Sen, PriyabrataDevi, Kamalakshi2017-09-082017-09-082015http://krishikosh.egranth.ac.in/handle/1/5810030816Citronella (Cymbopogon winterianus) is an aromatic and medicinal grass grown for commercial and industrial purpose due to its large repository of isoprenoids compounds known for its anti-tumoral, antibacterial, anti-fungal, antiviral, anti-cancerous and detoxifying properties as well as use as natural insect repellent properties. Despite of having numbers of valuable utilities, the genes involved in the biosynthetic pathway of these terpenes in citronella are not yet clearly elucidated. Keeping these points in view, the present study is formulated as a pioneering attempt to generate a large amount of molecular level information for citronella followed by identification of secondary metabolite associated gene. The whole transcriptome sequencing of leaf and root tissues of citronella (Cv. Jorlab-1) was carried out on Illumina MiSeq NGS plateform using Paired end (PE) 2x250bp library. A total of 11.7 Gb data were generated which upon de novo assembly yielded 68,986 unigenes. The complete bioinformatic analysis of these genes resulted : functional annotation of 63,454 unigenes along with 5478 novel genes; GO annotation of 56,621 unigenes; GO enrichment analysis of 218 secondary metabolite associated genes; detection of 3,851 putative SSR motifs and KEGG pathway analysis annotated 3,362 unigenes. The RT and qRT PCR analysis of the 20 secondary metabolite associated genes were in line with the findings from differential expression study. The regulatory relation of miRNA & TF with secondary metabolite associated genes revealed 14 pre-miRNA targets 29 TFs and 4 genes. There were three key regulatory enzymes of terpenoid backbone synthesis pathway viz.1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) (GenBank: KJ749651), 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGR) (GenBank: KM504513) and Geranylgeranyl pyrophosphate synthase (GGPPS) (GenBank: KP017769) of citronella analyzed further. The full length gene sequence of these genes were obtained by performing 5’ RACE PCR. The complete in silico proteomics analysis of the three proteins included analysis of primary protein sequence, phylogeny, physico-chemical property of the proteins, homology modeling and model validation along with molecular dynamic simulation and finally docking of co factor (NADPH). The 3D structure analysis of the proteins gave a clear insight about the catalytic sites, substrate and cofactor binding pockets and various conserved motifs of these proteins. It can be concluded that, this work sets the stage for multi-faceted future improvement of the plant, through discovery of new genes, marker-assisted breeding or genetic engineering, on this species as well as for other species of Poaceae and terpene producing medicinal plants.ennullThesis