Cloning And Functional Confirmation Of Key Enzyme Genes Of Flavonoids Metabolic Pathway In Dryopteris Fragrans(L) Schott

Dryopteris fragrans(L.) Schott is a perennial natural medicinal plant which is widely distributed in Wudalianchi of Heilongjiang Province. The habitat of D. fragrans is extremely special, mostly the lava area formed by volcanic eruption. Although D. fragrans has certain adaptability to special environment, it takes a long time for it to recover once its natural population is destroyed. So far, researchers have mainly paid attention to morphological and anatomical observation, extraction and separation of chemical composition and pharmacological activity of D. fragrans, whereas there is limited study on key enzyme genes involved in secondary metabolic pathways. With the constantly exploitation of D. fragrans, wild D. fragrans resource is becoming endangered. Therefore, research on obtaining plant with a high content of active substances by artificial planting is needed urgently.In this paper, the main material is the tissue culture plant of D. fragrans. According the conservative region of sequences obtained in silico cloning and other known sequences, specific primers were designed, and the conservative fragments of CHI, F3 H, PAL and C4 H gene in D.fragrans were cloned. Meanwhile, three members of PAL gene family in D.fragrans were obtained. The expression patterns of four genes including Df PAL1, Df PAL2 and Df PAL3 of Df PAL gene family and Df C4 H in different D. fragrans tissues and different treatment conditions was analyzed by Real-Time PCR, and Df PAL3 and Df C4 H were determined for further study. The full-length c DNA of Df PAL3 and Df C4 H were obtained by RACE technology. Their gene structures, protein structures, and protein physicochemical properties were studied by bioinformatics analysis, and the evolutionary status of D. fragrans was determined by building phylogenetic tree. Moreover, plant expression vectors p BI121-Df C4 H and p BI121-Df CHS were successfully constructed, and they were transformed into wild-type Arabidopsis by floral dip method. Transgenic plants were detected by PCR method, and T1 generation seeds were harvested. The first and final metabolites in the transgenic plants were measured to initially verify the Df C4 H and Df CHS gene function in flavonoid biosynthesis pathway, so as to provide a theoretical basis for enhancing metabolic production by gene regulation, and lay a foundation for further research on the function of Df C4 H and Df CHS genes in D. fragrans.The main results obtained in this study are as follows:1. In this paper, we screened out four flavonoid pathway key enzyme genes, Df CHI, Df F3 H, Df PAL and Df C4 H through analyzing D. fragrans transcriptome data, and further performed their silico cloning.2. A conserved 452-bp fragment of Df CHI gene was cloned from D. fragrans, with 55%-57% sequence identity to other known sequences. Its Gen Bank sequence number was KP658975.3. A conserved 693-bp fragment of Df F3 H gene was cloned from D. fragrans, with 63%-62% sequence identity to other known sequences. Its Gen Bank sequence number was KP658976.4. Three members of phenylalanine ammonia lyase(PAL) gene family, Df PAL1, Df PAL2, and Df PAL3 were isolated from D. fragrans. The conservative fragments were 880 bp in length, and the sequence identities among the three was 84.30%. Their Gen Bank sequence numbers were KF830704, KJ634145, and KJ634146, respectively. Blastx analysis showed that the sequence identities of Df PAL1, Df PAL2 and Df PAL3 to known sequences were 97%, 96%, and 97%, respectively.5. By Real-Time PCR method, in different tissues and different treatment conditions, Df PAL gene family and Df C4 H gene expression levels were detected. The result showed that Df PAL3 had the highest expression in gametophyte, followed by petiole, and the lowest was in the spores; Df PAL1 and Df PAL2 appeared low expression levels in these parts, and there were no significant differences; Df C4 H displayed similar expression trend to that of Df PAL3; after low temperature, high temperature or UV treatment, both Df C4 H and Df PAL2 showed acute change in expression level, while there were no significant changes in the expression of either Df PAL3 or Df PAL1, and they have similar expression levels.6. The full-length c DNA(2370 bp) of Df PAL3 was cloned from D. fragrans by RT-PCR and RACE for the first time, which had an ORF of 1608 bp encoding a protein of 535 amino acids. Gen Bank sequence number of Df PAL3 was KJ634146.2. Df PAL3 protein ID was AID16057.2. Bioinformatics analysis showed that the molecular weight, isoelectric point and molecular formula of Df PAL3 protein were 57.1966 k Da, 5.88 and C2537H4081N697O767S18, respectively, belonging to stable hydrophobic protein with α-type secondary structure; protein domain analysis had shown that Df PAL3 protein contained PAL domain, and it was one member of the PAL family; subcellular localization analysis indicated that Df PAL3 protein was most probably localized in the endoplasmic reticulum; no signal peptide was found in Df PAL3, and it was a non-secreted protein; Df PAL3 protein was not transmembrane protein; there was a coiled-coil structure between 515 and 528 amino acids of Df PAL3 protein; the prediction of glycosylation and phosphorylation sites showed Df PAL3 had one potential Nglycosylation site, 62 potential O-sugar glycosylation sites and 25 protein kinase phosphorylation sites; multiple sequence alignment analysis showed the sequence identities of Df PAL3 to those known PALs in Psilotum nudum, Isoetes lacustris, Herba Botrychii, and Pteridium aquilinum were 73%, 74%, 77%, and 93%, respectively; the phylogenetic tree showed that D.fragrans was most closely related to Herba Botrychii and Psilotum nudum, less closely related to gymnosperms, and least closely related to angiosperms.7. The full-length c DNA(2063 bp) of Df C4 H was cloned from D. fragrans by RT-PCR and RACEfor the first time, which had an ORF of 1527 bp encoding a protein of 508 amino acids. Gen Bank sequence number of Df C4 H was KF830705.2. Df C4 H protein ID was AHI17493.2. Bioinformatics analysis showed that the molecular weight, isoelectric point and molecular formula of Df C4 H were 58.1908 k Da, 8.92, and C2649H4189N719O719S18, respectively; it was unstable hydrophilic protein; secondary structure prediction showed Df C4 H was a mixed type protein; protein domain analysis had shown that Df C4 H protein contained cells cytochrome P450 domain, and it was one member of the cytochrome P450 family; subcellular localization analysis indicated that Df C4 H protein was most probably localized on the plasma membrane; signal peptide prediction result showed Df C4 H protein may contain a signal peptide at the N-terminus, and it also may had a coiled-coil structure; the prediction of glycosylation and phosphorylation sites showed Df C4 H protein had a potential Nglycosylation site, 36 potential O-glycosylation sites points and 19 protein kinase phosphorylation sites; phylogenetic analysis showed that D. fragrans was most closely related to moss Physcomitrella patens, less closely related to gymnosperms, and least related to angiosperms.8. In this paper, plant expression vector p BI121-Df C4 H was constructed, and transformed into wild-type Arabidopsis by floral dip method. PCR method was used to detect Df C4 H transgenic plants, and T1 generation seeds were obtained. HPLC analysis showed that the content of the first metabolite pcoumaric acid in T1 generation plants was significantly higher than that in wild-type plants. Moreover, the determination of the final product of the flavonoid biosynthetic pathway showed that the anthocyanin content of the Df C4 H transgenic plants was significantly higher than that of the wild type plants.9. In this paper, plant expression vector p BI121-Df CHS was conducted, and transformed into wildtype Arabidopsis by floral dip method. PCR method was used to detect Df CHS transgenic plants, and T1 generation seeds were obtained. HPLC analysis showed that the content of the first metabolite chalcone of T1 transgenic plants was significantly higher than that of wild-type plants. The determination of the final product of flavonoid biosynthetic pathway showed that the anthocyanin content of the Df CHS transgenic plants was significantly higher than that of the wild type plants.