Comparison of Five Endogenous Reference Genes for Specific PCR Detection and Quantification of Rice

doi:10.1016/j.rsci.2019.04.005

摘要/Abstract

. [J]. Rice Science, 2019, 26(4): 248-256.

图/表 12

参考文献 21

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Species	GM variety	Non-GM variety
Rice	Huahui 1, II Kefeng 6, Kefeng 6, Bar 68-1, Wan 21B, Kangyou 97	II Minghui 86, Minghui 86, Minghui 63, Shanyou 10, Shanyou 63, 80-4B, Donglong, Songjing 6, Zhonghua 11, Zhongzuo 58, Sanjiang 1, Liaojing 371
Cotton	LLcotton25, Mon15985, Mon1445, Mon88913	Non-GM variety
Maize	Bt176, TC1507, Mon810, Mon863	Non-GM variety
Soybean	Mon89788, A5547-127, GTS40-3-2	Non-GM variety
Beet	H7-1	Non-GM variety
Rapeseed	GT73, ToPas19/2
Papaya	Huanong 1
Tomato		Tomato
Potato		Potato
Gramineous plant		Wheat, oats, barley, mung bean, buckwheat
Model plant		Arabidopsis, cauliflower, tobacco
GM, Genetically modified.

Species	GM variety	Non-GM variety
Rice	Huahui 1, II Kefeng 6, Kefeng 6, Bar 68-1, Wan 21B, Kangyou 97	II Minghui 86, Minghui 86, Minghui 63, Shanyou 10, Shanyou 63, 80-4B, Donglong, Songjing 6, Zhonghua 11, Zhongzuo 58, Sanjiang 1, Liaojing 371
Cotton	LLcotton25, Mon15985, Mon1445, Mon88913	Non-GM variety
Maize	Bt176, TC1507, Mon810, Mon863	Non-GM variety
Soybean	Mon89788, A5547-127, GTS40-3-2	Non-GM variety
Beet	H7-1	Non-GM variety
Rapeseed	GT73, ToPas19/2
Papaya	Huanong 1
Tomato		Tomato
Potato		Potato
Gramineous plant		Wheat, oats, barley, mung bean, buckwheat
Model plant		Arabidopsis, cauliflower, tobacco
GM, Genetically modified.

Gene	Primer	Primer sequence (5′-3′)	Product size (bp)	Annealing temperature (ºC)	Reference
PLD	PLD-F PLD-R PLD-P	TGGTGAGCGTTTTGCAGTCT CTGATCCACTAGCAGGAGGTCC FAM-GTTGTGCTGCCAATGTGGCCTG-TAMRA	68	58	JRC, 2006
	PLD-F1 PLD-R1	CGGCTTGGGATGTGCTCTA TGGATGCTCCTGTCAATGGTG	268	54
	PLD-F2 PLD-R2	CTGAGTGTCGTCTGTGGGTGTTCC CCGTGCTAAAGATGGGTGAGG	245	58
	PLD-F3 PLD-R3	GAACAACACCATTGACAGGAGCAT CCACATTGGCAGCACAACATAGA	241	56
SPS	SPS-F SPS-R SPS-P	CGGTTGATCTTTTCGGGATG TTGCGCCTGAACGGATAT FAM-GACGCACGGACGGCTCGGA-TAMRA	81	54	Ding et al, 2004
	SPS-F1 SPS-R1	TTGCGCCTGAACGGATAT GGAGAAGCACTGGACGAGG	277	54	Jiang et al, 2009
	SPS-F2 SPS-R2	GCCATGGATTACATATGGCAAGA ATCTGTTTACTCGTCAAGTGTCATCTC	287	56
RBE4	RBE4-F RBE4-R RBE4-P	GTTTTAGTTGGGTGAAAGCGGTT CCTGTTAGTTCTTCCAATGCCCTTA FAM-TCTGGTTGGGAATAGATACT-MGBNFQ	106	54	Jeong et al, 2007
	RBE4-F1 RBE4-R1	TCCAATGCCCTTAAACTTTATCA AACGATCTAATTCTGGGCTTTAT	206	54	Jeong et al, 2007
	RBE4-F2 RBE4-R2	AGGGCATTGGAAGAACTAACAG CACCACCATCTTGAACAGGAAT	264	54
	RBE4-F3 RBE4-R3	TAGTTGGGTGAAAGCGGTTAG CACCACCATCTTGAACAGGAAT	343	54
GOS9	GOS9-F GOS9-R GOS9-P	TTAGCCTCCCGCTGCAGA AGAGTCCACAAGTGCTCCCG FAM-CGGCAGTGTGGTTGGTTTCTTCGG-TAMRA	68	56	Hernández et al, 2005
	GOS9-F1 GOS9-R1	ACTTACGGGATGGTCTTTGCA GCAATCAATCCATTACCTGCTC	255	54
	GOS9-F2 GOS9-R2	TAAGTTGCTTCGACGTACAGGA TAGATGACGGTGACGAGGATAGAGT	303	56
	GOS9-F3 GOS9-R3	GCTGCCCGTTAATACATGTTCCT CTAAAAGCAGTTCCACCACCTTGT	301	56
PEPC	PEPC-F PEPC-R PEPC-P	TAGGAATCACGGATACGCA TGAACTCAGGTTGCTGGAC FAM-AGGGAGATCCTTACTTGAGGCAGAGAC-TAMRA	80	54
	PEPC-F1 PEPC-R1	GGTCCCTGATACGCTTTA TCTGCCCACTCACTGCTA	153	54
	PEPC-F2 PEPC-R2	GCCGTTGTCGCTTGTTGTGTTC CAGAGACTGCGTATCCGTGATTCCT	183	60
	PEPC-F3 PEPC-R3	TCCCTCCAGAAGGTCTTTGTGTC GCTGGCAACTGGTTGGTAATG	271	56

Gene	Primer	Primer sequence (5′-3′)	Product size (bp)	Annealing temperature (ºC)	Reference
PLD	PLD-F PLD-R PLD-P	TGGTGAGCGTTTTGCAGTCT CTGATCCACTAGCAGGAGGTCC FAM-GTTGTGCTGCCAATGTGGCCTG-TAMRA	68	58	JRC, 2006
	PLD-F1 PLD-R1	CGGCTTGGGATGTGCTCTA TGGATGCTCCTGTCAATGGTG	268	54
	PLD-F2 PLD-R2	CTGAGTGTCGTCTGTGGGTGTTCC CCGTGCTAAAGATGGGTGAGG	245	58
	PLD-F3 PLD-R3	GAACAACACCATTGACAGGAGCAT CCACATTGGCAGCACAACATAGA	241	56
SPS	SPS-F SPS-R SPS-P	CGGTTGATCTTTTCGGGATG TTGCGCCTGAACGGATAT FAM-GACGCACGGACGGCTCGGA-TAMRA	81	54	Ding et al, 2004
	SPS-F1 SPS-R1	TTGCGCCTGAACGGATAT GGAGAAGCACTGGACGAGG	277	54	Jiang et al, 2009
	SPS-F2 SPS-R2	GCCATGGATTACATATGGCAAGA ATCTGTTTACTCGTCAAGTGTCATCTC	287	56
RBE4	RBE4-F RBE4-R RBE4-P	GTTTTAGTTGGGTGAAAGCGGTT CCTGTTAGTTCTTCCAATGCCCTTA FAM-TCTGGTTGGGAATAGATACT-MGBNFQ	106	54	Jeong et al, 2007
	RBE4-F1 RBE4-R1	TCCAATGCCCTTAAACTTTATCA AACGATCTAATTCTGGGCTTTAT	206	54	Jeong et al, 2007
	RBE4-F2 RBE4-R2	AGGGCATTGGAAGAACTAACAG CACCACCATCTTGAACAGGAAT	264	54
	RBE4-F3 RBE4-R3	TAGTTGGGTGAAAGCGGTTAG CACCACCATCTTGAACAGGAAT	343	54
GOS9	GOS9-F GOS9-R GOS9-P	TTAGCCTCCCGCTGCAGA AGAGTCCACAAGTGCTCCCG FAM-CGGCAGTGTGGTTGGTTTCTTCGG-TAMRA	68	56	Hernández et al, 2005
	GOS9-F1 GOS9-R1	ACTTACGGGATGGTCTTTGCA GCAATCAATCCATTACCTGCTC	255	54
	GOS9-F2 GOS9-R2	TAAGTTGCTTCGACGTACAGGA TAGATGACGGTGACGAGGATAGAGT	303	56
	GOS9-F3 GOS9-R3	GCTGCCCGTTAATACATGTTCCT CTAAAAGCAGTTCCACCACCTTGT	301	56
PEPC	PEPC-F PEPC-R PEPC-P	TAGGAATCACGGATACGCA TGAACTCAGGTTGCTGGAC FAM-AGGGAGATCCTTACTTGAGGCAGAGAC-TAMRA	80	54
	PEPC-F1 PEPC-R1	GGTCCCTGATACGCTTTA TCTGCCCACTCACTGCTA	153	54
	PEPC-F2 PEPC-R2	GCCGTTGTCGCTTGTTGTGTTC CAGAGACTGCGTATCCGTGATTCCT	183	60
	PEPC-F3 PEPC-R3	TCCCTCCAGAAGGTCTTTGTGTC GCTGGCAACTGGTTGGTAATG	271	56

	Reaction system (µL)					Reaction program
	2×FastStart Universal Probe Master	dNTP (10 Mm)	Primer Each (10 µM)	Probe (10 µM)	DNA (50 ng/μL)	Step1	Step 2		Cycles of Step 2
SPS-F/R/P	10	0.4	0.8	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	40
PLD-F/R/P	10	0.4	0.4	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	45
RBE4-F/R/P	10	1.0	1.0	0.4	1.0	95 °C 10 min	95 °C 30 s	60 °C 60 s	40
GOS-F/R/P	10	1.6	0.6	0.4	1.0	95 °C 10 min	95 °C 30 s	60 °C 60 s	40
PEPC-F/R/P	10	0.4	0.8	0.4	1.0	95 °C 10 min	95 °C 15 s	60 °C 60 s	40

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图/表 12

参考文献 21

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Metrics

Primer	Annealing temperature (ºC)	Final concentration (µmol/L)
GOS9-F3/R3	58	0.1
SPS-F1/R1	56	0.4
SPS-F2/R2	58	0.1
PEPC-F1/R1	56	0.2
PEPC-F3/R3	58	0.1

Species	Variety	PLD	SPS	GOS9	RBE4	PEPC
Cotton	LLcotton25	39.60	-	-	-	-
Maize	Non-GM variety	-	39.69	36.96/37.76/37.79	-	39.31
	TC1507	-	-	-	38.28/36.97/36.80	-
	Mon863	-	-	-	38.51	-
Soybean	Non-GM variety	-	-	38.98	38.19	-
	Mon89788	-	39.31	-	39.19	-
	GTS40-3-2	-	-	-	-	39.14
Rapeseed	GT73	-	-	-	39.05	-
Beet	Non-GM variety	39.36/38.50/38.42	-	38.16/39.81/38.19	38.28/37.99/37.43	39.98
	H7-1	-	-	39.28	-	-
Tomato	Non-GM variety	-	-	39.94/37.68	-	-
Oat	Non-GM variety	-	-	38.03	-	-
Barley	Non-GM variety	-	-	38.29	-	-
Tobacco	Non-GM variety	-	-	36.58/37.48/38.54	-	-
Cauliflower	Non-GM variety	-	-	39.49	38.19	-
Potato	Non-GM variety	-	-	39.55	39.35	-
Buck wheat	Non-GM variety	-	-	39.26	39.24	39.28
Arabidopsis	Non-GM variety	-	-	39.32/39.51	37.27/38.30/37.68	-
Rice	Kefeng 6	30.11/30.09/29.92	33.86/34.21/34.43	30.16/30.00/30.07	29.79/29.88/29.67	31.02/30.76/31.43
‘-’ means data are not available. Species with no Ct values for all the genes are not shown.

Gene	Parameter	Ct value						R²	Efficiency (%)
Gene	Parameter	50 ng (1.06 × 10⁵)	5 ng (1.06 × 10⁴)	0.5 ng (1.06 × 10³)	0.05 ng (106)	0.005 ng (10.6)	0.0025 ng (5.03)	R²	Efficiency (%)
PLD	Mean ± SD	21.90 ± 0.10	25.27 ± 0.08	28.64 ± 0.13	32.11 ± 0.10	35.41 ± 0.59	37.09 ± 0.75	0.997	94.90
	RSD	0.432	0.314	0.469	0.299	1.665	2.015
SPS	Mean ± SD	25.05 ± 0.02	28.05 ± 0.15	31.45 ± 0.10	34.81 ± 0.16	37.85 ± 0.17	39.29 ± 0.30	0.998	101.90
	RSD	0.061	0.054	0.322	0.468	0.451	0.775
PEPC	Mean ± SD	21.17 ± 0.01	24.55 ± 0.08	28.06 ± 0.01	31.34 ± 0.12	34.79 ± 0.25	35.86 ± 0.23	0.999	96.90
	RSD	0.047	0.333	0.036	0.373	0.725	0.064
RSD, Relative standard deviation. Data in the parenthesis are the copy number of DNA.

Sample	Gene	Positive Number				Average
Sample	Gene	1	2	3	4	Average
Huahui 1	RBE4	1142	1136	1104	1109	1122.75
Huahui 1	PEPC	1276	1251	1249	1234	1252.5
Wan 21B	RBE4	1006	1014	1036	1033	1022.25
Wan 21B	PEPC	989	975	992	1005	990.25

Variety	PLD	SPS	PEPC
Huahui 1	26.98	29.44	27.77
II Kefeng 6	27.01	29.49	27.73
II Minghui 86	26.58	29.08	27.32
Shanyou 10	27.27	29.97	28.08
Wan 21B	27.45	29.94	28.15
Shanyou 63	27.10	29.80	27.69
Kangyou 97	27.01	29.61	27.62
80-4B	27.28	29.98	27.99
Minghui 86	26.15	28.56	27.01
Bar 68-1	26.58	29.11	27.47
Donglong	26.60	29.10	27.29
Kefeng 6	26.50	29.22	27.41
Minghui 63	27.04	29.82	28.12
Songjing 6	26.09	29.32	27.51
Zhonghua 11	26.66	29.31	27.56
Zhongzuo 58	27.27	29.78	27.91
Sanjiang 1	27.17	29.52	27.82
Liaojing 371	27.05	29.57	27.59
Average	26.92	29.48	27.67
SD value	0.34	0.38	0.31
RSD (%)	1.26	1.29	1.13
RSD, Relative standard deviation.

Variety	PLD	SPS	PEPC
Huahui 1	26.98	29.44	27.77
II Kefeng 6	27.01	29.49	27.73
II Minghui 86	26.58	29.08	27.32
Shanyou 10	27.27	29.97	28.08
Wan 21B	27.45	29.94	28.15
Shanyou 63	27.10	29.80	27.69
Kangyou 97	27.01	29.61	27.62
80-4B	27.28	29.98	27.99
Minghui 86	26.15	28.56	27.01
Bar 68-1	26.58	29.11	27.47
Donglong	26.60	29.10	27.29
Kefeng 6	26.50	29.22	27.41
Minghui 63	27.04	29.82	28.12
Songjing 6	26.09	29.32	27.51
Zhonghua 11	26.66	29.31	27.56
Zhongzuo 58	27.27	29.78	27.91
Sanjiang 1	27.17	29.52	27.82
Liaojing 371	27.05	29.57	27.59
Average	26.92	29.48	27.67
SD value	0.34	0.38	0.31
RSD (%)	1.26	1.29	1.13
RSD, Relative standard deviation.