AcknowledgmentHussain T, Plunkett B, Ejaz M, Espley RV and Kayser O (2018) Identification of Putative Precursor Genes for the Biosynthesis of Cannabinoid-Like Compound in Radula marginata. Front. Plant Sci. 9:537.doi: 10.3389/fpls.2018.00537
For further details:This study was supported by the DISCO under the grant agreement 613513. We thank Mr. Sven Buijssen for providing the computationl resource, LiDong HPC. We also thank Dr. Felix Stehle for ESI-MS/MS and Dr. Pawel Rodziewicz for GC/MS analysis. Thanks to Dr. Richard ESpley for providing the Radula material and expressionl analysis.
Transcriptomic analysis of Radula marginata revealed identification of genes for cannabinoid-like compounds
Tajammul Hussain and Oliver KayserDepartment of Technical Biochemiestry, Faculty of Bio-Chemical Engineering, Technical University Dortmund
[email protected]; [email protected]
Poster presented: "Emerging Trends in Natural Product Biotechnology" at Technical University Dortmund on 20-21 September 2018
This is the first transcriptomic study for the Radulaceae family. We captured, assembled and annotate the R.marginata transcriptome. Identification of the precursor genes for the cannabinoid-like compound, validationof these genes through quantitative real time expressional analysis, characterization of the central precursorand finally detection of compound from metabolomics approach lead us to speculate that the cannabinoid-likepathway is likely to be conserved in lower and high land plants with the exception of first intermediate.However, these findings require further experimental work to confirm this proposed novelty. Overall, this studywould serve as a reference transcriptomic resource for Radulaceae family to further explore especially Radulamarginata. Moreover, this study also proposes R. marginata as an alternate to Cannabis sativa forcannabinoid-like compounds.
1. Toyota, M. et al. New bibenzyl cannabinoid from the New Zealand liverwort Radula marginata. Chem.Pharm. Bull. (Tokyo). 50, 1390–1392 (2002).2. Park, B. H. & Lee, Y. R. Concise synthesis of perrottetinene with bibenzyl cannabinoid. Bull. KoreanChem. Soc. 31, 2712–2714 (2010).3. Russo, E. B. Beyond Cannabis: Plants and the Endocannabinoid System. Trends Pharmacol. Sci. 37,594–605 (2016).
Similarity % Transcripts %
30-50 1.7650-59 13.9360-69 23.3770-79 24.8880-89 21.59
90-100 14.47
Sequence similarityB
E-value Transcripts %
0 ~ 1e-100 1.921e-100 ~ 1e-60 6.091e-60 ~ 1e-45 13.831e-45 ~ 1e-30 23.311e-30 ~ 1e-15 29.771e-15 ~ 1e-5 21.70
E-value distributionC
Marchantia polymorpha
Physcomitrella patens
Sorangium cellulosum
Pseudonocardia
Selaginella moellendorffi
i
Pseudonocardia autotrophica
Vitis vinife
ra
Nelumbo nucifera
Glycine max
Gossypium raim
ondii
Picea sitchensis
Theobroma cacao
Elaeis guineensis
Klebsormidium fla
ccidum
Gossypium hirsutum
Nicotiana ta
bacum
Eucalyptus grandis
Phoenix dactylifera
Noccaea caerulescens
Zea mays
Musa acuminata
Daucus carota
Gossypium arboreum
Num
ber o
f HS
P h
its
(Tho
usan
ds)
A
2
4
6
8
10
0
Species distribution
BLAST analysis: A)Blast species distribution of closely related bryophyte species againstNCBI non redundant (nr) database with an E-value of 1e -03. B) Sequence similarity and C)E-valuedistribution of BLAST results.
a) BLAST homology search
Gene Ontology analysis A) Gene ontology functional annotation for biologicalprocess (BP), Molecular function (MF) and Cellular component (CC) B)Overall annotationresults from BLAST, InterProScan (IPR), Gene ontology (GO) and KEGG pathway databases.
b) Gene ontology term and intrproscan mapping
iii) Identification of precursor genes for the bioynthesis ofcannabinoid-like compound and validation by qPCR
Candidate precursor genes for cannabinoid biosynthesis in Radula marginata: A)Identification of candidate genes for cannabinoid biosynthesis in R. marginata. GPP transcripts as well as all the upstream genes were identified and transcriptomic expression of identified genes was determined from FPKM B) Relative expression of candidate gene by real time quantitative PCR (RT-qPCR).
A
FP
KM
100
80
60
40
20
0
120
Rm_DXS
Rm_DXR
Rm_ISPD
Rm_ISPE
Rm_ISPF-1
Rm_ISPF-2
Rm_ISPG
Rm_ISPH
Rm_GPPS
Rm_STS
B
Rm_GPPS
Rel
ativ
e ex
pre
ssio
n
1.0
0.8
0.6
0.4
0.2
0.0
1.2
Rm_DXS
Rm_DXR
Rm_ISPD
Rm_ISPE
Rm_ISPG
Rm_ISPH
Rm_STS
i) de novo transcriptomic assembly Paired-end Illumina Hiseq-2000 platform generated 30 million raw reads for each pair. Raw reads filtered and quality trimmed. Trinity was used for de novo assembly of raw reads and resulted in 87,460 transcripts with a median contig size of 303 bp, and a maximum of 23,874 bp (Table 1).
RESULTS
Table 1: Sequencing Summary
ii) Annotation of assembled trasncriptomeTo annotate, all the assembled transcripts (87460) were BLASTed against NCBI publically availabale "nr" and "nt" databases (a) , followed by scanning with InterProScan (IPR) for protein signatures, assigning Gene ontology (GO) term (b) and finally map against KEGG database (c) for active pathways in Radula marginata.
c) KEGG Pathway mapping
Identified enzymes for respective pathway category
Metabolism 2464
No. of pathways
15
14
01
12
17
02
09
20
14
07
17
11
01
02
02
144
No. of unigenes
21158
2309
1573
3491
879
1456
941
5917
524
595
227
248
241
37
256
Pathway category
Carbohydrate metabolism
Amino acid metabolismBiosynthesis of antibiotics (Global and overview maps)
Metabolism of cofactors and vitamins
Lipid metabolism
Energy metabolism
Xenobiotics biodegradation and metabolism
Nucleotide metabolism
Metabolism of other amino acids
Biosynthesis of other secondary metabolites
Glycan biosynthesis and metabolism
Metabolism of terpenoids and polyketides
Translation
Immune systemSignal transduction
323
275
164
135
114
113
104
97
62
53
48
29
22
12
03
1554
No. of enzymesMain category type
Genetic Information Processing
Environmental Information Processing
Organismal Systems
Terpenoid backbone biosynthesis map00900 12 84 ec:2.7.4.2 Kinaseec:4.1.1.33 Decarboxylaseec:1.17.7.1 Synthase(ferredoxin)ec:2.2.1.7 Synthaseec:2.7.1.148 5'-diphospho-2-C-methyl-D-erythritolkinaseec:4.6.1.12 2-4-cyclodiphosphatesynthaseec:5.3.3.2 Delta-isomeraseec:2.3.1.9 C-acetyltransferaseec:2.3.3.10 Synthaseec:1.1.1.267 Reductoisomeraseec:2.1.1.100 O-methyltransferase
The liverwort Radula marginata belongs to the bryophyte division of land plants. Bryophytes are an early-diverged group of land plants. The division comprising of around 20,000 species and are divided into three phyla: mosses, hornworts and liverworts. Among them liverworts are the most abundant consisting of 6000-8000 species. Radulales is an order of subclass Jungermanniidae in this phylum. Radulaceae is the only family in this order, comprises Radula genus and 283 species. Radula marginata is of significant interest since perrottetinene and perrottetinenic acid 1,2 had been reported. These compounds are structural analogs to tetrahydrocannabinol (Δ9-THC), a psychopharmacological compound in Cannabis sativa L. Recently, agonistic activity of these compounds with cannabinoid receptor 1 (CB1) 3 further confirmed the potential of this specie in cannabinoid-based pharmaceutical industry. Therefore genetic-level understanding of secondary metabolic pathways that lead to the synthesis of cannabinoid-like natural compounds would be desirable.
INTRODUCTION
gene identification
cDNA library preparation
Next generation sequencing
de novo assembly
RNA extractionSample collection from native habitate in New Zealand
Illumina- Highseq 2000
Methodology
Conclusion and future prospects References