Rupesh Kumar Singh

Isolation of cold stress-responsive genes from Lepidium latifolium by suppressive subtraction hybridization

Suppression subtraction hybridization (SSH) libraries were constructed from RNA isolated from leaves of control and cold stress-induced Lepidium latifolium, a cold-tolerant plant species from high altitudes for isolation of cold-responsive genes. A total of 500 clones were obtained from the cold stress library. Dot blot expression analysis identified 157 clones that were upregulated and 75 that were downregulated during cold stress. These clones selected on the basis of their expression patterns on dot blot were sequenced. As much as 27 and 17 genes were identified from the forward and reverse libraries, respectively. The genes identified revealed homology with genes involved in diverse processes such as gene regulation/signaling, photosynthesis, DNA damage repair protein, pathogenesis-related protein, senescence-associated proteins and proteins with unknown functions.

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.

Construction of Hypericin Gland-Specific cDNA Library via Suppression Subtractive Hybridization

Hypericin, an important determinant of the pharmacological properties of the genus Hypericum, is considered as a major molecule for drug development. However, biosynthesis and accumulation of hypericin is not well understood. Identification of genes differentially expressed in tissues with and without hypericin accumulation is a useful strategy to elucidate the mechanisms underlying the development of the dark glands and hypericin biosynthesis. Suppression Subtractive Hybridization (SSH) is a unique method for PCR-based amplification of specific cDNA fragments that differ between a control (driver) and experimental (tester) transcriptome. This technique relies on the removal of dsDNA formed by hybridization between a control and test sample, thus eliminating cDNAs of similar abundance, and retaining differentially expressed or variable in sequence cDNAs. In our laboratory we applied this method to identify the genes involved in the development of dark glands and accumulation of hypericin in Hypericum perforatum. Here we describe the complete procedure for the construction of hypericin gland-specific subtracted cDNA library.