Screening of Internal Reference Gene for Pholidota chinensis and Their Applications

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

Abstract

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

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.

Figures/Tables 10

References 34

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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

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

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

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

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