Reference Genes Selection for Quantitative Real-time PCR in Antrodia cinnamomea Mycelium of Different Development Stages

doi:10.3969/j.issn.1000-2561.2020.06.013

Abstract

Abstract:

Quantitative Real-time PCR (qRT-PCR) technology is widely used in the analysis of gene expression because of its high sensit-ivity, high accuracy and high specificity. Therefore, it is important to select appropriate reference genes to measure the expression and accuracy of the sample. In this study, the expressions of 18 candidate internal reference genes of Actin, TPK, Floxuridine, CAP-Gly, α-Amylase, Phosphatase, HA2- helicase and MAGT1 were detected by qRT-PCR with A. cinnamomea Mycelium under 7, 21 and 35 d liquid fermentation time. The stability of the internal reference genes were analyzed using softwares of geNorm, NormFinder and BestKeeper. Floxuridine/ HA2-helicase, TPK, CAP-Gly genes were found more stable with geNorm. TPK, CAP-Gly, Floxuridine, HA2-helicase were found more stable with NormFinder. HA2-helicase was found to be best expressed with BestKeeper, followed by TPK and MAGT1. The comprehensive analysis showed that TPK and HA2-helicase had the best stability and were more suitable as the internal reference genes for A. camphorata. The results of this study would have a practical guiding role in the analysis of the selection of internal reference genes in the gene expression of A. cinnamomea by qRT-PCR.

Key words: Antrodia cinnamomea, reference gene, quantitative Real-time PCR

CLC Number:

S567.3
R282

LI Jing,WANG Zehui,CHEN Li,LIU Yanling,XIA Shuning,LIN Zhanxi. Reference Genes Selection for Quantitative Real-time PCR in Antrodia cinnamomea Mycelium of Different Development Stages[J]. Chinese Journal of Tropical Crops, 2020, 41(6): 1160-1166.

Figures/Tables 9

References 26

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基因名称 Gene name	引物序列（5°-3°） Primer sequences (5°-3°)	Genbank accession No.
肌动蛋白 (Actin)	F: TATTCTCGTCTCATGGCCTTTC R: GTCGCAGCTGGAGTTGATTA	MK968953
酪氨酸激酶(TPK)	F: CATGTCAGGCTCCAACGATAA R: GAGGACAAGTCCACGAGAAAG	MK968954
二氢尿嘧啶核苷(Floxuridine)	F: AGACGCCCACAACAACTAAA R: TAGTCTCGGCGGACATAAGA	MK968955
CAP甘氨酸(CAP-Gly)	F: ACGTTCTGAGAGGCGATTTG R: GCATGCCAGTAGTGGGATTTA	MK968956
α水解酶(α-Amylase)	F: CGACACCCAAGAGTAAGAGAAG R: CCGGTTTGACTTGTGGATTTG	MK968957
磷酸酯酶(Phosphatase)	F: ACCAGATTGTTGACCGTTACTC R: GCTAATGTTGGTGGCGTTATTG	MK968958
HA2解旋酶(HA2- helicase)	F: CACCAGATCGGCACAGTAAA R: GAATGGAGGATGGCCGTAAA	MK968959
Mg2⁺转运蛋白(MAGT1)	F: CATGTCAGGCTCCAACGATAA R: GAGGACAAGTCCACGAGAAAG	MK968960

基因名称 Gene name	引物序列（5°-3°） Primer sequences (5°-3°)	Genbank accession No.
肌动蛋白 (Actin)	F: TATTCTCGTCTCATGGCCTTTC R: GTCGCAGCTGGAGTTGATTA	MK968953
酪氨酸激酶(TPK)	F: CATGTCAGGCTCCAACGATAA R: GAGGACAAGTCCACGAGAAAG	MK968954
二氢尿嘧啶核苷(Floxuridine)	F: AGACGCCCACAACAACTAAA R: TAGTCTCGGCGGACATAAGA	MK968955
CAP甘氨酸(CAP-Gly)	F: ACGTTCTGAGAGGCGATTTG R: GCATGCCAGTAGTGGGATTTA	MK968956
α水解酶(α-Amylase)	F: CGACACCCAAGAGTAAGAGAAG R: CCGGTTTGACTTGTGGATTTG	MK968957
磷酸酯酶(Phosphatase)	F: ACCAGATTGTTGACCGTTACTC R: GCTAATGTTGGTGGCGTTATTG	MK968958
HA2解旋酶(HA2- helicase)	F: CACCAGATCGGCACAGTAAA R: GAATGGAGGATGGCCGTAAA	MK968959
Mg2⁺转运蛋白(MAGT1)	F: CATGTCAGGCTCCAACGATAA R: GAGGACAAGTCCACGAGAAAG	MK968960

基因名称Gene name	熔解温度Melting temperature/℃	斜率（k） Slope (k)	线性相关系数（R²） Linear correlation coefficient (R²)	扩增效率（E）Amplification efficiency (E)/%
Actin	53	3.103	0.984	90.9
TPK	54	3.515	0.925	92.5
Floxuridine	52	3.142	0.996	92.5
CAP-Gly	52	3.517	0.994	92.5
α-Amylase	55	3.241	0.996	96.6
Phosphatase	53	3.689	0.990	86.7
HA2- helicase	52	3.145	0.995	92.6
MAGT1	54	3.526	0.997	92.1

基因名称Gene name	熔解温度Melting temperature/℃	斜率（k） Slope (k)	线性相关系数（R²） Linear correlation coefficient (R²)	扩增效率（E）Amplification efficiency (E)/%
Actin	53	3.103	0.984	90.9
TPK	54	3.515	0.925	92.5
Floxuridine	52	3.142	0.996	92.5
CAP-Gly	52	3.517	0.994	92.5
α-Amylase	55	3.241	0.996	96.6
Phosphatase	53	3.689	0.990	86.7
HA2- helicase	52	3.145	0.995	92.6
MAGT1	54	3.526	0.997	92.1

基因名称Gene name	稳定值Expression stability values
Actin	0.970
TPK	0.204
Floxuridine	0.573
CAP-Gly	0.465
α-Amylase	1.626
Phosphatase	1.267
HA2- helicase	0.640
MAGT1	1.810