Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 Sep 12:5:e3781.
doi: 10.7717/peerj.3781. eCollection 2017.

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

Xiwu Qi  1   2 , Xu Yu  1   2 , Daohua Xu  1 , Hailing Fang  1   2 , Ke Dong  3 , Weilin Li  1   2 , Chengyuan Liang  1   2
Affiliations

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

Xiwu Qi et al. PeerJ. .

Abstract

Background: Lonicera japonica is an important medicinal plant that has been widely used in traditional Chinese medicine for thousands of years. The pharmacological activities of L. japonica 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 L. japonica transcriptome and analyzed CYP450s that may be involved in chlorogenic acid (CGA) biosynthesis.

Methods: The recent availability of L. japonica transcriptome provided opportunity to identify CYP450s in this herb. BLAST based method and HMM based method were used to identify CYP450s in L. japonica 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 L. japonica 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 L. japonica. The differences of typical motif sequences between A-type and non-A type CYP450s in L. japonica 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 LjC3Hs (CYP98A subfamily) and three LjC4Hs (CYP73A subfamily) that may be involved in biosynthesis of CGA, the major ingredient for pharmacological activities of L. japonica. qRT-PCR results indicated that two LjC3Hs exhibited oppositing expression patterns during the flower development and LjC3H2 exhibited a similar expression pattern with CGA concentration measured by HPLC. The expression patterns of three LjC4Hs were quite different and the expression pattern of LjC4H3 was quite similar with that of LjC3H1.

Discussion: Our results provide a comprehensive identification and characterization of CYP450s in L. japonica. Five CGA biosynthesis related CYP450s were cloned and their expression patterns were explored. The different expression patterns of two LjC3Hs and three LjC4Hs may be due to functional divergence of both substrate and catalytic specificity during plant evolution. The co-expression pattern of LjC3H1 and LjC4H3 strongly suggested that they were under coordinated regulation by the same transcription factors due to same cis elements in their promoters. In conclusion, this study provides insight into CYP450s and will effectively facilitate the research of biosynthesis of CGA in L. japonica.

Keywords: C3H; C4H; CGA biosynthesis; CYP450; Lonicera japonica.

PubMed Disclaimer

Conflict of interest statement

The authors declare there are no competing interests.

Figures

Figure 1
Figure 1. Phylogenetic analysis of predicted CYP450s in L. japonica and the representative members of CYP450 families.
Figure 2
Figure 2. Weblogos of conserved motifs identified in A-type (A) and non-A type (B) CYP450s from L. japonica.
Figure 3
Figure 3. Gene ontology annotation of A-type and non-A type CYP450s in L. japonica.
Figure 4
Figure 4. KEGG pathway analysis of predicted CYP450s in L. japonica.
Figure 5
Figure 5. Phylogenetic analysis of C3Hs and C4Hs from L. japonica and other plants.
LjC3Hs were labeled by black dots and LjC4Hs were labeled by black triangles. Protein sequences were downloaded from UniProt with accession numbers as follows: C3H1|Cc (A4ZKM5), CYP98A3|At (O22203), C3dH1-2|Sm (D8SCG3), C3dHi2|Ob (Q8L5H7), CYP98A44|Tp (C9EGT6), C3dH|Ot (T1NXG3), C4H|At (P92994), C4H|Cr (P48522), C4H|Ps (Q43067), C4H|Ms (P37114), C4H|Ca (O81928), C4H|Gm (Q42797).
Figure 6
Figure 6. CGA contents of buds and flowers in different developmental stages L. japonica.
YA-young alabastrum, GA-green alabastrum, WA-white alabastrum, SF-silvery flower, and GF-golden flower.
Figure 7
Figure 7. Transcriptional analyses of CGA biosynthetic pathway genes in buds and flowers of L. japonica at different developmental stages.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Bailey TL, Boden M, Buske FA, Frith M, Grant CE, Clementi L, Ren J, Li WW, Noble WS. MEME SUITE: tools for motif discovery and searching. Nucleic Acids Research. 2009;37:W202–W208. doi: 10.1093/nar/gkp335. - DOI - PMC - PubMed
    1. Bi C, Chen F, Jackson L, Gill BS, Li W. Expression of lignin biosynthetic genes in wheat during development and upon infection by fungal pathogens. Plant Molecular Biology Reporter. 2011;29:149–161. doi: 10.1007/s11105-010-0219-8. - DOI
    1. Boerjan W, Ralph J, Baucher M. Lignin biosynthesis. Annual Review of Plant Biology. 2003;54:519–546. doi: 10.1146/annurev.arplant.54.031902.134938. - DOI - PubMed
    1. Chai XY, Li SL, Li P. Quality evaluation of Flos Lonicerae through a simultaneous determination of seven saponins by HPLC with ELSD. Journal of Chromatography A. 2005;1070:43–48. doi: 10.1016/j.chroma.2005.02.031. - DOI - PubMed
    1. Chen J, Li SL, Li P, Song Y, Chai XY, Ma DY. Qualitative and quantitative analysis of active flavonoids in Flos Lonicerae by capillary zone electrophoresis coupled with solid–phase extraction. Jorunal of Separation Science. 2005;28:365–372. doi: 10.1002/jssc.200400024. - DOI - PubMed

LinkOut - more resources