACS基因在芍药切花开放和衰老过程均发挥重要的作用。研究结果将为切花寿命的改良提供参考。
[Objective]This study aims to select the most suitable reference genes,which are stably expressed during the process from the opening to senescence and different tissues in blooming period of peony cut flowers,and to deeply analyze the function of ACS and ACO genes in ethylene-mediated process of peony cut flower opening to senescence.[Method](1)With cultivar‘Taohuafeixue’as materials,the variations of expression levels of 13 candidate internal reference genes at different stages from blooming to senescence and in different tissues of the full bloom stage in herbaceous peony cut flowers were analyzed by qRT-PCR.The expression stability of 13 candidate internal reference genes was assessed byΔCt and 3 softwares including geNorm,NormFinder,and BestKeeper.The comprehensive rankings of expression stability of candidate internal reference genes were obtained by online RefFinder analysis.(2)Based on the selected double internal reference genes,the expression profile of ACS and ACO,key genes of ethylene biosynthesis were quantitatively analyzed at different stages from blooming to senescence and in different tissues of full bloom stage in herbaceous peony cut flowers.[Result](1)geNorm analysis showed that the expression stability of GAPDH and RAN3 at different stages was best.NormFinder analysis indicated that the expression stability of RAN3 was optimal.BestKeeper analysis illustrated that 18S rRNA expression was the most stable.RefFinder analysis indicated that RAN3 and GAPDH were the best suitable internal reference genes at different stages.(2)geNorm analysis indicated that the expression of ARF and PP2A in different tissues had the best expression stability.NormFinder analysis showed that the expression of UBQ was the most stable.BestKeeper analysis illustrated that GAPDH was best.RefFinder analysis indicated that UBQ and ARF were the best internal reference genes in different tissues in herbaceous peony cut flowers.(3)From opening to senescence process of peony cut flowers,the expression of ACS gene was significantly lower than that of ACO gene,and ACS was the‘switch’for the regulation of ethylene synthesis.ACO mainly played a role in pre-opening stage of cut flowers(0−12 h).ACS expression played an important role both in the bloom stage and in the senescence stage(6 h and 144 h),and played a limiting role in ethylene synthesis during the bloom stage.(4)ACS expression was significantly lower than ACO in different tissues of peony cut flowers during blooming period,there was tissue variability in the expression of ACS and ACO,and both of them were higher in stems and calyxes.[Conclusion]This study screens double internal reference gene combinations for qPCR quantitative expression analysis in different tissues of herbaceous peony cut flowers at varied periods from opening to senescence(RAN3,GAPDH)and at full bloom(UBQ,ARF).ACO gene,a key gene of ethylene biosynthesis mainly plays a role in the blooming process,and ACS plays an important role in both blooming and senescence process in herbaceous peony cut flowers.The finding will provide a reference for the improvement of cut flower longevity.
作者
戴睿
段帅帅
肖士奎
卫志鹏
吕淑芳
史国安
吴疆
范丙友
Dai Rui;Duan Shuaishuai;Xiao Shikui;Wei Zhipeng;LüShufang;Shi Guoan;Wu Jiang;Fan Bingyou(College of Agriculture,Henan University of Science and Technology,Luoyang 471023,Henan,China;Luoyang Lüzhu Horticulture Co.,Ltd,Luoyang 471131,Henan,China;College of Information Engineering,Yulin 719000,Shaanxi,China)
出处
《北京林业大学学报》
北大核心
2025年第1期106-115,共10页
Journal of Beijing Forestry University
基金
国家重点研发计划(2018YFD1000400)
国家自然科学基金项目(U1204323)。
关键词
芍药
内参基因
表达稳定性
开花
衰老
乙烯生物合成
Paeonia lactiflora
internal reference genes
expression stability
blooming
senescence
ethylene biosynthesis
