Molecular cloning and heterologous expression analysis of 1-Deoxy-D-Xylulose-5-Phosphate Synthase gene in Centella asiatica L.

Many genes involved in triterpenoid saponins in plants control isoprenoid flux and constitute the precursor pool, which is channeled into various downstream pathways leading to the synthesis of triterpenoid saponins in C. asiatica. Full-length 1-Deoxy-D-Xylulose-5-Phosphate-Synthase (CaDXS) gene was isolated for the study from the previously annotated Centella asiatica leaves transcriptomic data. The CaDXS gene sequence was submitted to the NCBI databases with GenBank accession number MZ997832. The full-length CaDXS gene contained a 2244 base pair open reading frame that encoded a 747 amino acid polypeptide. The predicted molecular weight (MW) and theoretical pI of DXS are 76.28 kDa and 6.86, respectively. Multiple amino acid sequence alignment of amino acids and phylogenetic studies suggest that CaDXS shares high similarities with DXS from other plants DXS belonging to different families. A phylogenetic tree was constructed using Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6. Structural analysis provided fundamental information about the three-dimensional features and physicochemical parameters of the CaDXS protein. Quantitative expression analysis showed that CaDXS transcripts were maximally expressed in leaf, followed by petiole, roots, and node tissues. CaDXS was cloned into the expression vector pET28a, expressed heterologously in DH5α bacteria, confirmed by sequencing, and subsequently characterized by protein expression and functional complementation. The study focused on understanding the protein structure, biological significance, regulatory mechanism, functional analysis, and gene characterization of the centellosides biosynthetic pathway gene DXS for the first time in the plant. It would provide new information about the metabolic pathway and its relative contribution to isoprenoid biosynthesis.

Molecular cloning and characterization of Triterpenoid Biosynthetic Pathway Gene HMGS in Centella asiatica (Linn.).

BACKGROUND: The flux of isoprenoids and the total accumulation of triterpenoid saponins known as centellosides in C. asiatica are controlled by the key genes of the Mevalonate pathway (MVA). These genes were reported to have positive regulation of the pathway in providing isoprenoid moieties. Though, some information is available on the pathway and secondary metabolites. However, most of the pathway steps are not characterized functionally. METHODOLOGY AND RESULTS: For the study, full-length pathway gene Hydroxymethyl glutaryl-CoA-synthase (CaHMGS; GenBank accession number: MZ997833), was isolated from previously annotated transcriptome data of Centella asiatica leaves. HMGS has been successfully cloned and heterologously expressed in bacteria E. coli strain DH5α. The cloned gene has been sequenced and further characterized through in silico studies by different bioinformatics tools. Also, the gene sequences have been submitted in NCBI. In silico studies of isolated gene sequence revealed the nature, characteristics of genes. The ORF of HMGS is 1449 bp encoding 482 amino acids. Predicted molecular weight (MW) of HMGS was 48.09 kDa and theoretical pI was 5.97. Blast results and Multiple sequence alignments of the gene showing the similarity with HMGS of other plants of their respective families. The Molecular Evolutionary Genetic Analysis (MEGA) version 10.1.6 was used to construct a phylogenetic tree. Differential tissue-specific expression of different plant parts was also checked. Tissue expression patterns unveiled that the highest expression level of the CaHMGS had been seen in the roots and lowest in the node of the plant. Functional complementation experiment of the CaHMGS in Saccharomyces cerevisiae wild strain YSC1021 and haploid strain YSC1021 which lack HMGS protein confirmed that the CaHMGS gene encodes functional CaHMGS that catalyzed the biosynthesis of mevalonate in yeast. CONCLUSIONS: The gene was reported, cloned and characterized first time in Centella asiatica. Understanding this biosynthetic pathway gene will further help in the improvement of plants for enhanced secondary metabolites production.