biologia plantarum

Biologia plantarum 57:97-104, 2013 | DOI: 10.1007/s10535-012-0250-8

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

S. R. Kumar¹, S. Anandhan², S. Dhivya¹, A. Zakwan², R. Sathishkumar^1,*

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

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

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.

Keywords: cyclin; Daucus carota; low temperature; profilin; sqRT-PCR; upregulated genes

Subjects: cold inducible genes; carrot; suppression subtractive hybridization; cyclin; temperature low; profilin; gene expression

Species: Daucus carota

Received: November 23, 2011; Accepted: January 12, 2012; Published: March 1, 2013Show citation