石仙桃内参基因筛选与应用

doi:10.13560/j.cnki.biotech.bull.1985.2025-0378

摘要/Abstract

摘要：

目的石仙桃（Pholidota chinensis）系兰科（Orchidaceae）石仙桃属（Pholidota）珍稀濒危的多年附生草本植物，为了研究该物种及近源种功能基因的表达量，迫切需要筛选其稳定的内参基因用于实时荧光定量PCR（RT-qPCR）分析。方法采用RT-qPCR检测11个管家基因在石仙桃不同组织（根、根状茎、假鳞茎、叶片、芽、花序）和非生物胁迫（20 mmol/L MeJA和50 mmol/L NaCl喷洒叶片、不同光照强度）的表达量，用geNorm、NormFinder、BestKeeper、ΔCt和RefFinder几何平均值综合分析11个内参基因表达的稳定性。结果选取的11个基因在石仙桃的不同组织中具有单一的扩增条带和峰图，扩增效率及表达丰度基本达到了内参基因的要求，表达稳定性表明，Actin/f58p0可作为不同组织基因表达的最佳内参基因，而TUA3/f11p0可作为非生物胁迫的最佳内参基因。分别以Actin/f58p0和TUA3/f11p0作为内参基因对石仙桃中天麻素合成关键基因GT1、GT2、GT3-01、GT3-02、GT4、ADH-01、ADH-02、ADH-03表达分析，在不同组织中，二者表达趋势基本一致，但HCT基因表达则更适宜以Actin/f58p0作为内参基因；非生物胁迫中，以TUA3/f11p0作为内参基因更佳，尤其是在NaCl和MeJA处理中，但在光照胁迫下，Actin/f58p0和TUA3/f11p0皆可作为内参，MeJA较NaCl对石仙桃基因表达量影响时间长。结论 Actin/f58p0和TUA3/f11p0分别可作为石仙桃不同组织、活性成分和非生物胁迫的内参基因，为石仙桃及近缘物种功能基因的发掘、表达量分析等研究奠定基础。

关键词: 石仙桃, 内参基因, 组织, 光照, NaCl, MeJA, RT-qPCR, 基因表达

Abstract:

Objective Pholidota chinensis is a rare and endangered perennial epiphytic medicinal plant of the Orchidaceae family. To study the expressions of functional genes in this species and its closely related species, it is timely to screen for stable housekeeping genes in real-time fluorescent quantitative PCR (RT-qPCR) analysis. Method The RT-qPCR was used to detect the expression of 11 housekeeping genes in different tissues (roots, rhizomes, pseudobulbs, leaves, buds, and flowers) and abiotic stresses (20 mmol/L MeJA sprayed leaves for 24 h and 48 h, 50 mmol/L NaCl sprayed leaves for 24 h and 48 h, and different light intensities) in P. chinensis. The expression stability of 11 candidate internal reference genes was analyzed by geNorm, NormFinder, BestKeeper, ΔCt, and geometric mean values from RefFinder. Result The results revealed single extended bands and peak maps for eleven genes. Although the amplification efficiency and expression abundance of eleven genes met the requirements expected of internal reference genes, the expression stability analysis indicated that Actin/f58p0 was the best internal reference gene for gene expression analysis in different tissues. Conversely, TUA3/f11p0 was favored as the internal reference gene for abiotic stress analysis. The expression trends of key genes involved in gastrodin biosynthesis, namely GT1, GT2, GT3-01, GT3-02, GT4, ADH-01, ADH-02, and ADH-03, were generally consistent across different tissues by using Actin/f58p0 and TUA3/f11p0 as internal reference genes. However, the Actin/f58p0 was more suitable as an internal reference gene for the shikimate O-hydroxycinnamoyltransferase gene (HCT). Under abiotic stress conditions, the TUA3/f11p0 was a better internal reference gene, especially under NaCl and MeJA treatments. However, under light stress, the Actin/f58p0 and TUA3/f11p0 were both as internal references. MeJA had a longer-lasting effect on gene expressions than NaCl. Conclusion This study identifies Actin/f58p0 and TUA3/f11p0 as reliable internal reference genes for analyzing gene expression in different tissues and under abiotic stresses in P. chinensis. These findings lay a foundation for research on functional gene discovery and expression analysis in P. chinensis and related species.

Key words: Pholidota chinensis, internal reference genes, tissue, light, NaCl, MeJA, RT-qPCR, gene expression

刘保财, 胡学博, 张武君, 赵云青, 黄颖桢, 陈菁瑛. 石仙桃内参基因筛选与应用[J]. 生物技术通报, 2026, 42(2): 158-168.

LIU Bao-cai, HU Xue-bo, ZHANG Wu-jun, ZHAO Yun-qing, HUANG Ying-zhen, CHEN Jing-ying. Screening of Internal Reference Gene for Pholidota chinensis and Their Applications[J]. Biotechnology Bulletin, 2026, 42(2): 158-168.

图/表 10

表1 候选内参基因RT-qPCR引物序列

Table 1 Prime sequences of RT-qPCR primers for candidate internal reference genes

基因 Gene	引物名称 Primer name	引物序列 Primer sequence （5′‒3′）	产物长度 Product length （bp）
UBQ	UBQ/f2p0	F： GTGACTGGTGATCCTTGGCA R： ACCAACTTTCAACAGGCCAGA	121
TUA	TUA/f11p0	F： CTGGGAAGGAAGATGCTGCA R： TCTAACCCGGTCTAGGCACA	89
TUA	TUA/f3p01	F： GGCAAGTACATGGCATGCTG R： TCGTCTTGATTGTGGCGACA	85
TUA	TUA/f3p02	F： GAATCAACTACCAACCTCCTACAG R： CTTCCATTCCTTCTCCGACATAC	183
EF-1α	EF-1α/f74p0	F： GAAGGATCCAACTGGTGCCA R： TCGAGCACCATTGGAACGAA	84
EF-1α	EF-1α/f11p0	F： GGTTCTCGACTGTCACACGT R： CCTTACCAGAGCGCCTATCG	80
Actin	Actin/f10p0	F： GGATTTGCCGGTGATGATGC R： ATGCCAACCATGACACCAGT	83
Actin	Actin/f58p0	F： CTCTTCTCTCCGCTGCCAAA R： CTTGGGAATACAGCCCTGGG	142
18S rRNA	18S/f2p0	F： CGCTCCTACCGATTGAATGG R： TTGTTACGACTTCTCCTTCCTCTA	125
GAPDH	GAPDH/f24p0	F： GCTCTGTTGAGGAGGGCATT R： TGATCTGAACCGTGCCAGTC	129
GAPDH	GAPDH/f94p0	F： AGTATGACACTGTGCACGGG R： GGGATCTCCTCAGGGTTCCT	121

表2 天麻素合成通路中9个基因RT-qPCR引物序列

Table 2 Primer sequences of RT-qPCR for 9 key candidate genes of gastrodin biosynthesis pathway in P. chinensis

基因 Gene	引物名称 Primer name	引物序列 Primer sequence （5′‒3′）
GT1	16563/f4p0/2237-F	TGACCTCGCAACAGATTATGAAC
	16563/f4p0/2237-R	TTCCAGTGCCTCAACAATAATTCT
GT2	261/f5p0/5355-F	TCTGTAACAATCCAATGACCAAGG
	261/f5p0/5355-R	TCCAACTCTTCGCCTAGTATTCT
GT3-01	17418/f3p0/2174-F	GTTAATAAGTGGAAGAGCGTAGCA
	17418/f3p0/2174-R	CACAACCTCCGCATCATCTC
GT3-02	28360/f2p0/1592-F	GAAGGAGTTGATGGAAGGAGAG
	28360/f2p0/1592-R	TCACTTAGTAGCCGTTGAATCC
GT4-01	20627/f2p0/1989-F	GTCAATGCTGGAGAATGCTTAATG
	20627/f2p0/1989-R	AAGAATGCCGCTGTTGAAGAT
ADH-01	30246/f18p0/1465-F	AGTTGCCCATCTTCCCTCTAAT
	30246/f18p0/1465-R	GCATCGCTTCGTTCACATAGT
ADH-02	32130/f3p0/1407-F	ATTGAGACAGTGAGACCAGAAGA
	32130/f3p0/1407-R	TTAGAACTAAGCGACGCCATTG
ADH-03	30741/f57p0/1339-F	GGAAGCCTCATTGGTGGAATAAG
	30741/f57p0/1339-R	CGACATCAACAACGAACCGATAT
HCT	26704/f5p0/1679-F	GGGCACCGCAATTACTAATACTT
	26704/f5p0/1679-R	CACATCCACCAACAACCTCTC

图1 石仙桃不同部位RNA的琼脂糖凝胶电泳

Fig. 1 Agarose gel electrophoretogram of total RNA from different tissues of P. chinensis

图2 11个候选内参基因PCR产物电泳检测

Fig. 2 Electrophoresis detection of PCR products for 11 candidate reference genes

表3 11个候选内参基因RT-qPCR的扩增参数

Table 3 Amplification parameters for RT-qPCR of 11 candidate reference genes

候选基因 Candidate gene	标准曲线斜率 Standard curve slope （k）	扩增效率 Amplification efficiency （E）（%）	相关系数 Correlation coefficient （R²）
UBQ/f2p0	-3.435 8	95.46	0.998 1
TUA/f11p0	-2.523 7	109.03	0.987 9
TUA/f3p01	-3.188 3	105.90	0.993 7
TUA/f3p02	-3.492 5	93.34	0.997 2
EF-1α/f74p0	-3.509 9	92.71	0.993 8
EF-1α/f11p0	-3.239 2	103.57	0.994 3
Actin/f10p0	-3.148 9	100.78	0.991 8
Actin/f58p0	-3.312 8	100.38	0.995 6
18S/f2p0	-3.646 5	88.03	0.992 9
GAPDH/f24p0	-3.374 4	97.86	0.997 9
GAPDH/f94p0	-3.330 2	99.66	0.985 6

图3 11个候选内参基因的熔解曲线

Fig. 3 Melting curves of 11 candidate reference genes

图4 候选内参基因Ct值箱线图A：不同组织；B：非生物胁迫

Fig. 4 Boxplots of candidate reference gene Ct valuesA: Different tissues; B: abiotic stresses

图5 石仙桃中11个候选参考基因的表达稳定性A、B：geNorm对不同组织（A）和非生物胁迫下（B）内参候选基因表达稳定性分析；C、D：NormFinder对不同组织（C）和非生物胁迫下（D）内参候选基因表达稳定性分析；E、F：BestKeeper对不同组织（E）和非生物胁迫下（F）内参候选基因表达稳定性分析；G、H：ΔCt对不同组织（G）和非生物胁迫下（H）内参候选基因表达稳定性分析

Fig. 5 Expression stabilities of 11 candidate reference genes in P. chinensisA, B: Stability analysis of geNorm on the expressions of internal candidate reference genes in different tissues (A) and under abiotic stress (B). C, D: Stability analysis of NormFinder on the expressions of internal candidate reference genes in different tissues (C) and under abiotic stress (D). E, F: Stability analysis of BestKeeper on the expressions of internal candidate reference genes in different tissues (E) and under abiotic stress (F). G, H: Stability analysis of ΔCt on the expressions of internal candidate reference genes in different tissues (G) and under abiotic stress (H)

图6 石仙桃不同组织中ADH、HCT、GT基因家族的9个基因的RT-qPCR相对表达量和Illumina测序表达量RT-qPCR1：以Actin/f58p0为内参基因；RT-qPCR2：以TUA3/f11p为内参基因；RNA-Seq：用Illumina测序获得的表达量。B1：根；B2：根状茎；B3：假鳞茎；B4：叶

Fig. 6 RT-qPCR relative expressions and Illumina sequencing expressions of nine genes within ADH, HCT and GT gene families in different tissues of P. chinensisRT-qPCR1 with Actin/f58p0 as the internal reference gene; RT-qPCR2 with TUA3/f11p as the internal reference gene; RNA-Seq: sequencing with Illumina expression obtained. B1: Roots; B2: rhizomes; B3: pseudobulbs; B4: leaves

图7 石仙桃非生物胁迫中ADH、HCT、GT基因家族的9个基因的RT-qPCR相对表达量A：以Actin/f58p0为内参基因；B：以TUA3/f11p为内参基因；Control：对照；GZ1C：1层遮阳网；GZ2C：2层遮阳网；GZ3C：3层遮阳网；NaCl-24 h：氯化钠处理后24 h；NaCl-48 h：氯化钠处理后48 h；MeJA-24 h：茉莉酸甲酯处理后24 h；MeJA-48 h：茉莉酸甲酯处理后48 h

Fig. 7 RT-qPCR relative expressions of nine genes on ADH, HCT, and GT gene families, in abiotic stress of P. chinensisA: Actin/f58p0 as the internal reference gene; B: TUA3/f11p as the internal reference gene. Control: Control; GZ1C: one layer of sunshade; GZ2C: two layers of sunshade; GZ3C: three layers of sunshade; NaCl-24 h: 24 h after NaCl treatment; NaCl-48 h: 48 h after NaCl treatment; MeJA-24 h: 24 h after methyl jasmonate treatment; MeJA-48 h: 48 h after methyl jasmonate treatment

参考文献 34

[1]	Zhang LY, Zhang RM, Hu XL, et al. Reference gene selection for qRT-PCR analysis in the shoots and roots of Camellia sinensis var. sinensis under nutritional stresses [J]. Sci Hortic, 2023, 320: 112237.
[2]	Yang C, Lin YS, Qiu ZW, et al. Reference gene selection for qRT-PCR normalization of gene expression analysis in Melaleuca bracteata F. Muell. under abiotic stresses and hormonal stimuli [J]. Sci Hortic, 2023, 319: 112184.
[3]	Moshchenskaya YL, Galibina NA, Korzhenevskiy MA, et al. High-quality RNA extraction and evaluation of reference genes for qPCR assay of Pinus sylvestris L. trunk tissues [J]. Russ J Dev Biol, 2023, 54(1): 24-36.
[4]	Paul S, Singh S, Chakrabarti A, et al. Selection and evaluation of appropriate reference genes for RT-qPCR based expression analysis in Candida tropicalis following azole treatment [J]. Sci Rep, 2020, 10(1): 1972.
[5]	Tang J, Li EJ, Liu JX, et al. Selection of reliable reference genes for gene expression normalization in Sagittaria trifolia [J]. Genes, 2023, 14(7): 1321.
[6]	Chen XQ, Wood JJ. Flora of China [M]. Beijing: Sciences Press, 2009, 335-339.
[7]	吴晓婷, 刘凯良. 闽东传统畲药草本植物资源调查与分析 [J]. 亚太传统医药, 2025, 21(5): 193-199.
	Wu XT, Liu KL. Investigation and analysis of native plant resources of traditional she medicinal herbs in eastern Fujian [J]. Asia Pac Tradit Med, 2025, 21(5): 193-199.
[8]	Li DL, Zheng XL, Duan L, et al. Ethnobotanical survey of herbal tea plants from the traditional markets in Chaoshan, China [J]. J Ethnopharmacol, 2017, 205: 195-206.
[9]	刘保财, 黄颖桢, 赵云青, 等. 野生石仙桃立地环境与生长特性研究 [J]. 福建农业学报, 2017, 32(8): 864-869.
	Liu BC, Huang YZ, Zhao YQ, et al. Natural habitat and growth characteristics of wild Pholidota chinensis Lindl [J]. Fujian J Agric Sci, 2017, 32(8): 864-869.
[10]	Liu L, Zou MJ, Zeng KW, et al. Chemical constituents and their antioxidant, anti-inflammatory and anti-acetylcholinesterase activities from Pholidota cantonensis [J]. Plant Foods Hum Nutr, 2021, 76(1): 105-110.
[11]	吴秀彩, 梁爽, 粟正英. 石仙桃属植物化学成分和药理活性的研究概况 [J]. 中国民族民间医药, 2020, 29(4): 60-62.
	Wu XC, Liang S, Su ZY. Advances in chemical constituents and pharmacological activity of Pholidota chinensis Lindl [J]. Chin J Ethnomed Ethnopharmacy, 2020, 29(4): 60-62.
[12]	Luo DH, Wang ZJ, Li ZM, et al. Structure of an entangled heteropolysaccharide from Pholidota chinensis Lindl and its antioxidant and anti-cancer properties [J]. Int J Biol Macromol, 2018, 112: 921-928.
[13]	Liu BC, Chen JY, Zhang WJ, et al. The gastrodin biosynthetic pathway in Pholidota chinensis Lindl. revealed by transcriptome and metabolome profiling [J]. Front Plant Sci, 2022, 13: 1024239.
[14]	刘保财, 张武君, 赵云青, 等. 石仙桃中4种转录因子鉴定及其组织表达的研究 [J]. 生物技术通报, 2024, 40(5): 141-152.
	Liu BC, Zhang WJ, Zhao YQ, et al. Identification and tissue expression of four transcription factors in Pholidota chinensis [J]. Biotechnol Bull, 2024, 40(5): 141-152.
[15]	Ruijter JM, Ramakers C, Hoogaars WMH, et al. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data [J]. Nucleic Acids Res, 2009, 37(6): e45.
[16]	Larsen B, Fuller VL, Pollier J, et al. Identification of iridoid glucoside transporters in Catharanthus roseus [J]. Plant Cell Physiol, 2017, 58(9): 1507-1518.
[17]	徐圆圆, 赵国春, 郝颖颖, 等. 无患子RT-qPCR内参基因的筛选与验证 [J]. 生物技术通报, 2022, 38(10): 80-89.
	Xu YY, Zhao GC, Hao YY, et al. Reference genes selection and validation for RT-qPCR in Sapindus mukorossi [J]. Biotechnol Bull, 2022, 38(10): 80-89.
[18]	Xie FL, Xiao P, Chen DL, et al. miRDeepFinder: a miRNA analysis tool for deep sequencing of plant small RNAs [J]. Plant Mol Biol, 2012, 80: 75-84.
[19]	Andersen CL, Jensen JL, Ørntoft TF. 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 Res, 2004, 64(15): 5245-5250.
[20]	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 Biol, 2002, 3(7): RESEARCH0034.
[21]	Pfaffl MW, 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]. Biotechnol Lett, 2004, 26(6): 509-515.
[22]	Silver N, Best S, Jiang J, et al. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR [J]. BMC Mol Biol, 2006, 7: 33.
[23]	Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method [J]. Methods, 2001, 25(4): 402-408.
[24]	Pei YF, Leng L, Sun W, et al. Whole-genome sequencing in medicinal plants: current progress and prospect [J]. Sci China Life Sci, 2024, 67(2): 258-273.
[25]	Chaturvedi T, Gupta AK, Lal RK, et al. March of molecular breeding techniques in the genetic enhancement of herbal medicinal plants: present and future prospects [J]. Nucleus, 2022, 65(3): 413-436.
[26]	Clark JW. Genome evolution in plants and the origins of innovation [J]. New Phytol, 2023, 240(6): 2204-2209.
[27]	Liu S, Zhang Q, Kollie L, et al. Molecular networks of secondary metabolism accumulation in plants: Current understanding and future challenges [J]. Ind Crops Prod, 2023, 201: 116901.
[28]	Lin Y, Liu GF, Rao Y, et al. Identification and validation of reference genes for qRT-PCR analyses under different experimental conditions in Allium wallichii [J]. J Plant Physiol, 2023, 281: 153925.
[29]	Wang QZ, Guo CQ, Yang SP, et al. Screening and verification of reference genes for analysis of gene expression in garlic (Allium sativum L.) under cold and drought stress [J]. Plants, 2023, 12(4): 763.
[30]	Zhang BB, Zhu HJ, Zhang LH, et al. Evaluation of quality characteristics of 15 Gastrodia elata germplasm resources based on HS-SPME-GC-MS, HPLC and electronic nose techniques [J]. Food Biosci, 2025, 71: 107158.
[31]	Cao H, Li H, Lu L, et al. Screening and validation of internal reference genes for quantitative real-time PCR analysis of leaf color mutants in Dendrobium officinale [J]. Genes, 2023, 14(5): 1112.
[32]	陈龙, 张淼, 秦慧真, 等. 石仙桃及其混伪品的DNA条形码鉴定 [J/OL]. 分子植物育种, 2023. .
	Chen L, Zhang M, Qin HZ, et al. DNA barcoding identification of Pholidota chinensis lindl. and its fakes [J/OL]. Mol Plant Breed, 2023. .
[33]	Mehralian M, Bidabadi SS, Azad M, et al. Melatonin-mediated alleviation of drought stress by modulation of physio-biochemical and metabolic status in Dracocephalum kotschyi Boiss. (Lamiaceae) [J]. Ind Crops Prod, 2023, 204: 117321.
[34]	Nazir F, Jahan B, Iqbal N, et al. Methyl jasmonate influences ethylene formation, defense systems, nutrient homeostasis and carbohydrate metabolism to alleviate arsenic-induced stress in rice (Oryza sativa) [J]. Plant Physiol Biochem, 2023, 202: 107990.