Cloning and Functional Characterization of 6-OMT Gene Related to Isocorydine Biosynthesis in Dactylicapnos scandens

doi:10.13560/j.cnki.biotech.bull.1985.2024-0544

Abstract

Abstract:

Objective (S)-norcoclaurine O-methyltransferase (6-OMT) is the key rate-limiting enzyme in the biosynthesis of isocorydine. This study is aimed to verify the function of the 6-OMT gene from Dactylicapnos scandens through cloning and in vitro enzyme activity assays, thereby laying the foundation for elucidating the isocorydine biosynthetic pathway in D. scandens. Method The DsOMT gene was mined from the transcriptome data of D. scandens. The full-length cDNA sequence was obtained via PCR amplification, and the protein structure of DsOMT was analyzed using bioinformatics. The expressions of the DsOMT gene in different tissues were examined. A pET-28a-DsOMT prokaryotic expression vector was constructed and transferred into Escherichia coli BL21(DE3) for induced expression. The protein was then purified and subjected to in vitro enzyme assays to characterize its function. Result Four DsOMT candidate genes, named DsOMT07, DsOMT08, DsOMT010, and DsOMT012, were identified from the transcriptome data, and their full-length cDNA sequences were successfully amplified. All four DsOMT proteins lacked transmembrane domains and signal peptides, classifying them as extramembrane proteins. Phylogenetic analysis indicated that these genes were closely related to the 6-OMT subfamily. Amino acid sequence analysis suggested potential catalytic activity at the 6-OH site of the upstream pathway for (S)-norcoclaurine. Expression profiling revealed that these four DsOMT genes were highly expressed in the roots. SDS-PAGE results showed that DsOMT proteins were highly soluble and efficiently expressed in Escherichia coli. In vitro enzyme assays demonstrated that DsOMT010 catalyzed the O-methylation of the C6 position of (S)-norcoclaurine, forming (S)-coclaurine. Conclusion Four DsOMT genes are successfully cloned and identified as extramembrane proteins closely related to the 6-OMT subfamily. These genes show high expressions in the roots. Additionally, heterologous expression of DsOMT is achieved in E. coli, and the purified proteins are characterized for their in vitro enzyme functions. Among them, DsOMT010 is identified as a (S)-norcoclaurine C-6 O-methyltransferase.

Key words: Dactylicapnos scandens, O-methyltransferase, bioinformatics analysis, prokaryotic expression, functional characterization

LI Ming, LIU Xiang-yu, WANG Yi-na, HE Si-mei, SHA Ben-cai. Cloning and Functional Characterization of 6-OMT Gene Related to Isocorydine Biosynthesis in Dactylicapnos scandens[J]. Biotechnology Bulletin, 2025, 41(2): 309-320.

Figures/Tables 13

Fig. 1 Structure of tissue samples and compounds of D. scandensA-C refers to the roots, stems and leaves of D. scandens, respectively. D and E are the structure and position numbers of isovioladinine and (S)-norcoclaurine, respectively

Table 1 Website links on bioinformatics analysis

项目列表 Analysis item	网址 Website
NCBI同源基因比对分析	https：//blast.ncbi.nlm.nih.gov/Blast.cgi
蛋白理化性质分析	https：//web.expasy.org/protparam/
蛋白信号肽预测	https：//services.healthtech.dtu.dk/services/SignalP-6.0/
蛋白跨膜结构域预测	https：//services.healthtech.dtu.dk/services/TMHMM-2.0/
蛋白二级结构预测	https：//npsa-prabi.ibcp.fr/cgi-bin/npsa
蛋白三级结构预测	https：//swissmodel.expasy.org/
蛋白保守基序分析	https：//meme-suite.org/meme/
蛋白保守结构域分析	https：//www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi
蛋白亚细胞定位	http：//www.csbio.sjtu.edu.cn/bioinf/Cell-PLoc-2/
多序列比对	https：//espript.ibcp.fr/ESPript/cgi-bin/ESPript.cgi

Table 2 PCR primer sequences

基因 Gene	正向引物序列 Forward primer sequence （5′-3′）	反向引物序列 Reverse primer sequence （5′-3′）
DsOMT08	cagcaaatgggtcgcggatccATGGAAGTGAAGAAGAGTGAAC	gacggagctcgaattcggatccTTAATATGGATAAACCTCAATAACAG
DsOMT07	cagcaaatgggtcgcggatccATGGAGATGATCATCAATGAAG	acggagctcgaattcggatccCTAAGGATATGCAACAATAACTG
DsOMT010	cagcaaatgggtcgcggatccATGGGTGTCAGTGATATAGCTG	acggagctcgaattcggatccCTATGGGAAGGCTTCAATGACAG
DsOMT012	cagcaaatgggtcgcggatccATGGGTGCCAATGAGATAGCAG	acggagctcgaattcggatccCTATGGGAAGGCTTCAATGACAG
PCR-pET-28a	taatacgactcactataggggaatt	aaacgggtcttgaggggttttttg

Fig. 2 Candidate gene heat map of D. scandens, phylogenetic tree and conserved motif analysis of methyltransferase genes in other plantsA: Analysis of MT family phylogenetic tree. B: Expression heat map of candidate genes in different tissues of D. scandens. C: Phylogenetic tree (pre) and conserved motif (post) analysis of candidate genes and OMT genes of other species

Fig. 3 Multiple sequence alignment of four screened DsOMT genes with OMT genes of other species

Table 3 Prediction and statistics of physicochemical properties of DsOMT protein

基因 Gene	氨基酸数目 Amino acid number	分子量 Molecular weight/Da	理论等电点 Theoretical pI	不稳定系数 Instability index	脂肪系数 Fat coefficient	亲水系数 Hydrophilicity coefficient	亚细胞定位 Subcellular localization
DsOMT07	356	39 577.72	4.95	28.65	105.96	0.024	叶绿体
DsOMT08	353	39 349.51	5.61	33.31	93.34	-0.150	叶绿体
DsOMT010	349	38 582.51	5.23	36.41	102.23	0.034	叶绿体
DsOMT012	348	38 346.22	5.47	38.58	102.53	0.025	叶绿体

Fig. 4 Transmembrane structural domains of four screened DsOMT proteins (top) and signaling peptide prediction (bottom)A-D are the predicted transmembrane structural domains (top) and signaling peptides (bottom) of DsOMT07, DsOMT08, DsOMT010, and DsOMT012 proteins

Fig. 5 Secondary structure prediction of four DsOMT proteinsA-D are the predicted secondary structures of DsOMT07, DsOMT08, DsOMT010 and DsOMT012 proteins in order. E: Secondary structure prediction statistics of four DsOMT proteins

Fig. 6 Prediction of the tertiary structure of four DsOMT proteinsA-D predicted tertiary structure of DsOMT07, DsOMT08, DsOMT010 and DsOMT012 proteins in order; blue: spiral; green: extension chain; silver-white: irregular curl

Fig. 7 Extraction of total RNA from D. scandens and cloning of DsOMT geneA: Total RNA electrophoresis gel diagram of each tissue of D. scandens, lane 1-3 are total RNA electrophoresis gel diagram of root, stem and leaf tissues of D. scandens, respectively. B: Clone 4 candidate DsOMT gene electrophoresis gels, lane 4-7are cloned for DsOMT07, DsOMT08, DsOMT010 and DsOMT012 genes, respectively. Lane M: DNA marker 5 000 electrophoresis band

Fig. 8 Single enzyme digestion of pET-28a vector and verification of DsOMT recombinant pET-28aA: Gel diagram of single-digested pET-28a linear vector, lane 1 is the undigested pET-28a vector, and lane 2 and 3 are the digested pET-28a vector. B: Electrophoresis gel diagram of pET-28a-DsOMT(+) vector with positive bacterial water test, and pET-28a-DsOMT 07(+), pET-28a-DsOMT 08(+), pET-28a-DsOMT 010(+) and pET-28a-DsOMT 012(+) in lane 4-5, 6-7, 8-9 and 10-11, respectively. Lane M is the DNA marker 5 000 electrophoresis band

Fig. 9 Purification of DsOMT proteinA-D are electropherograms of 250 mmol/L imidazole purification of DsOMT07, DsOMT08, DsOMT010 and DsOMT012 proteins, respectively. Lane 1-3 in A-D are duplicate protein sample bands, lane M in panel A, C, and D is protein marker 150, and lane M in panel B is protein marker 310

Fig. 10 Protein enzymatic reaction in vitro and LC-MS detectionA: Catalytic steps of DsOMT010 in vitro enzymatic reaction. B: TIC diagram of LC-MS detection and analysis. C: LC-MS detection of (S)-coclaurine standard EIC mass spectrometry. D: LC-MS detection of DsOMT010+(S)-norcoclaurine product EIC mass spectrometry

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