Selection and Validation of Reference Genes for qRT-PCR Analysis of the Correlated Genes in Flower Pigments Biosynthesis Pathway of Anemone obtusiloba

doi:10.16420/j.issn.0513-353x.2020-0306

Acta Horticulturae Sinica ›› 2021, Vol. 48 ›› Issue (2): 377-388.doi: 10.16420/j.issn.0513-353x.2020-0306

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Selection and Validation of Reference Genes for qRT-PCR Analysis of the Correlated Genes in Flower Pigments Biosynthesis Pathway of Anemone obtusiloba

MA Lulin^1,2,*, DUAN Qing^1,*, CUI Guangfen¹, DU Wenwen¹, JIA Wenjie¹, WANG Xiangning¹, WANG Jihua¹^,^**(), CHEN Fadi²^,^**()

¹Flower Research Institute,Yunnan Academy of Agricultural Sciences,Yunnan Flower Breeding Key Lab,National Engineering Research Center for Ornamental Horticulture,Kunming 650205,China
²College Horticulture of Nanjing Agricultural University,Nanjing 210095,China

Received:2020-09-27 Revised:2020-12-10 Online:2021-02-25 Published:2021-03-09
Contact: WANG Jihua,CHEN Fadi E-mail:wangjh0505@sohu.com;chenfd@njau.edu.cn

Abstract

Abstract:

In order to screen the appropriate reference genes for qRT-PCR analysis of the flavonoids/anthocyanins biosynthetic pathway related genes of Anemone obtusiloba,eight traditional reference genes including polyubiquitin（UBQ）,β-tubulin（β-TUB）,aquaporin（AQP）,actin（ACT）,glyceraldehyde-3-phosphate dehydrogenase（GAPDH）,histone（HIS）,elongation factor 1-β（EF-1β）and 60S ribosomal protein L13-1（RPL13）were selected as candidate reference genes based on the RNA-seq data of A. obtusiloba blue/white flowers. The expression of eight candidate reference genes were assessed by qRT-PCR in different tissues such as leaves,stems and blue/white different color flowers of A. obtusiloba,and the stability of them were analyzed by geNorm,NormFinder and BestKeeper programs. The relative expression of some pigments synthesis related genes in the biosynthetic pathway of flavonoids/anthocyanins were assessed to confirm the utility of the most stable reference gene. The results showed that UBQ was the most stable reference gene,while the stability of β-TUB was the lowest among all candidate reference genes. The qRT-PCR results of several pigments synthesis related genes using UBQ as the reference gene were in accordance with the results of RNA-seq. Thus,it was concluded that UBQ was the most suitable reference gene for gene expression analysis of flower pigments biosynthetic pathway in A. obtusiloba.

Key words: Anemone obtusiloba, qRT-PCR, reference gene, selection, stability, flavonoids/anthocyanins biosynthetic pathway

CLC Number:

S682

MA Lulin, DUAN Qing, CUI Guangfen, DU Wenwen, JIA Wenjie, WANG Xiangning, WANG Jihua, CHEN Fadi. Selection and Validation of Reference Genes for qRT-PCR Analysis of the Correlated Genes in Flower Pigments Biosynthesis Pathway of Anemone obtusiloba[J]. Acta Horticulturae Sinica, 2021, 48(2): 377-388.

Figures/Tables 8

References 41

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基因ID Gene ID	FPKM						比对结果 Blast result	基因 Gene
	蓝花 Blue flower			白花 White flower
		1	2	3	1	2			3
DN35855_c0_g1_i10_1	354.26	385.74	422.52	336.35	354.00	390.89	Glyceraldehyde 3-phosphate dehydrogenase [Gynura bicolor] （GenBank：AB550243.1）	GAPDH
DN24449_c0_g1_i2_1	314.43	331.50	366.20	272.08	300.62	327.47	Elongation factor 1-beta 2-like[Pistacia vera]（NCBI reference sequence：XM_031416658.1）	EF-1β
DN38564_c0_g2_i1_1	434.91	450.67	464.33	473.54	517.63	522.32	Actin 7 [Prunus dulcis] （GenBank：BBH05534.1）	ACT
DN25341_c0_g2_i1_2	153.63	173.57	173.87	143.18	160.60	185.16	Tubulin beta chain[Ricinus communis] （NCBI reference sequence： XM_025156136.1）	β-TUB
DN24929_c0_g1_i4_2	1 016.78	975.95	933.45	1 206.08	1 170.53	1 067.26	Polyubiquitin,partial [Wolffia australiana]（GenBank：AEZ49160.1）	UBQ
DN34836_c0_g2_i6_2	1 052.09	1 172.91	1 294.86	1 140.09	1 193.46	1 301.07	Aquaporin TIP1-2[Brassica rapa] （NCBI reference sequence： XP_009129499.）	AQP
DN24035_c0_g1_i2_1	131.24	122.16	141.00	106.95	108.04	119.61	Histone H2A variant 1[Phalaenopsis equestris]（NCBI reference sequence：XP_020573071.1）	HIS
DN25248_c0_g2_i7_1	197.63	210.20	236.21	157.42	172.19	188.42	60S ribosomal protein L13-1 [Camellia sinensis]（NCBI reference sequence：XP_028086825.1）	RPL13

基因ID Gene ID	FPKM						比对结果 Blast result	基因 Gene
	蓝花 Blue flower			白花 White flower
		1	2	3	1	2			3
DN35855_c0_g1_i10_1	354.26	385.74	422.52	336.35	354.00	390.89	Glyceraldehyde 3-phosphate dehydrogenase [Gynura bicolor] （GenBank：AB550243.1）	GAPDH
DN24449_c0_g1_i2_1	314.43	331.50	366.20	272.08	300.62	327.47	Elongation factor 1-beta 2-like[Pistacia vera]（NCBI reference sequence：XM_031416658.1）	EF-1β
DN38564_c0_g2_i1_1	434.91	450.67	464.33	473.54	517.63	522.32	Actin 7 [Prunus dulcis] （GenBank：BBH05534.1）	ACT
DN25341_c0_g2_i1_2	153.63	173.57	173.87	143.18	160.60	185.16	Tubulin beta chain[Ricinus communis] （NCBI reference sequence： XM_025156136.1）	β-TUB
DN24929_c0_g1_i4_2	1 016.78	975.95	933.45	1 206.08	1 170.53	1 067.26	Polyubiquitin,partial [Wolffia australiana]（GenBank：AEZ49160.1）	UBQ
DN34836_c0_g2_i6_2	1 052.09	1 172.91	1 294.86	1 140.09	1 193.46	1 301.07	Aquaporin TIP1-2[Brassica rapa] （NCBI reference sequence： XP_009129499.）	AQP
DN24035_c0_g1_i2_1	131.24	122.16	141.00	106.95	108.04	119.61	Histone H2A variant 1[Phalaenopsis equestris]（NCBI reference sequence：XP_020573071.1）	HIS
DN25248_c0_g2_i7_1	197.63	210.20	236.21	157.42	172.19	188.42	60S ribosomal protein L13-1 [Camellia sinensis]（NCBI reference sequence：XP_028086825.1）	RPL13

KEGG途径 KEGG pathway	基因表达情况（白花为对照） Gene expression（White flower was the control group）	基因ID Gene ID	FPKM
			蓝花 Blue flower			白花 White flower
						1	2	3	1	2	3
类黄酮生物合成 Flavonoid biosynthetic	上调Up	DN26579_c0_g1_i3_2	569.86	469.94	406.45	109.46	98.07	113.44
	上调Up	DN27520_c0_g1_i2_2	341.41	278.90	233.47	38.08	29.97	27.08
	上调Up	DN27888_c0_g1_i1_1	1 023.47	839.09	694.98	89.89	74.08	65.41
	上调Up	DN34928_c0_g1_i4_1	237.71	182.14	158.62	19.84	21.50	18.22
	上调Up	DN37635_c0_g1_i7_1	390.62	309.90	252.52	27.24	22.26	25.06
	无差异No-difference	DN29460_c0_g1_i3_1	1.66	2.73	1.54	1.08	0.85	0.93
	无差异No-difference	DN29383_c0_g1_i2_1	16.90	15.95	20.66	9.54	11.30	13.71
	无差异No-difference	DN32877_c0_g1_i3_2	64.45	55.33	49.30	53.18	40.37	68.46
	无差异No-difference	DN32877_c0_g2_i5_2	132.45	137.58	136.64	119.78	124.61	149.95
	无差异No-difference	DN33447_c0_g1_i2_2	99.69	101.66	84.61	93.18	87.51	108.33
	无差异No-difference	DN34032_c0_g1_i1_1	100.37	101.35	92.38	92.69	86.81	93.82
	无差异No-difference	DN34871_c2_g3_i1_2	34.07	41.26	40.60	34.36	36.08	45.69
	无差异No-difference	DN35887_c0_g1_i1_2	133.36	129.04	121.26	96.60	102.29	115.96
	无差异No-difference	DN36570_c0_g2_i4_1	81.57	91.62	87.44	81.32	80.35	109.16
花青素生物合成 Anthocyanin biosynthetic	上调Up	DN36200_c0_g1_i3_1	589.72	479.41	394.60	57.97	47.95	45.82
花青素生物合成 Anthocyanin biosynthetic	无差异No-difference	DN35376_c0_g1_i4_2	31.79	31.73	30.26	37.08	37.12	34.80

KEGG途径 KEGG pathway	基因表达情况（白花为对照） Gene expression（White flower was the control group）	基因ID Gene ID	FPKM
			蓝花 Blue flower			白花 White flower
						1	2	3	1	2	3
类黄酮生物合成 Flavonoid biosynthetic	上调Up	DN26579_c0_g1_i3_2	569.86	469.94	406.45	109.46	98.07	113.44
	上调Up	DN27520_c0_g1_i2_2	341.41	278.90	233.47	38.08	29.97	27.08
	上调Up	DN27888_c0_g1_i1_1	1 023.47	839.09	694.98	89.89	74.08	65.41
	上调Up	DN34928_c0_g1_i4_1	237.71	182.14	158.62	19.84	21.50	18.22
	上调Up	DN37635_c0_g1_i7_1	390.62	309.90	252.52	27.24	22.26	25.06
	无差异No-difference	DN29460_c0_g1_i3_1	1.66	2.73	1.54	1.08	0.85	0.93
	无差异No-difference	DN29383_c0_g1_i2_1	16.90	15.95	20.66	9.54	11.30	13.71
	无差异No-difference	DN32877_c0_g1_i3_2	64.45	55.33	49.30	53.18	40.37	68.46
	无差异No-difference	DN32877_c0_g2_i5_2	132.45	137.58	136.64	119.78	124.61	149.95
	无差异No-difference	DN33447_c0_g1_i2_2	99.69	101.66	84.61	93.18	87.51	108.33
	无差异No-difference	DN34032_c0_g1_i1_1	100.37	101.35	92.38	92.69	86.81	93.82
	无差异No-difference	DN34871_c2_g3_i1_2	34.07	41.26	40.60	34.36	36.08	45.69
	无差异No-difference	DN35887_c0_g1_i1_2	133.36	129.04	121.26	96.60	102.29	115.96
	无差异No-difference	DN36570_c0_g2_i4_1	81.57	91.62	87.44	81.32	80.35	109.16
花青素生物合成 Anthocyanin biosynthetic	上调Up	DN36200_c0_g1_i3_1	589.72	479.41	394.60	57.97	47.95	45.82
花青素生物合成 Anthocyanin biosynthetic	无差异No-difference	DN35376_c0_g1_i4_2	31.79	31.73	30.26	37.08	37.12	34.80

用途 Purpose	基因 Gene	引物序列（5′-3′） Primer sequence	产物长度/bp Product length
内参基因 Reference gene	GAPDH	F：ATGGGTGTGAATGAGAAGGA;R：CGTGGACGGTTGTCATAAG	142
	EF1-β2	F：GAAAGAGAGTGGCAAATCCT;R：GCATCTCGATGCTGCGAA	101
	ACT	F：GCCGTGCTTTCTCTCTATG;R：TTGGGACTGTGTGGCTAAC	82
	β-TUB	F：ATATCCCACCAACTGGACT;R：ACTGTTCTGAAACTCTGCG	90
	UBQ	F：CTTTGCCGGGAAACAATTAG;R：GACGGAGCACCAAGTGAA	86
	AQP	F：CCGGCGGAAAGTCTTATAAT;R：AATGTTTCCACCGAGAAGAG	135
	HIS	F：AGCTTGATACACTGATCAAAGG;R：GGTAGACTTGTTGATGAGTGAC	83
	RPL13	F：TGCAGTTACTCAGCTCACAG;R：CTTTGAAGGCTTTCATCTCCTC	98
鉴定基因 Identification gene	DN26579_c0_g1_i3_2	F：AAGACGTGGATCACGGTTA;R：CTTGAACCTCCCATTGCT	90
	DN27520_c0_g1_i2_2	F：GCACCGAAGAGGAATGCAA;R：GTCGATAACTGGAATCTGCG	86
	DN27888_c0_g1_i1_1	F：GTGGTGGCGGAGATGTTA;R：CCTTTGGTGAGAAACACTCG	83
	DN34928_c0_g1_i4_1	F：CCTAGTGACAAAGGTGCTG;R：TTAGTAAGTCGTCCATGCCG	95
	DN37635_c0_g1_i7_1	F：ATGCCTCCTAGCCTGCTAA;R：TACCAGTTGAACCTGTTTCAGA	84
	DN29460_c0_g1_i3_1	F：ATCAACCTCTCCCTTATAGACC;R：TTTCGTTCTTCTTCCCTGAGT	82
	DN29383_c0_g1_i2_1	F：ACCAAGATCAAGAATGGCG;R：TTGATATTTGTGTCGGTGAGC	122
	DN32877_c0_g1_i3_2	F：GAACGGATCTGTTGTCGC;R：GGTAGCTGGCAAATCTCAAT	126
	DN32877_c0_g2_i5_2	F：TGGCAATGGACATAAACCG;R：CTTTGAACTCAATCTTGTGACC	87
	DN33447_c0_g1_i2_2	F：TGACCACTGGTACAACGC;R：GACTGTAGTCCGATGAAACAC	115
	DN34032_c0_g1_i1_1	F：GGAGGAGGAATCATTTGCC;R：TCAACGAAATGTTGCTTAGCG	117
	DN34871_c2_g3_i1_2	F：ACCAAGATCAAGAATGGCG;R：TTGATATTTGTGTCGGTGAGC	122
	DN35887_c0_g1_i1_2	F：CACCATGATCTTGCCATTAAC;R：CATTAGCTTTCCAGTAGGCG	80
	DN36570_c0_g2_i4_1	F：GAACGGATCTGTTGTCGC;R：AGCCAAAGCCTTGTTAAGT	94
	DN36200_c0_g1_i3_1	F：CAGGAATGTCATCTGTACGG;R：ATGCAACAAGTTACCCAAGTAG	80
	DN35376_c0_g1_i4_2	F：TGCTTCTTCCCTCACCTG;R：TTCTCGTGGCAAATCCCG	130

Selection and Validation of Reference Genes for qRT-PCR Analysis of the Correlated Genes in Flower Pigments Biosynthesis Pathway of Anemone obtusiloba

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内参基因 Reference gene	最大值 Maximum value	最小值 Minimum value	平均值 Mean	标准差 SD	变异系数/% CV
GAPDH	22.86	21.81	22.315	0.381	1.709
EF1-β2	31.99	30.38	31.269	0.614	1.963
ACT	21.77	20.60	21.075	0.349	1.658
β-TUB	27.02	23.03	24.289	1.523	6.270
UBQ	22.96	21.51	22.184	0.586	2.641
AQP	22.06	20.51	21.150	0.599	2.831
HIS	27.82	26.03	27.006	0.714	2.645
RPL13	25.66	23.04	24.303	1.091	4.490

排序 Ranking	geNorm		NormFinder		BestKeeper
排序 Ranking	内参基因 Reference gene	M值 M	内参基因 Reference gene	稳定值 SV	内参基因 Reference genes	标准差 SD
1	UBQ	0.413	UBQ	0.068	ACT	0.282
2	EF1-β2	0.425	AQP	0.071	GAPDH	0.356
3	AQP	0.441	EF1-β2	0.113	AQP	0.531
4	HIS	0.460	HIS	0.160	UBQ	0.567
5	GAPDH	0.535	ACT	0.287	EF1-β2	0.574
6	ACT	0.543	GAPDH	0.311	HIS	0.678
7	RPL13	0.644	RPL13	0.325	RPL13	1.055
8	β-TUB	1.128	β-TUB	0.784	β-TUB	1.283

排序 Ranking	内参基因 Reference gene	几何平均数 Geomean of ranking values
1	UBQ	1.587
2	AQP	2.621
3	EF1-β2	3.107
3	GAPDH	3.107
5	ACT	3.915
6	HIS	4.579
7	RPL13	7.000
8	β-TUB	8.000

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