Screening of Reference Genes for Quantitative Real-time PCR of Rubber Saplings under Aluminum Stress

doi:10.3969/j.issn.1000-2561.2020.05.015

Abstract

Abstract:

Aluminum toxicity is a major factor inhibiting crop growth on tropical acidic soils. However, the study on the effects of aluminum toxicity on rubber tree growth and latex production is limited, the mechanism of rubber trees tolerate to aluminum has not been reported. To screen the reference genes stably expressed in rubber trees while aluminum stress and elucidate the molecular mechanism of rubber tree aluminum tolerance by real-time quantitative PCR assay, ten candidate internal reference genes, i.e. Actin, Actin7, 18S rRNA, 40S rRNA, YLS8, UBC2, UBC4, GAPDH, FP and ADF were selected in this study. The expression stability was analyzed with geNorm, NormFinder, BestKeeper and Delta Ct and RefFinder. The expression stability of the ten candidate internal reference genes were different. The top three stable reference genes were UBC4, 40S rRNA and FP. Meanwhile, the expression of two aluminum-induced malate transporter genes (HbALMT-1 and HbALMT-2) in rubber trees were verified with UBC4, 40S rRNA, FP, the combination of top three stable genes (UBC4+40S rRNA+FP) and the unstable gene GAPDH. The expression profiles of the two target genes were consistent when normalized by three reference genes and the combinations. The unstable reference gene (GAPDH) failed to standardize the expression data. In conclusion, UBC4, 40S rRNA and FP genes and the combinations were selected as the most stable reference genes, which could be normalized the expression of the relative genes under aluminum stress.

Key words: rubber tree, aluminum toxicity, quantitative real-time PCR, reference gene

CLC Number:

S718.43

MA Xiaowei,AN Feng,LIU Zifan,XIE Guishui. Screening of Reference Genes for Quantitative Real-time PCR of Rubber Saplings under Aluminum Stress[J]. Chinese Journal of Tropical Crops, 2020, 41(5): 955-963.

Figures/Tables 9

References 27

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基因Gene	NCBI登录号 Accession numbers for NCBI	引物序列（5°-3°） Primer sequence (5°-3°)	扩增长度 Amplicon length/bp
40S rRNA	XM_021803004	F: ACAGGCTCATCACCTCCAAG	283
40S rRNA	XM_021803004	R: CAACCACAAAAGTGCAATGG	283
18S rRNA	AB268099	F: GCTCGAAGACGATCAGATACC	146
18S rRNA	AB268099	R: TTCAGCCTTGCGACCATAC	146
YLS8	HQ323250	F: CCTCGTCGTCATCCGATTC	327
YLS8	HQ323250	R: GGAGCAATAACCAAACCACGA	327
Actin	JF270598	F: GATTCCGTTGCCCAGAAGTC	144
Actin	JF270598	R: CACCACTCAGCACAATGTTACC	144
Actin7	HQ260074	F: GGCACTTTGGTACTCAAGTC	102
Actin7	HQ260074	R: GAAGCATCCCAATCACTCTC	102
UBC2	HQ323246	F: CATTTATGCGGATGGAAGCA	125
UBC2	HQ323246	R: CAGGGGAGTTTGGATTTGGA	125
UBC4	HQ323249	F: TCCTTATGAGGGCGGAGTC	82
UBC4	HQ323249	R: CAAGAACCGCACTTGAGGAG	82
GAPDH	XM_021825341	F: GACCGATTTGGCATTGTTGAGGGTC	141
GAPDH	XM_021825341	R: TCCAGTGCTACTAGGAATGATGTTG	141
FP	HQ268026	F: ATCAGCTTGACGCAGGTTAC	99
FP	HQ268026	R: GAAACAGACGCTTCTGAGTG	99
ADF	HM126477	F: GCATATCGGCACTCATCAG	99
ADF	HM126477	R: GGAGAAGGCAAAGCAAGTC	99

基因Gene	NCBI登录号 Accession numbers for NCBI	引物序列（5°-3°） Primer sequence (5°-3°)	扩增长度 Amplicon length/bp
40S rRNA	XM_021803004	F: ACAGGCTCATCACCTCCAAG	283
40S rRNA	XM_021803004	R: CAACCACAAAAGTGCAATGG	283
18S rRNA	AB268099	F: GCTCGAAGACGATCAGATACC	146
18S rRNA	AB268099	R: TTCAGCCTTGCGACCATAC	146
YLS8	HQ323250	F: CCTCGTCGTCATCCGATTC	327
YLS8	HQ323250	R: GGAGCAATAACCAAACCACGA	327
Actin	JF270598	F: GATTCCGTTGCCCAGAAGTC	144
Actin	JF270598	R: CACCACTCAGCACAATGTTACC	144
Actin7	HQ260074	F: GGCACTTTGGTACTCAAGTC	102
Actin7	HQ260074	R: GAAGCATCCCAATCACTCTC	102
UBC2	HQ323246	F: CATTTATGCGGATGGAAGCA	125
UBC2	HQ323246	R: CAGGGGAGTTTGGATTTGGA	125
UBC4	HQ323249	F: TCCTTATGAGGGCGGAGTC	82
UBC4	HQ323249	R: CAAGAACCGCACTTGAGGAG	82
GAPDH	XM_021825341	F: GACCGATTTGGCATTGTTGAGGGTC	141
GAPDH	XM_021825341	R: TCCAGTGCTACTAGGAATGATGTTG	141
FP	HQ268026	F: ATCAGCTTGACGCAGGTTAC	99
FP	HQ268026	R: GAAACAGACGCTTCTGAGTG	99
ADF	HM126477	F: GCATATCGGCACTCATCAG	99
ADF	HM126477	R: GGAGAAGGCAAAGCAAGTC	99

基因 Gene	几何平均值 Geometric mean (Cq)	算术平均值 Arithmetic mean (Cq)	最小值 Min (Cq)	最大值 Max (Cq)	标准差 Standard deviation (±Cq)	变异系数 CV(Cq)/%	排名 Rank
Actin7	23.75	23.78	22.12	25.82	0.96	4.02	1
18S rRNA	18.51	18.56	16.42	20.84	1.08	5.80	2
UBC4	26.54	26.57	24.85	28.24	1.17	4.39	3
FP	29.68	29.70	28.11	31.78	1.20	4.05	4
40S rRNA	22.64	22.68	21.06	24.95	1.36	6.00	5
Actin	22.41	22.46	20.58	25.06	1.36	6.08	6
YLS8	24.16	24.22	21.36	26.07	1.42	5.85	7
ADF	24.99	25.05	23.28	27.62	1.64	6.56	8
UBC2	25.77	25.85	23.36	29.16	1.91	7.41	9
GAPDH	23.42	23.53	20.79	26.59	2.25	9.55	10

基因 Gene	几何平均值 Geometric mean (Cq)	算术平均值 Arithmetic mean (Cq)	最小值 Min (Cq)	最大值 Max (Cq)	标准差 Standard deviation (±Cq)	变异系数 CV(Cq)/%	排名 Rank
Actin7	23.75	23.78	22.12	25.82	0.96	4.02	1
18S rRNA	18.51	18.56	16.42	20.84	1.08	5.80	2
UBC4	26.54	26.57	24.85	28.24	1.17	4.39	3
FP	29.68	29.70	28.11	31.78	1.20	4.05	4
40S rRNA	22.64	22.68	21.06	24.95	1.36	6.00	5
Actin	22.41	22.46	20.58	25.06	1.36	6.08	6
YLS8	24.16	24.22	21.36	26.07	1.42	5.85	7
ADF	24.99	25.05	23.28	27.62	1.64	6.56	8
UBC2	25.77	25.85	23.36	29.16	1.91	7.41	9
GAPDH	23.42	23.53	20.79	26.59	2.25	9.55	10