Preparations of a number of plants which contain hydroxyanthraquinones as active constituents are used worldwide for their laxative effect. Anthraquinone glycosides of Cassia angustifolia and C. The endpoints screened were chromosomal aberrations and frequency of aberrant cells. Oral exposure to doses of these anthraquinones and their equivalent amount in leaf and pod extracts did not induce significant numbers of chromosomal aberrations or aberrant cells.
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Senna Cassia angustifolia Vahl. Laxative properties are due to sennosides anthraquinone glycosides natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides.
We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6.
The sequence assembly resulted in and transcripts with an average length of bp and bp for young and mature leaf, respectively. Out of the total transcripts, We used InterProscan to see protein similarity at domain level, a total of young leaf and mature leaf transcripts were annotated against the Pfam domains. All transcripts from young and mature leaf were assigned to KEGG pathways. There were and CDS, respectively, from young and mature leaf involved in metabolism of terpenoids and polyketides.
Many CDS encoding enzymes leading to biosynthesis of sennosides were identified. A total of 10, CDS differentially expressing in both young and mature leaf libraries of which 2, Several differentially expressed genes found functionally associated with sennoside biosynthesis. CDS encoding for many CYPs and TF families were identified having probable roles in metabolism of primary as well as secondary metabolites.
We developed SSR markers for molecular breeding of senna. We have identified a set of putative genes involved in various secondary metabolite pathways, especially those related to the synthesis of sennosides which will serve as an important platform for public information about gene expression, genomics, and functional genomics in senna.
Editor: Prasanta K. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files.
Competing interests: The authors have declared that no competing interests exist. The drug senna is mentioned in various texts of indigenous systems of medicine Ayurveda, Siddha Unani and Homoeopathy in India, pharmacopeias of United States, United Kingdom, Germany, and other counties [ 2 — 5 ].
The drug senna is widely used as a purgative, laxative, expectorant, wound dresser, antidysentric, and carminative. Senna leaves are commonly used as natural laxative both in modern as well as in traditional systems of medicine. The calcium sennoside is a popular form of dispensation useful in habitual constipation in modern medicine [ 6 ].
However, leaves are having international demand and preferred as ingredient of herbal tea in Europe [ 7 ]. Senna plant is a small, 1—2 m height under-shrub and belongs to family Caesalpiniaceae.
The stem is erect, smooth, and pale green to light brown with long spreading branches. Leaves are compound with four to eight pairs of leaflets. It is mucilaginous with sweet taste and peculiar odour. The flowers are small and yellow. The pods are broadly oblong, about 5—8 cm long and 2—3 cm broad bearing about six to nine seeds. Cassia angustifolia is cultivated mainly in India and Pakistan [ 6 ] and is native to tropical Africa and cultivated in Egypt, Sudan, and elsewhere [ 8 ].
Sennosides are the anthraquinone glycosides; four types of Sennoside A, B, C, and D found in large quantities in leaves 2. Sennosides A and B are homo-dianthrones dimers of two similar anthrone moieties of Rhein anthrone whereas Sennoside C and D are hetro-dianthrones of Rhein and Aloe-emodin anthrones. Sennosides act on the large intestine to stimulate peristalsis the muscular activity of the colon leading to elimination [ 11 — 13 ]. In plant, the sennosides are absent in fresh parts and form only during post harvest drying [ 14 ].
The dried leaflets and pods are main tissue used in herbal medicine and the pharmaceutical industry. The biosynthetic pathway leading to biosynthesis of sennosides in plants is unknown and relevant pathways are difficult to elucidate.
The knowledge of sennoside biosynthesis in the plants is derived from studies on Anthraquinone biosynthesis in other species. Biosynthesis of anthraquinones was studied in the plant of family Rubiaceae such as those for Morinda , Rubia and Galium species [ 15 — 17 ]. One of the remarkable features of Anthraquinone biosynthesis in higher plants is that they are derived from a variety of different precursors and pathways [ 15 , 18 ] and hence difficult to elucidate.
Anthraquinone are thought to be biosynthesized in the plant by a combination of isochorismate and plastidic hemiterpenoid 2-C-methyl-D-erthriolphosphate MEP pathways [ 19 , 20 ].
The mevalonate pathway is an important cellular metabolic pathway present in all higher eukaryotes and many bacteria. However, the enzymes and genes involved in the biosynthesis of these complex molecules are largely uncharacterized.
Anthraquinone is made up of three benzene rings namely A, B and C. Only a limited number of genes encoding for enzymes of each step have been identified and characterized that play an important role in the modification of the anthraquinone backbone structure.
A few cloned and characterized genes of the MEP pathway in plants were reported [ 23 ] like. Traditional approaches to gene cloning often require the isolation and partial sequencing of the appropriate enzyme in an attempt to obtain a genetic probe. This is often expensive, difficult, time-consuming, and futile.
Recently, genome-wide studies of model plant species have resulted in an explosive increase in our knowledge and capacity to understand, basic biological processes.
NGS technology, allow holistic profiling of RNA expression [ 38 , 39 ] in non-model plant species in which limited molecular genetics studies have been performed. RNA sequencing RNA-seq , provides whole-transcriptome expression profiles of selected plant tissues or cells, thereby permitting the integrated analysis of transcriptomes and metabolomes in any plant species. Transcriptome analysis followed by identification of potential candidate genes involved in the secondary metabolic pathway will lead to a better understanding of biosynthesis, regulation and chemical diversity of secondary metabolites in a plant species.
Transcriptome analysis by using NGS sequencing has been used extensively to unravel genes encoding enzyme involved in various steps of biosynthetic pathways of active principles in medicinal plants. Some includes the identification of genes encoding metabolic steps involved in the biosynthetic pathway of artemisinin in Artemisia annua [ 40 , 41 ], withanolides in Withania somnifera [ 42 , 43 ], cannabinoids in Cannabis sativa [ 44 ], ginsenosides in Panax ginseng [ 45 ], glycyrrhizin in Glycyrrhiza uralensis [ 46 ], picrosides in Picrorhiza kurrooa [ 47 ], hypericin in Hypericum perforatum [ 48 ], steroidal saponins in Chlorophytum borivilianum [ 49 ], camptothecin and anthraquinones in Ophiorrhiza pumila [ 50 ] and steroidal sapogenin biosynthesis in Asparagus racemosus [ 51 ].
Simple sequence repeats SSRs , also termed microsatellites, are nucleotide motifs consisting of tandem repeats of two to six base pairs. SSRs are ubiquitous and are found in both protein coding and non-coding regions affecting gene expression [ 52 ]. They are favoured for a variety of applications in plant breeding because of their multi-allelic nature, reproducibility, co-dominant inheritance, high abundance, and extensive genome coverage [ 53 ]. These markers are used in high-throughput genotyping and thus in the development of high density genetic maps, gene mapping, and marker-assisted selection MAS.
In the present study, in the well known medicinal plant senna, for the first time, we performed a paired-end transcriptome sequencing of young and mature leaf tissues differing for sennoside content using NGS technology. The main objective of our study is to identify candidate genes encoding the enzymes involved in the biosynthetic pathway of sennosides in senna. Our ultimate goal is to engineer the biosynthetic pathways for enhanced production of sennosides.
Through our combined analyses, we identified differentially expressed transcripts that are presumed to be associated with the biosynthesis of sennosides. These data sets are useful resources for further studies of the molecular genetics and functional genomics of this species. Samples and solutions were filtered with 0. Leaf samples were collected at flowering from Cassia angustifolia var.
Top 25 fresh leaves were collected in triplicates, dried, and powdered separately were used for estimation of sennoside content. The samples were filtered through 0. The mobile phase consisted of methanol and 1. The raw data was filtered using Trimmomatic v0. High quality data of young and mature leaf plant samples were assembled separately using Trinity RNA-Sequence assembler Version [ 56 ] on optimized parameters K mer size for the assembly was set to GC counts of transcripts was determined using a custom-made perl script.
The GO mapping also provided ontology of defined terms representing gene product properties which were grouped into three main domains: biological process BP , molecular function MF and cellular component CC. Identified gene names or symbols were then searched in the species specific entries of the gene-product tables of GO database.
Gene Ontology analysis helps in specifying all the annotated nodes comprising of GO functional groups. CDS were compared against Pfam database for higher-level groupings of related protein families, known as clans and the identification of domains that occurs within proteins.
The high quality reads for each sample was mapped on their respective set of CDS using CLC Genomic workbench to get the read counts which were used in DESeq 1 [ 59 ] to obtain significantly DE genes between young and mature leaf samples.
A complete linkage hierarchical cluster analysis was performed on top differentially expressed genes using Multiple Experiment Viewer MEV v4. Levels of expression were represented as log2 ratio of transcript abundance between young and mature leaf samples.
Differentially expressed gene identified in young and mature leaf samples were analyzed by hierarchical clustering. A heat map was constructed using the log-transformed and normalized value of genes based on Pearson uncentered correlation distance as well as based on complete linkage method. The sequences were initially processed and mined for SSR motifs dimers to hexamers with a length of 12 bp and above for di-, tri-, tetra, and hexa-nucleotide repeats, and 15 bp and above for penta-nucleotide repeats, using a program MISA MIcroSAtellite written in the Perl 5 script language that locates microsatellite patterns in FASTA formatted sequence files and reports the GenBank ID, microsatellite motifs dimers to hexamers , number of repeats and sequence coordinates for each microsatellite.
The rational for choosing the small cutoff value was that the SSRs are often disrupted by single base substitutions [ 60 ]. Microsatellites were classified into class I 20 nucleotides , class II 12—20 nucleotides and stochastic markers class III, repeat length of 6—12 nucleotides based on the length of the microsatellite motifs [ 61 ].
SSRs with a motif length of 20 bp and above were selected for designing primers. The canonical name proposed for designating markers includes function [unknown X ], lab designator DMAPR, Anand da ], species [ Cassia angustifolia ca ], type of marker [EST-microsatellite em ] and serial no.
Workflow for Illumina sequencing, de novo assembly, annotation, and other analysis carried out in the leaf transcriptome of Cassia angustifolia is given in Fig 1. High quality sequence of young and matured leaf samples were assembled de novo using Trinity RNA-Seq assembler. The version described in this paper is the first version.
Assembled transcript contigs were validated using CLC Bio Genomics workbench by mapping high quality reads back to the assembled transcript contigs. The assembly resulted in a total of 42, non-redundant transcripts with N 50 value of bp, largest contig length of bp and the average conting length of bp in young leaf whereas, it was non-redundant transcripts with N 50 value of bp, largest contig length of bp and the average contig length of bp in mature leaf Table 2. Assembled transcript contigs were validated by mapping high quality reads back to the assembled transcript contigs.
We observed Due to low expression of certain transcripts, the reads belonging to them might be either partially assembled or left out completely during the assembly process. This leads to a small fraction of reads unused during the assembly process.
In our study, 8. A total of 42, and 37, CDS were obtained for young and mature leaf samples, respectively Table 2 with a maximum CDS length of bp in young and bp in mature leaf.
The size distribution CDS according to their length was computed wherein the maximum number of CDS were in range of and above bp CDS which was followed by to CDS in the transcriptome of young leaf. A large number C. The GO mapping also provides ontology of defined terms representing gene product properties which were grouped into three main domains: Biological process, Molecular function, and Cellular component.
Cytogenetic study of some Thai species of flowering plants 
Senna Cassia angustifolia Vahl. Laxative properties are due to sennosides anthraquinone glycosides natural products. However, little genetic information is available for this species, especially concerning the biosynthetic pathways of sennosides. We present here the transcriptome sequencing of young and mature leaf tissue of Cassia angustifolia using Illumina MiSeq platform that resulted in a total of 6. The sequence assembly resulted in and transcripts with an average length of bp and bp for young and mature leaf, respectively. Out of the total transcripts,
Genotoxicity of Sennosides on the Bone Marrow Cells of Mice
Written Paper. Faculty of Sciences. Lookup at Google Scholar. Cytogenetic study of some Thai species of flowering plants. Chromosome numbers of 39 varieties of 33 Thai species belonging to 9 genera of Amaryllidaceae, Bignoniaceae, Caesalpiniaceae, Fabaceae and Lilliaceae were investigated.