De Novo transcriptome sequencing reveals important molecular networks and metabolic pathways of the plant, Chlorophytum borivilianum.

Chlorophytum borivilianum, an endangered medicinal plant species is highly recognized for its aphrodisiac properties provided by saponins present in the plant. The transcriptome information of this species is limited and only few hundred expressed sequence tags (ESTs) are available in the public databases. To gain molecular insight of this plant, high throughput transcriptome sequencing of leaf RNA was carried out using Illumina's HiSeq 2000 sequencing platform. A total of 22,161,444 single end reads were retrieved after quality filtering. Available (e.g., De-Bruijn/Eulerian graph) and in-house developed bioinformatics tools were used for assembly and annotation of transcriptome. A total of 101,141 assembled transcripts were obtained, with coverage size of 22.42 Mb and average length of 221 bp. Guanine-cytosine (GC) content was found to be 44%. Bioinformatics analysis, using non-redundant proteins, gene ontology (GO), enzyme commission (EC) and kyoto encyclopedia of genes and genomes (KEGG) databases, extracted all the known enzymes involved in saponin and flavonoid biosynthesis. Few genes of the alkaloid biosynthesis, along with anticancer and plant defense genes, were also discovered. Additionally, several cytochrome P450 (CYP450) and glycosyltransferase unique sequences were also found. We identified simple sequence repeat motifs in transcripts with an abundance of di-nucleotide simple sequence repeat (SSR; 43.1%) markers. Large scale expression profiling through Reads per Kilobase per Million mapped reads (RPKM) showed major genes involved in different metabolic pathways of the plant. Genes, expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community, interested in the molecular genetics and functional genomics of C. borivilianum.

Characterization of Squalene synthase gene from Chlorophytum borivilianum (Sant. and Fernand.).

Saponins are important group of secondary metabolites known for their pharmacological properties. Chlorophytum borivilianum contains high amount of saponins and is thus, recognized as an important medicinal plant with aphrodisiac properties. Though the plant is well known for its pharmaceutical properties, there is meager information available about the genes and enzymes responsible for biosynthesis of saponins from this plant. Squalene synthase (SqS) is the key enzyme of saponin biosynthesis pathway and here, we report cloning and characterization of SqS gene from C. borivilianum. A full-length CbSqS cDNA consisting of 1,760 bp was cloned which contained an open reading frame (ORF) of 1,233 bp, encoding a protein of 411 amino acids. Analysis of deduced amino acid sequence of CbSqS predicted the presence of conserved isoprenoid family domain and catalytic sites. Phylogenetic analysis revealed that CbSqS is closer to Glycine max and monocotyledonous plants. 3D structure prediction using various programs showed CbSqS structure to be similar to SqS from other species. C-terminus truncated recombinant squalene synthase (TruncCbSqS) was expressed in E. coli M15 cells with optimum expression induced with 1 mM IPTG at 37 °C. The gene expression level was analyzed through semi-quantitative RT-PCR and was found to be higher in leaves as compared to the roots.

Differentially expressed transcripts from leaf and root tissue of Chlorophytum borivilianum: a plant with high medicinal value.

Chlorophytum borivilianum is one of the important medicinal plants used for treating different health problems such as diabetes, arthritis, physical weakness, etc. Saponins present in C. borivilianum are the primary source of its significant medicinal properties and are synthesized by mevalonate and non-mevalonate pathways in plants. However, the biosynthesis of these compounds at molecular level is not studied in C. borivilianum. Cloning and sequencing of genes involved in metabolic processes are prerequisite to study the gene expression, their regulation and genetic engineering experiments. Expressed sequence tags (ESTs) provide a quick insight into various genes and their tissue specific expression. Suppression subtractive hybridization (SSH) libraries were constructed using mRNA from leaf and root tissues of C. borivilianum. High quality non-redundant 506 and 303 ESTs were generated from leaf and root specific libraries respectively. These sequences were analyzed using bioinformatics tools and grouped into different categories based on their similarity and cellular functions such as photosynthesis, metabolism, transcription factors, cell signaling, defense, stress response etc. ESTs also showed similarity with genes involved in saponins biosynthesis such as squalene synthase, squalene epoxidase, cytochrome p450, glycosyltransferase, etc. Semi-quantitative analysis of some of the ESTs involved in saponins biosynthesis confirmed their differential regulation in leaves and roots. These ESTs will provide an efficient resource to accelerate gene discovery in C. borivilianum and will help in determining promising targets for genetic engineering of saponins pathway.