PCR-based cloning, characterization, and stress-induced expression of chitinase genes in Kentucky bluegrass (Poa pratensis L.)

Chitinase is an enzyme that catalyzes the hydrolysis of chitin, an essential component of the cell walls of many fungi. Plant chitinases have been implicated in plant defense against pathogens. In plants, chitinase often exists as isoforms and three classes of chitinases have been proposed. In this study, isolation, characterization and expression of chitinase genes in Kentucky bluegrass (Poa pratensis L.) were studied.

With primers designed from conserved regions of chitinase genes from other plant species, a 710 bp fragment (CH710) containing a partial chitinase gene sequence was amplified from Kentucky bluegrass by PCR. Using cassette ligation mediated PCR, we amplified four 5’ and five 3’ unknown sequences flanking CH710. The sequence information of these flanking fragments led us to the amplification of three genomic sequences from Kentucky bluegrass by PCR, KBCH1, KBCH2 and KBCH3, which contain full coding regions of chitinase genes. The chitinase genes carrying KBCH1, KBCH2 and KBCH3 were designated as chi1, chi2 and chi3, respectively. Southern blot hybridization indicated the presence of more than seven chitinase genes in the Kentucky bluegrass genome.

Chi1 and chi2 each contain an open reading frame with no introns, encoding polypeptides of 340 and 320 amino acids, respectively. Both CHI1 and CHI2, the predicted proteins encoded by chi7 and chi2, are class I chitinases and share 94% amino acid identity. CHI1 has a short C-terminal extension, implicating that this protein may be a vacuolar protein. Although chi3 has high sequence similarity to chi7 and chi2, the potential open reading frame of chi3 is interrupted by a translation termination codon at the 51st amino acid indicating that it does not encode a functional chitinase.

In this study, the expression of chitinase genes in Kentucky bluegrass under various stress conditions was also investigated. RNA blot hybridization showed that stresses such as cold, heat, salicylic acid and ethephon all induced an increased accumulation of chitinase MRNA in Kentucky bluegrass leaves with ethephon leading to the highest induction. After ethephon treatment, the accumulation of chitinase transcripts at a high level was observed from 2 days to 5 days. The expression of two individual chitinase genes, chi1 and chi2, was shown to be stimulated, while being coordinately regulated, by ethephon.