三褶虾脊兰花发育相关基因RT-qPCR内参基因的筛选

doi:10.16420/j.issn.0513-353x.2023-0954

摘要/Abstract

摘要：

为了推进三褶虾脊兰（Calanthe triplicata）花发育相关基因表达模式的研究，使用geNorm、NormFinder、BestKeeper和RefFinder等4种算法在整花、花部各结构和混合样品中鉴定和评估了9个候选内参基因。结果显示，尽管各处理之间内参基因的稳定性排名略有差异，但UBQ1和UBQ2表现最为稳定。通过RT-qPCR扩增了与花发育相关的4个靶基因，结果佐证了UBQ1和UBQ2是研究三褶虾脊兰花发育基因表达的最佳内参基因。

关键词: 三褶虾脊兰, RT-qPCR, 内参基因, 稳定性评价

Abstract:

To facilitate studies on gene expression in Calanthe triplicata，nine candidate reference genes were identified and evaluated across three different groups（the whole flower group，the floral structures group and the mixed group）using geNorm，NormFinder，BestKeeper and RefFinder. The results showed although there were slight differences in the ranking of RGs between groups，UBQ1 and UBQ2 were the most stable. To valid the accuracy of the evaluation results，four target genes related to flower development were amplified by RT-qPCR，the results further supported that UBQ1 and UBQ2 were the most optimal for reference genes.

Key words: Calanthe triplicata, RT-qPCR, reference gene, stability evaluation

吴悠, 冀宏宇, 邓淑雯, 梁翠怡, 万志庭, 吴沙沙, 翟俊文. 三褶虾脊兰花发育相关基因RT-qPCR内参基因的筛选[J]. 园艺学报, 2024, 51(9): 2063-2074.

WU You, JI Hongyu, DENG Shuwen, LIANG Cuiyi, WAN Zhiting, WU Shasha, ZHAI Junwen. Selection and Validation of Reference Genes for RT-qPCR of Flower Development in Calanthe triplicata[J]. Acta Horticulturae Sinica, 2024, 51(9): 2063-2074.

图/表 11

参考文献 44

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基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	扩增长度/bp Amplicon length	扩增效率/% PCR efficiency	R²	序列号 Accession
ACTB	GCTTTCCCTTTATGCGAGT CATCAGTCAAATCCCTACCAG	57.0 57.1	145	98.72	0.9996	MZ005277
ADF	GGAGAAAACTGGAGGTGCT TGCGTCACAAAATCAAAATC	56.9 57.2	101	93.41	0.9964	MZ005280
EF1-α	TCATCATCGACTCCACCAC TTGCATCCATCTTATTGCAG	57.8 57.3	131	99.97	0.9996	MZ005274
GAPDH	GCGAGTCAGAACATCATCC GGCAAGTCAAGTCCACAAC	56.6 56.6	138	98.04	0.9992	MZ005278
PP2A	TCCGGTTGTTTGAATATGG GATAGGAGGAGGCAGATGG	56.8 57.2	108	101.76	0.9988	MZ005275
TIP41	TGACTACACATTCACGACACC TCACAATCCTCCCAGTTTG	57.0 57.0	107	92.42	0.9982	MZ005279
TUB	GCCTCACCCAGACTTTTATG TGTATGCCTTGCTTCCTTG	57.3 57.4	128	90.41	0.9901	MZ005276
UBQ1	CACCTTGTTCTCCGTCTCA GATGCCCTCCTTATCTTGG	57.2 57.1	97	103.44	0.9950	MZ005281
UBQ2	CCACTCTTCATCTGGTGCT GAATGCCCTCCTTATCCTG	57.7 57.4	143	98.16	0.9973	MZ005282

基因 Gene	引物序列（5′-3′） Primer sequence	退火温度/℃ Annealing temperature	扩增长度/bp Amplicon length	扩增效率/% PCR efficiency	R²	序列号 Accession
ACTB	GCTTTCCCTTTATGCGAGT CATCAGTCAAATCCCTACCAG	57.0 57.1	145	98.72	0.9996	MZ005277
ADF	GGAGAAAACTGGAGGTGCT TGCGTCACAAAATCAAAATC	56.9 57.2	101	93.41	0.9964	MZ005280
EF1-α	TCATCATCGACTCCACCAC TTGCATCCATCTTATTGCAG	57.8 57.3	131	99.97	0.9996	MZ005274
GAPDH	GCGAGTCAGAACATCATCC GGCAAGTCAAGTCCACAAC	56.6 56.6	138	98.04	0.9992	MZ005278
PP2A	TCCGGTTGTTTGAATATGG GATAGGAGGAGGCAGATGG	56.8 57.2	108	101.76	0.9988	MZ005275
TIP41	TGACTACACATTCACGACACC TCACAATCCTCCCAGTTTG	57.0 57.0	107	92.42	0.9982	MZ005279
TUB	GCCTCACCCAGACTTTTATG TGTATGCCTTGCTTCCTTG	57.3 57.4	128	90.41	0.9901	MZ005276
UBQ1	CACCTTGTTCTCCGTCTCA GATGCCCTCCTTATCTTGG	57.2 57.1	97	103.44	0.9950	MZ005281
UBQ2	CCACTCTTCATCTGGTGCT GAATGCCCTCCTTATCCTG	57.7 57.4	143	98.16	0.9973	MZ005282

基因 Gene	引物序列（5′-3′） Primer sequence
CtARF3	F：TGTATCCTTCGTTGTTCCCT；R：ATCTTCCCTGAGCCACATT
CtARF16	F：AAAGCTCAAGTGGTCGGAT；R：AAAGCCTCTCTAAGCTCCTCA
CtARF17	F：GAAGACACCGAACTAAGGCT；R：CCTCTCGAACTCAGAATCATC
CtARF22	F：GTGATTTGTCGGGTCGTAG；R：TCCACTTCTTCCCCTTCTC

基因 Gene	引物序列（5′-3′） Primer sequence
CtARF3	F：TGTATCCTTCGTTGTTCCCT；R：ATCTTCCCTGAGCCACATT
CtARF16	F：AAAGCTCAAGTGGTCGGAT；R：AAAGCCTCTCTAAGCTCCTCA
CtARF17	F：GAAGACACCGAACTAAGGCT；R：CCTCTCGAACTCAGAATCATC
CtARF22	F：GTGATTTGTCGGGTCGTAG；R：TCCACTTCTTCCCCTTCTC

排名 Rank	整花组 Flower group		花部结构组 Floral structure group		混合组 Mixed group
排名 Rank	基因 Gene	M	基因 Gene	M	基因 Gene	M
1	UBQ1	0.004	UBQ1	0.024	UBQ1	0.022
2	UBQ2	0.004	UBQ2	0.024	UBQ2	0.022
3	ADF	0.013	ADF	0.069	PP2A	0.073
4	ACTB	0.029	PP2A	0.083	EF1-α	0.084
5	TUB	0.048	EF1-α	0.089	TUB	0.087
6	PP2A	0.055	TUB	0.092	GAPDH	0.094
7	EF1-α	0.059	GAPDH	0.098	ACTB	0.098
8	GAPDH	0.063	ACTB	0.102	TIP41	0.113
9	TIP41	0.085	TIP41	0.114	ADF	0.129