钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

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

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

钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

马璐琳^1,2,*, 段青^1,*, 崔光芬¹, 杜文文¹, 贾文杰¹, 王祥宁¹, 王继华¹^,^**(), 陈发棣²^,^**()

¹云南省农业科学院花卉研究所,云南省花卉育种重点实验室,国家观赏园艺工程技术研究中心,昆明 650205
²南京农业大学园艺学院,南京 210095

收稿日期:2020-09-27 修回日期:2020-12-10 出版日期:2021-02-25 发布日期:2021-03-09
通讯作者: 王继华,陈发棣 E-mail:wangjh0505@sohu.com;chenfd@njau.edu.cn
基金资助:
国家自然科学基金项目(31960612);云南省农业科学院应用基础研究重点项目(YJZ201701);云南省科技领军人才计划项目(2016HA005);云南省现代农业花卉苗木产业技术体系建设项目(2017KJTX0010);云南省重大科技专项计划项目(2019ZG006)

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

摘要/Abstract

摘要：

为了筛选适合于钝裂银莲花类黄酮/花青素合成途径中相关基因qRT-PCR表达分析时的内参基因,根据钝裂银莲花蓝/白不同花色花器官组织的转录组测序结果,选取了多聚泛素酶基因（polyubiquitin,UBQ）、微管蛋白基因（β-tubulin,β-TUB）、水通道蛋白基因（aquaporin,AQP）、肌动蛋白基因（actin,ACT）、甘油醛-3-磷酸-脱氢酶基因（glyceraldehyde-3-phosphate dehydrogenase,GAPDH）、组蛋白基因（histone,HIS）、转录延伸因子基因（elongation factor 1-β,EF-1β）和60S核糖体蛋白基因（60S ribosomal protein L13-1,RPL13）等8个常用内参基因作为候选基因,以钝裂银莲花的叶片、茎杆和蓝/白色花器官等不同组织为试验材料,通过qRT-PCR检测这8个候选内参基因的表达情况,利用geNorm、NormFinder和BestKeeper等软件对其稳定性进行分析评价。结果表明：8个候选内参基因中,UBQ表现最稳定,而β-TUB相对稳定性最差。以最稳定的UBQ为内参对钝裂银莲花类黄酮/花青素合成途径中16个相关基因表达情况进行qRT-PCR分析,结果与前期转录组测序结果一致。UBQ为钝裂银莲花花色素合成途径相关基因表达分析的最适内参基因。

关键词: 钝裂银莲花, 实时荧光定量PCR, 内参基因, 筛选, 稳定性, 类黄酮/花青素合成途径

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

中图分类号:

S682

马璐琳, 段青, 崔光芬, 杜文文, 贾文杰, 王祥宁, 王继华, 陈发棣. 钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选[J]. 园艺学报, 2021, 48(2): 377-388.

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.

图/表 8

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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

钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

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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钝裂银莲花花色素合成相关基因qRT-PCR内参基因的筛选

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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摘要/Abstract

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图/表 8

参考文献 41

相关文章 15

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