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Acta Agronomica Sinica ›› 2022, Vol. 48 ›› Issue (10): 2546-2559.doi: 10.3724/SP.J.1006.2022.14148

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

Biochemical characteristics and expression differences of three members of CCRs in ramie (Boehmeria nivea)

TANG Ying-Hong1,3,4(), LIU Fang2, CHEN Jian-Rong2,*(), MAO Kai-Quan2, LI Hui1,3, WAN Hai-Qing1   

  1. 1College of Life and Environmental Science, Hunan University of Arts and Science, Changde 415000, Hunan, China
    2School of Biological and Environmental Engineering, Changsha University, Changsha 410022, Hunan, China
    3Changde Research Centre Agricultural Biological Macromolecule, Changde 415000, Hunan, China
    4Changde Research Centre for Artificial Intelligence and Biomedicine, Changde 415000, Hunan, China
  • Received:2021-08-15 Accepted:2022-01-05 Online:2022-10-12 Published:2022-02-22
  • Contact: CHEN Jian-Rong E-mail:yinghongtang2019@163.com;z20080849@ccsu.edu.cn
  • About author:First author contact:

    ** Contributed equally to this work

  • Supported by:
    Youth Fund Project of National Natural Science Foundation(31301374);Youth Fund Project of Hunan Natural Science Foundation(2021JJ40378);Doctoral Research Start Project of Hunan University of Arts and Science(19BSQD35)

Abstract:

Cinnamoyl-CoA reductases (CCR) is a key enzyme in lignin biosynthesis. CCR mostly exists in the form of gene family in plants. In this study, four family members of CCR (BnCCR, BnCCR-1, BnCCR-2, and BnCCR-3) were screened and cloned by ramie transcriptome information. Sequence analysis revealed that the four BnCCRs can be divided into three categories. BnCCR belonged to bona fide CCR group, BnCCR-1 was a CCR like protein, while BnCCR-2 and BnCCR-3 sequences were different from bona fide CCR in NADPH binding domain, catalytic triad, and CCR substrate binding site, and formed a new group in ramie. Transmembrane structure analysis showed that BnCCR contained two transmembrane regions, and the other three BnCCRs didn’t contain transmembrane regions. Structural analysis indicated that the secondary and tertiary structure of the four BnCCRs were different, and the BnCCR-2 homologous modeling template was different from the other three BnCCRs. The qRT-PCR analysis demonstrated that BnCCR had higher expression levels in xylem at rapid growth stage, BnCCR-2 had the highest expression levels in phloem at rapid growth stage. In vitro enzyme activity analysis further found that BnCCR had typical bona fide CCR catalytic activity and substrate adaptability, while BnCCR-2 was different from the substrate adaptability of typical CCR, and had substrate specificity for cinnamoyl CoA and sinapoyl CoA. Therefore, BnCCR was involved in lignin biosynthesis, while BnCCR-2 was a new group with structure, tissue expression and in vitro biochemical function different from typical CCR. BnCCR-2 may not participate or not only participate in lignin biosynthesis, which provides a new reference for studying the evolution of CCR protein family.

Key words: ramie (Boehmeria nivea), cinnamoyl-CoA reductase, expression difference, catalytic specificity

Table 1

Primers for gene cloning and qRT-PCR used in this study"

引物用途
Primer function
引物名称
Primer name
引物序列
Primer sequence (5'-3')
产物长度
Amplication size (bp)
退火温度
Tm (℃)
基因克隆
Gene cloning
BnCCR-F
BnCCR-R
AAGCGAAAATGCCAGTAGAC
TCAACCATACAAGTTACAACCG
1343 55.0
BnCCR-1F
BnCCR-1R
AATGGTTTAGGATTGAGAAAGTGG
GCTGAACAACAACTGCATCACAATA
1195 63.0
BnCCR-2F
BnCCR-2R
CTCTCATCACAATTTACAACCC
ATTCACTCTACGTGAGCCACT
944 52.3
BnCCR-3F
BnCCR-3R
ATGGACTGTGAAAAACCTGTCG
GATAAATCAACCCACCACAACAT
979 50.9
荧光定量PCR
qRT-PCR
BnCCR-P1
BnCCR-P2
CCCGATGTTGTGGTTGATGAGTC
ACCAAATCCACGCCTTTCTCC
137 60.0
BnCCR-1P1
BnCCR-1P2
AAGACGAGACGCTGGTCAGTT
GACGCCGATTCCCTCATAA
110 58.9
BnCCR-2P1
BnCCR-2P2
GAATTTATGGCAGATGTGGAGGTA
AACAACGGCTGTGATTGAAGAAG
111 61.2
BnCCR-3P1
BnCCR-3P2
TGGATTGGGAGGTAAGAGGA
TGCTAGTGCTTGCCACAACT
197 56.8
内参基因
Reference gene
Actin I-P1
Actin I-P2
CGTTGAACCCTAAGGC
ATCCAGCACGATACCAG
137

Table 2

Primers of construction of prokaryotic expression vector used in this study"

引物名称
Primer name
引物序列
Primer sequence (5'-3')
蛋白分子量
Molecular weight (kD)
退火温度
Tm (℃)
BnCCR-BamH I
BnCCR-Hind III
CGCGGATCCATGCCAGTAGACAGCGCTTTGCCAG
CCCAAGCTTCTAAGATTGGATCTTGATGGAATCTTCTTGGG
37 70.7
BnCCR-1 BamH I
BnCCR-1 Sma I
CGCGGATCCATGATGGGGGAGAGAGTGTGTGTG
TCCCCCGGGTTAGCTTAGGGGTAGGACGCCGA
35 66.4
BnCCR-2 BamH I
BnCCR-2 Hind III
CGCGGATCCATGGCACCAACAGTCTGTGTAATG
CCCAAGCTTTCACTCTACGTGAGCCACTCTTTC
33 61.6
BnCCR-3 Sac I
BnCCR-3 Sal I
CGAGCTCATGGACTGTGAAAAACCTGTCG
ACGCGTCGACCTAGCAACAAGCAGTGCTCTCA
34 59.2

Table S1

Unigene annotation of CCR gene"

基因 ID
Gene ID
基因长度
Gene length (bp)
Nr ID Nr得分
Nr score
Nr 注释
Nr annotation
CL21616 1530 gi|316939060|gb|ADU64758.1| 610 肉桂酰辅酶A还原酶(巴西橡胶树)
Cinnamoyl-CoA reductase (Hevea brasiliensis)
CL14670 1357 gi|270315112|gb|ACZ74588.1| 352 肉桂酰辅酶A还原酶-类似蛋白1 (柳枝稷)
Cinnamoyl CoA reductase-like 1 (Panicum virgatum)
CL3762 1221 gi|229368454|gb|ACQ59093.1| 403 肉桂酰辅酶A还原酶1 (陆地棉)
Cinnamoyl-CoA reductase 1 (Gossypium hirsutum)
CL15530 1120 gi|21553548|gb|AAM62641.1| 424 肉桂酰辅酶A还原酶-类似蛋白(拟南芥)
Cinnamoyl-CoA reductase-like (Arabidopsis thaliana)

Fig. 1

Sequence alignment of BnCCRs gene Conserved sites involved in NADP(H) binding motifs are marked with triangles and the NADP+ specificity motifs are marked with full triangles. Sites involved in catalytic process are marked with full circles. Signature sites are marked with stars."

Fig. 2

Phylogenetic analysis of BnCCRs amino acid sequences Red solid dots indicate four genes of cinnamoyl CoA reductase in Boehmeria nivea: BnCCR, BnCCR-1, BnCCR-2, and BnCCR-3."

Fig. 2

Transmembrane helices analysis of BnCCRs protein based on TMHMM Red represents the transmembrane domain; the blue line represents the cytoplasmic loop; the red line represents the extracellular ocean."

Fig. 3

Deduced structural prediction of BnCCRs protein Extended strand is indicated by yellow ribbon; Alpha helix is indicated by pink ribbon; Beta Turn and Random coil is indicated by white ribbon. The R38(X)5K44 motif is indicated by red stick amino acid residues; the conserved sites S123-Y157-K161 of the short chain dehydrogenase superfamily is indicated by blue, earthy yellow, and purple stick amino acid residues, respectively. The important substrate binding site H202 of CCR is indicated by green stick amino acid residue; NAP in BnCCR is shown in wathet ball."

Fig. 4

Relative expression patterns of BnCCRs genes R: rapid growth period; M: maturity stage; L: late maturity."

Fig. 5

Purification of recombinant BnCCRs proteins M: protein 170 molecular marker; CK: control; 1: bacteria; 2: supernatant protein; 3: the target protein."

Fig. 6

Chromatogram of recombinant BnCCR catalysis with five mixed substrates"

Fig. 7

Chromatogram of recombinant BnCCR-2 catalysis with five mixed substrates"

Table 3

Kinetic analysis of recombinant BnCCR and BnCCR-2"

底物
Substrate
Km (μmol L-1) Vmax (nkat mg-1 protein) Kcat (s-1) Kcat/Km (mmol L-1 s-1)
BnCCR BnCCR-2 BnCCR BnCCR-2 BnCCR BnCCR-2 BnCCR BnCCR-2
肉桂酰CoA
Cinnamoyl CoA
183.77 168.01 3.82 8.06 0.15 0.29 0.83 1.71
芥子酰CoA
Sinapoyl CoA
391.62 197.68 7.68 4.35 0.31 0.16 0.78 0.79
对香豆酰CoA
p-coumaroyl CoA
123.90 ND 4.55 ND 0.18 ND 1.47 ND
阿魏酰CoA
Feruloyl CoA
806.38 ND 13.01 ND 0.52 ND 0.65 ND
咖啡酰CoA
Caffeoyl CoA
ND ND ND ND ND ND ND ND

Fig. S2

Chromatogram of recombinant BnCCR catalysis with four substrates respectively"

Fig. S3

Chromatogram of recombinant BnCCR-2 catalysis with two substrates"

Fig. S4

Michaelis-Menten curve of recombinant BnCCR and BnCCR-2 catalysis with different substrates"

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[1] TANG Ying-Hong, CHEN Jian-Rong, LIU Fang, YUAN You-Mei, GUO Qing-Quan, CHANG Hong-Tao. cDNA Cloning and Analysis of Cinnamoyl-CoA Reductase Gene from Boehmeria nivea [J]. Acta Agron Sin, 2015, 41(09): 1324-1332.
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