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De novo sequencing and analysis of Lophophora williamsii transcriptome, and searching for putative genes involved in mescaline biosynthesis.

Ibarra-Laclette E, Zamudio-Hernández F, Pérez-Torres CA, Albert VA, Ramírez-Chávez E, Molina-Torres J, Fernández-Cortes A, Calderón-Vázquez C, Olivares-Romero JL, Herrera-Estrella A, Herrera-Estrella L - BMC Genomics (2015)

Bottom Line: A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots.High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases.This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN, 36500, Irapuato, Guanajuato, México. enrique.ibarra@inecol.mx.

ABSTRACT

Background: Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites.

Results: In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR.

Conclusions: High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

No MeSH data available.


Pathway assignment based on KEGG. a Major categories based on molecular interaction and reaction networks. b Subclasses and gene distribution in the ‘Biosynthesis of other secondary metabolites’ category
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Fig1: Pathway assignment based on KEGG. a Major categories based on molecular interaction and reaction networks. b Subclasses and gene distribution in the ‘Biosynthesis of other secondary metabolites’ category

Mentions: The 40,436 L. williamsii unigenes were annotated by sequence similarity BLASTx searches against Arabidopsis thaliana (www.arabidopsis.org) and reference plant proteins (Refseq; comprising all green plants, ftp://ftp.ncbi.nlm.nih.gov/refseq/release/plant/) datasets. A total of 25,149 (62.19 %) unigenes showed significant similarity (e-value 10−3) to Arabidopsis or the RefSeq databases (Additional file 3: Table S3). The high percentage of L. williamsii unigenes obtained in this study that did not match the RefSeq protein database (37.81 %) indicates that there is potential for the discovery of as-yet undescribed and novel plant genes in peyote, although many of these unigenes may represent non-coding RNAs or untranslated regions (UTRs). In addition, the significance of a BLAST search depends on the length of the query sequence; therefore, short sequences are rarely matched to known genes [15], or these sequences may represent rapidly evolving parts of genes that have diverged substantially from their homologs [16]. Based on the Arabidopsis top hits, we obtained the gene ontology annotations (GOs) for 23,729 L. williamsii unigenes (Additional file 3: Table S4). Using the WEGO software [17] unigenes with Arabidopsis hits were assigned to gene ontology classes with 85,988 functional terms. Biological processes comprised the majority of the functional terms (39,957; 46.47 %), followed by cellular components (23,619; 27.47 %) and molecular functions (22,412; 26.06 %) (Additional file 2: Figure S2; see also Additional file 3: Table S4). In addition, using the KEGG Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/) from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [18], Enzyme Commission (EC) numbers [19] and KEGG Orthology (KO) codes were also associated to each annotated L. williamsii unigene. 5,008 unigenes having KO codes were assigned to metabolic, genetic and environmental information processing pathways (Additional file 3: Tables S3 and S5). KEGG metabolic pathways that are well-represented by L. williamsii unigenes belong to carbohydrate, amino acid, energy and lipid metabolism (Fig. 1a). In the subclass secondary metabolism, the greatest number of unique sequences was mapped to phenylpropanoid biosynthetic pathways, for which tyrosine is the initial precursor (Fig. 1b). This result was expected considering that L. williamsii classes I and II alkaloids are also derived from this amino acid.Fig. 1


De novo sequencing and analysis of Lophophora williamsii transcriptome, and searching for putative genes involved in mescaline biosynthesis.

Ibarra-Laclette E, Zamudio-Hernández F, Pérez-Torres CA, Albert VA, Ramírez-Chávez E, Molina-Torres J, Fernández-Cortes A, Calderón-Vázquez C, Olivares-Romero JL, Herrera-Estrella A, Herrera-Estrella L - BMC Genomics (2015)

Pathway assignment based on KEGG. a Major categories based on molecular interaction and reaction networks. b Subclasses and gene distribution in the ‘Biosynthesis of other secondary metabolites’ category
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4557841&req=5

Fig1: Pathway assignment based on KEGG. a Major categories based on molecular interaction and reaction networks. b Subclasses and gene distribution in the ‘Biosynthesis of other secondary metabolites’ category
Mentions: The 40,436 L. williamsii unigenes were annotated by sequence similarity BLASTx searches against Arabidopsis thaliana (www.arabidopsis.org) and reference plant proteins (Refseq; comprising all green plants, ftp://ftp.ncbi.nlm.nih.gov/refseq/release/plant/) datasets. A total of 25,149 (62.19 %) unigenes showed significant similarity (e-value 10−3) to Arabidopsis or the RefSeq databases (Additional file 3: Table S3). The high percentage of L. williamsii unigenes obtained in this study that did not match the RefSeq protein database (37.81 %) indicates that there is potential for the discovery of as-yet undescribed and novel plant genes in peyote, although many of these unigenes may represent non-coding RNAs or untranslated regions (UTRs). In addition, the significance of a BLAST search depends on the length of the query sequence; therefore, short sequences are rarely matched to known genes [15], or these sequences may represent rapidly evolving parts of genes that have diverged substantially from their homologs [16]. Based on the Arabidopsis top hits, we obtained the gene ontology annotations (GOs) for 23,729 L. williamsii unigenes (Additional file 3: Table S4). Using the WEGO software [17] unigenes with Arabidopsis hits were assigned to gene ontology classes with 85,988 functional terms. Biological processes comprised the majority of the functional terms (39,957; 46.47 %), followed by cellular components (23,619; 27.47 %) and molecular functions (22,412; 26.06 %) (Additional file 2: Figure S2; see also Additional file 3: Table S4). In addition, using the KEGG Automatic Annotation Server (KAAS; http://www.genome.jp/tools/kaas/) from the Kyoto Encyclopedia of Genes and Genomes (KEGG) database [18], Enzyme Commission (EC) numbers [19] and KEGG Orthology (KO) codes were also associated to each annotated L. williamsii unigene. 5,008 unigenes having KO codes were assigned to metabolic, genetic and environmental information processing pathways (Additional file 3: Tables S3 and S5). KEGG metabolic pathways that are well-represented by L. williamsii unigenes belong to carbohydrate, amino acid, energy and lipid metabolism (Fig. 1a). In the subclass secondary metabolism, the greatest number of unique sequences was mapped to phenylpropanoid biosynthetic pathways, for which tyrosine is the initial precursor (Fig. 1b). This result was expected considering that L. williamsii classes I and II alkaloids are also derived from this amino acid.Fig. 1

Bottom Line: A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots.High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases.This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

View Article: PubMed Central - PubMed

Affiliation: Laboratorio Nacional de Genómica para la Biodiversidad (LANGEBIO), Centro de Investigación y Estudios Avanzados del IPN, 36500, Irapuato, Guanajuato, México. enrique.ibarra@inecol.mx.

ABSTRACT

Background: Lophophora williamsii (commonly named peyote) is a small, spineless cactus with psychoactive alkaloids, particularly mescaline. Peyote utilizes crassulacean acid metabolism (CAM), an alternative form of photosynthesis that exists in succulents such as cacti and other desert plants. Therefore, its transcriptome can be considered an important resource for future research focused on understanding how these plants make more efficient use of water in marginal environments and also for research focused on better understanding of the overall mechanisms leading to production of plant natural products and secondary metabolites.

Results: In this study, two cDNA libraries were generated from L. williamsii. These libraries, representing buttons (tops of stems) and roots were sequenced using different sequencing platforms (GS-FLX, GS-Junior and PGM, respectively). A total of 5,541,550 raw reads were generated, which were assembled into 63,704 unigenes with an average length of 564.04 bp. A total of 25,149 unigenes (62.19 %) was annotated using public databases. 681 unigenes were found to be differentially expressed when comparing the two libraries, where 400 were preferentially expressed in buttons and 281 in roots. Some of the major alkaloids, including mescaline, were identified by GC-MS and relevant metabolic pathways were reconstructed using the Kyoto encyclopedia of genes and genomes database (KEGG). Subsequently, the expression patterns of preferentially expressed genes putatively involved in mescaline production were examined and validated by qRT-PCR.

Conclusions: High throughput transcriptome sequencing (RNA-seq) analysis allowed us to efficiently identify candidate genes involved in mescaline biosynthetic pathway in L. williamsii; these included tyrosine/DOPA decarboxylase, hydroxylases, and O-methyltransferases. This study sets the theoretical foundation for bioassay design directed at confirming the participation of these genes in mescaline production.

No MeSH data available.