| Carthami flos is the dried flowers of Carthamus tinctorius L.which is clas sified as compositae.It is blood circulation drug and can promot blood circulati on,dissipate blood stasis and analgesia.It can be used for amenorrhea,dysmenorr hea,persistent lochia,mass,lump and so on.It is widely used in chinese patent m edicine and clinical prescriptions,for example,Xuefuzhuyu pill,Rupixiao tablets,Sh angkejiegu piece,Shujinhuoxue pill and Jinshu particle in directory of national essential drugs.In this study,we started with the first key enzyme gene called c halcone synthase gene which exist in safflower flavonoids biosynthetic pathway and the safflor yellow,cloned, analysised and expressed of chalcone synthase g ene,analyzed the accumulation of the safflower yellow,laid the foundation for th e study of biosynthetic mechanisms of the safflower yellow.The results are as f ollows:1. The cloning, analysising and expressing of the chsRNA was extracted from the fresh flower during flowering days (1d-7d).T he qualified RNA was reverse transcribed to cDNA.Designed specific primers,in vestigated PCR conditions,obtained PCR products and sequenced PCR product s equences.The results are as follows.The best method to safflower RNA extracti on is improved Trizol.The best PCR primers is:Forward-Primer:ATGGCATCCT TAACCGATATT, Reverse-Primer:TTAAGCGGCAATGGGGGT.The optimum re action condition is:94℃,3min,30cycles (94℃,30sec;64.2℃,30sec;72℃,10min),72℃,5min.The PCR product is1149bp with a1041bp complete open reading frame and can encode346amino acids.This protein bel ongs to the CHS family comparisoned by blastp in NCBI.CHS of safflower ha ve similarities with more than100species in NCBI,it has high similarities wit h Silybum marianum,Callistephus chinensis,Chrysanthemum x morifolium,Gynura bicolor,Rudbeckia hirta and Dahlia pinnata respectively are95%、95%、94%、94%、93%、92%. Analysised the biological characteristics of chs,the results are as follows:C onstructed the phylogenetic tree by MEGA5.1showed the CHS of safflower ha s the closest relationship with the CHS of silymarin, both of which belong to the Cynareae of the Carduoideae in Compositae.Forecasted the protein by ProtP aram showed the molecular formula is C1678H2693N451O493S20, molecular weight i s37.7kD, isoelectric point is6.10, negatively charged amino acid residues (A sp+Glu) is42, positively charged amino acid residues (Arg+Lys) is38,unst able parameter is39.39, belonging to stabilize proteins.Predicted hydrophobic m ap by Protscale showed the hydrophobic peptide chain highest peak is2.218,th e highest hydrophilic peak is-2.164,hydrophilic amino acids in relatively evenl y distributed throughout the peptide chain, slightly more hydrophobic amino aci ds.Predicted the secondary structure of CHS of safflor by Protscale showed tha t about142α-helix,45%,48extended clip,14%,156free-curl,41%.Modeled the three-dimensional structure of CHS of safflor by ProtscaleProtscale amino a cid sequence of the protein were safflower CHS homology, access to tertiary structure of CHS.Through the investigation of standard curve, the amplification curve, the di ssolve curve,established the real-time PCR methods of chs and the housekeepin g gene25s,obtained the relative expression of chs,decreasing order is3d (2.80)>6d (1.12)>2d (1.10)>1d (0.85)>5d (0.82)>1d (0.28)>4d (0.13).Showi ng four stages:1d-3d is rapid rise stage,3d is the highest, up to2.%;3d-4d is t he turning stage,4d is the lowest,only0.13;4d-6d is the slow rise stage;6d-7d is the slow reducting stage.2. The study of the accumulation of HSYAThe content of HSYA is the highest in safflor yellow and also the main active ingredien, research of the accumulation of HSYA contribite to the analyzethe rules of safflor yellow. Therefore,determined the content of HSYA of1d-7d by HPLC, measured results are3d (1.71%)>2d (1.53%)>4d (1.42%)>5d (1.14%)>6d (0.68%)>7d (0.66%)>1d (0.46%)2d-5d are in line with the "Chinese Pharmacopoeia"2010edition a requirement.As can be seen from the results, HSYA dynamic changes of accumulation showing increase and decreaset, in which the highest3d, up1.71%, the lowest1d,only0.46%, the sample2d-4d HSYA with higher levels, but safflower medicinal parts are the open tubular flowers,2d with fewer open tubular flowers, not harvested, so the flowers on the third day, fourth day of the harvest the best.3. The study of HPLC fingerprintTo fully grasp the accumulation rule of safflore yellow, by measuring the wavelength of the mobile phase, isocratic, gradient study, established HPLC fingerprint chromatographic conditions:chromatographic column:ZORBAX SB-C18(4.6x250mm,5μm);moving phase:A is acetonitrile, B is0.01%trifluoroacetic acid, gradient elution:0～10min,2%～10%A;10～25min,10%～15%A;25～45min,15%～20%A;45～65min,20%～25%A;65～70min,25%～2%A;determine wavelength:403nm;column temperature:25℃;flow velocity:1mL/min;sample volume:20μL;collected fingerprint of samples1d-1d,and established a total safflower fingerprint pattern diagram.The results showed the samples1d-1d have12common peaks and with a similarity at least0.97, a very high similarity.The higher peaks are the7th and the10th peak which about80%of the total peak area.Analysis the larger peak7and peak10by LC-MS.In the negative ion mode,the mass-to-charge ratio of peak7is611,fragment ions are473,403,325,207.Compared with the literature and identified as hydroxysafflor yellow A(C27H32O16,612).In the negative ion mode,the mass-to-charge ratio of peak10is1043,fragment ions are1025,923,593,449,287.Compared with the literature and identified as anhydrosafflor yellow B(C48H52O26,1044).In descending order of total peak area are:3d>2d>4d>5d>6d>\d>7d,1d-3d showing increasing and3d-7d showing decreasing1d and7d unchanged, indicating that the total amount of safflor yellow is conservation.peak7(HSYA) and peak10in descending order of peak area are:3d>2d>4d>5d>6d>7d>1d,1d-3d showing increasing and3d-7d showing decreasing.The rest of the peak area ratio of the total peak area is small, and the laws vary.4. The correlation analysis of the relative expression of chs and the accumulation of safflor yellowAnalysis the correlation of the relative expression of chs and the accumulation of safflor yellow:1d-3d is rapid rise stage,its rapid rise to the highest point,and with a rapid rise to the highest of safflor yellow;3d-4d is the turning stage,its sharp decline to the lowest, and with a dicrease of safflor yellow,but to a lesser extent;4d-6d is the slow rise stage,safflor yellow showed a slow decline in the accumulation period;6d-7d is the slow reducting stage,safflor yellow showed a flat.Since the synthesis of plant secondary metabolites is a complex system, which is affected by many factors, such as environmental factors, regulatory factors, that is why showing a positive correlation at first four days and a weak correlation relevance at the following three days.The study successfully cloned, analysised and expressed the chalcone synthase gene, obtained a accumulation rule HSYA, established HPLC fingerprint of safflor, identifed anhydrosafflor yellow B, got the dynamic accumulation rule of safflor yellow and analyzed the correlation of the relative expression of chs and the accumulation of safflor yellow. |
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