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Isolation and characterization of cold inducible genes in carrot by suppression subtractive hybridization

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Biologia Plantarum

Abstact

Daucus carota is cultivated widely but grows best in cool climates. Suppression subtractive hybridization (SSH) is a PCR based method used to selectively amplify differentially expressed cDNAs and simultaneously suppress non-target cDNA. A subtraction forward library was constructed using RNA isolated from the leaves of unstressed and cold stressed carrot plants to determine the genes upregulated during cold stress. Out of the hundreds of clones obtained, sequences of 41 promising clones were submitted to the NCBI EST database. Sequence analyses revealed that these genes have significant roles in signal transduction, osmolyte synthesis and transport, regulation of transcription, translation and protein folding. Semiquantitative real-time polymerase chain reaction analysis (sqRT-PCR) of Dc cyclin, Dc WD and Dc profilin shows that the first two genes were upregulated while Dc profilin was constitutively expressed, but the analyses of the same with SSH, a much more sensitive technique showed an upregulation of all three genes.

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Abbreviations

DREBs:

dehydration-responsive element-binding proteins

EST:

expressed sequence tag

MS:

Murashige and Skoog

qRT-PCR:

quantitative real-time polymerase chain reaction

RACE:

rapid amplification of cDNA ends

SAGE:

serial analysis of gene expression

SSH:

suppression subtractive hybridization

SUMO:

small ubiquitin like modifier

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  1. S. Anandhan

    Present address: Directorate of Onion and Garlic Research, Pune, 410505, India

Authors and Affiliations

  1. Plant Genetic Engineering Laboratory, Department of Biotechnology, Bharathiar University, Coimbatore, 641046, India

    S. R. Kumar, S. Dhivya & R. Sathishkumar

  2. Molecular Biology and Genetic Engineering Laboratory, Defence Institute of Bio-Energy Research, Haldwani, 263139, India

    S. Anandhan & A. Zakwan

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  1. S. R. Kumar
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Correspondence to R. Sathishkumar.

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Acknowledgements: The authors thank M. Aslam and V. Sinha for the help in library construction work. Thanks to the DRDODIBER, India for the financial support through a research grant to RS and for a fellowship support to SRK.

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Kumar, S.R., Anandhan, S., Dhivya, S. et al. Isolation and characterization of cold inducible genes in carrot by suppression subtractive hybridization. Biol Plant 57, 97–104 (2013). https://doi.org/10.1007/s10535-012-0250-8

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