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

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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.

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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

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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.

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  1. Rajender S. Sangwan

    Present address: Center of Innovative and Applied Bioprocessing (CIAB), A National Institute under DBT (Govt. of India), C-127, Phase-8, Industrial Area, S.A.S. Nagar, Mohali, 160071, Punjab, India

Authors and Affiliations

  1. CSIR-Central Institute for Medicinal and Aromatic Plants (CIMAP), Kukrail Picnic Spot Road, P.O. CIMAP, Lucknow, 226015, India

    Lokesh K. Narnoliya, Raja Rajakani, Neelam S. Sangwan, Vikrant Gupta & Rajender S. Sangwan

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  1. Lokesh K. Narnoliya
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  3. Neelam S. Sangwan
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Correspondence to Neelam S. Sangwan.

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Narnoliya, L.K., Rajakani, R., Sangwan, N.S. et al. Comparative transcripts profiling of fruit mesocarp and endocarp relevant to secondary metabolism by suppression subtractive hybridization in Azadirachta indica (neem). Mol Biol Rep 41, 3147–3162 (2014). https://doi.org/10.1007/s11033-014-3174-x

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