Isolation and characterization of class I chitinases from cotton (Gossypium hirsutum)

The objective of this dissertation was to isolate and characterize chitinases from cotton (Gossypium hirsutum). While chitinases have been isolated from a variety of agriculturally important crops, this research would lead to a more complete understanding of the defensive responses of cotton. The Polymerase Chain Reaction was used to amplify a 574 bp fragment of a chitinase gene from cotton genomic DNA. This fragment was used as a homologous probe to screen a cotton genomic library. Ultimately, the entire coding region of a class I chitinase gene was isolated. A Reverse Transcriptase-Polymerase Chain Reaction was used to isolate two class I chitinase cDNAs from ethylene-treated leaf tissue. The cDNAs were found to be 98% identical at the nucleotide level. Southern blotting analysis indicates that class I chitinases are members of a small multigene family with at least 3 members per haploid genome. The responsiveness of cotton chitinase activity to ethylene was studied at the protein and RNA levels. These results indicate that a wound response precludes ethylene induction of chitinase activity in detached cotton leaves. Conversely, lower levels of ethylene were able to moderately induce chitinolytic activity in plantlets.