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Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species

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Abstract

The smallest 32 amino acid α-amylase inhibitor from Amaranthus hypochondriacus (AAI) is reported. The complete gene of pre-protein (AhAI) encoding a 26 amino acid (aa) signal peptide followed by the 43 aa region and the previously identified 32 aa peptide was cloned successfully. Three cysteine residues and one disulfide bond conserved within known α-amylase inhibitors were present in AhAI. Identical genomic and open reading frame was found to be present in close relatives of A. hypochondriacus namely Amaranthus paniculatus, Achyranthes aspera and Celosia argentea. Interestingly, the 3′UTR of AhAI varied in these species. The highest expression of AhAI was observed in A. hypochondriacus inflorescence; however, it was not detected in the seed. We hypothesized that the inhibitor expressed in leaves and inflorescence might be transported to the seeds. Sub-cellular localization studies clearly indicated the involvement of AhAI signal peptide in extracellular secretion. Full length rAhAI showed differential inhibition against α-amylases from human, insects, fungi and bacteria. Particularly, α-amylases from Helicoverpa armigera (Lepidoptera) were not inhibited by AhAI while Tribolium castaneum and Callosobruchus chinensis (Coleoptera) α-amylases were completely inhibited. Molecular docking of AhAI revealed tighter interactions with active site residues of T. castaneum α-amylase compared to C. chinensis α-amylase, which could be the rationale behind the disparity in their IC50. Normal growth, development and adult emergence of C. chinensis were hampered after feeding on rAhAI. Altogether, the ability of AhAI to affect the growth of C. chinensis demonstrated its potential as an efficient bio-control agent, especially against stored grain pests.

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Acknowledgements

AJB and SKR thank Council for Scientific and Industrial Research (CSIR) for Senior Research Fellowship and Dept. of Science and Technology (DST), New Delhi for Ramanujan Fellowship, respectively. Authors thanks Dt. Bhushan Dholakia for critically reading and providing suggestions in the manuscript drafts. This project is supported by the Department of Biotechnology, Goverment of India (BT/PR2295/AGR/5/552/2011) to CSIR-National Chemical Laboratory (GAP 298126), Pune, and North Maharashtra University, Jalgaon. This project is also supported by CSIR under XII five year plan projects (BSC0107 and BSC0120) to CSIR-National Chemical Laboratory, Pune.

Author contributions

APG, SKR, PKP and VLM designed the experiments. AJB, SMC, and KB conducted the experiments. YY performed homology modelling and docking studies. AJB, VSG and APG wrote the manuscript. All authors read and approved the final manuscript.

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  1. Plant Molecular Biology Unit, Division of Biochemical Sciences, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune, 411 008, India

    Amey J. Bhide, Sonal M. Channale, Yashpal Yadav, Kabita Bhattacharjee, Vidya S. Gupta, Sureshkumar Ramasamy & Ashok P. Giri

  2. Department of Biochemistry, Shivaji University, Kolhapur, 416 004, India

    Pankaj K. Pawar

  3. School of Life Sciences, North Maharashtra University, Jalgaon, 425 001, India

    V. L. Maheshwari

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11103_2017_609_MOESM1_ESM.tif

Supplementary Figure 1—Homology modeling and circular-dichroism (CD) spectroscopy of AhAI. Homology modeling was performed using I-TASSER server. The most suitable model was selected based on CD spectroscopic data. CD spectroscopy was performed at concentration of 50 µg/mL rAhAI in 20 mM Tris (pH 8.5) buffer (TIF 3570 KB)

11103_2017_609_MOESM2_ESM.tif

Supplementary Figure 2—Sub-cellular localization and detection of GFP in tobacco epidermal cells. a CBB stained gel showing proteins extracted from apoplast and total cellular leaf extract devoid of apoplast before transfer to the PVDF membrane. An equal amount of protein (28 µg) was loaded in each well. Rubisco protein present in the total cellular extract was represented by ‘R’. The absence of Rubisco in apoplastic fluid indicated no cellular damage caused during the extraction procedure. b Complete transfer of the proteins (except few very high molecular weight proteins) to the membrane was confirmed by silver staining (TIF 8271 KB)

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Bhide, A.J., Channale, S.M., Yadav, Y. et al. Genomic and functional characterization of coleopteran insect-specific α-amylase inhibitor gene from Amaranthus species. Plant Mol Biol 94, 319–332 (2017). https://doi.org/10.1007/s11103-017-0609-5

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