Cloning and analysis of open reading frame(ORF)of 4,5-DOPA dioxygenase in Bougainvillea peruviana
[Objective]DOPA dioxygenase extradiol is one of the key enzymes in the beet pigment biosynthesis pathway,which can catalyze the decomposition of dopa to form beet aldehydanine.And it is also the most likely enzyme protein related to the final flower color formation of Bougainvillea peruviana.The open reading frame(ORF)of B.peruviana dioxygenase gene was cloned,and the amino acid sequence encoded by it was analyzed by bioinformatics to explore the mechanism of flower color change and the metabolic pathway of beet pigment.[Method]Taking the bracts of B.peruviana as materials,RNA was extracted,reverse transcription was carried out,and then gene cloning and gene PCR amplification were completed by rapid transformation.Finally,conserved region prediction,signal peptide analysis,phosphorylation site prediction,transmembrane signal prediction,homology comparison,phylogenetic tree construction and secondary and tertiary structure prediction were performed for the encoded amino acid sequence,the structure and function of amino acid sequence were studied.[Result]The 4,5-DOPA dioxygenase gene ORF sequence of B.peruviana was 801 bp in length,encoding 266 amino acids.The amino acid sequence encoded by the ORF sequence had a conserved domain of cd07363.The encoded protein was a stable acidic hydrophilic protein with a molecular weight of 29734.76 kD and an isoelectric point of 6.26.The secondary structure contained 11 α-helixes,7 β-turns,and 20 β-sheets.It had 4 phosphorylation sites and no transmembrane structure.The sequence was also highly homologous to 66 sequences,with B.peruviana had the same evolutionary branch as B.peruviana Thimma,B.stipitata Griseb and Pisonia umbellifera,which was consistent with the law of natural evolution.[Conclusion]The amino acid sequence of the dopa dioxygenase open reading frame ORF of B.peruviana belongs to the third type of estradiol dioxygenase gene family,which has the conserved amino acid sequence of the gene family,indicating that the dopa dioxygenase gene has a unique conservatism in the production of beet pigment plants,and belongs to a stable,acidic hydrophilic protein.There is no transmembrane structure,and it may only play a role in the cytoplasm.Beet pigment is the main factor controlling the change of flower color in B.peruviana.Studying the key enzymes in the synthesis pathway of beet pigment in B.peruviana and analyzing the synthesis and metabolism mechanism of beet pigment can provide theoretical reference for the formation mechanism of flower color in B.peruviana.It is expected to enrich the flower color of B.peruviana by transgenic means in the future work.
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