Identification and analysis of regulatory elements and associated transcription factors of costunolide biosynthetic pathway genes in Saussurea lappa

Abstract

The PhD work included the comparative analysis of root and leaf tissue transcriptomes (three replicates each) to study the costunolide pathway genes and the related TFs in Saussurea lappa. Various bioinformatics tools were employed to study the transcriptomic data, revealing about 162,539 assembled unigenes. These were functionally annotated, classified and used in tissue-specific differential expression analysis studies. The differentially expressed costunolide pathway genes and putative TF transcripts were identified and annotated. Putative transcription factors were identified using PlantTFDB and TF-gene co-expression network was generated followed by clustering using module based analysis to observe their coordinated behavior. Further, the analysis of key regulatory genes i.e. diphosphomevalonate decarboxylase (SlDPD) and costunolide synthase (SlCOS) involved in the costunolide biosynthesis was performed and their promoter region were cloned using genome walking technique to study the existing cis-regulatory elements. Bioinformatics tools were employed to predict the cis regulatory elements, leading to the identification of bHLH, Dof and WRKY family of TFs as the interacting partners of SlDPD gene, and MYB family of TFs as an interacting partner of SlCOS gene. The transcripts of TF families were identified and annotated using various in silico tools to reveal potential candidates that could be interacting with the genes of interest. Yeast one-hybrid technique was appointed to identify the interacting TFs and revealed SlbHLH, SlDof and SlWRKY TFs interacting with SlDPD gene promoters and SlMYB TF intecating with SlCOS gene promoter. RT-PCR was performed to obtain the comparative gene and TF candidates expression patterns. newline