4-coumarate: coenzyme A ligase (4CL) is one of the key enzymes for the biosynthesis of flavonoids and lignin and is located at the key point of the transition from the main pathway to the branch pathway of phenylalanine metabolism. It controls the metabolic pathways into different phenylpropane compounds and produces different secondary metabolites (Li Shanshan, 2015). As reported, 4CL genes have been cloned from various plants, such as arabidopsis (Ehlting et al., 1999), soybean (Lindermayr et al., 2002), aspen (Hu et al., 1998), salviae (Zhao et al., 2006), prunella (Kim et al., 2014) and bee balm (Weitzel and Petersen, 2010). 4CL genes usually exist in the form of gene family in plants. Different 4CL genes can control different metabolic pathways in different directions. Therefore, the study of 4CL gene family members is of great significance for the regulation of secondary metabolites and the accumulation of secondary metabolites.

Dracocephalum tanguticum Maxim is a perennial herb of the genus Cymbidium in the labiform family, and grows on the banks of dry river valleys, fields, grassy plains or the edges of pine forests at an altitude of 1,900~4,000m and other places (Shen et al., 2009). It is called “Zhiyangge” in Tibetan; the whole herb can be used as medicine, has the functions of clearing liver heat, drying yellow water, healing sores and hemostasis, and can treat liver and stomach heat, unhealed yellow water sore, hemorrhage and other symptoms (Xue et al., 2010). It mainly contains flavonoids, volatiles, amino acids, steroids, terpenes and other substances (Zhang et al., 1994), flavonoids, terpenes and volatile substances have been frequently studied in recent years (Gao et al., 2020). Flavonoids, however, are important secondary metabolites of polyphenols in plants, and has anti-inflammatory, antibacterial, anti-oxidation and other important functions (Qiao et al., 2009). Therefore, it is of great significance to study the effects of 4CL genes on the metabolic pathway of phenylalanine of Dracocephalum tanguticum Maxim on the accumulation of flavonoids.

1 Results and Analysis

1.1 Extraction of RNA of Dracocephalum tanguticum Maxim and construction of overexpression vector

The total RNA from the root of Dracocephalum tanguticum Maxim; and the concentration, purity and integrity of RNA were detected. As detected by ultramicro nucleic acid analyzer, the concentration of RNA was 558.4 ng/ L. The OD₂₆₀/OD₂₈₀ value of 1.96 indicates that the purity of RNA is high; the detection by 1% agarose gel electrophoresis showed that the electrophoretic bands are clear and bright (Figure 1D), indicating that the RNA was not degraded and could be used for subsequent tests.

PCR amplification with specific primers was performed with the cDNA reversed-transcribed from the total RNA from the root of Dracocephalum tanguticum Maxim as template; and the specific bands of about 2,000bp close to expectations were obtained. The target band was connected to the pJET vector and sent for sequencing. A 1,947bp-long gene segment was obtained and named Dt4CL1. The 1,947bp-long gene segment with restriction enzyme cutting site was amplified, connected to the overexpression vector PHB and sent for sequencing. The sequencing results showed that Dt4CL1 was successfully connected to the PHB and that the construction of the Dt4CL1-PHB overexpression vector was completed (Figure 1A; Figure 1B; Figure 1C).

Figure 1 Construction of Dt4CL1-PHB overexpression vector and RNA isolation from Dracocephalum tanguticum Maxim roots Note: M: Trans 2K Plus II DNA Marker; A: Amplification of the 4CL precursor sequence, 1: PCR product; B: PCR detection recombinant plasmid of Dt4CL1-pJET, 1: PCR product of Dt4CL1-pJET recombinant plas mid; C: PCR detection recombinant plasmid of Dt4CL1-PHB, 1: Dt4CL1-PHB digestion verification; D: RNA isolation from Dracocephalum tanguticum Maxim roots

1.2 Dt4CL1 gene sequencing

The ORF Finder online tool of NCBI was used to analyze the open reading frame (ORF) of Dt4CL1 gene. The results showed that Dt4CL1 gene has a 1 719 bp-long ORF, with a total of 572 amino acids encoded (Table 1). It was found by sequence alignment that Dt4CL1 gene has typical conservative domains BoxI (SSGTTGL- PKGV)and BoxII (GEICIRG) (Figure 2).

Table 1 Analysis of Dt4CL1 gene open reading frames (ORFs)

Figure 2 cDNA sequence and encoded amino acid sequence of Dt4CL1

1.3 Domain, physical and chemical properties and hydrophilicity and hydrophobicity of protein encoded by Dt4CL1 gene

The Blast of NCBI was used to predict and analyze the domain of the amino acid sequence of Dt4CL1. The results showed that Dt4CL1 protein have a PLN02246 family domain (Figure 3).

Figure 3 The amino acid conserved domain prediction of Dt4CL1

The online software Protparam was used to analyze the physical and chemical properties of the protein encoded by Dt4CL1 gene. Dt4CL1gene encodes a protein sequence containing 572 amino acid residues with a predicted molecular weight (MW) of around 61.61 kD. The molecular formula is C₂₇₆₁H₄₃₉₅N₇₂₁O₈₂₆S₂₂. The theoretical isoelectric point (pI) is 5.39, indicating that Dt4CL1 protein is weakly acidic. The instability coefficient is 34.52%, indicating that the protein is stable. The total mean hydrophilicity of Dt4CL1 protein is 0.107 and the fat index is 98.86.

The online software ProtScale was used to predict the hydrophilicity/hydrophobicity of Dt4CL1 protein. The results showed that the 26th amino acid in the Dt4CL1 polypeptide chain is the most hydrophilic with the lowest score of -2.789 and that the 260th amino acid is the most hydrophobic with the highest score of 3.078 (Figure 4), indicating that the hydrophobic region of Dt4CL1 protein is larger than the hydrophilic region. Dt4CL1 protein is predicted to be hydrophobin.

Figure 4 Hydrophobicity prediction of protein encoded by Dt4CL1

1.4 Analysis of the transmembrane region and protein signal peptide of Dt4CL1 protein

The online software TMHMM Server v.2.0 was used to perform transmembrane domain prediction analysis on Dt4CL1 protein. According to the results, Dt4CL1 protein has no transmembrane domain (Figure 5A). The online software SignalP 3.0 was used to perform signal peptide prediction for the protein encoded by Dt4CL1 gene. The analysis showed that the C value is 0.076, Y value 0.038 and S value 0.077, predicting that the protein encoded by the gene has no signal peptide and that the protein may be a non-secretory protein (Figure 5B).

Figure 5 Transmembrane region prediction of protein encoded by Dt4CL1 (A), signal peptide prediction of protein encoded by Dt4CL1 (B)

1.5 Analysis of secondary and tertiary structures of Dt4CL1 protein

SOPMA was used to predict the secondary structure of Dt4CL1 protein. The results showed that the α-helix in the secondary structure of Dt4CL1 protein accounted for 29.90% and β-turn 6.29%, random coil 42.66% and extended strand 21.15% (Figure 6), speculating that α-helix and random coil are main structural element in the whole protein structure.

Figure 6 Secondary structure analysis of protein encoded by Dt4CL1

The online software SWISS-MODEL was used for the predictive analysis of the 3D structure of Dt4CL1 protein and for homologous modeling (Figure 7). According to the comparison of Dt4CL1 protein and PDB, the amino acid sequence consistency of 5bsr.1.A reached 65.52%. The encoding protein of 5bsr.1.A sequence was 4-coumarate-CoA ligase.

Figure 7 3D structure of protein encoded by Dt4CL1 gene Note: A: PDB: 5bsr.1.; B: Dt4CL1

1.6 Phylogenetic analysis of Dt4CL1 protein

The software MEGA 7 was used for protein evolutionary tree prediction for Dt4CL1 protein and the 4CL protein of other species. The results showed that, in evolution, Dt4CL1 protein is in the same family as salvia miltiorrhiza and prunella, indicating that it has the closest genetic relationship and that the protein of plants of the genus Cymbidium has high homology (Figure 8). The genes on the 4CL evolutionary tree can be roughly divided into two categories, which is consistent with the distribution of 4CL genes involved in lignin and flavonoid metabolic pathways. As analyzed, the Dt4CL1 protein of Dracocephalum tanguticum Maxim has the closest genetic relationship with the 4CL3 protein of Salvia miltiorrhiza of the genus Cymbidium, the 4CL protein of prunella and the 4CL protein of Ocimum tenuiflorum.

Figure 8 Phylogenic analysis of 4CL Proteins from different plants

2 Discussion

4CL is located at the key point of the transition from the main pathway to the branch pathway of phenylalanine metabolism and is one of the key enzymes for the biosynthesis of flavonoids and lignin. It controls the metabolic pathways into different phenylpropane compounds and produces different secondary metabolites. Flavonoids are important secondary metabolites of polyphenols in plants, are distributed in almost all tissues of plants and have important anti-inflammatory, anti-bacterial and anti-oxidant functions. The synthesis pathway of flavonoid compounds starts from the phenylpropane metabolic pathway and enters into the flavonoid synthesis pathway after the multi-step catalysis of PAL, C4H and 4CL to produce 4-coutoxyl coa (Li, 2019). The 4CL gene in plants usually exists in the form of gene family. Different plants have different number of gene family; and there are differences in structure and function among different members (Ru, 2017). Some studies have shown that the evolution of 4CL is generally divided into two classes: Class I and Class II. Class I is mainly involved in the biosynthesis of lignin and other phenylpropane derivatives and Class II mainly in the biosynthesis of flavonoids (Gui et al., 2011). Studies showed that there are 2 conserved polypeptide sequences in the protein sequence of 4CL (SSGTTGLPKGV) (GEICIRG) (Yang et al., 2014). However, contrastive analysis found that Dt4CL also has Box I and Box II, indicating that there is high sequence conservatism between different species and different types of 4CL genes (Gao et al., 2020). In the three 4CL genes in the genome of arabidopsis, 4CL1 and 4CL2 belong to Class I and 4CL3 belongs to Class II (Raes et al., 2003). In the genome of Salvia miltiorrhiza, the 4CL3 belongs to Class II and 4CL1 and 4CL2 belong to Class I (Wang et al., 2015). It is speculated, through the systematic evolutionary analysis of Dt4CL1 protein, that the function of Dt4CL1 protein may be related to the biosynthesis of flavonoid-related compounds, which is of great significance for studying the accumulation of flavonoid-related polyphenolic compounds in Dracocephalum tanguticum Maxim plants.

The 4CL1 of Dracocephalum tanguticum Maxim is a key gene in the phenylpropane metabolic pathways of Dracocephalum tanguticum Maxim and may play an important role in the synthesis of flavonoids in Dracocephalum tanguticum Maxim. In this study, it was verified by the bioinformatics analysis of Dt4CL1 that Dt4CL1 gene is a member of 4CL gene family and constructs a plant expression vector, which helps further understand the role of Dt4CL1 in the biosynthesis of flavonoids in Dracocephalum tanguticum Maxim.

3 Materials and Methods

3.1 Experimental materials

The Dracocephalum tanguticum Maxim planted in the Science and Technology Park of Tibet Agriculture and Animal Husbandry University (29°40′22.48″N, 94°20′13.91″E) and identified by Professor Lan Xiaozhong was used as the material in this test.

The agents used in this test include total RNA extraction kit (RNAsimple Total RNA Kit, TIANGEN), reverse transcription kit RevertAid^TM first-strand cDNA Synthesis kit (Thermo Scientific), Phanta Max Super-Fidelity DNA Polymerase kit, AxyPrep DNA gel extraction kit, AxyPrep plasmid DNA small-dose kit, T4 DNA Ligase (Invitrogen), Hind III, Xba I restriction enzyme (Thermo Scientific) and Clone JET PCR Cloning Kit (Thermo Scientific), Trans 2K Plus II DNA Marker and E. coli DH5α.

3.2 Cloning and sequencing of Dt4CL1 gene

The root of Dracocephalum tanguticum Maxim was taken and frozen quickly with liquid nitrogen. The total RNA was extracted with plant total RNA extraction kit from the root. Blast comparison was performed in the genome database of Dracocephalum tanguticum Maxim according to the 4CL gene sequence of prunella to obtain the Dt4CL1 sequence of Dracocephalum tanguticum Maxim (c76971_g1#c76971_g1_i1). The software primer 5.0 was used to design primers. Forward primer Dt4CL1-F： 5'-CCAAGCTTGATCAACCATACTCTGTAACGG-3’ (the underlined part is the protective base and the italic part Hind III restriction enzyme cutting site); reverse primer Dt4CL1-R: 5'-GCTCTAGACGATGAACAGTTTGCTTATCGC-3' (the underlined part is the protective base and the italic part Xba I restriction enzyme cutting site). PCR reaction system: 2×Buffer 25 μL, dNTPs Mix 1μL, forward and reverse primers (2 μL for each), cDNA template 2 μL, Phanta Max Super-Fidelity DNA Polymerase 1 μL, ddH2O 7 μL and the total system 50 μL. PCR reaction program: Predegeneration at 95℃ for 3 min (degeneration at 95℃ for 15 s, annealing at 58℃ for 15 s, extension at 72℃ for 1 min) for a total of 35 cycles; thorough extension at 72℃ for 5 min. The product was recovered after PCR amplification, connected to pJET vector, transferred to DH5α competence and sent to Beijing Liuhe Huada Gene Technology Co., Ltd. for sequencing. It was correctly named Dt4CL1-pJET according to the sequencing results.

3.3 Construction of Dt4CL1-PHB overexpression vector

Dt4CL1-F and Dt4CL1-R以Dt4CL1-pJET plasmid were used as the template for PCR amplification. The target product was recovered and purified. The target product and PHB vector were digested by enzyme, purified and recovered with restriction enzymes Hind III and Xba I; the products recovered were mixed in proportion, after which T4 ligase was used for enzymatic connection at 23℃ for 1h. The recombinant vector was transferred to DH5α competent cells and sent for sequencing. It was correctly named Dt4CL1-PHB according to the sequencing results.

3.4 Bioinformatics analysis of Dt4CL1 gene

The Blast database of NCBI was used to analyze the conserved domain of 4CL1 gene of Dracocephalum tanguticum Maxim. The software MEGA7.0 was used to construct 4CL phylogenetic tree. Such bioinformatics software as ORF Finder, ExPASy-Protparam tool, ExPASy-Protscale, TMHMM Server v.2.0, SignalP 3.0, SOPMA and SWISS-MODEL was used for the sequencing and analysis of the protein encoded by Dt4CL1 gene.

Authors’ contributions

Hao Peiyu completed the data analysis and wrote the first draft of this paper as the executer of the experimental design and study in this study; Xu Yuanjiang and Quan Hong participated in experimental design and analyzed test results; Lan Xiaozhong was the designer and the person in charge of the project and guided experimental design, data analysis, writing and revision of the paper. All authors read and agree to the final text.

Acknowledgements

This study was jointly funded by the Major Science and Technology Project of Tibet Autonomous Region (XZ201901-GA-04), the Fourth National Survey of Traditional Chinese Medicine Resources-Survey of Tibetan Medicine Resources of Tibet Autonomous Region (Central) (20200501-542301), the Postgraduate Innovation Project of Tibet Agriculture and Animal Husbandry University (YJS2020-39), and the Budget Project of Tibet Autonomous Region (2019-44).

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