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stilbenoid (anticancer) (antiviral) (antimicrobial) (cardio-protector) stilbenoid (Stemona collinsae) stilbenoid plant (key enzyme) stilbene synthase polyketide

ene synthase polyketide synthase type III stilbene synthase stilbenoid stilbenoid Stilbene synthase 5 Pinus sylvestris (ACC. no. Q02323), Arachis hypogaea (ACC. no. BAA78617), Rheum tataricum (ACC. no. AAP13782), Bauhinia variegata (ACC. no. ABF59517) Vitis vinifera (ACC. no. ABV82966) (conserved region) Chalcone/Stilbene synthase (CHS/STS) like gene (Stemona collinsae) RT-PCR 585 Chalcone synthase 5 3 RACE 5 3 CHS/STS like gene 1352 (open reading frame, ORF) 1185 394 Naringenin-Chalcone synthase Elaeis oleifera (% identity) 75% Chalcone synthase Stilbene synthase Chalcone synthase Stilbene synthase (function) CHS/STS-like gene stilbenoid chitosan sodium acetate pinosylvin STS/Chalcone like gene 24 CHS/STS-like gene pinosylvin chitosan 50 mg/l sodium acetate 5 mM

Abstract The biosynthesis of interest of stilbenoids studying hasve been increasing significantly interest dueowing to theirdiscovery of anticancer, antiviral, antimicrobial and cardio-protector properties. The previous reports showed that the root of Stemona cCollinsae containsed various stilbenoids having being an important antimicrobial activitiesy. Stilbene synthase (STS) is a member of type III plant polyketide synthase (PKS), which and plays a key role in the stilbenoid biosynthesis. Therefore, cloning of this gene represents To obtain aa useful biotechnological toogenetic materiall for to improvinge the production of the valuable stilbenoid metabolites, the STS gene was cloned from S. collinsae using RT-PCR technique, . The primers were designed from the conserved sequences of the stilbene synthaseSTS genes of were retrieved from the database as Pinus sylvestris (ACC. no. Q02323), Arachis hypogaea (ACC. no. BAA78617), Rheum tataricum (ACC. no. AAP13782), Bauhinia variegata (ACC. no. ABF59517) and Vitis vinifera (ACC. no. ABV82966). These sequences were aligned for designing primers from conserve domain to use as primers in Reverse-transcription polymerase chain reaction (RT-PCR).To clone the full length gene, A 585 bp PCR product of a central part of Chalcone/Stilbene synthase (CHS/STS) like gene were obtained from Stemona collinsea. The nucleotide sequence showed high similarity to chalcone synthase gene in the Genebank database.t The 5 end and 3 ends of this gene were then amplified identified by RACE technique. The result showed that the cDNA fragment of 1185 bp codes for an open reading frame of 394 amino acid residues. The complete sequence of CHS/STS like gene were assembled into 1413 bp containing an open reading frame (ORF) of 1185 bp encoding 394 amino acids. This deduced amino acid sequencessequence had high homology to chalcone synthase (CHS) and STS and was compared to other genes in Genbank database using Blastx program. The complete CHS/STS like gene of Stemona collinsae showed 75% identity to Naringenin-Chalcone synthase of Elaeis oleifera. Indeed, Due to the high homology of Chalcone and Stilbene synthase, the is cloned gene of S. collinsae might code for a putativeencode a CHS or STS/CHS. Subsequently, based on the deduced amino acid primary sequence. Therefore, an activity assay of this protein encoded from a cloned gene is necessary to confirm its function. In order to examine the expression of STS/CHS-chalcone synthase like gene was studied using semi-quantitative RT-PCR analysis. Due to theand amount level of pinosylvin iswere induced effected by the induction of elicitors such as chitosan and sodium acetate, . Specific primers of S. collinsae STS/chalcone synthase like gene and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were used in semi-quantitative RT-PCR analysis. After the treatments of S. collinsae root tissue was treated with

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different concentrations of chitosan and sodium acetate., After the exposure of ure of root tissue to 50 mg/l chitosan and 5 mM sodium acetate for 24 hrs, the expression of STS/CHS-like gene in S. collinsae was up-regulated when compared with the control without the elicitor treatment. resulted in significant elevation of STS/chalcone synthase like gene expression in comparison to control.In addition, The increases in the maximum production of pinosylvin were also accordingly occurredfound after the treatment by the induction at 50 mg/l chitosan and 5 mM sodium acetate for 24 hours of incubation period.

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