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

Abstract

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.

Genomic Analysis Based on Chromosome-Level Genome Assembly Reveals an Expansion of Terpene Biosynthesis of Azadirachta indica

“…Azadirachtin is a triterpenoid compound of neem tree, which has effective insecticidal activities against a wide range of insect species, but has very low toxicity to mammals. Terpene synthase (TPS), cytochrome P450 (CYP450), alcohol dehydrogenase (ADH), acyltransferase (ACT), and esterase (EST) were proposed to be involved in biosynthesis of azadirachtin ( Wang et al, 2020 ). In this study, a large number of genes encoding CYP 450s (78), TPSs (58), and ACTs (34) were identified in secondary metabolite biosynthesis gene clusters.…”

Genomic analysis based on chromosome-level genome assembly reveals an expansion of terpene biosynthesis of Azadirachta indica

“…Azadirachtin is a triterpenoid compound of neem tree, which has effective insecticidal activities against a wide range of insect species, but has very low toxicity to mammals. Terpene synthase (TPS), cytochrome P450 (CYP450), alcohol dehydrogenase (ADH), acyltransferase (ACT), and esterase (EST) were proposed to be involved in biosynthesis of azadirachtin (Wang et al, 2020). In this study, a large number of genes encoding CYP P450s (78), TPSs (58), and ACTs (34) were identified in secondary metabolite biosynthesis gene clusters.…”

“…Molecular docking was performed with Autodock 4.0. Four triterpenoids (azadirone, nimbin, nimbolide, and tirucalla-7,24-dien-3β-ol) were selected as potential substrates for CYPs (Wang et al, 2020).…”

“…The accuracy and integrity of the genome assembly was evaluated using BUSCO v3.0.2, based on the OrthoDB (http://cegg.unige.ch/orthodb) database. The transcriptomic NGS short reads from 5 tissues of A.indica (SRR12709585, SRR12709584, SRR12709583, SRR12709582, and SRR12709581) (Wang, Wang, & Huo, 2020) were mapped against the assemblies using Hisat2 (Kim, Langmead, & Salzberg, 2015) with default parameters. The genomic NGS short reads were also mapped to the assemblies using Bowtie2.…”

“…We further investigated the expression pattern of TPS genes in A. indica. Transcriptome datasets from five tissues of A. indica were obtained from our previous study (Wang et al, 2020) and remapped to the chromosome-level genome assembly in this study. More than 88% of the RNAseq reads were mapped uniquely to the genome assembly across all samples (Table S19).…”