Flavonoid 3’- Hydroxylase(F3’H) Gene Cloning And Expression Analysis Based On The Transcriptome Sequencing Of Dryopteris Erythrosora

Flavonoid is a kind of secondary metabolites which widely exists in the plants. Ithas the functions of antioxidation, anti inflammation, inhibition of tumor cell activity,anti HIV activity, and less toxicity. The content and types of flavonoid in fern is higer than other plants.While the metabolic pathway of flavonoids in ferns is still unknown.Flavonoid 3’-hydroxylase(F3’H) is one of key enzymes in the flavonoid synthesis pathway which belongs to P450 family. F3’H mainly catalytics naringenin and two hydrogen kaempferol generation, eriodictyol and two hydrogen in oak bark, and these two products are of important intermediates in the biosynthetic pathway of flavonoids.This study takes Dryopteris erythrosora as experimental material, transcriptome sequencing technologies have the possibility of F3’H gene sequence 62, because of the repetitive sequences or other gene sequence, through the NCBI sequence alignment and with external things for a gene tree, selected from seven sequences and design specific primers and cloned full-length. Then, through the construction of prokaryotic expression vector p ET32 a, the cloned 7 genes into BL21 was induced to express. Finally, it was confirmed that the cloned gene was F3 ’H gene, and the gene structure, protein structure and physical and chemical properties were analyzed by bioinformatics analysis.The results of this study are mainly in the following aspects:1. Using Hi-seq 2000 sequencing platform, sequence assembly, splicing, filtering to obtain a total of 143604 Unigene, these Unigene annotations to the protein database,can be compared to the 69917. Unigene clustering analysis to obtain F3’H possibility is 62, the 62 genes and species F3’H gene construct gene tree, from screening to 7 as F3’H candidate genes.2. Transcriptome sequencing to obtain sequence information, extraction to thehigh quality of the RNA, combining race technology, 7 pairs of specific primers were designed and successfully cloned got the full sequence of seven F3’H candidate gene, named for the De F3’H-1, De F3’H-2, De F3’H-3, De F3’H-4, De F3’H-5,De F3’H-6 and De F3’H-7, the gene sequence of the open reading frame(ORF) length length respectively 1533 bp,1599bp,1539 bp,1575bp,1455 bp,1590bp and 1491 bp,coding 510,532, 512,524, 484,529,496 amino acids.3. This experiment successfully constructed p ET32 a vector construction, to transfer and De F3’H-3 gene sequence of recombinant expression vector BL21,expression was induced by substrate dihydrokaempferol, eventually won the predicted protein, the identification of the protein for flavonoid 3’- hydroxylase, De F3’H-3gene belongs to F3’H gene family.4. Using bioinformatics to predict the gene structure and protein properties of the obtained De F3’H-3 gene. The results showed that the open reading frame(ORF)length of this gene was 1539 bp, encoding 512 amino acids, the protein molecular weight was 50653.48 Da, and the isoelectric point was 8.05In summary, this study with Dryopteris erythrosora as research material, on the flavonoid biosynthesis key enzyme of flavonoid 3 ’- hydroxylase gene for cloning and expression analysis, thus for flavonoids approach to the study of gene function pave the way. In addition, the research on the origin and evolution of the gene provide basic data.