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Molecular Biology Reports

, Volume 41, Issue 5, pp 3147–3162 | Cite as

Comparative transcripts profiling of fruit mesocarp and endocarp relevant to secondary metabolism by suppression subtractive hybridization in Azadirachta indica (neem)

  • Lokesh K. Narnoliya
  • Raja Rajakani
  • Neelam S. Sangwan
  • Vikrant Gupta
  • Rajender S. Sangwan
Article

Abstract

Azadirachta indica (neem) is a medicinally important plant that is valued for its bioactive secondary metabolites. Higher levels of the bioactive phytochemicals are accumulated in fruits than in other tissues. In the present study, a total of 387 and 512 ESTs, respectively, from endocarp and mesocarp of neem fruits were isolated and analyzed. Out of them 318 ESTs (82.17 %) clones from endocarp and 418 ESTs (81.64 %) from mesocarp encoded putative proteins that could be classified into three major gene ontology categories: biological process, molecular function and cellular component. From the analyses of contigs, 73 unigenes from the forward subtracted library and 35 unigenes from the reverse subtracted library were obtained. The ESTs from mesocarp encoded cytochrome P450 enzymes, which indicated hydroxylation to be a major metabolic event and that biogeneration of hydroxylated neem fruit phytochemicals was differentially regulated with developmental stage-specificity of synthesis. Through this study, we present the first report of any gene expression data in neem tissues. Neem hydroxy–methyl glutaryl–coenzyme A reductase (NHMGR) gene was used as expressing control vis-a-vis subtracted tissues. NHMGR was present in fruit, endocarp and mesocarp tissues, but absent in subtractive libraries, revealing that it was successfully eliminated during subtraction. Eight genes of interest from subtracted libraries were profiled for their expression in fruit, mesocarp and endocarp. Expression profiles validated the quality of the libraries and functional diversity of the tissues. The subtractive cDNA library and EST database described in this study represent a valuable transcript sequence resource for future research aimed at improving the economically important medicinal plant.

Keywords

Azadirachta indica Cytochrome P450 Legumin Limonoids Mesocarp and endocarp Suppressive subtraction hybridization Neem, plant secondary metabolism 

Abbreviations

ACPs

Acyl carrier proteins

CTAB

Cetyl trimethyl ammonium Bromide

CYP

Cytochrome P450

EST

Expressed sequence Tag

GO

Gene ontology

IPS

Inter pro scan

KEGG

Kyoto encyclopedia of genes and genomes

NCBI

National Centre for Biotechnology Information

NHMGR

Neem 3-hydroxy-3-methyl glutaryl coenzyme A reductase

RT-PCR

Real time-polymerase chain reaction

RQ

Relative quantification

SSH

Suppressive subtraction hybridization

SSR

Short sequence repeat

UTR

Un translated region

Notes

Acknowledgments

This work was financially supported by Council of Scientific and Industrial Research, Govt. of India, under the Grant HCP002. LKN is thankful to University Grants Commission, Govt of India, for financial assistance in form of senior research fellowship. The authors wish to express their sincere thanks to the Director, CSIR-CIMAP, for encouragement and providing necessary facilities.

Supplementary material

11033_2014_3174_MOESM1_ESM.ppt (1.2 mb)
Supplementary material 1 (PPT 1,199 kb)
11033_2014_3174_MOESM2_ESM.docx (16 kb)
Supplementary material 2 (DOCX 16 kb)

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Copyright information

© Springer Science+Business Media Dordrecht 2014

Authors and Affiliations

  • Lokesh K. Narnoliya
    • 1
  • Raja Rajakani
    • 1
  • Neelam S. Sangwan
    • 1
  • Vikrant Gupta
    • 1
  • Rajender S. Sangwan
    • 1
    • 2
  1. 1.CSIR-Central Institute for Medicinal and Aromatic Plants (CIMAP)LucknowIndia
  2. 2.Center of Innovative and Applied Bioprocessing (CIAB)A National Institute under DBT (Govt. of India)MohaliIndia

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