Construction of cold induced subtracted cDNA library from Cicer microphyllum and transcript characterization of identified novel wound induced gene
A forward cold induced subtracted cDNA library was constructed to identify the stress regulated genes in a high altitude cold desert-adapted species Cicer microphyllum, a wild relative of cultivated chickpea, distributed in western and trans-Himalayas. A total of 1,040 clones were obtained from the subtracted cDNA library. These clones were screened for presence of insert with colony PCR. A total of 523 clones were picked by colony PCR among which 300 clones were observed differentially expressed as per dot blot analysis. Differentially expressed clones were sequenced and assembled into clusters based on the presence of overlapping, identical, or similar sequences. A total of 283 ESTs were submitted in gene bank (accession numbers GO241043 to GO241326). BLAST analysis of these ESTs revealed its similarity for regulatory proteins like kinases, metallothionin, and enzymes/proteins with unknown functions. A cDNA encoding wound induced like protein, identified from this cold induced subtraction cDNA library, was full-length cloned using RACE and sequenced (accession number GQ914056). Southern blot of C. microphyllum indicated single copy of the gene in genome. Transcript expression profiling of this gene by quantitative real-time PCR and northern blot confirmed its up-regulation during low temperature stress. Further, in situ RNA hybridization also revealed cold (4°C) induced expression of the gene.
KeywordsCicer microphyllum Low temperature Suppression subtraction hybridization (SSH) Wound induced gene qRT PCR In situ hybridization
The authors thank the Director, DIHAR, Leh for providing seeds of Cicer microphyllum. Dr. Patade Vikas Yadav is gratefully acknowledged for critical review during manuscript preparation. Financial support to RKS, SS and PP in the form of Research Fellowship by DRDO is gratefully acknowledged.
It is certified that the authors do not have any conflict of interest in publishing the manuscript.
- Chaurasia OP, Basant B, Pande PC, Ahmed Z (2007) Raw edible plants of cold desert Ladakh. Ind J Trad Knowledge 6(1):182–184Google Scholar
- Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bull 19:11–15Google Scholar
- Elstner EF (1991) Mechanism of oxygen activation in different compartments of plant cells. In: Pell EJ, Steffen KL (eds) Active oxygen/oxidative stress and plant metabolism. American Society of Plant Physiologists, Rockville, pp 13–25Google Scholar
- Farmer EE, Ryan CA (1990) Interplant communication: air borne methyl jasmonate induces synthesis of preotinase inhibitors in plant leaves. Proc Natl Acad Sci USA 87:7713–7716Google Scholar
- Lam JD, Arteca RN, Pell EJ (2006) Senescence associated gene expression during ozone induced leaf senescence in Arabidopsis. Plant Physiol 120:1015–1023Google Scholar
- Liu M, Lu C, Shen X, Yin W (2006) Characterization and function analysis of a cold-induced AmCIP gene encoding a dehydrin-like protein in Ammopiptanthus mongolicus. DNA Seq 5:342–349Google Scholar
- McKersie BD (1991) The role of oxygen free radicals in mediating freezing and desiccation stress in plants. In: Pell EJ, Steffen KL (eds) Active oxygen/oxidative stress and plant metabolism. American Society of Plant Physiologists, Rockville, pp 107–118Google Scholar
- Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York, pp 72–74Google Scholar
- Singh S, Anandhan S, Arun MB, Goyary D, Singh NK, Das SC (2007) In vitro establishment of Cicer microphyllum, a wild relative of cultivated chickpea from cold desert of Ladakh. J Arid Leg 4(1):66–68Google Scholar
- Tottempudi KP (1997) Role of catalase in inducing chilling tolerance in pre-emergent maize seedlings. Plant Physiol 114(1369-1):376Google Scholar