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林业科学 ›› 2016, Vol. 52 ›› Issue (8): 29-37.doi: 10.11707/j.1001-7488.20160804

• 论文与研究报告 • 上一篇    下一篇

光皮桦实时荧光定量PCR内参基因的筛选

刘文哲, 牛明月, 李秀云, 林二培, 黄华宏, 童再康   

  1. 浙江农林大学亚热带森林培育国家重点实验室培育基地 临安 311300
  • 收稿日期:2016-01-29 修回日期:2016-04-20 出版日期:2016-08-25 发布日期:2016-09-19
  • 通讯作者: 林二培
  • 基金资助:
    国家自然科学基金项目(31100494);浙江省竹木农业新品种选育重大科技专项(2012C12908-8)。

The Selection of Reference Genes for Quantitative PCR in Betula luminifera

Liu Wenzhe, Niu Mingyue, Li Xiuyun, Lin Erpei, Huang Huahong, Tong Zaikang   

  1. The Nurturing Station for the State Key Laboratory of Subtropical Silviculture Zhejiang A & F University Lin'an 311300
  • Received:2016-01-29 Revised:2016-04-20 Online:2016-08-25 Published:2016-09-19

摘要: [目的]利用定量PCR和表达分析软件研究候选内参基因在光皮桦不同组织中的表达稳定性,并通过目的基因的表达分析验证其可靠性,以获得可用于光皮桦定量PCR的稳定内参基因。[方法]选取16个常用的看家基因作为候选内参基因,设计引物,通过普通PCR、溶解曲线及琼脂糖凝胶电泳验证引物的特异性;通过实时荧光定量PCR及软件geNorm、NormFinder和BestKeeper分析候选内参基因在光皮桦16个不同组织中的表达稳定性,根据分析结果筛选出最佳的内参基因和组合,并进一步通过2个目的基因CSLDKOR对内参基因的稳定性进行验证。[结果]PCR和电泳结果显示,候选内参基因PCR目的片段条带清晰单一,熔解曲线也呈现明显的单一峰,表明这些引物的特异性良好。利用特异引物,对这些候选基因在16个不同组织的表达进行分析,发现除了18S,其他基因的Ct值均集中在25~30之间,表明内参基因在不同组织中表达较稳定,它们之间的表达水平差异不明显。NormFinder和BestKeeper分析结果均表明基因EF1α的稳定性最好,geNorm计算得出最稳定的是TATA,而UBi-lp在所有候选内参基因中表达最不稳定。进一步选取3个稳定表达候选基因(EF1α,TATAUBi4)及稳定性较差的UBi-lp作为内参基因,对2个目的基因CSLDKOR在不同组织中的相对表达进行分析,结果表明这2个目的基因相对于3个稳定的内参基因显示出一致的相对表达量,而不稳定的内参基因UBi-lp并没有对表达数据进行有效的标准化,结果存在明显偏差。[结论]通过实时荧光定量PCR及综合3种软件分析的结果,在光皮桦不同的组织中,EF1α,TATAUBi4内参基因的表达稳定性高,对这3个基因进行验证并明确其作为荧光实时定量PCR内参的可靠性。因此,EF1α,TATAUBi4可作为研究基因在光皮桦不同组织器官中表达的稳定内参。

关键词: 光皮桦, 内参基因, 实时荧光定量PCR

Abstract: [Objective] Real-time quantitative PCR (RT-qPCR) has become the preferred approach to the quantification of gene expression. Besides its simplicity and efficiency, this method requires suitable reference genes to guarantee the accuracy of the quantification. Betula luminifera is not only a high-quality timber species with excellent wood properties, but also an ideal species for genetic study. Stability of candidate reference genes in different tissues was investigated using RT-qPCR and expression analysis software to select suitable reference genes, the reliability of the reference genes was further verified through expression analysis of two functional genes in different tissues.[Method] 16 housekeeping genes were chosen as candidate reference genes, and specificity of the primers was investigated by agarose gel electrophoresis and melting curve of target amplified fragments. The software of geNorm, NormFinder and BestKeeper were used to analyze expression stability of reference genes in 16 different tissues. Furthermore, two functional genes were selected to verify the reliability of the suitable reference genes.[Result] Electrophoresis results showed distinct PCR products with the expected size, and the single-peak melting curves further indicated the specificity of the selected primers. Except 18S, the Ct values of the candidate genes were all between 25 to 30, indicating the expressions of these candidate genes were quite stable in different tissues. EF1α was characterized as the most stable gene based on NormFinder and BestKeeper analysis, while TATA was ranked in the first place according to geNorm. UBi-lp showed the most unstable expression. Furthermore, three top-stable genes (EF1α, TATA and UBi4) and the unstable gene UBi-lp were chosen to verify the reliability of the reference genes. As results, the two target genes showed consistent expression profiles when normalized by the three top-ranked reference genes, and UBi-lp failed to standardize the expression data.[Conclusion] EF1α, TATA and UBi4 were most stable reference genes in different tissues, the reliability of these reference genes were further confirmed by expression analysis of the target genes. Therefore, EF1α, TATA and UBi4 could serve as reference genes for gene expression among different tissues in B. luminifera.

Key words: Betula luminifera, reference gene, real-time quantitative PCR

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