Triterpenoid profiling and functional characterization of the initial genes involved in isoprenoid biosynthesis in neem (Azadirachta indica)

Abstract

BackgroundNeem tree (Azadirachta indica) is one of the richest sources of skeletally diverse triterpenoids and they are well-known for their broad-spectrum pharmacological and insecticidal properties. However, the abundance of Neem triterpenoids varies among the tissues. Here, we delineate quantitative profiling of fifteen major triterpenoids across various tissues including developmental stages of kernel and pericarp, flower, leaf, stem and bark using UPLC-ESI(+)-HRMS based profiling. Transcriptome analysis was used to identify the initial genes involved in isoprenoid biosynthesis. Based on transcriptome analysis, two short-chain prenyltransferases and squalene synthase (AiSQS) were cloned and functionally characterized.ResultsQuantitative profiling revealed differential abundance of both total and individual triterpenoid content across various tissues. RNA from tissues with high triterpenoid content (fruit, flower and leaf) were pooled to generate 79.08 million paired-end reads using Illumina GA ΙΙ platform. 41,140 transcripts were generated by d e novo assembly. Transcriptome annotation led to the identification of the putative genes involved in isoprenoid biosynthesis. Two short-chain prenyltransferases, geranyl diphosphate synthase (AiGDS) and farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) were cloned and functionally characterized using transcriptome data. RT-PCR studies indicated five-fold and ten-fold higher relative expression level of AiSQS in fruits as compared to leaves and flowers, respectively.ConclusionsTriterpenoid profiling indicated that there is tissue specific variation in their abundance. The mature seed kernel and initial stages of pericarp were found to contain the highest amount of limonoids. Furthermore, a wide diversity of triterpenoids, especially C-seco triterpenoids were observed in kernel as compared to the other tissues. Pericarp, flower and leaf contained mainly ring-intact triterpenoids. The initial genes such as AiGDS, AiFDS and AiSQS involved in the isoprenoids biosynthesis have been functionally characterized. The expression levels of AiFDS and AiSQS were found to be in correlation with the total triterpenoid content in individual tissues.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0593-3) contains supplementary material, which is available to authorized users.

Gene expression of glycyrrhizin acid and accumulation of endogenous signaling molecule in Glycyrrhiza uralensis Fisch adventitious roots after Saccharomyces cerevisiae and Meyerozyma guilliermondii applications

“…GPP produces farnesyl diphosphate (FPP) through farnesyl diphosphate synthase ( FPS ) . The biosynthesis of squalene from FPP is completed by squalene synthase ( SQS ) . The squalene is further oxidized by squalene epoxidase ( SE ) to (S)‐2, 3‐oxidosqualene .…”

Effect of Plant Growth Regulators on Coloured Callus Formation and Accumulation of Azadirachtin, an Essential Biopesticide in Azadirachta indica

“…Although AZA has been recognized as an effective biodegradable pesticide [61], the current method of solvent extraction of this compound from the seeds of this species is unable to produce a sufficient economic yield of AZA. This is largely due to low variable yield, poor quality control, the presence of impurities and occurrence of diseases in the species [62].…”

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

“…Several resources on neem genome, such as the complementary DNA (cDNA) library [9], expressed sequence tag library [10], draft genome [11,12] and transcriptome data [13,14,15] are available in public databases such as the National Center for Biotechnology Information (NCBI) and the European Bioinformatics Institute (EBI). Genes involved in the mevalonate (MVA) pathway, methylerythritol phosphate (MEP) pathway [14,16], or 2,3-oxidosquanlene biosynthesis [11,15,16,17] in neem have been identi ed. Several neem-speci c genes were found after comparative genome analysis of neem with Arabidopsis thaliana, Oryza sativa, and Citrus sinensis [11].…”

“…Hence, ve neem tissues (fruit, leaf, stem, ower, and root) were sampled for transcriptome sequencing. Among the ve tissues, fruit with the green hard seed has been reported to contain the highest amount of azadirachtin A throughout fruit development [14,19] Leaf sample from a neem tree in China contained azadirachtin A at a concentration of 969.9 μg/g [20]. The percentage azadirachtin content in different tissues was consistent with that in a previous report (seed kernels, 0.03%; leaves, 0.9x10 −3 %; bark, 0.5x10 −3 %; root, 0.3x10 −3 %; stem 0.2x10 −3 %) [21].…”