RESUMO
Roots of Euphorbia fischeriana and Euphorbia ebracteolata are recorded as the source plant of traditional Chinese medicine "Langdu," containing active ingredients with anticancer and anti-AIDS activity. However, the two species have specific patterns in the graphic distribution. Compared with E. ehracteolata, E. fischeriana distributes in higher latitude and lower temperature areas and might have experienced cold stress adaptation. To reveal the molecular mechanism of environmental adaptation, RNA-seq was performed toward the roots, stems, and leaves of E. fischeriana and E. ehracteolata. A total of 6,830 pairs of putative orthologs between the two species were identified. Estimations of non-synonymous or synonymous substitution rate ratios for these orthologs indicated that 533 of the pairs may be under positive selection (Ka/Ks > 0.5). Functional enrichment analysis revealed that significant proportions of the orthologs were in the TCA cycle, fructose and mannose metabolism, starch and sucrose metabolism, fatty acid biosynthesis, and terpenoid biosynthesis providing insights into how the two closely related Euphorbia species adapted differentially to extreme environments. Consistent with the transcriptome, a higher content of soluble sugars and proline was obtained in E. fischeriana, reflecting the adaptation of plants to different environments. Additionally, 5 primary or secondary metabolites were screened as the biomarkers to distinguish the two species. Determination of 4 diterpenoids was established and performed, showing jolkinolide B as a representative component in E. fischeriana, whereas ingenol endemic to E. ebracteolate. To better study population genetics, EST-SSR markers were generated and tested in 9 species of Euphorbia. A total of 33 of the 68 pairs were screened out for producing clear fragments in at least four species, which will furthermore facilitate the studies on the genetic improvement and phylogenetics of this rapidly adapting taxon. In this study, transcriptome and metabolome analyses revealed the evolution of genes related to cold stress tolerance, biosynthesis of TCA cycle, soluble sugars, fatty acids, and amino acids, consistent with the molecular strategy that genotypes adapting to environment. The key active ingredients of the two species were quantitatively analyzed to reveal the difference in pharmacodynamic substance basis and molecular mechanism, providing insights into rational crude drug use.
RESUMO
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.