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

橡胶灵芝菌内参基因筛选与分析

赵欣阳^{1, 2,},
罗佑红^{1, 2},
蔡海滨²,
周燚^1, ,,
涂敏²

1.
长江大学农学院，湖北　荆州　434000
2.
中国热带农业科学院橡胶研究所，海南　海口　571101

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
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出版历程
- 收稿日期: 2024-02-27
- 修回日期: 2024-04-24
- 网络出版日期: 2024-06-26

Screening and analysis on reference genes of Ganoderma pseudoferreum

ZHAO Xinyang^{1, 2
,},
LUO Youhong^{1, 2},
CAI Haibin²,
ZHOU Yi^{1
, ,},
TU Min²

1.
College of Agriculture, Yangtze University, Jingzhou, Hubei,　434000, China
2.
Rubber Research Institute, Chinese Academy of Tropical Agricultural Sciences, Haikou, Hainan　571101, China

摘要

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（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。
- 橡胶树 /
- 红根病 /
- 橡胶灵芝菌 /
- 内参基因 /
- 实时荧光定量PCR
Abstract: : Objective The stability of candidate reference genes of Ganoderma pseudoferreum under different stress treatments were evaluated to provide reference for exploring the gene function and molecular mechanism of G.pseudoferreum infestation of rubber tree roots. Methods The G. pseudoferreum mycelia under five abiotic stresses ( temperature, salt, oxidation, pH, drought ) and one biotic stress (biocontrol bacteria) were collected to isolate RNA and reverse transcribed into cDNA. Real-time fluorescence quantitative PCR technology was used to amplify the six candidate reference genes (UBC, ACT, RPL6, β-TUB, APT, 28s), and the softwares of geNorm, NormFinder, BestKeeper and RefFinder were used to evaluate genes stability to find the most suitable reference genes for different stresses. Results The RNA of all samples showed two clear bands, and the melting curves of the six candidate reference genes showed obvious single peak. Combined analysis of geNorm, NormFinder, Bestkeeper and RefFinder, found that the stability of gene expression under temperature stress was UBC ＞ ACT ＞ RPL6 ＞ β-TUB ＞ 28s ＞ APT, and the stability of gene expression under salt stress was ACT ＞ RPL6 ＞ UBC ＞ APT ＞ β-TUB ＞ 28s; the stability of gene expression under oxidative stress was UBC ＞ ACT ＞ 28s ＞ APT ＞ β-TUB ＞ RPL6; the stability of gene expression under pH stress was RPL6 ＞ UBC ＞ APT ＞ ACT ＞ β-TUB ＞ 28s, the stability of gene expression under drought stress was ACT ＞ UBC ＞ β-TUB ＞ RPL6 ＞ APT ＞ 28s, and the stability of gene expression under biotic stress was UBC ＞ ACT ＞ RPL6 ＞ 28s ＞ APT ＞ β-TUB. Conclusion Combining all genes and experimental conditions, we recommend genes ACT and UBC as the best reference genes for normalizing gene expression under drought, oxidation, temperature and biotic stress experimental conditions. Genes ACT and RPL6 are the most suitable reference genes under salt stress, and UBC and RPL6 are the most suitable reference genes under pH stress.
- Rubber tree /
- Red root rot disease /
- Ganoderma pseudoferreum /
- Reference genes /
- RT-qPCR

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图 1 总RNA提取

注：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

Note: 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.

下载: 全尺寸图片幻灯片

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.

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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

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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.

下载: 全尺寸图片幻灯片

图 5 geNorm软件分析所需内参基因的数目

Figure 5. The required number of reference genes in by geNorm

下载: 全尺寸图片幻灯片

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. Stabilities 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.

下载: 全尺寸图片幻灯片

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
UBC	KAF9027008	TGGCATTGATGTTCGGGTGG	106
ACT	KAF9050787	CATCGAGCACGGTATTGTCA	167
ACT	KAF9050787	TCTCGAACATGATTTGGGTC	167
β-TUB	KAF9039628	CAAATGCAGAACGTCCAGAAC	159
β-TUB	KAF9039628	GTGAACTCCATCTCGTCCATAC	159
RPL6	KAF9033237	CTGTACCTCGTCGGTGTCGG	137
RPL6	KAF9033237	GTTGGCGTCTCCACCTTTGC	137
APT	GL18178	GAGTACGGTGTGGATGTCTTC	130
APT	GL18178	CGAGCTTGGCTACGAGTTC	130
28s	无登录号	GCATATCAATAAGCGGAGGA	130
28s	无登录号	GCACTTCTCCAGACTACAAC	130

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	RPL6	UBC	UBC	UBC	UBC
GroupSD	0.02	0.05	0.05	0.02	0.04	0.04
2	APT	β-TUB	ACT	APT	ACT	ACT
GroupSD	0.02	0.05	0.05	0.02	0.04	0.06
3	UBC	APT	β-TUB	ACT	RPL6	RPL6
GroupSD	0.11	0.05	0.07	0.02	0.12	0.07
4	β-TUB	UBC	RPL6	RPL6	β-TUB	APT
GroupSD	0.11	0.07	0.07	0.1	0.19	0.08
5	ACT	ACT	APT	β-TUB	28s	28s
GroupSD	0.11	0.07	0.15	0.11	0.46	0.11
6	28s	28s	28s	28s	APT	β-TUB
GroupSD	0.53	0.24	0.24	0.15	0.56	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	RPL6	UBC	UBC	UBC	UBC
CV±SD	2.17±0.37	0.89±0.25	1.53±0.23	2.90±0.37	0.44±0.06	0.53±0.07
2	RPL6	APT	RPL6	ACT	RPL6	ACT
CV±SD	2.30±0.65	1.11±0.26	1.84±0.49	3.29±1.46	1.32±0.44	1.11±0.20
3	UBC	ACT	ACT	APT	ACT	RPL6
CV±SD	3.02±0.50	1.71±0.30	2.50±0.42	3.59±0.82	3.72±0.48	1.50±0.43
4	β-TUB	UBC	β-TUB	β-TUB	β-TUB	β-TUB
CV±SD	3.07±0.74	1.74±0.29	4.23±1.07	3.86±1.21	4.35±1.03	4.46±1.45
5	APT	β-TUB	APT	RPL6	28s	APT
CV±SD	3.68±0.87	2.73±0.67	5.19±1.23	5.47±1.45	25.65±4.14	6.79±1.72
6	28s	28s	28s	28s	APT	28s
CV±SD	25.41±1.95	23.73±1.88	10.06±1.21	6.82±0.59	32.66±5.95	7.41±0.97

下载: 导出CSV

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