Transcriptome analysis to identify putative genes involved in 20 hydroxyecdysone synthesis in Sesuvium portulacastrum L.

Sesuvium portulacastrum L. a facultative halophyte belonging to family Aizoaceae, thrives in coastal parts around the world. The plant contains many secondary metabolites, among which phytoecdysteroids are of great importance, namely 20-hydroxyecdysone (20HE) which is being used in Sericulture industry on a large scale and has potential use as a biopesticide in agriculture. However, till date the biosynthetic pathway for 20HE is not known or partially postulated. In an attempt to identify the biosynthetic genes and metabolites involved in 20HE synthesis we investigated a putative mutant (20M4-16) with enhanced ability to accumulate 20HE in leaves. An untargeted LC-MS based metabolomics approach was used to do biochemical analysis of 20M4-16 leaf tissue along control plant to investigate the metabolic profiles. The final peak intensity file generated by R package analysis was subjected for statistical analysis using Metaboanalyst 3.0, PCA and OPLS-DA plot showed clear difference of metabolic profiles between control and 20M4-16 plant. Furthermore, to identify the biosynthetic genes of 20HE pathway, 20M4-16 (Irradiated) Sesuvium leaf tissue along with control were subjected for RNAseq analysis using Illumina platform.A total of 44,410,438 clean reads for control and 23,620,986 for 20M4-16 libraries were obtained and assembled into 157,208 unigenes. Out of which we found 61,656 (>40% similarity index) of assembled transcripts have at least one significant hit in NCBI database. Moreover, we found 5450 differentially expressed genes (DEG; p value <0.05) among which 4642 were upregulated in 20M4-16 and 808 were downregulated in control. Subsequently the DEGs encoding for key enzymes in MVA and MEP pathway, transcription factors and transporter genes were analyzed and summarized. rtPCR analysis revealed that HMGR, the first rate limiting enzyme involved in MVA pathway showed 5.5-fold increase in 20M4-16 leaf tissue while DXS first committed enzyme in MEP pathway showed 2.3-fold increase in 20M4-16. Additionally, CYP71D44 a cytochrome P450 which is recently reported to be involved in 20HE biosynthesis was probed from Sesuvium transcriptome with 67% similarity. Expression of CYP71D443 like P450 cytochrome showed 5.5-fold higher expression in 20M4-16 compared to control.