RESUMO
3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR) plays an important role in catalyzing the first committed step of isoprenoids biosynthesis in mevalonic acid (MVA) pathway. Here, we cloned a full-length transcript of Paris fargesii Franch. The full-length cDNA of P. fargesii HMGR (Pf-HMGR, GenBank accession no. JX508638) was 1,973 bp and contained a 1,728 bp ORF encoding 576 amino acids. Sequence analysis revealed that the deduced Pf-HMGR had high similarity with HMGRs from other plants, including Ricinus communis (77%), Litchi chinensis (76%), Michelia chapensis (75%) and Panax quinquefolius (72%). It had a calculated molecular mass of about 62.13 kDa and an isoelectric point (pI) of 8.47. It contained two transmembrane domains, two putative HMGR binding sites and two NADP(H)-binding sites. The predicted 3-D structure revealed that Pf-HMGR had a similar spatial structure with other plant HMGRs. Three catalytic regions, including L-domain, N-domain and S-domain were detected by structural modeling of HMGR. Tissue expression analysis revealed that Pf-HMGR was strongly expressed in roots and stems than in leaves. Taken together, our data laid a foundation for further investigation of HMGR's functions and regulatory mechanisms in plants.
Assuntos
Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Biologia Computacional , Hidroximetilglutaril-CoA Redutases/química , Hidroximetilglutaril-CoA Redutases/genética , Hidroximetilglutaril-CoA Redutases/metabolismo , Liliaceae/enzimologia , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , /genética , Reação em Cadeia da Polimerase em Tempo Real , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Objective: Paris fargesii is an important medicinal plant and its secondary metabolites, steroidal saponins, synthesized by the mevalonate pathway are the active ingredients. The study on the gene of the key enzyme 3-hydroxy-3-methyl- glutaryl-coenzyme A reductases (HMGR) in the mevalonate pathway is helpful for the regulation of steroidal saponins syntheses. Methods: In the basis of degenerate primers designed according to mRNA homologous conserved regions of 11 HMGRs from 11 plant species published on NCBI and total RNA extracted from the seedling of P. fargesii, a 404 bp fragment of HMGR was amplified by reverse transcription PCR technology, which is a new cDNA as searching in the Genbank. Results: Sequence analysis showed that the obtained fragment had 76%-81% identification compared with other seven plant species. The deduced amino acid seqence was predicted to have a wider and higher homology with other plants. Conclusion: Protein characteristic zones, conserved regions, and phylogenetic analysis by Prosite and ClustalX could give preliminary evidence for the existence of HMGR in P. fargesi, which is the report of HMGR gene isolated from the plants in Liliaceae for the first time.