Selection of Reference Genes in Ganoderma pseudoferreum for Studying a Rubber Tree Disease
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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，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（UBC、ACT、RPL6、β-TUB、APT、28s），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UBC＞ACT＞RPL6＞β-TUB＞28s＞APT，盐胁迫下基因表达稳定性为ACT＞RPL6＞UBC＞APT＞β-TUB＞28s；氧化胁迫下基因表达稳定性为UBC＞ACT＞28s＞APT＞β-TUB＞RPL6；pH胁迫下基因表达稳定性为RPL6＞UBC＞APT＞ACT＞β-TUB＞28s，干旱胁迫下基因表达稳定性为ACT＞UBC＞β-TUB＞RPL6＞APT＞28s，生物胁迫下基因表达稳定性为UBC＞ACT＞RPL6＞28s＞APT＞β-TUB。 结论 结合所有候选基因稳定性和胁迫条件，推荐基因ACT和UBC作为在干旱、氧化、温度和生物胁迫下的内参基因，基因ACT和RPL6为盐胁迫下的内参基因，UBC和RPL6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。 Abstract:Objective Stability of the reference genes of Ganoderma pseudoferreum under stresses was determined for studying the gene functions and mechanism of the red root rot disease on rubber trees. Methods RNA of G. pseudoferreum mycelia under a stress of temperature, salt, oxidation, pH, drought, or a biocontrol bacteria invasion were isolated and reverse-transcribed into cDNA. RT-qPCR was applied to amplify 6 candidate reference genes, i.e., UBC, ACT, RPL6, β-TUB, APT, and 28s. Software geNorm, NormFinder, BestKeeper, and RefFinder were used to determine the stability of the genes under stresses. Results The RNA of all samples displayed two clear bands and melting curves with a single peak. Analyses by geNorm, NormFinder, Bestkeeper, and RefFinder collectively ranked the expression stability of the genes under a temperature stress as UBC ＞ACT ＞RPL6 ＞β-TUB ＞28s ＞APT; that under a salt stress, ACT ＞RPL6 ＞UBC ＞APT ＞β-TUB ＞28s; that under an oxidative stress, UBC ＞ ACT ＞28s ＞APT ＞β-TUB ＞RPL6; that under a pH stress, RPL6 ＞UBC ＞APT＞ACT ＞ β-TUB ＞28s; that under a drought stress, ACT ＞ UBC ＞β-TUB ＞RPL6 ＞APT ＞28s; and that under the biotic stress, UBC ＞ACT ＞RPL6 ＞ 28s ＞APT ＞β-TUB. Conclusion It appeared that ACT and UBC to be the choice reference genes for normalizing the gene expression under drought, oxidation, temperature, and biotic stresses, while ACT and RPL6 under a salt stress, and UBC and RPL6 under a pH stress. -
Key words:
- rubber tree /
- red root rot disease /
- Ganoderma pseudoferreum /
- reference genes /
- RT-qPCR
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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–A7: salt stress; A8–A12: drought stress; A13–B2: pH stress; B3–B7: oxidation stress; B8–B11: temperature stress; B12–B14: biotic stress.
图 2 6个候选内参基因熔解曲线
A~F分别为 ACT、UBC、β-TUB、RPL6、APT、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 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
GeneGenBank登录号
GenBank No.引物序列
Primer sequence (5'-3')扩增长度
Amplification length /bpUBC 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 
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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）
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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）
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