Analysis Of Dynamic Accumulation Of Hydroxysafflor Yellow A In Carthamus Tinctorius L. And Molecular Cloning Of Relative Gene.

Flos Carthami, dried tubular flower of Carthamus tinctorius L., is an one or two years herb, is also an important crude drug in traditional Chinese medicine. Safflower yellow(SY) is one of chalcone compounds and it's considered as the active compound in Carthamus. It was proved that SY had effects of promoting blood circulation, improving microcirculation, anticoagulated blood and preventing and curing thrombosis. hydroxysafflor yellow A (HSYA) has the higher concertration in SY and it is considered as the main active compound of Carthamus. The quality of Flos Carthami was influenced by the content of the HSYA directly. It is significant to find out the rule of HSYA dynamic accumulation and the relative functional genes which may enhance the safflower quality.In this study, HSYA dynamic accumulation rule was analyzed through measuring contents of HSYA with high performance liquid chromatography (HPLC). Then, cross hybridized F1 and F2 individuals were measured by high performance liquid chromatography. The result showed that, HSYA was produced and accumulated during the forming of flores, and it reached the platform since the third day protanthesis. HSYA was detected in all the F1 individuals, and only some of the F2 individuals detected the HSYA. While in some of the F2 individuals, HSYA concentration was above 2.0%, in some F2 individuals, HSYA concentration was 0%, which means the segregation of character. In order to found out the closely linked gene termed HSya, which may control the qualitative character of HSYA, we used cDNA-AFLP technique and bulked segregant analysis method (BSA). The cDNA-AFLP reaction system was established. According to the HPLC result, we two near-isogenic pools with some individuals with and without HSYA respectively in order to screen the transcript-derived fragments (TDFs) closely linked to HSYA relative gene. After screening the gene pool by different pairs of primer combinations, analysis the TDFs with F2 individuals, independent RT-PCR expression analysis validated the expression pattern of the TDFs, 2 TDFs differentiated between present and absent pools with relatively low recombination rate were found closely linked to gene controlled the high quality trait of C. tinctorius. We termed that TDF-A, TDF-C. They are 478bp, 150bp respectively. After analysis genetic distance of the 2 TDFs, it shows that the genetic distance of TDF-A and TDF-C were 8.1cM and 2.1cM. Bioinformatics analysis showed TDF-A had somewhat homologous with the gene coding predicted protein of Populus trichocarpa and TDF-C had highly homologous with the gene coding protein of 17.66 kea class I small heat shock protein of Ageratina adenophora. We got a 697 bp full-length gene of TDF-C by RACE method. The complete gene sequence is predicted to contain a 411 bp open reading frame (ORF) coding for a 136 amino acids (aa) protein, which has homogenous with the heat shock protein in many plants. The heat shock protein is not only an expressed products which may induce by stress, is also a kind of molecular chaperones. It can assist polypeptides to self-assemble by inhibiting alternative assembly pathways that produce nonfunctional structures, this may has effect on the quality and enzymatic active of some enzyme, so that, it may affct the biosynthesis and accumulation of secondary metabolite to some extent.Our finding is the report of HSYA accumulating dynamic analysis to found the best collection time, is also the significant foundation of a transcriptome-wide association with quality of the safflower and may provid idea for target for medical plant functional studies.