Isolation and characterization of the third gene encoding a 16.9 kDa class I low-molecular-mass heat shock protein, Oshsp 16.9C, in rice

Using rice cDNA pTS1 encoding a 16.9kDa heat shock protein (HSP) as a probe for Southern hybridization analysis, we observed five prominent bands of 9.5, 5.9, 3.4, 2.5, and 1.7kb in the Eco RI digests of rice genomic DNA and found them to contain six individual genes. The 5.9kb DNA fragment was further digested with Hind III to generate three fragments of 3.5, 1.7 and 0.7kb, and we found, using pTS1 cDNA as a probe, the 3.5kb fragment contained a putative low-molecular-mass (LMM) HSP gene. The DNA sequencing of 3.5kb fragment revealed encoding of a presumptive 16.9kDa HSP (149 amino acid residues) with predicted pI value of 6.42. The nucleotide sequence of this gene was highly homologous to the coding regions of two rice class I LMM HSP genes, Oshsp 16.9A and Oshsp l6.9B, sharing 93.1% and 94.3% sequence identity, respectively, as published previously in our laboratory. The deduced amino acid sequence of this gene is similar to those of the Oshsp l6.9A and Oshsp l6.9B genes with a difference of only 11 and 10 amino acids, respectively. It was hence designated as Oshsp l6.9C (accession no. U81385). We used 3′ UTRs (untranslated regions) for analysis of Oshsp l6.9C gene expression since the 3′ UTRs of these three genes showed very low sequence homology.