黄麻内参基因筛选及次生细胞壁合成相关基因的表达分析

doi:10.3724/SP.J.1006.2022.14119

Abstract

Abstract:

Selecting suitable reference genes for calibration and standardization can not only improve the accuracy of real-time quantitative PCR (qRT-PCR), but also lay the foundation for analyzing the expression pattern of genes related to secondary cell wall synthesis in jute. In the present work, 8 candidate reference genes were screened on the homologs of common reference genes by using available genomic and transcriptomic data of the research group. To evaluate the stability of candidate genes, we used different tissues (roots, stems and leaves) of ‘Huangma 179’ at the 14 days after germination (DAG) as materials for qRT-PCR. Then qRT-PCR data were analyzed by coefficient of variation (CV) of Ct value, the softwares of geNorm and NormFinder, respectively. The results showed that the most stablest reference gene was CcDnaJ, and the best combination of reference genes was ‘CcDnaJ + CcUBQ + CcUBI’. The expression patterns of secondary wall synthesis related genes were analyzed by transcriptomic data of hypocotyls at the 10 DAG, stem barks at the 60 DAG and 90 DAG. The electronic expression of lignin synthase genes, Cc4CL1 and CcCCoAOMT1, in stem barks at the 60 DAG were the highest, and then decreased at the 90 DAG. The expression of cellulose synthase genes, CcCesA4, CcCesA7, CcesA8, and xylan biosynthesis genes, CcIRX8, CcIRX9, CcFRA, in hypocotyls at the 10 DAG were the highest, and decreased in stem barks at the 60 DAG and 90 DAG. These findings indicate that these genes were involved in jute fiber development. Furthermore, the expression patterns of 5 secondary wall synthesis related genes, Cc4CL1, CcCCoAOMT1, CcCesA4, CcCesA7, and CcesA8, were analyzed by qRT-PCR using the reference gene combination of ‘CcDnaJ + CcUBQ + CcUBI’ at different stem developmental stages, i.e. hypocotyls at the 7 DAG and stems at the 14 DAG. The results revealed that relative expression of Cc4CL1, CcCesA4, CcCesA7, and CcesA8 genes in stems at the 14 DAG were higher than that in hypocotyles at the 7 DAG, suggesting that jute fiber formation starts at between 10 and 14 DAG, and the reference gene combination of ‘CcDnaJ + CcUBQ + CcUBI’ was available as well.

Key words: Corchorus L., qRT-PCR, reference gene, secondary cell wall, cellulose, lignin

YANG Xin, LI Yu, LIU Chuan-Bing, ZHANG Li-Lan, HE Qin-Yao, QI Jian-Min, ZHANG Li-Wu. Reference genes screening for expression analysis of secondary cell wall synthesis related genes in jute (Corchorus capsularis)[J].Acta Agronomica Sinica, 2022, 48(7): 1614-1624.

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基因简称 Gene abbreviation	基因名称 Gene name	拟南芥 Arabidopsis thaliana (AT)	黄麻数据库编号 Database number of C. capsularis (Cc)	引物名称 Primer name	引物序列 Primer sequence (5°-3°)
CcACT	Actin	AT5G09810.1	Cc.02G0001300	CcACT-F CcACT-R	GGAGCTGAGAGATTCCGTTG CTTGCTCATACGGTCTGCAA
CcDanJ	Chaperone protein	AT3G07590.1	Cc.04G0010010	CcDanJ-F CcDanJ-R	TCCTGCCTGACAGCATAAATCTTG TTAACGGCCTCTACCACGTCCACG
CcEF1α	Elongation factor 1-α	AT1G07940.1	Cc.v30140180	CcEF1α-F CcEF1α-R	GTTTGAGACCACCAAATACTACTGCA TCAATAATGAGGACAGCACAATCAGC
CcTUBα	α-Tubulin	AT1G04820.1	Cc.05G0004250	CcTUBα-F CcTUBα-R	AATCAACTATCAGCCACCCACAGT CCTTCCTCCATTCCTTCACCTACG
CcTUBβ	β-Tubulin	AT2G29550.1	Cc.v30016860	CcTUBβ-F CcTUBβ-R	GAATGTATGGTGTTGGACAATGAGGC AAAAAGTGTAACCGTGGGAAGGGGAT
CcUBC	Ubiquitin-conjugating enzyme	AT3G52560.4	Cc.04G0035920	CcUBC-F CcUBC-R	TGGTCCTCCTAATACTGTCCACGAA ATCCTCCATTGTATACTCCCTTTGC
CcUBI	Ubiquitin	AT2G47110.1	Cc.04G0041210	CcUBI-F CcUBI-R	AGGAACGTGAAGCCATAGAACG ATCTAAAAGGTAGTTTGCCGCC
CcUBQ	Polyubiquitin	AT5G20620.1	Cc.04G0014390	CcUBQ-F CcUBQ-R	GCATCTCCACCATGCTCCTTAA GTGCCTCTCGAACCATTTCCTC

基因简称 Gene abbreviation	上游引物和下游引物 Forward and reverse sequences (5°-3°)	扩增片段大小 Amplified fragments (bp)
Cc4CL1	F: TGGGACGACGGGTCAGATTTAT R: CGTTGGAAGCTTTGGCTTGCTT	236
CcCCoAOMT1	F: GGAGCCTGAGGCCATGAAAGAG R: CAAGGGCAGTAGCAAGGAGAGA	176
CcCesA4	F: ACAAGGATGACTCAGATCATCGCC R: TTTCTTGCCTGACTTTCCATTTCT	186
CcCesA7	F: ACAATCGCAATGAACTCGTCGT R: GATCTCCATCCACTGTCAGCCC	120
CcCesA8	F: GCAAGCACTTTATGGCTATGGACCT R: TCTCAATCTCTCTCAGGTTGAAAATT	176

基因简称 Gene abbreviation	电子表达量平均数 Mean of electronic expression	标准差 Standard deviation	变异系数 Coefficient of variation (%)
CcACT	10.477	0.689	6.6
CcDnaJ	8.151	0.509	6.2
CcEF1α	11.820	0.491	4.2
CcTUBα	9.041	0.838	9.3
CcTUBβ	9.064	0.555	6.1
CcUBC	8.500	0.318	3.7
CcUBI	8.982	0.249	2.8
CcUBQ	7.485	0.366	4.9

基因简称 Gene abbreviation	扩增效率 Efficiency (%)	相关系数 Correlation coefficient	扩增片段大小 Amplified fragments (bp)
CcACT	84.5	0.9984	195
CcDnaJ	96.4	0.9946	140
CcEF1α	86.3	0.9939	114
CcTUBα	80.2	0.9960	186
CcTUBβ	90.0	0.9936	206
CcUBC	87.1	0.9911	199
CcUBI	105.6	0.9904	116
CcUBQ	95.6	0.9967	126

基因简称 Gene abbreviation	平均数 Means of Ct value	标准差 Standard deviation	变异系数 Coefficient of variation (%)
CcACT	28.385	4.052	14.3
CcDnaJ	30.698	2.957	9.6
CcEF1α	27.668	4.525	16.4
CcTUBα	30.107	4.768	15.8
CcTUBβ	30.378	4.385	14.4
CcUBC	29.752	2.174	7.3
CcUBI	30.000	3.164	10.5
CcUBQ	30.995	3.348	10.8

Reference genes screening for expression analysis of secondary cell wall synthesis related genes in jute (Corchorus capsularis)

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基因简称 Gene abbreviation	稳定值 Stability value	排名 Rank
CcDnaJ	0.273	1
CcUBQ	0.712	2
CcUBI	0.786	3
CcTUBβ	0.934	4
CcUBC	1.108	5
CcEF1α	1.142	6
CcTUBα	1.335	7
CcACT	1.439	8

基因简称 Gene abbreviation	黄麻数据库编号 Database number of C. capsularis	结构域 Domain	SMART			CDD (protein)
基因简称 Gene abbreviation	黄麻数据库编号 Database number of C. capsularis	结构域 Domain	起始 Start	终止 End	E值 E-value	区间 Interval		E值 E-value
Cc4CL1	Cc.06G0029430	AMP-binding	34	446	2.2e-115	6	542	—
Cc4CL1	Cc.06G0029430	AMP-binding_C	454	529	3.4e-18	6	542	—
CcCCoAMOT1	Cc.02G0006600	Methyltransf_24	84	191	2.3e-11	1	247	—
CcCesA4	Cc.07G0013090	Cellulose_synthase	318	1058	—	49	1062	—
CcCesA4	Cc.07G0013090	Cellulose_synthase	1097	1837	—	49	1062	—
CcCesA7	Cc.07G0011040	Cellulose_synthase	335	1040	—	1	1044	—
CcCesA8	Cc.06G0026520	Cellulose_synthase	256	972	—	1	978	—
CcFRA8	Cc.07G0001000	Exostosin	95	397	4.5e-71	97	395	1.09e-75
CcIRX8	Cc.01G0038940	Glycosyl transferases _8	173	516	1.2e-76	1	543	—
CcIRX9	Cc.05G0004240	Glycosyl transferase _43	135	254	2.8e-37	1	288	5.39e-159

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