Conference Proceedings The 5th Thailand-Japan International Academic Conference 2012

Molecular Cloning and Characterization of Stilbene synthase (STS) gene in Stemona collinsae
Supaporn Passorn
Lecturer, School of Agriculture and Natural Resources, University of Phayao, Thailand (ppapon@up.ac.th)

ABSTRACT: The biosynthesis of stilbenoids has been increasing interest due to their anticancer, antiviral, antimicrobial and cardio-protector properties. The previous reports showed that the root of Stemona collinsae contains various stilbenoids having antimicrobial activities. Stilbene synthase (STS) is a member of type III plant polyketide synthase (PKS), which plays a key role in the stilbenoid biosynthesis. To obtain a useful genetic material for improving 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 STS genes of Pinus sylvestris, Arachis hypogaea, Rheum tataricum, Bauhinia variegata and Vitis vinifera. To clone the full length gene, the 5 end and 3 ends of this gene were then 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. This deduced amino acid sequences had high homology to chalcone synthase (CHS) and STS and showed 75% identity to Naringenin-Chalcone synthase of Elaeis oleifera. Due to the high homology of Chalcone and Stilbene synthase, an activity assay of this protein encoded from a cloned gene is necessary to confirm its function. KEY WORDS: Stemona collinsae, stilbene synthase (STS), stilbenoid

1. INTRODUCTION S. collinsae is a well-known Thai herb having different biological activities and provides an important source of new pharmaceutical and agrochemical agents such stilbenoid compounds [1]. Stilbenoid formation is controlled by STS which catalyzes condensation reaction of malonyl-CoA and 4-coumaroyl-CoA. STS are classified into two groups depending on substrate specific requirement such as resveratrol synthase corresponding to a p-coumaroyl-CoA-specific type and pinosylvin synthase to a cinnamoyl-CoA specific type [2]. This report presents the cloning of stilbene synthase gene being a useful tool in the future to improve the production of valuable stilbenoid metabolites in S. collinsae. 2. MATERIALS AND METHODS 2.1 Plant materials and total RNA extraction The tissue cultures of S. collinsea were grown on agar Murashige and Shoog (MS) Medium at 25 2 C with 16 hours light/day photoperiod for 8 weeks. Total RNA was extracted from Stemona colinsae tissue culture using a TRIzol kit (Invitrogen Carlsbad, CA, USA) according to the manufacturers instructions. 2.2 Cloning of Stilbene synthase cDNA from S. collinsae Reverse transcription-polymerase chain reaction (RT-PCR) was used for partial stilbene synthase cDNA amplification. Total RNA was reverse transcribed into first-stand cDNA using oligo(dT)

primer. The first-strand cDNA was used as template for PCR using degenerated primer, 5-AA(A/G)GC IAT(N)CAIGA(A/G)TGGGG-3 (forward) and 5-CCACCIGG(A/G)TGI(A/G)CAA(C/T)CC-3 (reverse), which contain inosine (stand by I). These primers were designed on the STS conserved regions of P. sylvestris (ACC. no. Q02323), A. hypogaea (ACC. no. BAA78617), R. tataricum (ACC. no. AAP13782), B. variegata (ACC. no. ABF59517) and V. vinifera (ACC. no. ABV82966), corresponding to the amino acid sequences KAIKEW and DLAENN respectively. Nucleotide sequences of 5 and 3 ends of this gene were amplified by the method of rapid amplification of cDNA ends (RACE). 2.3 Bioinformatic analyses Analysis of the sequence was carried out with GENETYXE-WIN Version 3.1. Sequence homology was verified by database searching at the National Center for Biotechnology Information. 3. RESULTS AND DISCUSSION 3.1 Isolation and sequencing of the full-length cDNA A 585 bp PCR product of a partial cDNA of Stilbene synthase were obtained from S. collinsea. The 5 end and 3 end of this gene were amplified by RACE technique. The complete sequence of STS gene were assembled into 1413 bp containing an open reading frame (ORF) of 1185 bp encoding 394 amino acids polypeptide with calculated molecular

mass of 42.9 kDa and a pI of 5.71 as shown in Fig. 1. The nucleotide sequence showed high similarity to chalcone synthase gene in the Genebank database having 75% identity to Naringenin-Chalcone synthase of Elaeis oleifera. However, the noticeable properties of stilbenes synthase were high homologies to chalcone synthase and resveratrol synthase [3]. Therefore, an activity assay of this protein encoded from a cloned gene is essential to prove its function.

Fig. 2 A phylogenetic tree based on the deduced amino acid sequences of various STSs. 4. CONCLUSIONS This work proposed the first molecular cloning involving in biosynthesis of stilbenoid from S. collinsae which obtained the full-length cDNA sequence of the STS gene. For future research, the functional expression of this gene could be further investigated to elucidate its property and reveal its role in defense mechanism of S. collinsae. ACKNOWLEDGEMENTS
This work was supported by a grant from National Research Council of Thailand. I would like to thank Dr. Kobkul Laoteng for her kindly suggestions and for supplying the plant material from Sukalya Poothong.

REFERENCES [1] T. Pacher, , C. Seger, D. Engelmeier, S. Vajrodaya, O. Hofer and H. Greger. Antifungal stilbenoids from Stemona collinsae J. Nat. Prod, No. 65, 2002, pp. 820-827. [2] P. H. Goodwin, T. Hsiang and L. Erickson, A comparison of stilbene and chalcone synthase inclulding a new stilbene synthase gene from Vitis riparia cv. Gloire de Montpellier Plant Science, No. 151, 2000, pp. 1-8. [3] S. Troft, T. Lanz, , S.A. Rensing, J. Schroder, and G. Schroder, Evidence that stilbene synthase have developed from chalcone synthase several times in the course of evolution, J. Mol. Evol, No. 38, 1994, pp. 610-618 [4] J. D. Thompson, D.G. Higgins, T. J. Gibson, CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice, Nucleic Acid Res., No. 22, 1994, pp. 4673-4680.

Fig. 1 Nucleotide and deduced amino acid sequence of

the full length cDNA of a S. collinsae STS gene.

3.2 Comparison of the deduced amino acid sequence with the structure of other stilbene synthase The sequences were aligned with CLUSTAL-W using default parameters and the UPGMA algorithm for phylogenetic analysis [4]. Sequence identity of S. collinsae STS was 65, 62.7, 62.3 and 61% respectively with B. variegate, A. hypogaea, P. sylvestris and V. vinifera. This finding suggest that S. collinsae STS properly clustered with the STS derived from other plants as shown in Fig. 2.