Cloning, Identification Of FmSTS2Gene Of Fallopia Multiflora And The Relationship Between Its Expression With Stilbene Glycoside’Expression

Fallopia multiflora (Thunb.) is a perennial herbaceous climbing plant of Polygonaceae family.The extract of Fallopia Multiflora contains stilbenes, anthraquinones and lecithin. While2,3,5,4’-tetrahydroxystilbene-2-beta-D-glucoside (TSG) is the most important one.TSG possess very effective activities for treatment of cardiovascular diseases, hyperlipidemia, neurosis, and other diseases commonly associated with aging. Much effort has been directed at extraction,structure determination, biological activity of TSG over the past decades.In recent years,some success also has been achieved in the metabolic regulation and gene engineering of stilbenes. However, detailed biosynthesis pathways and metabolic regulation of TSG, especially complicated regulation mechanism and expressing of genes and enzymes are unknown. So it is significant to shift focus from previous research priorities to search relevant enzymes,genes, abiotic stress and biotic signals so as to elucidate its detailed biosynthesis pathway and understand metabolic regulation networks. The elucidating of biosynthesis pathway and regulation mechanism of TSG is believed to contribute to improve the disease resistance of plants and health caring quality of Fallopia multiflora and also provide a opportunity to know more about global regulation networks and coordination between each pathway of secondary metabolism.Resveratrol and other trihydroxy stilbenes is derived from phenylalanine via the general phenylpropanoid pathway. PAL, the first and key enzyme of the phenylpropanoid sequence,it catalyzes the formation of transcinnamicacid by nonoxidative deamination of L-phenylalanine, which could be the rate-limiting step in the phenylalanine metabolism pathway. The second step in the phenylpropanoid pathway is the hydroxylation of trans cinnamic acid to4-coumaric acid, which is catalyzed by C4H,a cytochrome P450monooxygenase. The4-coumaric acid is then activated to its CoA thioester by4CL. The last step is catalysed by Stilbene synthase (STS) which is the key enzyme of the biosynthesis pathway. STS provides the first committed step by catalyzing the sequential decarboxylative addition of three acetate units from malonyl-CoA to a p-coumaroyl-CoA starter molecule derived from phenylalanine via the general phenylpropanoid pathway. Downstream modification enzymes in branching pathways produce a number of biologically important stilbenes.It is not clear whether Stilbene synthase is the key enzyme of the biosynthesis pathway of TSG. In this study, we focused on studying the STS gene in Fallopia multiflora in order to figure out whether STS is a relevant enzyme in the biosynthesis pathway of TSG.ObjectiveIn order to provides information for further research on the functions of stilbene synthase(STS) in the biosynthesis pathway and relevant enzymes,genes of TSG of Fallopia multiflora, the full-length cDNA of a new gene designated as FmSTS2(GenBank accession number:JX914503) was cloned and identified,and the relationship between The content of Stilbene Glycoside and expression of FmSTS2in different Fallopia multiflora tissues was studied.Methods1.Isolation of RNA from Fallopia multiflora:Total RNA was extracted from rhizome tissues of Fallopia multiflora using the Plant Total RNA Isolation kit (Bioteke, China). Reverse transcription was carried out with Oligo dT18using the Transcriptior First Strand cDNA Synthesis Kit (Roche, USA). 2.Cloning of full-length cDNA of STS in Fallopia multiflora by RACE:A pair of oligonucleotide primers designed based on the conservative amino sequences in STS and cDNA of Fallopia multiflora were used to isolate the conserved sequence of the STS gene using PCR. The primers of RACE were designed on the basis of the conserved sequence of the STS.5’-RACE was carried out using the5’RACE System for Rapid Amplification of cDNA Ends,Version2.0Kit(Invitrogen,USA).3’-RACE was carried out using SMARTerTM RACE cDNA Amplification Kit(Clontech,USA).The nested5’-RACE and3’-RACE products were purified, and gel-purified products were ligated into a pMD18-T Vector and sequenced.The full-length cDNA of the new gene designated as FmSTS2.3.Clone the open reading frame (ORF) of FmSTS2with the cDNA of Fallopia multiflora:The gel-purified products of FmSTS2gene was inserted into a prokaryotic expression vector pET28a(+) and was introduced into E. coli BL21and grown at200rpm and37℃in Luria-Bertani (LB) medium containing kanamycin (50μg/ml). At an absorbance at600nm (OD600) of0.6,1mM IPTG was added and the incubation temperature was30℃. After incubation for4h、5h、6h、7h、8h、9h, cells were harvested by centrifugation resuspended in2ml of potassium phosphate buffer(pH7.5), and sonicated on ice for10min. The soluble proteins was monitored by SDS-PAGE and the optimum induction time was gained. Expression of soluble protein induced for7h at20℃,23℃,25℃,28℃,30℃,35℃respectively were monitored by SDS-PAGE.4.The soluble proteins which were induced in optimum conditions were centrifuged at10,000g for10min at4℃. The supernatant was passed through a column of Ni-NTA His-BindTM Resin containing Ni2+as an affinity ligand. After washing with0.1M potassium phosphate buffer (pH7.5) containing0.5mM NaCl and40mM imidazole, the recombinant was eluted with0.1M potassium phosphate buffer (pH7.5) containing400mM imidazole. The sample was stored at-80℃. The efficiency of purification was monitored by SDS-PAGE.5.The standard assay (250μL) contained150μM starter coumaroyl-CoA,280μM malonyl-CoA,0.1M potassium phosphate (pH7.5), and10μg of protein, was incubated at35℃for30min, then extracted twice with250μL of ethylacetate and the product was dissolved in50μL of80%(v/v) ethanol after drying under vacuum.Analysis of the enzymatic products was performed by high-performance liquid chromatography (HPLC) on a C18reverse phase column (5μm,250mm×4.6mm). The eluents were acetonitrile-water(25:75) at a flow rate of1mL/min. The detection wavelengths were306nm.The temperature is25℃.6.We clean, dry, and chop or grind the raw different organization of Fallopia multiflora into powder,drying them for25h at55℃. Dissolve0.2g powder in25mL of50%(v/v) ethanol for12h and filter solution with0.45μM filterable membrane. Dissolve0.004g standard TSG powder in lOmL of50%(v/v) ethanol as control.C18column(4.6mm×250mm,5μm)was used with a mixture of acetonitrile:water(25:75,V/V)as mobile phase,with a flow rate of1mL/min and the UV detection wavelength was320nm.7. Total RNA was extracted from rhizome, old stem, young stem and leaf of Fallopia multiflora.RT-PCR was performed for FmSTS2gene expression in different tissues of Fallopia multiflora.Results1. The sequence of conserved sequence of the STS is780bp long. This fragment was then used to design gene-specific primers for amplifying the5’and3’ends of cDNA by5’-RACE and3’-RACE. Nested amplification for the5’and3’ends of the gene yielded bands of the anticipated lengths (850bp and250bp for the5’and3’ends, respectively). A gel-purified products were ligated into a pMD18-T Vector and sequenced.A full-length cDNA sequence designated as FmSTS2(GenBank accession No:JX914503) was obtained.The nucleotide sequence of the FmSTS2cDNA is1,644bp long, with an ORF of1,137bp, a441bp5’untranslated region and a66bp3’ untranslated region. The ORF predicted a polypeptide of378amino acids with a calculated molecular mass of42kDa.2.SDS-PAGE analysis indicated that soluble protein with molecular weight of about42kDa was expressed after the induction of IPTG and optimum conditions were induction temperature28℃,induction time7h. HPLC chromatograms showed the remaining time of enzymatic products was same with Resveratrol (13.24min).It indicated the main enzymatic products is Resveratrol.3. The content of TSG in different Fallopia multiflora tissues was determined by HPLC. The result revealed that the accumulation of TSG was most abundant in rhizomes(3.37%), followed by the old stems(0.78%), while young stems(0.06%), leaves (0.007%)exhibited the lowest content levels. RT-PCR was performed for FmSTS2gene expression in different tissues of Fallopia multiflora.The results show that internal control gene ActinI expressed consistent in different tissues,but FmSTS2expressed in rhizome most, followed by the old stem.Young stem and leaf express FmSTS2least.Conclusion:The full-length cDNA of a new gene designated as FmSTS2(JX914503) was cloned from Fallopia multiflora and its cDNA full-length is1644bp.The FmSTS2gene was transformed into prokaryotic expression cells E.coli BL21and successfully expressed in the form of soluble proteins. Soluble protein catalyzed malonyl-CoA and coumaroyl-CoA, of which enzymatic products was identified to be resveratrol. The recombinant protein shows the same bioactivity with natural Stilbene synthase.RT-PCR showed that the distribution of FmSTS2corresponds well with the accumulation of TSG, suggesting that FmSTS2might be the relevant enzyme in the biosynthesis pathway of TSG.Our study provides information for further research on the functions of FmSTS2in the biosynthesis pathway of TSG of Fallopia multiflora,and contributes to improve health caring quality of Fallopia multiflora using genetic engineering technology.