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Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant

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Abstract

In the present study, we have cloned a gene encoding JcMT2a protein from Jatropha curcas L., a promising biofuel tree species. Full length sequence of JcMT2a gene was isolated using RACE PCR. Heterologous expression of JcMT2a in Escherichia coli and its purification has shown distinct bands corresponding to the GST and GST-fused JcMT2a protein. Significant tolerance was observed in E. coli cells expressing recombinant GST-JcMT2a for zinc, copper and cadmium metals compared to cells expressing GST alone. JcMT2a also restored Cu and Cd tolerance in the metal sensitive yeast mutants. Quantitative real time PCR showed a significant increase in JcMT2a transcripts with Cu and Cd in the leaf compared to root tissue. Our Scanning electron microscopy and energy dispersive X-ray spectroscopy analysis clearly demonstrates that J. curcas L. could be a potential candidate for phytoremediation to clean heavy metals from the environment, in addition to its non-edible oil seed yields for biodiesel production.

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Abbreviations

MT:

Metallothionein

RACE:

Rapid amplification of cDNA ends

UTR:

Untranslated region

GST:

Glutathione S-transferase

SEM–EDAX:

Scanning electron microscopy and energy dispersive X-ray spectroscopy

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Acknowledgments

The work was supported by DST grant (DST/IS-STAC/CO2-SR-68/09) from Department of Science and Technology, Government of India. We are thankful to DST-FIST facility of department of Plant Sciences, DBT-CREBB facility of School of Life Sciences and CIL of University of Hyderabad for providing SEM facility. Shalini Mudalkar and Debashree Sengupta are indebted to UGC and CSIR, New Delhi, India, respectively for the fellowship. Ramesh Golla was supported by Dr. D.S. Kothari Postdoctoral fellowship from UGC.

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Authors and Affiliations

  1. Department of Plant Sciences, University of Hyderabad, Gachibowli, Hyderabad, 500046, Andhra Pradesh, India

    Shalini Mudalkar, Ramesh Golla, Debashree Sengupta & Attipalli Ramachandra Reddy

  2. Tree Oils India Limited, Zaheerabad, 502226, Andhra Pradesh, India

    Sreenivas Ghatty

Authors
  1. Shalini Mudalkar
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  2. Ramesh Golla
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  3. Debashree Sengupta
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  4. Sreenivas Ghatty
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  5. Attipalli Ramachandra Reddy
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Corresponding author

Correspondence to Attipalli Ramachandra Reddy.

Additional information

Shalini Mudalkar and Ramesh Golla have contributed equally to this work.

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Supplementary Fig. 1

Sequence alignment of MT2 from different plants which were used to design primers JcMTF and JcMTR from the conserved regions highlighted in blue using Clustal W (TIFF 648 kb)

Supplementary Fig. 2

Sequence alignment of genomic and cDNA regions of JcMT2a (TIFF 228 kb)

Supplementary Fig. 3

Protein parameters and ORF finder results of JcMT2a protein (TIFF 314 kb)

Supplementary Fig. 4

Multiple alignment of JcMT2a protein sequence with MT2 like proteins of Ricicnus, Nelumbo, Elaeis, Sesbania and Salvia (TIFF 181 kb)

Supplementary Fig. 5

BLAST analysis showing the sequence alignment and percentage similarity of JcMT2a with MTs of other plant species (TIFF 214 kb)

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Mudalkar, S., Golla, R., Sengupta, D. et al. Molecular cloning and characterisation of metallothionein type 2a gene from Jatropha curcas L., a promising biofuel plant. Mol Biol Rep 41, 113–124 (2014). https://doi.org/10.1007/s11033-013-2843-5

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