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OBJECTIVE: To investigate the chemical constituents of the seeds of Lepidium apetalum Willd. METHODS: The compounds were isolated and purified by Diaion HP-20, Toyopearl HW-40, MCI Gel CHP-20, ODS, silica gel chromatography combined with Pre-HPLC and the structures were identified on the basis of spectral data and physiochemical properties. RESULTS: Sixteen compounds were isolated and identified from the water extract as catechol(1), protocatechuic aldehyde(2), 2-phenyl acetamide(3), methyl-5-hydroxypyridine-2-carboxlate(4), benzylcarbamic acid(5), N-benzylacetamide(6), raphanuside C(7), 1-phenyl-1, 2-ethanediol(8), 2-(4-hydroxyphenyl)-ethanol(9), isorhamnetin-7-O-α-L-rhamnopyranoside(10), kaempferol(11), methyl 2, 4, 6-trihydroxybenzoate(12), 2-(4-hydroxyphenyl)acetonitrile(13), syringic acid(14), protocatechuic acid(15), and methyl sinapate(16). CONCLUSION: Compounds 1-16 are isolated from this plant for the first time.
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Objective To study the chemical constituents from the seeds of Lepidium apetalum. Methods Compounds were isolated from the water extract from the seeds of L. apetalum by using Diaion HP-20, Toyopearl HW-40, MCI Gel CHP-20, ODS, Silica gel chromatography and semi-preparative-HPLC. Results Nine compounds were isolated and identified as lepidiumlignan A (1), lepidiumlignan B (2), erythro-1-(4-O-β-D-glucopyranosyl-3-methoxyphenyl)-2-[4-(3-hydroxypropyl)-2,6-dimethoxyphenoxy]-1,3- propanediol (3), (7R,7’E,8S)-4,9-dihydroxy-3,3’,5-trimethoxy-4’,7-epoxy-8,5’-neolign-7’-en-9’-oic acid (4), spicatolignan B (5), (-)-pinoresinol-4-O-β-D-glucopyranoside (6), (-)-isolariciresinol (7), aegineoside (8), and (+)-syringaresinol-O-β-D-glucopyra- noside (9). Conclusion Compounds 1 and 2 are new compounds, named as lepidiumlignan A and lepidiumlignan B. Compounds 3-9 are isolated from the plants for the first time.
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Objective In order to study the key genes involved in flavonoid biosynthesis pathway, the flavanone-3-hydroxylase (F3H) gene was isolated from Lepidium apetalum, which is named as LaF3H. Meanwhile, the sequence analysis, prokaryotic expression, and purification were also performed. Methods Specific primers were designed according to LaF3H gene sequences in the transcriptome data of L. apetalum, and the cDNA sequence of LaF3H gene was isolated from L. apetalum. By construction the prokaryotic expression vector pET-32a-LaF3H, the recombinant LaF3H protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. Results The open reading frame (ORF) of LaF3H was 1 080 bp, which encoded a protein of 359 amino acid residues, with a predicted molecular mass of 40 320. Sequence analysis showed that LaF3H contains five conserved motifs of F3H protein. The phylogenetic analysis indicated that LaF3H protein showed the highest homology with F3H protein from cruciferous plants (such as AtF3H from Arabidopsis thaliana). The prokaryotic expression vector pET-32a-LaF3H was constructed and the recombinant LaF3H protein was successfully expressed in E. coli BL21 (DE3) cells. Furthermore, the recombinant LaF3H protein was purified through Ni2+ affinity chromatography. Conclusion The LaF3H gene was isolated from L. apetalum and the recombinant LaF3H protein was obtained. The results of this study provided the foundation for the further preparation of LaF3H antibody and detection of LaF3H enzyme activity, and were helpful for functional characterization of LaF3H gene involved in flavonoid biosynthesis pathway of L. apetalum.
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OBJECTIVE: To clone the mevalonate kinase (MK) gene involved in cardiac glycosides biosynthesis pathway of Lepidium apetalum, and carry out bioinformatic analysis, prokaryotic expression, purification, and tissue-specific expression analysis. METHODS: By analyzing the transcriptome data of L. apetalum and designing specific primers, the cDNA of LaMK gene was isolated from L. apetalum (GenBank accession No. KX290928).Escherichia coli BL21 (DE3) cells were transformed with the prokaryotic expression vector pET-32a-LaMK and used for prokaryotic expression of recombinant LaMK protein. RESULTS: The open reading frame (ORF) of LaMK was 1 137 bp, which encoded a protein of 379 amino acid residues, with a predicted molecular weight of 40.43×103. Bioinformatic analysis showed that LaMK protein may locate in cytoplasm, had no transmembrane domain and signal peptide, and exhibited specific N-terminal domain and C-terminal domain of GHMP kinase super family, as well as the binding site of ATP. Phylogenetic analysis indicated that LaMK protein had the highest homology with MK protein from cruciferous plants (such as AtMK from Arabidopsis thaliana). Through construction of the prokaryotic expression vector, the recombinant LaMK protein was successfully expressed in Escherichia coli BL21 (DE3) cells. Furthermore, the recombinant LaMK protein was purified by Ni2+ affinity chromatography. Real-time PCR analysis indicated that LaMK was expressed at a high level in flowers, low levels in leaves and roots, lower expression level in stems, and the lowest level in seedlings. CONCLUSION: In this study, the LaMK gene is cloned from L. apetalum, the prokaryotic expression system is established, and the purified recombinant LaMK protein is obtained. This study lays the foundation for preparation of the antibody of LaMK protein and research of the function of LaMK gene involved in cardiac glycosides biosynthesis pathway.
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Lepidium apetalum was used as an experimental material in this study. By analyzing the tran-scriptome data of L. apetalum and application of the specific primers, cDNA of cinnamate-4-hydroxylase (C4H) gene was isolated from L. apetalum and named as LaC4H (GenBank accession No. KX064050). Meanwhile, the bioinformatic analysis, prokaryotic expression, purification, tissue-specific expression analysis and expres-sion analysis after methyl jasmonate (MeJA) treatment were carried out. The results indicated that: ① The open reading frame (ORF) of LaC4H was 1 518 bp, which encoded a protein of 505 amino acid residues, with a predicted molecular mass of 57.73 kD. ② Bioinformatic analysis showed that LaC4H protein contained the conserved sequences of cytochrome P450 (CYP450) and 5 substrate recognition sites (SRSs) of CYP73A1, therefore LaC4H protein was a member of CYP450 superfamily. The phylogenetic analysis indicated that LaC4H protein showed the highest homology with C4H protein from cruciferous plants (such as AtC4H from Arabidopsis thaliana). ③ Through the construction of the prokaryotic expression vector pET-32a-LaC4H, the recombinant LaC4H protein was successfully expressed in E. coli BL21 (DE3) cells and the recombinant LaC4H protein was purified by Ni2+ affinity chromatography. ④ Real-time PCR analysis indicated that LaC4H was expressed in a high transcript level in stems, lower levels in leaves and flowers, the lowest level in roots. After MeJA treatment, the expression level of LaC4H in leaves was increased significantly to reach the highest level at 48 h. Furthermore, the expression levels of LaC4H were positively correlated with the flavonoids contents in leaves. The results of this study provide the fundamental information on LaC4H gene in the flavonoids biosyn-thesis pathway of L. apetalum.
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OBJECTIVE:To analyze the volatile components in Descurainia sophia and Lepidium apetalum and compare its dif-ferences. METHODS:HS-SPME was conducted for extracting volatile components in D. sophia and L. apetalum,GC-MS was used for detecting components,and area normalization method was adopted for calculating relative content of each component. RE-SULTS:The volatile components in D. sophia and L. apetalum were 25 and 18,accounting for 75.76% and 64.29% of total vola-tile components,respectively,and chemical components with the highest contents were β-caryophyllene and O-tolunitrile. CON-CLUSIONS:The method is simple,reliable,and can be used for the analysis of volatile components in D. sophia and L. apetalum. The volatile components show great differences in the kinds and contents,the study can provide basis for rapid identification of D. sophia and L. apetalum.
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Objective: To obtain the key enzyme gene involved in terpenoid biosynthesis pathway, phosphomevalonate kinase (PMK) gene was cloned from Lepidium apetalum, and sequence analysis and prokaryotic expression were performed. Methods: Based on the transcriptome data of L. apetalum, by designing specific primers of LaPMK gene, an open reading frame (ORF) of LaPMK gene was isolated from L. apetalum. Escherichia coli BL21 (DE3) cells were transformed with the prokaryotic expression vector pET32a-LaPMK and used for prokaryotic expression under IPTG induction. Results: LaPMK gene has ORF of 1 518 bp (GenBank accession number KT004541), which encoded a protein of 505 amino acid residues. Bioinformatic analysis indicated that LaPMK protein which located in cytoplasm had no transmembrane domain and signal peptide, and exhibited the specific N-terminal domain and C-terminal domain of GHMP kinase super family. Phylogenetic analysis indicated that LaPMK protein showed the highest homology, 92% similarity, with PMK protein from Brassica rapa. The recombinant LaPMK protein was successfully expressed in E. coli BL21 (DE3) cells. Conclusion: The LaPMK gene is cloned from L. apetalum, and the stable prokaryotic expression system of pET32a-LaPMK is constructed. This study will provide the fundamental information for the further purification and the antibody preparation of LaPMK protein be helpful for the functional researches of LaPMK gene in terpenoid biosynthesis pathway.
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The chemical constituents of the seeds of Lepidium apetalum Willd. were investigated using chromatographic methods, including Diaion HP-20, Toyopearl HW-40, MCI Gel CHP-20, ODS, silica gel chromatography and semi-preparative-HPLC. Three compounds were isolated and their structures were elucidated by spectral data and physicochemical properties, which were identified as lepidiumamide A (1), cis-desulfoglucotropaeolin (2), trans-desulfoglucotropaeolin (3). Among those, compound 1 is a new phenylacetamide, compound 2 and 3 were isolated from this plant for the first time, and their configurations were also identified for the first time.
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This study was aimed to clone the GGPS (geranylgeranyl pyrophosphate synthase) gene from Lepidium apetalum, to analyze its sequence, and to express the protein in E.coli expression system. Specific PCR cloning primers were designed for GGPS gene from Lepidium apetalum according to the full-length sequence from a previous transcriptome sequencing project. PCR amplification was performed with this primer pair on a leaf cDNA template. TA cloning, sequencing and sequence analysis were performed.GGPS gene from Lepidium apetalum was expressed in the E.coli expression system. The results showed that the full-lengthGGPS cDNA from Lepidium apetalum was 1 146 bp coding a protein of 381 amino acids. The LaGGPS protein had an isoprenoid synthase domain. According to a phylogenetic tree constructed with multiple alignment of GGPS protein sequences from various plant species, GGPS protein from Lepidium apetalum was the closest to Arabidopsis thaliana and Sinapis alba. The prokaryotic expression vectorpET-32a-LaGGPS was also constructed successfully. The protein was expressed in E.coli BL21 strain. It was concluded that the cloning and prokaryotic expression of LaGGPS gene provided a foundation for a follow-up research of its function with protein purification and activity analysis.