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

作物学报 ›› 2022, Vol. 48 ›› Issue (7): 1614-1624.doi: 10.3724/SP.J.1006.2022.14119

• 作物遗传育种·种质资源·分子遗传学 • 上一篇下一篇

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

杨昕¹^,²(), 李玉¹^,²(), 刘传兵³, 张力岚¹^,², 何青垚¹^,², 祁建民¹^,², 张立武¹^,²^,^*()

¹福建农林大学农学院 / 作物遗传育种与综合利用教育部重点实验室 / 福建省作物设计育种重点实验室, 福建福州 350002
²福建农林大学农业农村部东南黄红麻实验观测站 / 福建省麻类种质资源共享平台 / 福建省南方经济作物遗传育种与多用途开发国际科技合作基地, 福建福州 350002
³恩施土家族苗族自治州农业科学院, 湖北恩施 445000

收稿日期:2021-07-10 接受日期:2021-10-19 出版日期:2022-07-12 网络出版日期:2021-11-15
通讯作者: 张立武
作者简介:杨昕, E-mail: 295548772@qq.com
李玉, E-mail: 1349693117@qq.com第一联系人：
^** 同等贡献
基金资助:
国家自然科学基金项目(31771369);财政部和农业农村部国家现代农业产业技术体系建设专项(CARS-16)

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

YANG Xin¹^,²(), LI Yu¹^,²(), LIU Chuan-Bing³, ZHANG Li-Lan¹^,², HE Qin-Yao¹^,², QI Jian-Min¹^,², ZHANG Li-Wu¹^,²^,^*()

¹Key Laboratory of Ministry of Education for Genetics, Breeding and Multiple Utilization of Crops / Fujian Key Laboratory for Crop Breeding by Design / College of Agriculture, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
²Experiment Station of Ministry of Agriculture and Rural Affairs for Jute and Kenaf in Southeast China / Public Platform of Fujian for Germplasm Resources of Bast Fiber Crops / Fujian International Science and Technology Cooperation Base for Genetics, Breeding and Multiple Utilization Development of Southern Economic Crops, Fujian Agriculture and Forestry University, Fuzhou 350002, Fujian, China
³Academy of Agricultural Sciences, Enshi Tujia & Miao Autonous Prefecture of Hubei, Enshi 445000, Hubei, China

Received:2021-07-10 Accepted:2021-10-19 Published:2022-07-12 Published online:2021-11-15
Contact: ZHANG Li-Wu
About author:First author contact:
^** Contributed equally to this work
Supported by:
National Natural Science Foundation of China(31771369);China Agriculture Research System of MOF and MARA(CARS-16)

摘要/Abstract

摘要：

选择合适的内参基因进行校正和标准化, 既能提高实时定量荧光PCR的准确性, 也为分析黄麻次生细胞壁合成相关基因的表达模式奠定基础。本研究通过常用的内参基因, 结合课题组已有的基因组数据和转录组数据, 初步筛选出8个内参基因(CcTUBα、CcACT、CcDnaJ、CcTUBβ、CcUBQ、CcEF1α、CcUBC和CcUBI)。为验证这些候选内参基因的稳定性, 以优良品种‘黄麻179’为材料, 对种子萌发后14 d的根、茎、叶进行qRT-PCR分析。经Ct值的变异系数、软件geNorm和NormFinder的综合分析, 确定表达最稳定内参基因为CcDnaJ, 最佳内参基因组合方式为CcDnaJ+CcUBQ+CcUBI。通过种子萌发后10 d下胚轴、60 d和90 d茎皮的转录组数据分析次生细胞壁合成相关基因的表达模式, 发现木质素合成基因Cc4CL1、CcCCoAOMT1的表达量在种子萌发后60 d茎皮最高, 而种子萌发后90 d茎皮表现为下降; 纤维素合成酶基因CcCesA4、CcCesA7、CcesA8和木聚糖合成基因CcIRX8、CcIRX9、CcFRA8的表达量在种子萌发后10 d下胚轴最高, 而种子萌发后60 d、90 d茎皮的表达量均有下降, 表明这些基因参与了黄麻纤维发育。以CcDnaJ+CcUBQ+CcUBI作为内参基因组合, qRT-PCR分析5个次生细胞壁合成相关基因, 即Cc4CL1、CcCCoAOMT1、CcCesA4、CcCesA7、CcesA8, 在种子萌发后7 d下胚轴和14 d茎的表达模式, 发现这5个基因在种子萌发期后14 d茎的表达量均高于种子萌发期后7 d下胚轴, 暗示着黄麻纤维的形成开始于7~14 d之间, 同时表明CcDnaJ+CcUBQ+CcUBI的内参基因组合具有实用性。

关键词: 黄麻, qRT-PCR, 内参基因, 次生细胞壁, 纤维素, 木质素

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

杨昕, 李玉, 刘传兵, 张力岚, 何青垚, 祁建民, 张立武. 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[J]. 作物学报, 2022, 48(7): 1614-1624.

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

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

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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