Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis in Neolamarckia cadamba

doi:10.11983/CBB18003

Abstract

Abstract: In this study, root, shoot, leaf, flower, fruit, peel and cambium tissue of Neolamarckia cadamba was sampled to analyze the expression of 43 candidate reference genes in 21 housekeeping gene families, such as ACT, CAC, CYP, and EF1α, by RT-qPCR. The software geNorm, NormFinder and BestKeeper were used to analyze expression stability of these candidate reference genes in the seven different tissues. geNorm analysis revealed that the stability of the UPL gene was the highest (M=0.443) and the stability of UBQ was the lowest (M=2.859). NormFinder analysis revealed that the stability of UPL was the highest (E=0.223), UBQ was the lowest (M=4.759). BestKeeper analysis revealed that the standard deviation of UPL (SD=0.513) was the lowest. These findings suggest that UPL has the highest stability, and UBQ has the lowest stability. UPL gene could be selected as an internal reference gene for analysis of gene expression among different tissues of N. cadamba.

Key words: Neolamarckia cadamba, real-time quantitative PCR, reference gene

Zhang Deng, Li Jingjian, Zhang Mengjie, Bao Yutao, Yang Xiao, Xu Wuyun, Ouyang Kunxi, Chen Xiaoyang. Selection and Validation of Reference Genes for Quantitative RT-PCR Analysis in Neolamarckia cadamba[J]. Chinese Bulletin of Botany, 2018, 53(6): 829-839.

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URL: https://www.chinbullbotany.com/EN/10.11983/CBB18003

https://www.chinbullbotany.com/EN/Y2018/V53/I6/829

Figures/Tables 11

References 28

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Tissues	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀	Yield (ng?μL^-1)
Root	2.19	2.1	107.5
Bud	2.12	2.14	410.6
Leaf	2.13	1.96	258.5
Flower	2.19	2.22	114.8
Fruit	2.11	1.84	156.8
Bark	2.17	1.89	299.7
Cambium	2.14	2.11	331.8

Tissues	A₂₆₀/A₂₈₀	A₂₆₀/A₂₃₀	Yield (ng?μL^-1)
Root	2.19	2.1	107.5
Bud	2.12	2.14	410.6
Leaf	2.13	1.96	258.5
Flower	2.19	2.22	114.8
Fruit	2.11	1.84	156.8
Bark	2.17	1.89	299.7
Cambium	2.14	2.11	331.8

Name	UniGene ID	Reference gene (Rg) ID	F primer (5'-3') R primer (5'-3')	Amplicator length (bp)
Actin	comp52737_c0	g1	TGTAGTGGATGAATGCTTCTGTTAT	95
	comp52737_c0	g1	CTTCCTCCTACCAACTTCAAATG	95
	comp79635_c0	g2	CTTCTGAGGTTATGGAGCAATCT	101
	comp79635_c0	g2	CGATAAATCAAAACTTCAAGCC	101
Clathrin adaptor complexes medium	comp48976_c0	g3	CTCAGAGAACGCTGCTGACTAC	161
Clathrin adaptor complexes medium	comp48976_c0	g3	GAGCCAAGGGAAACAAGATAA	161
Cyclophilin	comp67418_c0	g4	GGGGTCTCACGCTCTTTACT	83
	comp67418_c0	g4	GGATTGGATTGGGTTGGTT	83
	comp75463_c0	g5	CCCCAGCAAGAAGACCACT	213
	comp75463_c0	g5	TTGACCATGAATCCCAACCA	213
	comp77969_c0	g6	ATAGCATCCCAACCGAACA	187
	comp77969_c0	g6	CCCTCTTGCCTCCTGTGTAT	187
Elongation factor 1α	comp87079_c1	g7	ACCAGCATCACCGTTCTTCA	123
	comp87079_c1	g7	GTCCTCGATTGCCACACCT	123
	comp87526_c0	g8	AATCAGACAGAAACCCCTCAA	245
	comp87526_c0	g8	GAACCTCTCAATCACACGCTT	245
Eukaryotic initiation factor	comp6386_c0	g9	GTTGAAACTTCTTGGACATCG	250
Eukaryotic initiation factor	comp6386_c0	g9	CTTGAGACACTGATTTGTATGAGA	250
Farnesyl pyrophosphate synthase 1	comp72548_c0	g10	TGATAATCTGGCTTCCACCTT	112
	comp72548_c0	g10	TGGGAGGAACTCAATCTCCTAC	112
	comp75377_c0	g11	TATCAGGCTCAGCATTCCACT	212
	comp75377_c0	g11	TTGCCACAATAACACATCCAT	212
F-boxkelch-repeat protein	comp78454_c0	g12	AAGGCCAATTCTGTTCAAGC	143
	comp78454_c0	g12	CCTAGAGGGAAAGACATGACTG	143
	comp78817_c0	g13	GCAAACGGGGTAAAAGGA	102
	comp78817_c0	g13	AAAGGGTAAGAGTGACGACAGC	102
GAPDH	comp78593_c0	g14	TGTTCCAAGTGGGCATTTAC	247
	comp78593_c0	g14	CGCTCTGAGGTGTTAATAAGTG	247
	comp80828_c1	g15	CTGAGCATTTTTTAGGCTTGTC	151
	comp80828_c1	g15	TCAGATTCATGTGGCAGTCG	151
GTP-binding nuclear protein	comp85262_c0	g16	TCTCGCAACCTGCCTCTT	257
GTP-binding nuclear protein	comp85262_c0	g16	TATCACTCCCATCTTCGCAC	257
Phosphoenolpyruvate carboxylase-related kinase 1	comp75525_c0	g17	CGACCTCACATTCCTCATTAC	291
	comp75525_c0	g17	ACATAGACCATCCAGAGCCCA	291
	comp80613_c0	g18	TACATAGACCATCCAGAGCCA	112
	comp80613_c0	g18	GCAAAAGGGCAAGCAACAG	112
Protein phosphatase 2A	comp81334_c1	g19	GGGCTTTCCATCCCATACC	128
	comp81334_c1	g19	AGCCTTAGGGGGATTGGAA	128
	comp52412_c0	g20	ATGTTGGATGATATTAGTGGTGTG	161
	comp52412_c0	g20	TCATAGGAAAATAGACCTCTGGTT	161
Ribosomal protein L	comp46755_c0	g21	CTGAGGATTGTTAGCAGTTGAC	119
	comp46755_c0	g21	ACCAGAAAACAGACCACCTAAG	119
	comp52434_c0	g22	AAGGAAGGTAAAGCAGGGAA	177
	comp52434_c0	g22	GCATGGGCAGGGATATAAAC	177
	comp87976_c0	g23	CACGCAGCATAGCCAAAC	157
	comp87976_c0	g23	AGGCAGTTCTCTGATTCTTTTG	157
表2 (续) Table 2 (continued)
Name	UniGene ID	Reference gene (Rg) ID	F primer (5'-3') R primer (5'-3')	Amplicator length (bp)
Ribosomal protein S	comp65909_c1	g24	GCTATGGTAGTCTCCCGAAAG	182
	comp65909_c1	g24	GGGGGAACAAGACTAAGGGT	182
	comp67276_c0	g25	TTTTGTTTCCCCTCTTTGC	97
	comp67276_c0	g25	AACCTTGAACAACCTGTGTAGAA	97
	comp71526_c0	g26	CGGTTACACAAGGTTGAATGA	117
	comp71526_c0	g26	AGAGGGTCTGGATTTGAGTGA	117
Ribulose 1,5-bisphosphate carboxylase	comp47386_c0	g27	CAGCACCGTAATCCATAAAAC	226
	comp47386_c0	g27	CAAGCAGCCCAGCAAGTC	226
	comp88001_c0	g28	ACAGGATGGGTAGAAAGAGGC	210
	comp88001_c0	g28	AGGATTGAGCCGAATACAACG	210
S-adenosylmethionine decarboxylase	comp44802_c0	g29	TCTTCGTGGCACTTCTCTCC	133
	comp44802_c0	g29	ACAGGGTGTTGACTTGTTTCC	133
	comp71874_c0	g30	ATAAGGTCTCTTCTTGTTCGTGTAG	178
	comp71874_c0	g30	GACTGAACAGCAACAGGAATAAT	178
	comp80075_c0	g31	GCTGCCTGTGGGTCTCCTA	85
	comp80075_c0	g31	GTAAACCCCAATGCTACTCCT	85
Translation elongation factor	comp65909_c1	g32	GCTATGGTAGTCTCCCGAAAG	184
	comp65909_c1	g32	CTGGGGGAACAAGACTAAGG	184
	comp70791_c0	g33	TCAACCAACCGTTCCTACC	195
	comp70791_c0	g33	ACAACAGTCCTTTGCCACC	195
Tubulin α	comp70323_c2	g34	GGTGGTGGAACTGGCTCTG	217
	comp70323_c2	g34	GGCAAATGTCATAGATGGCTT	217
	comp76448_c4	g35	AAGGAGGGAATGAGTGGAG	107
	comp76448_c4	g35	ACTATGGCAAGAAGTCAAAGC	107
Tubulin B	comp66056_c0	g36	GCAAGAAAGCCTTCCTCCTAA	153
	comp66056_c0	g36	TTCCCAACAATGTCAAATCAA	153
	comp79707_c1	g37	TTCAGGAGAGTCAGCGAGC	187
	comp79707_c1	g37	CATCGTCTTCATATTCCCCTT	187
Ubiquitin conjugating enzyme	comp79182_c1	g38	TCCTTGCTTGTGGCGTCA	213
Ubiquitin conjugating enzyme	comp79182_c1	g38	CACGGGTGTCAAATCTGGC	213
Ubiquitin	comp67366_c0	g39	GACGGGAGGACCTTAGCA	298
	comp67366_c0	g39	CTCGGAGACGGAGAACAA	298
	comp82561_c0	g40	GCATTTGTGTCTTGCCTCTTTAT	186
	comp82561_c0	g40	GCGATGAGCAACATTCCTTTA	186
	comp68357_c0	g41	TTTTTCAGCAAAGAACAACCG	135
	comp68357_c0	g41	TGAAGACCCTCACTGGAAAGA	135
Ubiquitin-protein ligase	comp87122_c0	g42	GGTTGGTGGTAGAGTTGTGACTC	182
	comp87122_c0	g42	CGAGCACTACCACGACACG	182
	comp87211_c0	g43	GCCCCTCCGTTAAACTCG	122
	comp87211_c0	g43	GCCATACTCCCACCGAAAT	122

Name	UniGene ID	Reference gene (Rg) ID	F primer (5'-3') R primer (5'-3')	Amplicator length (bp)
Actin	comp52737_c0	g1	TGTAGTGGATGAATGCTTCTGTTAT	95
	comp52737_c0	g1	CTTCCTCCTACCAACTTCAAATG	95
	comp79635_c0	g2	CTTCTGAGGTTATGGAGCAATCT	101
	comp79635_c0	g2	CGATAAATCAAAACTTCAAGCC	101
Clathrin adaptor complexes medium	comp48976_c0	g3	CTCAGAGAACGCTGCTGACTAC	161
Clathrin adaptor complexes medium	comp48976_c0	g3	GAGCCAAGGGAAACAAGATAA	161
Cyclophilin	comp67418_c0	g4	GGGGTCTCACGCTCTTTACT	83
	comp67418_c0	g4	GGATTGGATTGGGTTGGTT	83
	comp75463_c0	g5	CCCCAGCAAGAAGACCACT	213
	comp75463_c0	g5	TTGACCATGAATCCCAACCA	213
	comp77969_c0	g6	ATAGCATCCCAACCGAACA	187
	comp77969_c0	g6	CCCTCTTGCCTCCTGTGTAT	187
Elongation factor 1α	comp87079_c1	g7	ACCAGCATCACCGTTCTTCA	123
	comp87079_c1	g7	GTCCTCGATTGCCACACCT	123
	comp87526_c0	g8	AATCAGACAGAAACCCCTCAA	245
	comp87526_c0	g8	GAACCTCTCAATCACACGCTT	245
Eukaryotic initiation factor	comp6386_c0	g9	GTTGAAACTTCTTGGACATCG	250
Eukaryotic initiation factor	comp6386_c0	g9	CTTGAGACACTGATTTGTATGAGA	250
Farnesyl pyrophosphate synthase 1	comp72548_c0	g10	TGATAATCTGGCTTCCACCTT	112
	comp72548_c0	g10	TGGGAGGAACTCAATCTCCTAC	112
	comp75377_c0	g11	TATCAGGCTCAGCATTCCACT	212
	comp75377_c0	g11	TTGCCACAATAACACATCCAT	212
F-boxkelch-repeat protein	comp78454_c0	g12	AAGGCCAATTCTGTTCAAGC	143
	comp78454_c0	g12	CCTAGAGGGAAAGACATGACTG	143
	comp78817_c0	g13	GCAAACGGGGTAAAAGGA	102
	comp78817_c0	g13	AAAGGGTAAGAGTGACGACAGC	102
GAPDH	comp78593_c0	g14	TGTTCCAAGTGGGCATTTAC	247
	comp78593_c0	g14	CGCTCTGAGGTGTTAATAAGTG	247
	comp80828_c1	g15	CTGAGCATTTTTTAGGCTTGTC	151
	comp80828_c1	g15	TCAGATTCATGTGGCAGTCG	151
GTP-binding nuclear protein	comp85262_c0	g16	TCTCGCAACCTGCCTCTT	257
GTP-binding nuclear protein	comp85262_c0	g16	TATCACTCCCATCTTCGCAC	257
Phosphoenolpyruvate carboxylase-related kinase 1	comp75525_c0	g17	CGACCTCACATTCCTCATTAC	291
	comp75525_c0	g17	ACATAGACCATCCAGAGCCCA	291
	comp80613_c0	g18	TACATAGACCATCCAGAGCCA	112
	comp80613_c0	g18	GCAAAAGGGCAAGCAACAG	112
Protein phosphatase 2A	comp81334_c1	g19	GGGCTTTCCATCCCATACC	128
	comp81334_c1	g19	AGCCTTAGGGGGATTGGAA	128
	comp52412_c0	g20	ATGTTGGATGATATTAGTGGTGTG	161
	comp52412_c0	g20	TCATAGGAAAATAGACCTCTGGTT	161
Ribosomal protein L	comp46755_c0	g21	CTGAGGATTGTTAGCAGTTGAC	119
	comp46755_c0	g21	ACCAGAAAACAGACCACCTAAG	119
	comp52434_c0	g22	AAGGAAGGTAAAGCAGGGAA	177
	comp52434_c0	g22	GCATGGGCAGGGATATAAAC	177
	comp87976_c0	g23	CACGCAGCATAGCCAAAC	157
	comp87976_c0	g23	AGGCAGTTCTCTGATTCTTTTG	157
表2 (续) Table 2 (continued)
Name	UniGene ID	Reference gene (Rg) ID	F primer (5'-3') R primer (5'-3')	Amplicator length (bp)
Ribosomal protein S	comp65909_c1	g24	GCTATGGTAGTCTCCCGAAAG	182
	comp65909_c1	g24	GGGGGAACAAGACTAAGGGT	182
	comp67276_c0	g25	TTTTGTTTCCCCTCTTTGC	97
	comp67276_c0	g25	AACCTTGAACAACCTGTGTAGAA	97
	comp71526_c0	g26	CGGTTACACAAGGTTGAATGA	117
	comp71526_c0	g26	AGAGGGTCTGGATTTGAGTGA	117
Ribulose 1,5-bisphosphate carboxylase	comp47386_c0	g27	CAGCACCGTAATCCATAAAAC	226
	comp47386_c0	g27	CAAGCAGCCCAGCAAGTC	226
	comp88001_c0	g28	ACAGGATGGGTAGAAAGAGGC	210
	comp88001_c0	g28	AGGATTGAGCCGAATACAACG	210
S-adenosylmethionine decarboxylase	comp44802_c0	g29	TCTTCGTGGCACTTCTCTCC	133
	comp44802_c0	g29	ACAGGGTGTTGACTTGTTTCC	133
	comp71874_c0	g30	ATAAGGTCTCTTCTTGTTCGTGTAG	178
	comp71874_c0	g30	GACTGAACAGCAACAGGAATAAT	178
	comp80075_c0	g31	GCTGCCTGTGGGTCTCCTA	85
	comp80075_c0	g31	GTAAACCCCAATGCTACTCCT	85
Translation elongation factor	comp65909_c1	g32	GCTATGGTAGTCTCCCGAAAG	184
	comp65909_c1	g32	CTGGGGGAACAAGACTAAGG	184
	comp70791_c0	g33	TCAACCAACCGTTCCTACC	195
	comp70791_c0	g33	ACAACAGTCCTTTGCCACC	195
Tubulin α	comp70323_c2	g34	GGTGGTGGAACTGGCTCTG	217
	comp70323_c2	g34	GGCAAATGTCATAGATGGCTT	217
	comp76448_c4	g35	AAGGAGGGAATGAGTGGAG	107
	comp76448_c4	g35	ACTATGGCAAGAAGTCAAAGC	107
Tubulin B	comp66056_c0	g36	GCAAGAAAGCCTTCCTCCTAA	153
	comp66056_c0	g36	TTCCCAACAATGTCAAATCAA	153
	comp79707_c1	g37	TTCAGGAGAGTCAGCGAGC	187
	comp79707_c1	g37	CATCGTCTTCATATTCCCCTT	187
Ubiquitin conjugating enzyme	comp79182_c1	g38	TCCTTGCTTGTGGCGTCA	213
Ubiquitin conjugating enzyme	comp79182_c1	g38	CACGGGTGTCAAATCTGGC	213
Ubiquitin	comp67366_c0	g39	GACGGGAGGACCTTAGCA	298
	comp67366_c0	g39	CTCGGAGACGGAGAACAA	298
	comp82561_c0	g40	GCATTTGTGTCTTGCCTCTTTAT	186
	comp82561_c0	g40	GCGATGAGCAACATTCCTTTA	186
	comp68357_c0	g41	TTTTTCAGCAAAGAACAACCG	135
	comp68357_c0	g41	TGAAGACCCTCACTGGAAAGA	135
Ubiquitin-protein ligase	comp87122_c0	g42	GGTTGGTGGTAGAGTTGTGACTC	182
	comp87122_c0	g42	CGAGCACTACCACGACACG	182
	comp87211_c0	g43	GCCCCTCCGTTAAACTCG	122
	comp87211_c0	g43	GCCATACTCCCACCGAAAT	122

Rg ID	M	Rg ID	M	Rg ID	M	Rg ID	M	Rg ID	M
g25	0.443	g23	1.142	g36	1.649	g6	2.045	g40	2.365
g42	0.443	g31	1.183	g34	1.693	g4	2.080	g17	2.412
g21	0.471	g24	1.229	g28	1.737	g2	2.113	g16	2.458
g33	0.565	g13	1.287	g7	1.789	g32	2.148	g38	2.503
g15	0.704	g22	1.348	g12	1.836	g39	2.184	g5	2.566
g43	0.862	g14	1.404	g10	1.881	g19	2.217	g27	2.652
g29	0.966	g26	1.467	g11	1.927	g37	2.251	g41	2.859
g18	1.014	g9	1.533	g30	1.967	g1	2.285
g8	1.090	g35	1.597	g3	2.007	g20	2.321