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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (7): 1614-1624.doi: 10.3724/SP.J.1006.2022.14119

• OCROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

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,*()   

  1. 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 Online:2022-07-12 Published:2021-11-15
  • Contact: ZHANG Li-Wu E-mail:295548772@qq.com;1349693117@qq.com;lwzhang@fafu.edu.cn
  • 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)

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

Table 1

Information of candidate reference genes in jute"

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

Table 2

Primer sequences and product size of secondary cell wall synthesis genes in jute"

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

Table 3

Analysis of electronic expression of candidate reference genes in different tissues at different developmental stages in jute"

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

Fig. 1

Agarose gel electrophoresis of RNA quality detection of different tissues of ‘Huangma 179’ at 14 days after germination M: DL2000 DNA marker; 1: root; 2: stem; 3: leaf."

Fig. 2

Agarose gel electrophoresis of specific analysis of primers for qRT-PCR"

Table 4

Amplification characteristics of primers of 8 candidate reference genes in jute"

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

Fig. 3

Melting curves of 8 candidate reference genes in jute"

Table 5

Analysis stability of reference genes by Ct value in jute"

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

Fig. 4

Average expression stability (M) and pairwise variation (V) values of candidate reference genes by geNorm software"

Table 6

Stability ranking of candidate reference genes based on NormFinder software"

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

Table 7

Identification of secondary cell wall synthesis genes and their domains by SMART and NCBI (CDD) in jute"

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)
起始
Start
终止
End
E
E-value
区间
Interval
E
E-value
Cc4CL1 Cc.06G0029430 AMP-binding 34 446 2.2e-115 6 542
AMP-binding_C 454 529 3.4e-18
CcCCoAMOT1 Cc.02G0006600 Methyltransf_24 84 191 2.3e-11 1 247
CcCesA4 Cc.07G0013090 Cellulose_synthase 318 1058 49 1062
Cellulose_synthase 1097 1837
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

Fig. 5

Expression analysis of genes related to secondary cell wall synthesis in jute Hypocotyl-10 d: hypocotyls of 10 days after germination; Stem-60 d: stem barks of 60 days after germination; Stem-90 d: stem barks of 90 days after germination. Different lowercase letters above the bars indicate that there is significant difference at the 0.05 probability level in the relative expression of the same gene at different stages."

Fig. 6

Relative expression of five secondary cell wall synthesis related genes at different stem developmental stages of jute Hypocotyl-10 d: hypocotyls of 10 days after germination; Stem-60 d: stem barks of 60 days after germination; Stem-90 d: stem barks of 90 days after germination. Different lowercase letters above the bars indicate that the expression of the same gene at different stages is significant difference at the 0.05 probability level."

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