Glucosinolate profiling and molecular analysis of MYB28 gene for metabolome editing in Moringa oleifera Lam

Abstract

Moringa (Moringa oleifera Lam.), an indigenous herb native to South Asia, recognized for its health benefits, is often referred to as "the miracle tree." Thriving in the foothills of the Himalayas, moringa belongs to the Moringaceae genus, comprising 14 known species. Each part of the plant possesses medicinal properties, contributing to antimicrobial, anti-inflammatory, detoxifying, and anticancer activities. Glucosinolates (GSLs) are stable secondary metabolites derived from sulfur and nitrogen-rich amino acids. Myrosinase, the sole known β-thioglucosidase, is responsible for driving their breakdown. The resulting product, isothiocyanates (ITCs), possesses fungicidal, bacteriocidal, nematocidal, and herbivore-deterrent qualities. The R2R3-MYB class of transcription factors significantly influences glucosinolate (GSL) biosynthesis. Studies manipulating the MYB28 gene, a key transcription factor, have been reported to regulate GSL levels in plants. GSLs are suggested to be produced in green tissues and then transported to developing reproductive tissues through the phloem. While extensive research explores the control of the GSL pathway in Brassica and Arabidopsis, in members of Brassicales like moringa, the regulatory mechanism of GSLs is yet unclear. It is reported that the GSLs or ITCs in M. oleifera are probably responsible for many of the therapeutic benefits that have long been associated with them in traditional medicine. M. oleifera is home to several unusual GSLs with unique properties. Glucomoringin, also known as 4-(α-L-rhamnopyranosiloxy) benzyl glucosinolate (4RBGS) is the most prevalent GSL in all of M. oleifera's components, especially the pulp seed, followed by 3-hydroxy- 4-(α-L-rhamnopyranosyloxy) benzyl glucosinolate (4-OHBGS). Due to the presence of a second saccharide residue in the aglycon side chain, this chemical could show biological effects that are very different when compared to those of other GSLs because of its unusual structure. Thus, one of the main goals of M. oleifera breeding is to improve nutritional and medicinal characteristics by creating high GSL lines. The objective of the current research is glucosinolate profiling, isolation and expression analysis of MYB28 gene from M. oleifera variety PKM-1 and development of CRISPR/Cas construct for functional analysis of MYB28 gene. Desulphoglucosinolate profiling using High Performance Liquid Chromatography showed a major peak of 4RBGS and a minor peak of 4-OHBGS in aerial tissues, suggesting them as the predominant GSLs. However, in roots, in addition to the benzyl GSLs, indole GSL (indol-3-ylmethyl GSL, I3M) was also detected. The highest concentration of total GSLs was found in moringa seeds (399.48 µmoles/g dry weight). This was followed by stem (63.95 µmoles/g dry weight) and flowers (30.61 µmoles/g dry weight) had the least amount of GSL accumulation. Isolation and sequence analysis of MYB28 gene in moringa proved that the sequence is diverse from that of its related species coming under the order Brassicales. Homology search using NCBI BLASTn showed 86% identity with the predicted mRNA sequence of Abelmoschus esculentus transcription factor MYB28. Bioinformatic analysis of the genomic and CDS sequences were performed using softwares like NCBI Splign, Molbiotools, ORFfinder and InterPro to characterize the MYB28 gene isolated from moringa. Expression profiling of the MYB28 gene was performed in different tissues of moringa using Real-time qRT-PCR. Highest level of MYB28 expression was found in the stem followed by immature pod tissue, flower and leaf tissue. Lowest level of gene expression was found in seeds, where no GSL synthesis occur, which act as only sink for GSLs. In order to elucidate the role of the putative MYB28 in GSL biosynthesis, a gene knock-out cassette based on CRISPR/Cas9 system was generated in the study. The genomic sequence of MYB28 obtained in the study was used to design gRNA using Cas-designer of ‘CRISPR RGEN Tools’ software. The MYB28 CRISPR/Cas9 construct was ligated to CRISPR/Cas9 binary vector pKSE401 and cloned in to E. coli strain DH5α. The positive clones were confirmed by Sanger sequencing of the plasmid DNA. The construct was further mobilized to A. tumefaciens strain GV3101. Positive clones were identified by colony PCR using vector and gRNA specific primers. The construct will be used for moringa genetic transformation. Callus and cell suspension cultures of moringa was established for moringa genetic transformation in future.