Identification and analysis of CYP450 genes from transcriptome of and expression analysis of chlorogenic acid biosynthesis related CYP450s.

BACKGROUND

is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of are mainly due to its rich natural active ingredients, most of which are secondary metabolites. CYP450s are a large, complex, and widespread superfamily of proteins that participate in many endogenous and exogenous metabolic reactions, especially secondary metabolism. Here, we identified CYP450s in transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis.

METHODS

The recent availability of transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in transcriptome. Then, phylogenetic analysis, conserved motifs analysis, GO annotation, and KEGG annotation analyses were conducted to characterize the identified CYP450s. qRT-PCR was used to explore expression patterns of five CGA biosynthesis related CYP450s.

RESULTS

In this study, 151 putative CYP450s with complete cytochrome P450 domain, which belonged to 10 clans, 45 families and 76 subfamilies, were identified in transcriptome. Phylogenetic analysis classified these CYP450s into two major branches, A-type (47%) and non-A type (53%). Both types of CYP450s had conserved motifs in . The differences of typical motif sequences between A-type and non-A type CYP450s in were similar with other plants. GO classification indicated that non-A type CYP450s participated in more molecular functions and biological processes than A-type. KEGG pathway annotation totally assigned 47 CYP450s to 25 KEGG pathways. From these data, we cloned two (CYP98A subfamily) and three (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of . qRT-PCR results indicated that two exhibited oppositing expression patterns during the flower development and exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three were quite different and the expression pattern of was quite similar with that of .

DISCUSSION

Our results provide a comprehensive identification and characterization of CYP450s in . Five CGA biosynthesis related were cloned and their expression patterns were explored. The different expression patterns of two and three may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of and strongly suggested that they were under coordinated regulation by the same transcription factors due to same elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in .