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Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions

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Abstract

Key Message

The EF1α genes were stable in the large majority of soybean tissues during development and in specific tissues/conditions under stress.

Abstract

Quantitative real-time PCR (qPCR) analysis strongly depends on transcript normalization using stable reference genes. Reference genes are generally encoded by multigene families and are used in qPCR normalization; however, little effort has been made to verify the stability of different gene members within a family. Here, the expression stability of members of the soybean EF1α gene family (named EF1α 1a1, 1a2, 1b, 2a, 2b and 3) was evaluated in different tissues during plant development and stress exposure (SA and PEG). Four genes (UKN1, SKIP 16, EF1β and MTP) already established as stably expressed were also used in the comparative analysis. GeNorm analyses revealed different combinations of reference genes as stable in soybean tissues during development. The EF1α genes were the most stable in cotyledons (EF1α 3 and EF1α 1b), epicotyls (EF1α 1a2, EF1α 2b and EF1α 1a1), hypocotyls (EF1α 1a1 and EF1β), pods (EF1α 2a and EF1α 2b) and roots (EF1α 2a and UKN1) and less stable in tissues such as trifoliate and unifoliate leaves and germinating seeds. Under stress conditions, no suitable combination including only EF1α genes was found; however, some genes were relatively stable in leaves (EF1α 1a2) and roots (EF1α 1a1) treated with SA as well as in roots treated with PEG (EF1α 2b). EF1α 2a was the most stably expressed EF1α gene in all soybean tissues under stress. Taken together, our data provide guidelines for the selection of EF1α genes for use as reference genes in qPCR expression analyses during plant development and under stress conditions.

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Abbreviations

ABA:

Abscisic acid

ADP-RF :

ADP-ribosylation factor

C t :

Cycle threshold

EF1α :

Elongation factor 1α

EF1β :

Elongation factor 1β

ETIF :

Eukaryotic translation initiation factor

GA:

Gibberellin

MTP :

Metalloprotease, Insulin degrading enzyme

NAA:

Naphthylacetic acid

PEG:

Polyethylene glycol

SA:

Salicylic acid

SKIP :

16 SKP1/Ask-Interacting Protein 16

t m :

Melting temperature

UKN1 :

Hypothetical protein

UTR:

Untranslated region

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Acknowledgments

This research was supported by CAPES, CNPq and FUNCAP.

Conflict of interest

The authors declare that have no conflict of interest.

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Authors and Affiliations

  1. Department of Biochemistry and Molecular Biology, Federal University of Ceara, Campus do Pici, Cx., Postal 6033, Fortaleza, Ceará, 60451-970, Brazil

    Kátia D. C. Saraiva, Dirce Fernandes de Melo, Vanessa D. Morais, Ilka M. Vasconcelos & José H. Costa

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  1. Kátia D. C. Saraiva
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  2. Dirce Fernandes de Melo
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  3. Vanessa D. Morais
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  4. Ilka M. Vasconcelos
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  5. José H. Costa
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Corresponding author

Correspondence to José H. Costa.

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Communicated by J. Register.

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Saraiva, K.D.C., Fernandes de Melo, D., Morais, V.D. et al. Selection of suitable soybean EF1α genes as internal controls for real-time PCR analyses of tissues during plant development and under stress conditions. Plant Cell Rep 33, 1453–1465 (2014). https://doi.org/10.1007/s00299-014-1628-1

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