Cloning And Identification Of Fallopia Multiflora Stilbene Synthase Gene (FmSTS) And Its Genetic Transformation In Arabidopsis Thaliana

Fallopia multiflora (Thunb.) is a perennial herbaceous climbing plant of Polygonaceae family; both the vines and roots could be used as herbal medicine. The extract of F. multiflora contains many active components for human health, while stilbenes such as 2,3,5,4'-tetrahydroxystilbene-2-beta-D-glucoside (TSG) are the most important ones. Modern pharmacological and clinical studies have demonstrated that TSG possess very effective activities for treatment of cardiovascular diseases, hyperlipidemia, neurosis, and other diseases commonly associated with aging. However, there have been no reports on the biosynthetic pathways of TSG in F. multiflora until now.It has been reported that resveratrol and other trihydroxy stilbenes are synthesized through phenylalanine pathways and the last step of which is catalyzed by stilbene synthase (STS). However, the related research in F. multiflora has not been reported. In this study, we focused on studying the STS gene in F. multiflora, and a full-length STS cDNA sequence, designed as FmSTS, was cloned based on homology-based techniques and rapid amplification of cDNA ends (RACE). In order to identify the gene and determine its specific function clearly, FmSTS was expressed in E. coli cells, and in vitro activity assays showed that the recombinant FmSTS protein had the catalytic activity of STS. Besides, FmSTS was transformed into Arabidopsis thaliana and the analysis of metabolic profiles in transgenic Arabidopsis plants showed that a kind of stilbenes, resveratrol glucoside accumulated in transgenic lines, which confirms its specific role in the biosyntheses of stilbenes. These results will help to elucidate the metabolite pathway of TSG in F. multiflora. The main results were as follows:(1) In order to avoid the contamination of polysaccharides, polyphenols and other pigments, an improved CTAB method for the effective isolation of RNA from F. multiflora was developed. The yield of total RNA was more than 100μg/g of fresh weight, and the brightness of 28S was nearly twice as much as that of 18S, which indicated the obtained RNA was of sufficient quality and suitable for downstream application such as RT-PCR, RACE technology and Northern blot analysis.(2) Based on the conservative amino sequences in STS, the degenerate primers were designed, and an STS gene in F. multiflora named FmSTS (GenBank accession number: GQ411431) was cloned for the first time by RACE technology. The nucleotide sequence of the FmSTS cDNA is 1228 bp long, with an open reading frame (ORF)of 1137 bp. The analysis of the FmSTS cDNA sequence predicted a polypeptide of 378 amino acids with a calculated molecular mass of 41.25 kDa and a predicted isoelectric point of 5.90. Blast-P search in NCBI showed that the deduced amino acid sequence shared high identity with Polygonum cuspidatum STS (ACC76753) and Rheum tataricum STS (AAP13782), while the predicted STS protein had the absolutely conservertive Cys-His-Asn stucture in the active centre, the same as other members of the STS family.(3) The genomic region of FmSTS was cloned (GenBank accession number: GQ411432) and sequencing results showed that the coding sequence of FmSTS gene was interrupted by three introns, which is a different gene structure from most of the STS genes studied so far containing only one intron at a conserved Cys60 site. Southern blot analysis showed that there are at least three copies of the FmSTS gene in the genome, and it is a multiple gene family.(4) FmSTS gene was inserted into a prokaryotic expression vector pET28a(+) and transformed into E. coli BL21. SDS-PAGE analysis indicated that a specific protein with molecular weight of about 42 kDa was expressed after the induction of IPTG, but mostly in insoluble form. After optimization of expression conditions, a small amount of soluble FmSTS protein was expressed, and they were purified with Ni-NTA chelating sepharose affinity chromatography. The recombinant FmSTS protein showed the same bioactivity with natural STS protein separated from plants, which could catalyze malonyl-CoA and coumaroyl-CoA to form resveratrol. For further identified the recombinant FmSTS protein, Western blot analysis was carried out, and the antibody against Vitis vinifera STS could specifically recognize the FmSTS band expressed in pET28-FmSTS/BL21 cells, while no signal could be detected when hybridized with the protein expressed in the blank control of pET-28a(+)/BL21 cells. These results confirmed FmSTS as a real STS gene from the aspect of immunology.(5) To understand the metabolic function of FmSTS in planta, it was placed under the control of the cauliflower mosaic virus (CaMV) 35S promoter and overexpressed in transgenic Arabidopsis thaliana via Agrohacterium-mediated transfer methods. The transgenic plants were selected in cultures with 50mg/L hygromycin, while PCR and RT-PCR tests were conducted to confirm that the FmSTS gene had been successfully integrated into the Arabidopsis genome. HPLC and electrospray ionization (ESI)-tandem mass spectrometry analyses demonstrated that resveratrol glucoside accumulated as the predominant stilbene metabolite in most of the transgenic lines, and the highest content was up to 65.3μg /g fresh weight.(6) Northern blot analysis revealed that the accumulation of FmSTS transcripts was most abundant in rhizomes, followed by the old stems, young leaves, and young stems, while the old leaves exhibited the lowest expression levels. This distribution corresponds well with the accumulation of the major bioactive principles TSG, suggesting that FmSTS might play an important role in TSG biosynthesis.