Selection of Reference Genes in <i>Ganoderma pseudoferreum</i> for Studying a Rubber Tree Disease

ZHAO Xinyang; LUO Youhong; CAI Haibin; ZHOU Yi; TU Min

doi:10.19303/j.issn.1008-0384.2024.05.014

Volume 39 Issue 5

May 2024

Turn off MathJax

Article Contents

Article Navigation > Fujian Journal of Agricultural Sciences > 2024 > 39(5): 615-622

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）：615−622 doi: 10.19303/j.issn.1008-0384.2024.05.014

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）：615−622 doi: 10.19303/j.issn.1008-0384.2024.05.014

Citation:

PDF( 1179 KB)

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

doi: 10.19303/j.issn.1008-0384.2024.05.014

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

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

Received Date: 2024-02-27
Rev Recd Date: 2024-04-24

Available Online: 2024-06-26

Publish Date: 2024-05-28

Abstract

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.
- rubber tree,
- red root rot disease,
- Ganoderma pseudoferreum,
- reference genes,
- RT-qPCR

FullText(HTML)

References(28)

References

[1]	NANDRIS D, NICOLE M, GEIGER J P. Root rot diseases of rubber trees [J]. Plant Disease, 1987, 71(4): 298−306. doi: 10.1094/PD-71-0298
[2]	张运强, 张辉强, 邓晓东. 橡胶树红根病病原菌的鉴定 [J]. 热带作物学报, 1997, 18(1):16−23. ZHANG Y Q, ZHANG H Q, DENG X D. Identification of pathogenic fungi of rubber red root disease [J]. Chinese Journal of Tropical Crops, 1997, 18(1): 16−23. (in Chinese)
[3]	丁婧钰. 橡胶树与相思树病原灵芝种类鉴定及生物学特性研究[D]. 海口: 海南大学, 2018. DING J Y. Type identification of pathogenic Ganoderma and biological characteristics of the rubber tree and Acacia spp. [D]. Haikou: Hainan University, 2018. (in Chinese)
[4]	YANG Y T, ZHANG X, CHEN Y, et al. Selection of reference genes for normalization of microRNA expression by RT-qPCR in sugarcane buds under cold stress [J]. Frontiers in Plant Science, 2016, 7: 86.
[5]	ZHAO X, YANG H L, CHEN M J, et al. Reference gene selection for quantitative real-time PCR of mycelia from Lentinula edodes under high-temperature stress [J]. BioMed Research International, 2018, 2018: 1670328.
[6]	张越. 黑木耳qRT-PCR内参基因的筛选及功能基因表达水平的研究[D]. 长春: 吉林农业大学, 2020. ZHANG Y. Studies of screening of reference genes and functional genes expression levels of Auricularia heimuer for qRT-PCR[D]. Changchun: Jilin Agricultural University, 2020. (in Chinese)
[7]	刘英, 喻晓明, 蔡佺佑, 等. 巨大口蘑内参基因的筛选 [J]. 食用菌学报, 2017, 24(4):12−18. LIU Y, YU X M, CAI Q Y, et al. Reference gene selection for real-time quantitative PCR in Tricholoma giganteum [J]. Acta Edulis Fungi, 2017, 24(4): 12−18. (in Chinese)
[8]	赵建霞, 沈颖越, 冯伟林, 等. 双孢蘑菇内参基因的筛选与矫正 [J]. 浙江农业学报, 2019, 31(8):1312−1320. doi: 10.3969/j.issn.1004-1524.2019.08.12 ZHAO J X, SHEN Y Y, FENG W L, et al. Screening of internal reference gene of Agaricus bisporus [J]. Acta Agriculturae Zhejiangensis, 2019, 31(8): 1312−1320. (in Chinese) doi: 10.3969/j.issn.1004-1524.2019.08.12
[9]	XU J, XU Z C, ZHU Y J, et al. Identification and evaluation of reference genes for qRT-PCR normalization in Ganoderma lucidum [J]. Current Microbiology, 2014, 68(1): 120−126. doi: 10.1007/s00284-013-0442-2
[10]	武晨剑. 金针菇不同发育阶段差异基因筛选及转录因子FfMYB和FfGAL基因的原核表达与纯化[D]. 太谷: 山西农业大学, 2021. WU C J. Screening of differentially expression genes related to different developmental stages of Flammulina filiformis and prokaryotic expression and purification of transcription factors FfMYB and FfGAL[D]. Taigu: Shanxi Agricultural University, 2021. (in Chinese)
[11]	ANDERSEN C L, JENSEN J L, ØRNTOFT T F. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets [J]. Cancer Research, 2004, 64(15): 5245−5250. doi: 10.1158/0008-5472.CAN-04-0496
[12]	VANDESOMPELE J, DE PRETER K, PATTYN F, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes [J]. Genome Biology, 2002, 3(7): RESEARCH0034. doi: 10.1186/gb-2002-3-7-reports0034
[13]	PFAFFL M W, TICHOPAD A, PRGOMET C, et al. Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper–Excel-based tool using pair-wise correlations [J]. Biotechnology Letters, 2004, 26(6): 509−515. doi: 10.1023/B:BILE.0000019559.84305.47
[14]	DHEDA K, HUGGETT J F, CHANG J S, et al. The implications of using an inappropriate reference gene for real-time reverse transcription PCR data normalization [J]. Analytical Biochemistry, 2005, 344(1): 141−143. doi: 10.1016/j.ab.2005.05.022
[15]	XIE F L, XIAO P, CHEN D L, et al. miRDeepFinder: A miRNA analysis tool for deep sequencing of plant small RNAs [J]. Plant Molecular Biology, 2012, 80(1): 75−84. doi: 10.1007/s11103-012-9885-2
[16]	孙茜茜, 何其光, 靳鹏飞, 等. 橡胶树红根病菌GPFTR1基因的克隆、亚细胞定位与表达分析 [J]. 基因组学与应用生物学, 2019, 38(8):3646−3653. SUN Q Q, HE Q G, JIN P F, et al. Cloning, subcellular localization and expression analysis of GPFTR1 gene from Ganoderma pseudoferreum [J]. Genomics and Applied Biology, 2019, 38(8): 3646−3653. (in Chinese)
[17]	伏雪, 涂敏, 蔡海滨, 等. 橡胶树红根病病原菌木聚糖酶编码基因GpTR1774的克隆与表达分析 [J]. 热带作物学报, 2023, 44(12):2461−2468. doi: 10.3969/j.issn.1000-2561.2023.12.011 FU X, TU M, CAI H B, et al. Cloning and expression analysis of xylanase GpTR1774 gene from Ganoderma pseudoferreum [J]. Chinese Journal of Tropical Crops, 2023, 44(12): 2461−2468. (in Chinese) doi: 10.3969/j.issn.1000-2561.2023.12.011
[18]	林仕恺, 涂敏, 鲁红学, 等. 巴西橡胶树红根病病菌液体培养条件优化研究 [J]. 热带农业科学, 2014, 34(10):71−74. doi: 10.3969/j.issn.1009-2196.2014.10.016 LIN S K, TU M, LU H X, et al. Optimization of Ganoderma pseudoferreum liquid culture condition [J]. Chinese Journal of Tropical Agriculture, 2014, 34(10): 71−74. (in Chinese) doi: 10.3969/j.issn.1009-2196.2014.10.016
[19]	CAO L P, ZHANG Q, MIAO R Y, et al. Reference gene selection for quantitative real-time PCR analysis of Hymenopellis radicata under abiotic stress [J]. Fungal Biology, 2024, 128(1): 1567−1577. doi: 10.1016/j.funbio.2023.11.004
[20]	吴建阳, 何冰, 杜玉洁, 等. 利用geNorm、NormFinder和BestKeeper软件进行内参基因稳定性分析的方法 [J]. 现代农业科技, 2017, (5):278−281. doi: 10.3969/j.issn.1007-5739.2017.05.174 WU J Y, HE B, DU Y J, et al. Analysis method of systematically evaluating stability of reference genes using geNorm, NormFinder and BestKeeper [J]. Modern Agricultural Science and Technology, 2017(5): 278−281. (in Chinese) doi: 10.3969/j.issn.1007-5739.2017.05.174
[21]	张晓华, 孙达锋, 刘绍雄, 等. 食(药)用菌内参基因筛选研究进展 [J]. 中国食用菌, 2023, 42(5):1−6. ZHANG X H, SUN D F, LIU S X, et al. Research progress of internal reference gene screening for edible and medicinal fungi [J]. Edible Fungi of China, 2023, 42(5): 1−6. (in Chinese)
[22]	JAIN M, NIJHAWAN A, TYAGI A K, et al. Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR [J]. Biochemical and Biophysical Research Communications, 2006, 345(2): 646−651. doi: 10.1016/j.bbrc.2006.04.140
[23]	FANG P, LU R F, SUN F, et al. Assessment of reference gene stability in Rice stripe virus and Rice black streaked dwarf virus infection rice by quantitative Real-time PCR [J]. Virology Journal, 2015, 12: 175. doi: 10.1186/s12985-015-0405-2
[24]	HERIDE C, URBÉ S, CLAGUE M J. Ubiquitin code assembly and disassembly [J]. Current Biology, 2014, 24(6): R215−R220. doi: 10.1016/j.cub.2014.02.002
[25]	XIANG Q J, LI J, QIN P, et al. Identification and evaluation of reference genes for qRT-PCR studies in Lentinula edodes [J]. PLoS One, 2018, 13(1): e0190226. doi: 10.1371/journal.pone.0190226
[26]	WEI Y M, LIU Y, LI L, et al. Identification of s9ap used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of Pleurotus eryngii [J]. Molecular Biology Reports, 2023, 50(1): 621−629. doi: 10.1007/s11033-022-07562-3
[27]	QIAN J, GAO Y N, WÁNG Y, et al. Selection and evaluation of appropriate reference genes for RT-qPCR normalization of Volvariella volvacea gene expression under different conditions [J]. BioMed Research International, 2018, 2018: 6125706.
[28]	ZHANG C, LI T, HOU C L, et al. Selection of reference genes from Shiraia bambusicola for RT-qPCR analysis under different culturing conditions [J]. AMB Express, 2017, 7(1): 14. doi: 10.1186/s13568-016-0314-9