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园艺学报 ›› 2018, Vol. 45 ›› Issue (5): 919-930.doi: 10.16420/j.issn.0513-353x.2017-0780

• 研究论文 • 上一篇    下一篇

朱顶红实时荧光定量PCR中不同组织器官内参基因的筛选

刘晓婷1,2,*,王顺利2,*,薛璟祺2,薛玉前2,吕英民1,**,张秀新2,**   

  1. (1北京林业大学园林学院,国家花卉工程技术研究中心,北京100083;2中国农业科学院蔬菜花卉研究所,国家花卉改良中心,中国农业科学院牡丹研究中心,农业部园艺作物生物学与种质创制重点实验室,北京 100081)
  • 出版日期:2018-05-25 发布日期:2018-05-25

Selection of Reference Genes for Quantitative Real-Time PCR in Different Tissue and Organ of Barbadoslily

LIU Xiaoting1,2,*,WANG Shunli2,*,XUE Jingqi2,XUE Yuqian2,Lü Yingmin1,**,and ZHANG Xiuxin2,**   

  1. (1China National Floriculture Engineering Research Centre,College of Landscape Architecture,Beijing Forestry University,Beijing 100083,China;2Key Laboratory of Biology and Genetic Improvement of Horticultural Crops,Ministry of Agriculture,China;National Center for Flower Improvement;Department of Peony,Chinese Academy of Agricultural Sciences;Institute of Vegetables and Flowers,Chinese Academy of Agricultural Sciences,Beijing 100081,China)
  • Online:2018-05-25 Published:2018-05-25

摘要: 以朱顶红(Hippeastrum vittatum)不同组织器官为试验材料,通过实时荧光定量PCR技术检测肌动蛋白基因(ACT)、亲环蛋白基因(CYP)、转录延伸因子基因(EF-1α)、甘油醛–3–磷酸脱氢酶基因(GAPDH、GAPDH2)、α微管蛋白基因(TUA)和β微管蛋白基因(TUB)这7个常用的看家基因的表达水平,并利用geNorm、NormFinder和BestKeeper综合评价其表达稳定性。结果表明,EF-1α和GAPDH2的表达水平较高,而且相对稳定;其次为TUB、TUA和GAPDH。geNorm和NormFinder软件分析结果显示,在不同组织器官中CYP和GAPDH2的表达稳定性最高,其次是EF-1α;进一步分析表明,CYP的表达稳定性虽然较高,但是其表达丰度较低,故不适合作为内参基因。BestKeeper分析表明,EF-1α和GAPDH2在不同组织器官中的表达稳定性较好。综合分析表明,EF-1α和GAPDH2在所有样品中表达量较高,稳定性较好。以筛选出来的EF-1α、GAPDH2以及EF-1α + GAPDH2作为内参基因检测朱顶红花器官调控基因PI的表达水平,结果表明,以上述两个基因及其组合为内参基因得到的PI表达模式相同,而且在花器官中的表达高于茎盘和根,基本符合PI调控花模型的机理。因此,EF-1α、GAPDH2或EF-1α + GAPDH2可作为朱顶红的内参基因进行相关基因的表达调控研究。

关键词: 朱顶红, 实时荧光定量PCR, 内参基因, EF-1α, GAPDH2

Abstract: Expression levels of 7 commonly used housekeeping genes including Actin(ACT),Cyclophilin(CYP),Elongation factor-1α(EF-1α),Glyceraldehyde-3-phosphate(GAPDH),Glyceraldehyde-3-phosphate 2(GAPDH2),α-tubulin(TUA)and β-tubulin(TUB)were detected by qRT-PCR in different Hippeastrum tissues and organs in this study. The stability of expression of these reference genes were evaluated by geNorm,NormFinder and BestKeeper respectively. The results of qRT-PR showed that EF-1α and GAPDH2 had high expressed level and stability;the second were TUB,TUA and GAPDH,respectively. According to the analysis of geNorm and NormFinder,both CYP and GAPDH2 were the relatively stable genes in different tissues and organs of Hippeastrum,followed by EF-1α. Although CYP had high expression stability,while its expression abundance was low. Thus,GAPDH2 and EF-1α could be as candidate reference genes for gene expression analysis. BestKeeper analysis indicated that EF-1α and GAPDH2 were in the high stability in different tissues and organs. According to the qRT-PCR and the three software results,it was found that EF-1α and GAPDH2 were suitable for the candidate reference genes. Then,the relative expression levels of Pistillata(PI)were detected,using EF-1α,GAPDH2 and EF-1α + GAPDH2 as reference genes. The results showed that the expression patterns were similar by using the three types of reference genes,and the expression patterns were referred to the mechanism of floral model that were higher in floral organs. Above all,EF-1α,GAPDH2 or EF-1α + GAPDH2 as reference genes were appropriate for gene expression analysis in Hippeastrum.

Key words: Hippeastrum vittatum, quantitative real-time PCR, reference gene, EF-1α, GAPDH2

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