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Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein

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Abstract

In this study, we report the isolation and characterization of the mRNA encoding OPAQUE2 (O2) like TF of finger millet (FM) (Eleusine coracana) (EcO2). Full-length EcO2 mRNA was isolated using conserved primers designed by aligning O2 mRNAs of different cereals followed by 3′ and 5′ RACE (Rapid Amplification of cDNA Ends). The assembled full-length EcO2 mRNA was found to contain an ORF of 1248-nt coding the 416 amino acids O2 protein. Domain analysis revealed the presence of the BLZ and bZIP-C domains which is a characteristic feature of O2 proteins. Phylogenetic analysis of EcO2 protein with other bZIP proteins identified using finger millet transcriptome data and O2 proteins of other cereals showed that EcO2 shared high sequence similarity with barley BLZ1 protein. Transcripts of EcO2 were detected in root, stem, leaves, and seed development stages. Furthermore, to investigate nitrogen responsiveness and the role of EcO2 in regulating seed storage protein gene expression, the expression profiles of EcO2 along with an α-prolamin gene were studied during the seed development stages of two FM genotypes (GE-3885 and GE-1437) differing in grain protein content (13.8 and 6.2%, respectively) grown under increasing nitrogen inputs. Compared to GE-1437, the EcO2 was relatively highly expressed during the S2 stage of seed development which further increased as nitrogen input was increased. The Ecα-prolamin gene was strongly induced in the high protein genotype (GE-3885) at all nitrogen inputs. These results indicate the presence of nitrogen responsiveness regulatory elements which might play an important role in accumulating protein in FM genotypes through modulating EcO2 expression by sensing plant nitrogen status.

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Abbreviations

DAS:

Day after sowing

HPG:

High protein genotype,

LPG:

Low protein genotype

N:

Nitrogen

NUE:

Nitrogen use efficiency

O2:

Opaque2

PBF:

Prolamine-binding factor

qPCR:

Real-time PCR

RACE:

Rapid amplification of cDNA ends

SSP:

Seed storage proteins

UTR:

Untranslated region

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Acknowledgements

This work was supported by Science and Engineering Research Board (SERB), DST, New Delhi, India, in the form of “Fast Track Scheme for Young Scientist” awarded to VSG and SG (SR/FT/LS-33/2011 and YSS/2015/00536). The logistic support provided by the Director, Experiment Station, G.B. Pant University of Agriculture and Technology, Pantnagar is also thankfully acknowledged.

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Author notes

  1. Vikram Singh Gaur and Lallan Kumar have contributed equally.

Authors and Affiliations

  1. College of Agriculture, JNKVV, Waraseoni, Balaghat, Madhya Pradesh, India

    Vikram Singh Gaur

  2. Department of Molecular Biology and Genetic Engineering, College of Basic Sciences and Humanities, G. B. Pant University of Agriculture and Technology, Pantnagar, 263145, India

    Lallan Kumar, Supriya Gupta, Dinesh Pandey & Anil Kumar

  3. Department of Genetics and Plant Breeding, G. B. Pant University of Agriculture and Technology, Pantnagar, 263145, India

    J. P. Jaiswal

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  1. Vikram Singh Gaur
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  2. Lallan Kumar
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  3. Supriya Gupta
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  4. J. P. Jaiswal
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  5. Dinesh Pandey
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  6. Anil Kumar
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Contributions

AK conceived the idea and conceptualized and designed the manuscript. VSG and LK are involved in the lab work of the presented R & D data. VSG, LK, and SG wrote the manuscript. JPJ, DP, and AK contributed in critically revising the draft and updating the manuscript for publication.

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Correspondence to Anil Kumar.

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Gaur, V.S., Kumar, L., Gupta, S. et al. Identification and characterization of finger millet OPAQUE2 transcription factor gene under different nitrogen inputs for understanding their role during accumulation of prolamin seed storage protein. 3 Biotech 8, 163 (2018). https://doi.org/10.1007/s13205-018-1150-1

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