| Fagopyrum cymosum (Trev.) Meisn, one of perennial herbs of the genus Polygonaceae in the Fagopyrum Mill family, origins from southwest of china, and ranges from Shanxi, Jiangsu, Zhejiang, Jiangxi, Henan, Hunan, Hubei, Guangxi, Guangdong, Sichuan, Chongqing and Yunnan ect.. It is an important resource plant with rich nutrition and great medicinal values, and contains a large number of excellent genes. As an important traditional Chinese Medicinal Materials, the active extract of earthnut (secondary metabolites) of Fagopyrum cymosum is one of main constituents of many efficient drugs, and has prominent functions in anti-cancer, controlling tumour cell affect and transfering to lung, and anti-inflammatony antibacteria ect. At present, cultivated yield of Fagopyrum cymosum is restricted and cultivated species are lesser than wild type in nutrition value, efficient constituents and quantity for medicine, which leads to requesting for Fagopyrum cymosum largely, so that wild resource is destroyed greatly. In view of the conditions, it is ranked into the second degree of national protected plants. Domestically and abroad resource collection, diversity, physiological characteristic, breeding, pharmacodynamics and ecology environment and other aspects are outspread, however, in the light of function genes research relating to active components of medicine not reported.In order to further explore worth of Fagopyrum cymosum and its excellent function genes, and protect wild resource, in this paper, acquisition of high flavonoids-producing red callus lines and construction cDNA Library of F. cymosum were reported. Furthermore, the cloning,structure and function analysis of the key gene(DFR) and transcription regulational gene(MYBP) in the pathway of flavoloid metabolism were performed. This research not only provided a useful tool for conserving excellent genetic resource, cloning genes and regulating research in the pathway of flavonoids biosynthesis, but laid the groundwork for future producing medicinal second metabolic products on a large scale.1. Acquisition of high flavonoids-producing red callus linesExplants of stems and leaves were from sterile seedling of F. cymosum. When the stems were cultured on MS medium supplemented with 2.0mg/L 6-BA and 0.1 mg/L NAA, the induction rate was 100% after 12 days. When the leaves were cultured on MS medium supplemented with 1.0mg/L 6-BA and 0.5 mg/L NAA, the induction rate was 100% after 13 days. After continuous thrice clutures on respective medium of stems and leaves, the callus could be distinguished by colour with naked eyes into four cell lines , a red (stem1 callus line), a brown (stem2 callus line), a bright yellow (leaf1 callus line) and a white (leaf2 callus line) , representing respectively the different colour of metabolite accumulations. A sensitive spectrophotometric method has been developed for detecting the flavonoids in cultured cells. It revealed that the red line contained 6.3804 mg/g flavonoids which was 3.5 times more than the white line .2. Construction of F. cymosum callus cDNA libraryBy modified CTAB extraction method and the LiCl precipitation method, total RNA With higher purity and integrality were isolated from F. cymosum callus. cDNA library was constructed with SMART cDNA Library Construction Kit. The titer of the primary library was 1.5×10~6 pfu/mL , in which 98% clones were recombinant and the inserted cDNA fragments ranged from 0.5 kb to 2 kb, 66% of them more than 1 kb. The amplified library has a titer of 7.0×10~9 pfu/mL. The positive signals of P gene, belonging to Myb R2R3 family and expressed at low abundance, were detected by degenerate PCR in the amplified library. The data indicated that the cDNA Library had high quality. These meant that a high quality cDNA library of F. cymosum callus was obtained.3.The cloning and function analysis of FcDFRDihydroflavonol-4-reductase (DFR; EC1.1.1.219) catalyzes a key step late in the biosynthesis of anthocyanins, condensed tannins (proanthocyanidins), and other flavonoids. According to the obtained homologous probe from orther plant's DFRs, one DFR cDNA clones(FcDFR) were isolated from the F. cymosum using the RACE(rapid amplification of cDNA ends) methods to scanning cDNA library (GenBank accession No. EF522145/ EF522146).FcDFR contains a 201bp 3'-untranslated region and a 78bp 5'-untranslated region and a 1026-bp coding region encoding 341 amino acids with an MW and theoretical pI value of 38.60 kD and 5.91, respectively. The comparison between cDNA and genomic DNA sequences revealed that FcDFR is composed of two exons and one intron. Southern blot analysis indicated that DFR belongs to a small gene family, and FcDFR was a single copy in F. cymosum genomes.A NADP-binding site (VTGASGFVGSWLVMRLLEHGY) and a substrate specificity motif (TVNVEEKQKPVYDETCWSDVDFCRRV), were observed in the deduced amino acid sequence of FcDFR contained C-terminal region. So that, FcDFR was postulated to have an activity to convert DHQ or DHK to leucodelphinidin or leucopelargonidin. The bioinformation analysis indicatied that FcDFR did not have a signal peptide, was localized in the plasma membrane, and had a typical tertiary tructure of enzyme.FcDFR PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcDFR target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301-FcDFR. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301-FcDFR), recombinant plasmid were transformed into tobacco plants. 95 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .In 95 lines of transgenic plants, it was variable of total flavonoids contents in different lines. And many of them were improved in varied degree. The content of total flavonoids in leaves of 78 transgenic tobacco plants increased significantly, the highest being up to 1.8 folds, which indicated that the synthesis of total flavonoids was promoted by overexpress of FcDFR . However, the content of total flavonoids in 17 transgeniclines decreased.4. The cloning and function analysis of FcMYBP1According to the obtained homologous probe from the R2R3 conserved region in maize P gene and orther MYB genes, Using RACE on a F. cymosum callus cDNA library, we identified one clone, named FcMYBPl, encoding a putative R2R3 MYB protein. FcMYBP1 appeared to be a full-length cDNA of 1159 bp encoding a protein of 265 amino acids and contaied a 250 bp 3'-untranslated region and a 111 bp 5'-untranslated region. The comparison between cDNA and genomic DNA sequences revealed that FcMYBP1 is composed of two exons and one 81bp intron. Southern blot analysis indicated that FcMYBP1 belongs to a single copy gene in F. cymosum genomes. The FcMYBP1 protein sequence showed highly homology to PH4(61%) , VvMYB5b(55 %), BNLGHi233(66% ) , VvMYB5a(61%), and VvMYBPA1 (76%).Through structure and property analysis of FcMYBP1 with bioinformational methods, We found that the amino acid sequence of FcMYBP1 showed great homology to other MYBP in the N-terminus, but not in the C-terminus. The R2R3 repeat region of FcMYBP1 is highly conserved and contains the motif [D/E]Lx2[R/K]x3Lx6Lx3R for interaction with bHLH proteins. Although the C-terminal region shows little homology to other MYBs, FcMYBP1, PH4, AtMYB5 and VvMYB5a share two conserved motifs in their C-terminal domains, C1(130—138AA) and C2 (214-229AA) . Phylogenetic analysis revealed that FcMYBP1 and VvMYB5a from Vitis vinifera formed a closely related subgroup. The putative function of FcMYBP1 may be involved in the control of different branches of the phenylpropanoid pathway in F. cymosum.FcMYBP1 PCR products and pC2301 vector containing digestion sites were digested with Xba I and Sma I, and then FcMYBP1 target fragments and pC2301 vector were collected and performed the ligation. This generated an recombinant expression vector plasmid pC2301- FcMYBP1. Using the leaf disc transformation procedure mediated by Agrobacterium tumefaciens EHA105 (including pC2301- FcMYBP1), recombinant plasmid were transformed into tobacco plants. 80 transgenical tobacco plantlets were obtained, in which the transgenic lines were identified by GUS GUS histochemical staining and PCR and RT-PCR analysis .In 32 random selective lines of transgenic plants, it was greatly variable of total flavonoid contents in different lines. The content of total flavonoids in 20 transgenic tobacco leaves increased significantly, the highest being up to 3.2 folds, and 8 lines showed similar contents with controls, and 4 tobacco leaves decreased significantly. In summary, the flavoloid contents of 75 % transgenic plants were altered greatly, which indicated that FcMYBP1 was involved in the pathway of flavonoid metabolisms. |
