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.
Highlights
Neem 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
Amongst the predicted genes from this pathway, here we report, molecular cloning and functional characterization of full-length geranyl diphosphate synthase (AiGDS), farnesyl diphosphate synthase (AiFDS) and squalene synthase (AiSQS) from Neem
Using real-time PCR analysis, we showed that the expression level of one of the important genes in the pathway, AiSQS correlates with the triterpenoid content in respective tissues
Summary
Neem 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. Neem tree is one of the richest reserves of secondary metabolites, mainly tetranortriterpenoids (limonoids), which are known to be responsible for insecticidal and wide pharmaceutical activities [1, 2]. Various parts of this evergreen tree have been used as traditional medicine in day-to-day household remedies from ancient time. Ring-intact triterpenoids encompass 4,4,8-trimethyl17-furanylsteroidal skeleton such as azadirone, azadiradione, and gedunin (1-5) type of structures (Fig. 1). The biosynthetic pathway leading to the formation of triterpenoids (Fig. 2a) in Neem plant has been predicted [1, 7]
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