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橡胶灵芝菌内参基因筛选与分析

赵欣阳 罗佑红 蔡海滨 周燚 涂敏

赵欣阳,罗佑红,蔡海滨,等. 橡胶灵芝菌内参基因筛选与分析 [J]. 福建农业学报,2024,39(5):1−9
引用本文: 赵欣阳,罗佑红,蔡海滨,等. 橡胶灵芝菌内参基因筛选与分析 [J]. 福建农业学报,2024,39(5):1−9
ZHAO X Y, LUO Y H, CAI H B, et al. Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease [J]. Fujian Journal of Agricultural Sciences,2024,39(5):1−9
Citation: ZHAO X Y, LUO Y H, CAI H B, et al. Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease [J]. Fujian Journal of Agricultural Sciences,2024,39(5):1−9

橡胶灵芝菌内参基因筛选与分析

基金项目: 国家重点研发计划专项(2023YFD1200204);海南省重点研发计划项目(ZDYF2021XDNY291);海南省优秀人才团队项目(20210203)
详细信息
    作者简介:

    赵欣阳(2000 — ),女,硕士,主要从事分子植物病理学研究,E-mail:xyzhao1219@163.com

    通讯作者:

    周燚(1972 — ),男,博士,教授,主要从事分子植物病理学研究,E-mail: zhouyi@yangtzeu.edu.cn;涂敏(1980 — ),女,博士,副研究员,主要从事作物绿色防控研究,E-mail: tm_tumin@163.com

  • 中图分类号: S432

Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease

  • 摘要:   目的  分析评价不同胁迫处理的橡胶灵芝菌候选内参基因的稳定性,以期为探索橡胶灵芝菌基因的功能及其侵染橡胶树的分子机制等提供参考。  方法  以5个非生物因子(温度、盐、氧化、pH、干旱)和1个生物因子(生防细菌)胁迫下的橡胶灵芝菌作为材料,提取RNA并反转录为cDNA,采用实时荧光定量PCR技术,扩增6个候选内参基因(UBCACTRPL6β-TUBAPT28s),利用分析软件geNorm、NormFinder、BestKeeper和RefFinder根据基因表达的稳定性对基因进行排序,选择最适合不同胁迫的内参基因组合。  结果  所有样品RNA均为清晰的两条带,且6个候选内参基因的熔解曲线为明显单一峰。结合geNorm、NormFinder、Bestkeeper和RefFinder对其进行表达稳定性分析发现,温度胁迫下基因表达稳定性为UBCACTRPL6β-TUB28sAPT,盐胁迫下基因表达稳定性为ACTRPL6UBCAPTβ-TUB28s;氧化胁迫下基因表达稳定性为UBCACT28sAPTβ-TUBRPL6;pH胁迫下基因表达稳定性为RPL6UBCAPTACTβ-TUB28s,干旱胁迫下基因表达稳定性为ACTUBCβ-TUBRPL6APT>28s,生物胁迫下基因表达稳定性为UBCACTRPL628sAPTβ-TUB  结论  结合所有候选基因稳定性和胁迫条件,推荐基因ACTUBC作为在干旱、氧化、温度和生物胁迫下的内参基因,基因ACTRPL6为盐胁迫下的内参基因,UBCRPL6为pH胁迫下的内参基因。本研究结果为不同胁迫下橡胶灵芝菌相关基因的表达研究提供了合适的内参基因。
  • 图  1  总RNA提取

    A1:CK;A2~A7:盐胁迫;A8~A12:干旱胁迫;A13~B2:pH胁迫;B3~B7:氧化胁迫;B8~B11:温度胁迫;B12~B14:生物胁迫。

    Figure  1.  Extraction of total RNA

    A1: CK; A2-7: salt stress; A8-12: drought stress; A13-B2: pH stress; B3-7: oxidation stress; B8-11: temperature stress; B12-14: biotic stress.

    图  2  6个候选内参基因熔解曲线

    A: ACT; B: UBC; C: β-TUB; D: RPL6; E: APT; F: 28s。

    Figure  2.  Melting curves generated for 6 reference genes

    A: ACT; B: UBC; C: β-TUB; D: RPL6; E: APT; F: 28s.

    图  3  6个候选内参基因RT-qPCR分析的Ct值

    Figure  3.  Ct values of 6 candidate reference genes analyzed by RT-qPCR

    图  4  geNorm分析6个候选内参基因的表达稳定性值(M)

    A:盐胁迫;B:pH胁迫;C:干旱胁迫;D:氧化胁迫;E:温度胁迫;F:生物胁迫。

    Figure  4.  Expression stability values (M) of 6 candidate reference genes by geNorm

    A: Salt stress; B: pH stress; C: drought stress; D: oxidation stress; E: temperature stress; F: biotic stress.

    图  5  geNorm软件分析所需内参基因的数目

    Figure  5.  Required number of reference genes by geNorm

    图  6  候选参考基因稳定性的RefFinder排序

    A:盐胁迫;B:pH胁迫;C:干旱胁迫;D:氧化胁迫;E:温度胁迫;F:生物胁迫。

    Figure  6.  Stability of candidate reference genes ranked by RefFinder

    A: Salt stress; B: pH stress; C: drought stress; D: oxidation stress; E: Temperature stress; F: biotic stress.

    表  1  RT-qPCR引物

    Table  1.   RT-qPCR primers

    基因
    Gene
    Gene Bank登录号
    Gene Bank accessing number
    引物序列
    Primer sequence (5'-3')
    扩增长度/bp
    Amplification length /bp
    UBC KAF9027008 TCTGGCGGCGTCTTCTTCCT 106
    TGGCATTGATGTTCGGGTGG
    ACT KAF9050787 CATCGAGCACGGTATTGTCA 167
    TCTCGAACATGATTTGGGTC
    β-TUB KAF9039628 CAAATGCAGAACGTCCAGAAC 159
    GTGAACTCCATCTCGTCCATAC
    RPL6 KAF9033237 CTGTACCTCGTCGGTGTCGG 137
    GTTGGCGTCTCCACCTTTGC
    APT GL18178 GAGTACGGTGTGGATGTCTTC 130
    CGAGCTTGGCTACGAGTTC
    28s 无登录号 GCATATCAATAAGCGGAGGA 130
    GCACTTCTCCAGACTACAAC
    下载: 导出CSV

    表  2  NormFinder稳定性分析

    Table  2.   Stability analysis by NormFinder

    排名
    Rank
    盐胁迫
    Salt stress (GroupSD)
    pH胁迫
    pH stress (GroupSD)
    干旱胁迫
    Drought stress (GroupSD)
    氧化胁迫
    Oxidative stress (GroupSD)
    温度胁迫
    Heat stress (GroupSD)
    生物胁迫
    Biotic stress (GroupSD)
    1 RPL6(0.02) RPL6(0.05) UBC(0.05) UBC(0.02) UBC(0.04) UBC(0.04)
    2 APT(0.02) β-TUB(0.05) ACT(0.05) APT(0.02) ACT(0.04) ACT(0.06)
    3 UBC(0.11) APT(0.05) β-TUB(0.07) ACT(0.02) RPL6(0.12) RPL6(0.07)
    4 β-TUB(0.11) UBC(0.07) RPL6(0.07) RPL6(0.1) β-TUB(0.19) APT(0.08)
    5 ACT(0.11) ACT(0.07) APT(0.15) β-TUB(0.11) 28s(0.46) 28s(0.11)
    6 28s(0.53) 28s(0.24) 28s(0.24) 28s(0.15) APT(0.56) β-TUB(0.12)
    下载: 导出CSV

    表  3  Bestkeeper稳定性分析

    Table  3.   Stability analysis by Bestkeeper

    排名
    Rank
    盐胁迫
    Salt stress (CV±SD)
    pH胁迫
    pH stress (CV±SD)
    干旱胁迫
    Drought stress (CV±SD)
    氧化胁迫
    Oxidative stress (CV±SD)
    温度胁迫
    Heat stress (CV±SD)
    生物胁迫
    Biotic stress (CV±SD)
    1 ACT(2.17±0.37) RPL6(0.89±0.25) UBC(1.53±0.23) UBC(2.90±0.37) UBC(0.44±0.06) UBC(0.53±0.07)
    2 RPL6(2.30±0.65) APT(1.11±0.26) RPL6(1.84±0.49) ACT(3.29±1.46) RPL6(1.32±0.44) ACT(1.11±0.20)
    3 UBC(3.02±0.50) ACT(1.71±0.30) ACT(2.50±0.42) APT(3.59±0.82) ACT(3.72±0.48) RPL6(1.50±0.43)
    4 β-TUB(3.07±0.74) UBC(1.74±0.29) β-TUB(4.23±1.07) β-TUB(3.86±1.21) β-TUB(4.35±1.03) β-TUB(4.46±1.45)
    5 APT(3.68±0.87) β-TUB(2.73±0.67) APT(5.19±1.23) RPL6(5.47±1.45) 28s(25.65±4.14) APT(6.79±1.72)
    6 28s(25.41±1.95) 28s(23.73±1.88) 28s(10.06±1.21) 28s(6.82±0.59) APT(32.66±5.95) 28s(7.41±0.97)
    下载: 导出CSV
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  • 收稿日期:  2024-02-27
  • 修回日期:  2024-04-24
  • 网络出版日期:  2024-06-26

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