Multi-tissue transcriptome analysis using hybrid-sequencing reveals potential genes and biological pathways associated with azadirachtin A biosynthesis in neem (azadirachta indica)

Background: Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. The production of azadirachtin A depends on extraction from neem tissues, which is not an eco-friendly and sustainable process. The low yield and discontinuous supply of azadirachtin A impedes further applications. The biosynthetic pathway of azadirachtin A is still unknown and is the focus of our study. Results: We attempted to explore azadirachtin A biosynthetic pathway and identified the key genes involved by analyzing transcriptome data from five neem tissues through the hybrid-sequencing (Illumina HiSeq and Pacific Biosciences Single Molecule Real-Time (SMRT)) approach. Candidates were first screened by comparing the expression levels between the five tissues. After phylogenetic analysis, domain prediction, and molecular docking studies, 22 candidates encoding 2,3-oxidosqualene cyclase (OSC), alcohol dehydrogenase, cytochrome P450 (CYP450), acyltransferase, and esterase were proposed to be potential genes involved in azadirachtin A biosynthesis. Among them, two unigenes encoding homologs of MaOSC1 and MaCYP71CD2 were identified. A unigene encoding the complete homolog of MaCYP71BQ5 was reported. Accuracy of the assembly was verified by quantitative real-time PCR (qRT-PCR) and full-length PCR cloning. Conclusions: By integrating and analyzing transcriptome data from hybrid-seq technology, 22 differentially expressed genes (DEGs) were finally selected as candidates involved in azadirachtin A pathway. The obtained reliable and accurate sequencing data provided important novel information for understanding neem genome. Our data shed new light on understanding the biosynthesis of other triterpenoids in neem trees and provides a reference for exploring other valuable natural product biosynthesis in plants.