ГЕНЕТИКА, 2010, том 46, № 7, с. 932-939
ГЕНЕТИКА РАСТЕНИЙ
УДК 575:582.892
GENE ONTOLOGY STUDY OF METHYL JASMONATE-TREATED AND NON-TREATED HAIRY ROOTS OF Panax ginseng TO IDENTIFY GENES INVOLVED IN SECONDARY METABOLIC PATHWAY
© 2010 S. Sathiyamoorthy, J.-G. In, S. Gayathri, Y.-Ju Kim, D.-Ch. Yang
Korean Ginseng Center and Ginseng Genetic Resource Bank, Kyung Hee University, Yongin, 449-701
e-mail: dcyang@khu.ac.kr Received August 24, 2009
The roots of Panax ginseng C.A. Meyer, known as Korean ginseng have been a valuable and important folk medicine in East Asian countries. It mainly used to maintain the homeostasis of the human body, with the presence of ginsenosides and non-saponin compounds like phenol compounds, acidic polysaccharides and polyethylene compounds. Functional genomics aid to annotate based on gene ontology. In this study, we focused on the genes involving in secondary metabolic pathways and to visualize temporal changes of gene expression in ginseng hairy roots with methyl ester methyl jasmonate (MeJA) along with non-treated hairy roots. A 5.774 EST clones were clustered and assembled as 501 contigs and 2.955 singletons. Annotations categorized with molecular functions, biological processes, cellular compounds of gene ontological terms and biochemical functions, enzyme commission to sequences were assigned to metabolic pathways of Kyoto Encyclopedia of Genes and Genomes database. Comparatively, EST sequences are assigned to cellular process, metabolic process, biotic and abiotic stress stimuli, developmental and biological regulations and transports are up-regulated 2—3 fold in MeJA treated hairy roots. 46 different sub groups of enzymes found in the MeJA treated plants. These annotated ESTs represents a significant proportion of the P. ginseng and provides molecular resource for developmental of microarrays for gene expression studies concerning development, metabolism and reproduction.
Panax ginseng C.A. Meyer, a perennial herb from the Araliaceae family with thousands years of history, has been traditionally known as a medicinal plant with mysterious powers in the Oriental medicine [1]. Oriental people have traditionally used ginseng roots and its extracts to revitalize the body and mind, increase physical strength, prevent aging and increase vigor. Many scientists have introduced new pharmacological concepts for the tonic effect of ginseng, resulting in interest and attention by explaining the basic pharmacology of ginseng with adaptogen effects [2, 3]. Originally, the efficacy of Korean ginseng has been recognized based on Oriental medical science theory [3]. Despite the considerable commercial interest in the production of secondary metabolites for the development of pharmacological agents, the genes that are involved in the biosynthesis of secondary metabolites remain uncharacterized at the molecular level [4]. Jasmonate and its methyl ester methyl jasmonate (MeJA) acts as a signaling compounds that modulates various physiological processes in plants, including root growth, senescence, the defense response against pathogens and insects attack [5, 6]. MeJA induces or increases the biosynthesis of many secondary metabolites that plays an important role in the adaptation of the plant to particular environments [7, 8]. MeJA-treated hairy roots expressed more transcripts that involved in secondary metabolites biosynthesis in the non-treated hairy roots [9].
Functional annotation of novel sequence data is a primary requirement for the utilization of functional genomic approaches in plant research [10]. High-throughput molecular biological technologies along with the availability of user friendly bioinformatics tools allows the feasible evaluation, functional annotation and classification of a high number of expressed sequence. Such characterization would be useful for functional genomic research in plants, in-depth knowledge about molecules, such as nucleic acids and deduced proteins, gene regulatory networks, and metabolic pathways become possible to apply homology annotation [11, 12]. Hierarchically structured onto-logical terms can now be adopted to query sequences and to describe genes and their products at different levels of knowledge and specificity. The Gene Ontology (GO) is most widely used schema for the functional characterization of plants, animals, microbial genes and gene products [13]. The GO project has developed three structured vocabularies (ontologies) describing genes, transcripts and proteins of any organism in terms of their associated cellular components, biological processes and molecular functions in a species-independent manner. Moreover, the GO vocabularies can be queried at different levels, allowing annotators to assign properties to genes or gene products, depending on the depth of knowledge and specificity about that entity [14]. When we consider about species for which the genome sequence is not available, EST
are the most commonly used methods for genome and transcriptome level analysis. EST experiments may represent a valuable resource for functional genomics and genetics in non-model plants. In the present study, we produced 2.640 ESTs of non treated hairy roots (DC01) and 3.134 EST sequences of MeJA treated hairy roots from NCBI dbEST database (accession number CN845540-CN848674). EST analysis of cDNAs from specific plant tissues with different treatment is an efficient way to identify genes that are involved in the biosynthesis of secondary metabolites in specific tissues. Usually for the high throughput transcriptome data, microarray is a way to analyze the different level of gene expressions, here we choose the treated and non treated ESTs to make the comparative analysis based on GO terms [15]. Biochemical functional annotation is an alternative method of categorizing unique sequences by biochemical functions, sequences were assigned to metabolic pathways via KEGG [16] using enzyme commission (EC) numbers as the basis for assignment. The one dimensional annotation and biochemical analysis of ESTs, with the secondary metabolic pathway and enzymes subgroups were discussed.
MATERIALS AND METHODS
Plant materials and cDNA library construction. A ginseng (Panax ginseng C.A. Meyer) hairy root lines had been established through the infection with Agro-bacterium rhizogenes strain R-1000. The hairy roots were cultured in 1/2-strength SH liquid medium and subcultured for 4 weeks intervals. The total RNA was isolated from the hairy root tissue samples via the aqueous phenol extraction procedures [17]. The poly (A) + RNA was isolated via oligo-dT-cellulose chromatography using an mRNA isolation kit (Stratagene, http://www.stratagene.com). A commercial cDNA synthesis kit (Uni-Zap XR, Stratagene) and a Giga-packlll Gold packaging extract were used in the construction of the library, in accordance with the instruction as per the manual provided by the manufacturers (Stratagene, USA). The fractions containing cDNAs larger than 500 bp were recovered, and this cDNA was amplified once, yielding a primary titre of 106 pfuml—1. The plasmid library was then plated on LB plates with ampicillin. Individual colonies were propagated, and were in glycerol stock at —80°C until use.
Nucleotide sequencing and sequence analysis. pBluescript SK(+) Uni-ZAP phagemids were excised from the Uni-ZAP XR library using helper phage in E. coli XL-1 blue MRF. The phagemids harboring inserts were selected via blue and white colonies screening on IPTG/X-GAL/ampicillin plates. We then conducted single-run partial sequencing of these randomly selected cDNA clones. The 5' ends of the cDNA inserts were sequenced with sequencing primers, using an automatic DNA sequencer (ABI Prism 3700 DNA sequencer, Perkin-Elmer, http://las.perkinelmer.com/), in ac-
cordance with the thermal cycling protocol of the Big-Dye Terminator Cycle Sequencing kit.
Generation of the unigene dataset. The unigene dataset produces a set of non redundant sequences composed of singlets and contigs. Generating a unigene dataset requires several steps, all of them integrated in a freely accessible web-interface named EGassembler [18]. For the unigene dataset generation, EGassembler was used with default analysis parameters. This bioinformatics pipeline starts with a sequence cleaning stage, removing low quality sequence stretches, followed by the detection and removal of repetitive elements. The resulting output is then searched and cleaned from organelle sequences. The remaining sequences are compared to generate ho-mology clusters and deduce contig sequences using CAP3 software [19]. The sequences which are not included in clusters were classified in the singlets dataset.
Functional analysis and pathway assignments. Gene ontology (GO) term annotation and function-based analysis of unique sequences were performed using Blast2go [10—12], a sequence-based tool to assign GO terms, extracting them for each BLAST hit obtained by mapping to extant annotation associations. GO terms for each of the three main categories (biological process, molecular function, and cellular component) was obtained from sequence similarity using the application default parameters. From these annotations, pie charts were made using 2nd level GO terms based on biological process, molecular function, and cellular component. This annotation was simplified and focused on plant related to the functional categories by using the Plant GOslim. Pathway assignments, enzyme commission (EC) numbers were assigned to unique sequences that had BLASTX scores with a cut-offvalue of E = 10-5 or less upon searching protein databases. The sequences were mapped to KEGG biochemical pathways according to the EC distribution in the pathway database.
RESULTS AND DISCUSSION
Sequencing and assembly of ESTs
The cloned cDNA library of P. ginseng was constructed, total 2640 ESTs were produced from non-treated hairy roots (DC01) and 3134 ESTs of MeJA treated hairy roots (
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