植物学报 ›› 2015, Vol. 50 ›› Issue (6): 754-764.doi: 10.11983/CBB14173

• 技术方法 • 上一篇    下一篇

盐胁迫下大豆根组织定量PCR分析中内参基因的选择

姜琼1,2, 王幼宁1, 王利祥1,2, 孙政玺1,2, 李霞1,,A;*()   

  1. 1中国科学院遗传与发育生物学研究所农业资源研究中心, 石家庄 050021
    2中国科学院大学, 北京 100049
  • 收稿日期:2014-09-22 接受日期:2015-01-14 出版日期:2015-11-01 发布日期:2015-09-06
  • 通讯作者: 李霞 E-mail:xli@genetics.ac.cn
  • 作者简介:

    ? 共同第一作者

  • 基金资助:
    国家自然科学基金(No.31230050)和转基因生物新品种培育重大专项(No.2014ZX0800929B)

Validation of Reference Genes for Quantitative RT-PCR Analysis in Soybean Root Tissue under Salt Stress

Qiong Jiang1,2, Youning Wang1, Lixiang Wang1,2, Zhengxi Sun1,2, Xia Li1*   

  1. 1Center of Agricultural Research Resources, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Shijiazhuang 050021, China
    2University of Chinese Academy of Sciences, Beijing 100049, China
  • Received:2014-09-22 Accepted:2015-01-14 Online:2015-11-01 Published:2015-09-06
  • Contact: Li Xia E-mail:xli@genetics.ac.cn
  • About author:

    ? These authors contributed equally to this paper

摘要:

实时荧光定量PCR已广泛用于基因表达的分析, 适当的内参基因选择是获得准确分析结果的关键。在大豆(Glycine max)分子生物学研究中, 逆境响应基因和microRNA (miRNA)表达的内参辅助检测基因均有哪些目前尚不清楚。该研究选用不同盐梯度和时间点组合处理的大豆根组织为材料, 对已报道的其它条件下表达相对稳定的内参基因(ACTACT2/7CYP2ELF1AELF1BF-BoxTUAUBC2)以及miRNA内参基因(U6、miR1515a、miR1520c、miR1520d、miR171a和miR171b)的表达情况进行了全面检测; 并采用Δ-Ct、Bestkeeper、NormFinder和Genorm四种方法对检测结果进行了综合分析, 发现ELF1BCYP2适合作为大豆根系盐胁迫响应基因研究的内参基因, miR1515a和U6适合作为盐胁迫下大豆根组织miRNA研究的内参。上述研究结果为大豆盐胁迫响应基因和miRNA表达及其进一步的功能研究奠定了基础。

Abstract:

Quantitative RT-PCR (qRT-PCR) is a robust and commonly used technique to study gene expression. However, the reference genes and microRNA (miRNA) that can be used in soybean molecular research, in particular the stress responsive genes and miRNA in roots, are not well characterized. In this study, we systematically analyzed the expression stability of eight widely used reference genes (ACT, ACT2/7, CYP2, ELF1A, ELF1B, F-Box, TUA and UBC2) and miRNA internal references (U6, miR1515a, miR1520c, miR1520d, miR171a, and miR171b) in root tissues treated with different NaCl concentrations at specified times. The Δ-Ct method, Bestkeeper 1.0, NormFinder 0.953 and Genorm 3.5 were used to analyze the data, then the web-based tool RefFinder was used to integrate the results obtained. ELF1B/CYP2 and miR1515a/U6 were the most suitable genes for qRT-PCR analysis of soybean gene and miRNA expression analysis under salt stress, respectively. The highly ranked reference genes and miRNAs identified from this study can be used for detecting differences in expression rates of genes and miRNAs in response to salt stress in soybean.

图1

14种内参基因的PCR扩增产物"

表2

持家基因的稳定性排序"

Method Stability (high→low)
1 2 3 4 5 6 7 8
Δ-CT methods CYP2 ACT ELF1B UBC2 ACT2/7 ELF1A F-Box TUA
Bestkeeper ELF1B TUA UBC2 CYP2 ACT ACT2/7 ELF1A F-Box
NormFinder CYP2 ACT ELF1B UBC2 ELF1A ACT2/7 F-Box TUA
Genorm ELF1B /UBC2 CYP2 ACT ACT2/7 F-Box ELF1A TUA
Recommended
comprehensive ranking
ELF1B CYP2 UBC2 ACT ACT2/7 TUA ELF1A F-Box

图2

盐胁迫处理大豆根的8个持家基因Real-time PCR溶解曲线"

图3

盐胁迫处理大豆根的6个miRNAs Real-time PCR溶解曲线"

表3

miRNA的稳定性排序"

Method Stability (high→low)
1 2 3 4 5 6
Δ-CT methods miR1520c miR1515a miR171a U6 miR1520d miR171b
Bestkeeper miR1515a miR1520c U6 miR171a miR1520d miR171b
NormFinder miR1520c miR1515a miR171a U6 miR1520d miR171b
Genorm U6/miR1520c miR1515a miR171a miR1520d miR171b
Recommended
comprehensive ranking
miR1520c miR1515a U6 miR171a miR1520d miR171b

图4

候选内参基因在盐胁迫处理大豆根尖的表达水平。 1-14同图1。"

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