Cloning and expression analysis of CbuCYP71D15 gene from Catalpa bungei'jinsi'
[Objective]The molecular characteristics and expression patterns of CbuCYP71D15 gene in Catalpa bungei'jinsi'were analyzed to provide a reference for revealing the role of CbuCYP71D15 gene in the formation of'jinsiqiu'heartwood color.[Method]Based on the transcriptome data of'jinsi'wood samples,the full-length coding sequence(CDS)of CbuCYP71D15 gene was cloned by conventional PCR amplification technology.The bioinformatics analysis of CbuCYP71D15 gene was carried out by ProtParam,Protscale,PSORT Prediction and other online tools.Transcriptome data were analyzed to explore the expression level of CbuCYP71D15 gene in sapwood.At the same time,the expression pattern of this gene in different tissues of'jinsi'was analyzed by real-time fluorescence quantitative PCR(qRT-PCR).[Result]The CbuCYP71D15 gene sequence with a length of 1 497 bp was successfully cloned.Bioinformatics analysis showed that the gene encodes 498 amino acids,with a relative molecular weight of 56.5 kD and a theoretical isoelectric point of 9.16.It was a stable hydrophilic protein.Its secondary structure was mainly composed of alpha helix(46.18%)and random coil(34.34%),and there was a transmembrane segment.The encoded amino acid sequence was found to have the highest similarity to the homologous sequence of S.indicum by sequence alignment and phylogenetic analysis,and the genetic relationship of homologous proteins was the closest.Moreover,CYP71Ds are conserved during evolution,and CbuCYP71D15 protein had conserved domains and heme binding sites characterized by cytochrome P450 superfamily,it belonged to CYP71 family.Transcriptome data analysis showed that the expression level of CbuCYP71D15 gene in sapwood was consistent with the trend that 1,4-naphthoquinones were significantly higher in GS1 than in GS2 and GS3.The qRT-PCR analysis showed that CbuCYP71D15 gene was expressed in all tissues of'jinsi',and the expression abundance was the highest in sapwood.[Conclusion]The CbuCYP71D15 gene may be involved in the synthesis and metabolism of chromophoric substances in the heartwood of'jinsi'.This study can provide a basis for further analysis of the biological function of CbuCYP71D15 gene in'jinsiqiu'.
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