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林业科学 ›› 2017, Vol. 53 ›› Issue (10): 70-79.doi: 10.11707/j.1001-7488.20171008

• 论文与研究报告 • 上一篇    下一篇

盐和干旱胁迫下杨树新内参基因的筛选

储文渊1, 王玉娇1, 朱东悦1, 陈竹1, 严涵薇1,2, 项艳1,2   

  1. 1. 安徽农业大学林学与园林学院 合肥 230036;
    2. 安徽农业大学作物抗逆育种与减灾国家地方联合工程实验室 合肥 230036
  • 收稿日期:2017-01-16 修回日期:2017-06-03 出版日期:2017-10-25 发布日期:2017-11-29
  • 基金资助:
    国家自然科学基金项目(31370561);国家科技支撑计划子课题(2015BAD07B070104)。

Selection of Novel Reference Genes in Poplar under Salt and Drought Stresses

Chu Wenyuan1, Wang Yujiao1, Zhu Dongyue1, Chen Zhu1, Yan Hanwei1,2, Xiang Yan1,2   

  1. 1. School of Forestry and Landscape Architecture, Anhui Agricultural University Hefei 230036;
    2. National Engineering Laboratory of Crop Stress Resistance Breeding, Anhui Agricultural University Hefei 230036
  • Received:2017-01-16 Revised:2017-06-03 Online:2017-10-25 Published:2017-11-29

摘要: [目的]通过杨树的多个基因芯片数据,筛选出在盐和干旱胁迫下能够稳定表达的基因作为杨树的新内参基因,为挖掘出更稳定、更理想的内参基因提供途径。[方法]利用已公布的基因芯片数据库获取不同生长时期、不同逆境处理下的杨树表达数据,将这些数据进行归一化处理,对基因在不同试验条件下表达量的稳定性进行排序。结合基因的功能注释信息,筛选新的内参基因。为进一步验证基因表达的稳定性,以经过NaCl,PEG处理的南林95杨(组培苗)的叶片材料为研究对象,利用实时荧光定量PCR方法,对6个传统内参基因(PtUKN1,PtUBQActinEF1α,18S rRNA,TUA8)和本研究筛选的6个新内参基因在处理后的0,1,4,8,12,24 h的基因表达量进行分析,再利用geNorm,NormFinder和BestKeeper内参基因分析软件对试验数据进行统计和排序,以比较这12个基因的表达稳定性,从而筛选到合适的内参基因。[结果]筛选6个稳定表达的新内参基因(PtRG1,PtRG2,PtRG3,PtRG4,PtRG5PtRG6)。将这6个新内参基因与6个传统内参基因进行稳定性比较后发现,在geNorm程序分析中盐胁迫下PtRG2PtRG3稳定性较好,干旱胁迫下PtRG3PtRG5的稳定性较好;在NormFinder程序分析中盐胁迫下TUA8PtRG1稳定性较好,干旱胁迫下PtRG1PtRG2稳定性较好;在BestKeeper程序分析中盐胁迫下PtRG1PtRG5稳定性较好,干旱胁迫下PtRG3PtRG5稳定性较好。综合以上3个内参基因分析软件的结果,PtRG1PtRG3PtRG5的稳定性较好。为了进一步验证新内参基因的可靠性,以在NaCl和PEG处理条件下受诱导表达的PtVQ6PtVQ13PtVQ37作为对照进行荧光定量PCR试验,发现3个VQ基因的总体表达趋势与使用其他内参基因的结果一致。[结论]本研究鉴定的PtRG1PtRG3PtRG5适宜作为杨树在盐和干旱胁迫下的内参基因,这些新内参基因的挖掘对准确校正实时荧光定量PCR试验中目的基因的表达水平具有重要意义。

关键词: 杨树, 实时荧光定量PCR, 内参基因, 盐胁迫, 干旱胁迫

Abstract: [Objective] Choosing a suitable reference gene is an effective method to improve the accuracy and to reduce the experimental error of quantitative real-time PCR (qPCR). It has become an important technique to select stable expression of genes as novel reference genes using gene expression data in recent years. The purpose of our study is to select the novel reference genes which are expressed stably under salt and drought stresses in poplar using multiple microarray data. Our study will enrich the reference genes in poplar, and provide more channels for exploring stable and desirable reference genes.[Method] The gene microarray data of Populus trichocarpa in different growth periods under different stress treatments were collected from public gene chip database. The data were normalized to rank the stability of the expression of genes under different experimental conditions. Based on the functional annotation information of the genes, six novel reference genes were selected. In order to further verify the stability of gene expression, we have chosen the leaves of Populus deltoides ‘Nanlin95’ assessed by NaCl, PEG for analysis. Six traditional reference genes(PtUKN1, PtUBQ, Actin, EF1α, 18S rRNA, TUA8)and six novel reference genes were assessed by qPCR at six points (0, 1, 4, 8, 12, 24 h). Using the reference gene analyzing programs of geNorm, NormFinder and BestKeepe, the experimental data were counted and ranked in order to compare the expression stability of the 12 genes and accordingly select the suitable novel reference genes.[Result] In this study, six stable expression genes (PtRG1, PtRG2, PtRG3, PtRG4, PtRG5, PtRG6) were selected from the gene chip database. Comparisons of the stability between these six new reference genes and the six traditional reference genes displayed that, in the geNorm program analysis, the stability of PtRG2 and PtRG3 was better under salt stress and the stability of PtRG3 and PtRG5 was better under the drought stress; in NormFinder program analysis, the stability of TUA8 and PtRG1 was better under salt stress and the stability of PtRG1 and PtRG2 was better under the drought stress; in BestKeeper program analysis, the stability of PtRG1 and PtRG5 was better under salt stress and the stability of PtRG3 and PtRG5 was better under the drought stress. To further verify the stability of these gene expression, PtVQ6, PtVQ13 and PtVQ37 expressed highly in the poplar VQ gene family under NaCl and PEG treatment were selected as the target gene to conduct qPCR again and found the results were consistent with the previous study, suggesting PtRG1, PtRG3 and PtRG5 were stable.[Conclusion] PtRG1, PtRG3 and PtRG5 identified in our study are suitable as the reference genes in poplar under salt and drought stresses, which would contribute to a more accurate analysis for the expression of the resistance genes in qPCR.

Key words: poplar, quantitative real-time PCR, reference genes, salt stress, drought stress

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