Cloning and characterization of 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) gene from Paris fargesii Franch.

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.

Optimized Ultrasonic-Assisted Extraction of Polysaccharides from Chinese Traditional Medicinal Plant Paris fargesii Using Orthogonal Design.

Paris fargesii is a famous Chinese traditional medicinal plant used as antipyretic, antidotal, antiphlogistic and analgesic. This research aimed at the determination of the total polysaccharides contents in P. fargesii, which were collected from Baoxing country of Sichuan Province in order to get the optimized extraction technology. Ultrasonic method was adopted in the extraction of total polysaccharides from P. fargesii. Through single factor experiment and orthogonal test, the effects of ultrasonic extraction conditions on the total polysaccharides extraction were measured, including solid-liquid ratio, ultrasonic temperature, ultrasonic power and ultrasonic time. Then colorimetric method was applied for the assaying. The optimized extraction conditions of total polysaccharides from P. fargesii were as follows: ultrasonic power: 600w, solid-liquid ratio: 1:20, ultrasonic extraction time: 1.5h and ultrasonic temperature: 90℃. Under the optimum parameters, the total polysaccharides extraction efficiency was 0.0715%.

Cloning and analysis of HMGR gene conserved fragments in Paris fargesii / 中草药

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.