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

Huiyan Wang,Ning Wang,Yixin Huo

doi:10.1186/s12864-020-07124-6

Abstract

BackgroundAzadirachtin 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.ResultsWe 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.ConclusionsBy 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.

Highlights

Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture
To generate a comprehensive overview of neem transcriptome, total RNAs were extracted from leaves, fruits, roots, stems, and flowers
The unigenes categorized in the cellular component category were predominantly associated with membrane (GO: 0016020), cell part (GO: 0044464), and cell (GO: 0005623). These findings showed that the main Clusters of Orthologous Groups of proteins (COG) and Gene Ontology (GO) classifications for the fundamental biological processes were identified

Summary

Introduction

Azadirachtin A is a triterpenoid from neem tree exhibiting excellent activities against over 600 insect species in agriculture. Azadirachtin A, the major insecticidal ingredient in neem-based products, exhibits excellent bioactivity against over 600 insect species [1] in agricultural areas [2]. It is processed by insects as a natural hormone and it induces antifeedant, repellent, and growth inhibiting behavior in insects [3]. Azadirachtin A-based pesticides are biodegradable, environment-friendly, and non-toxic to mammals, plants, and birds. Due to these general superior characteristics of azadirachtin A, the agriculture segment accounts for the highest share (40%) in total revenues of neem extract product market. Due to the limitation of the distribution of neem materials along with the complex and low-yield azadirachtin Aextracting approach, the production of azadirachtin A is far from meeting the market demand

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