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[Cloning and expression analysis of 5-phosphomevalonate kinase gene (CcPMK) in Cinnamomum camphora]
Han Zheng1, Mu-Yao Yu2, Chun-Juan Pu2
1Tianjin University of Traditional Chinese Medicine Tianjin 301617, China State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences Beijing 100700, China.
Abstract:
The 5-phosphomevalonate kinase(PMK) is a key enzyme in mevalonate(MVA) pathway which reversibly catalyzes the phosphorylation of mevalonate 5-phosphate(MVAP) to form mevalonate-5-diphosphate(MVAPP) in the presence of ATP and divalent metal ion such as Mg~(2+). In this research, on the basis of the transciptome database of Cinnamomum camphora, the PMK was cloned by cDNA from C. camphora, and was named CcPMK(GenBank number KU886266). The ORF of CcPMK was composed of 1 545 bp, encoding 514 amino acids. The bioinformatics analysis of CcPMK indicated that the molecular weight of the encoded protein was 56.14 kDa, with a theoretically isoelectric point of 7.64, and there was no signal peptide and transmembrane structure in putative protein. By multiple sequence alignment and phylogenetic tree analysis, we found that similarity between CcPMK and PMK amino acid sequence of other plants was as high as 75%. Among the similar sequences, 45% of them belonged to the alpha helix, while 16% belonged to the beta strand. CcPMK obtained 3 PMK protein family motifs and 1 ATP binding site Gly-Leu-Gly-Ser-Ser-Ala-Ala, and its 3 D structure contained a catalytic pocket structure, proving CcPMK as a member of PMK gene family. The result of phylogenetic tree showed that CcPMK was closely related to monocotyledon plants such as Phonenix dactylifera. The results of the Real-time PCR indicated that the expression level of CcPMK in borneol type was higher than that in linalool type, cineol type, iso-nerolidol type and camphor type. CcPMK expressed highest in roots and lowest in branches. Our results revealed that the expression level of CcPMK was different among five chemical types and different plant tissues, and the research provides foundation for further study of the terpenoids biosynthetic pathway in C. camphora.
Insights
Researchers cloned and analyzed the 5-phosphomevalonate kinase (PMK) gene from Cinnamomum camphora, named CcPMK. Its expression varies across different chemical types and tissues, providing insights into terpenoid biosynthesis.
Area of Science:
- Biochemistry
- Plant Molecular Biology
- Genomics
Background:
- The mevalonate (MVA) pathway is crucial for synthesizing isoprenoids.
- 5-phosphomevalonate kinase (PMK) is a key enzyme in the MVA pathway, catalyzing the phosphorylation of mevalonate 5-phosphate (MVAP).
- Understanding plant-specific enzymes like PMK is vital for metabolic engineering and understanding secondary metabolite production.
Purpose of the Study:
- To clone and characterize the PMK gene from Cinnamomum camphora (CcPMK).
- To analyze the bioinformatics, structural, and phylogenetic properties of CcPMK.
- To investigate the expression patterns of CcPMK in different chemical types and tissues of C. camphora.
Main Methods:
- cDNA cloning of PMK from C. camphora transcriptome data.
- Bioinformatics analysis including ORF prediction, molecular weight, isoelectric point, and structural motifs.
- Multiple sequence alignment and phylogenetic tree construction.
- Real-time PCR for gene expression analysis in different chemical types and plant tissues.
Main Results:
- The CcPMK gene (GenBank KU886266) was successfully cloned, with an ORF of 1545 bp encoding 514 amino acids.
- Bioinformatics analysis revealed CcPMK's molecular weight (56.14 kDa), isoelectric point (7.64), and lack of signal peptide or transmembrane regions.
- Phylogenetic analysis showed CcPMK is closely related to monocotyledonous plants and shares high similarity (75%) with other plant PMKs, possessing conserved motifs and an ATP binding site.
- CcPMK expression was significantly higher in the borneol chemical type and predominantly in roots compared to other types and tissues.
Conclusions:
- CcPMK is a functional member of the PMK gene family with conserved structural and catalytic features.
- The differential expression of CcPMK across chemical types and tissues suggests its regulatory role in terpenoid biosynthesis in C. camphora.
- This study provides a foundation for further research into the terpenoid biosynthetic pathway and potential applications in C. camphora.
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