Characterization of Chitinase Activity and Gene Expression in Muskmelon Seeds

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Virginia Tech

Chitinase has been suggested to play a role in defense mechanisms. In this study, the activity and expression of chitinase in muskmelon seeds were investigated. Multiple chitinase isoforms were detected in muskmelon seeds from early development through radicle emergence. One acidic and three basic chitinase isoforms were detected in developing seeds at 40 days after anthesis (DAA). Both acidic and basic chitinase isoforms were detected in endosperm tissue during seed imbibition and after radicle emergence. Basic chitinase isoforms, but not acidic isoforms, were detected in embryo tissue. Basic chitinase isoforms were also detected in the embryonic axis or radicle tissue. Taken together, these observations indicate that chitinases are regulated developmentally and in a tissue-specific manner in muskmelon seeds. Therefore the potential function of chitinases in muskmelon seeds is discussed.

Two complete cDNAs, Cmchi1 and Cmchi2, and a partial genomic clone of Cmchi2 have been isolated from muskmelon seeds. Cmchi2 gene has two introns in the coding region while Cmchi1 is intronless. Cmchi1 cDNA encodes a class III chitinase while Cmchi2 cDNA encodes a class II chitinase. Cmchi1 and Cmchi2 proteins might be targeted to secretory pathways because they possess signal peptides.

Southern blotting suggested that there is at least one additional gene similar to Cmchi1 in the muskmelon seed genome, while there is only one copy of Cmchi2. Northern blotting analysis showed that both Cmchi1 and Cmchi2 are expressed in the radicle tissue at the time of radicle emergence. This indicates that the expression is regulated developmentally and in a tissue-specific manner. Salicylic acid (SA) and benzothiadiazole (BTH) stimulated the expression of Cmchi1 but not Cmchi2 in seeds after radicle emergence, indicating that SA might be involved in inducing the expression of Cmchi1, while a different signal might be involved in triggering the expression of Cmchi2.

The protein encoded by Cmchi1cDNA was expressed in E.coli. It did not show any enzymatic activity. Western blotting using an antibody raised against the class III chitinase protein in cucumber was inconclusive, as this antibody recognized the purified Cmchi1 fusion protein and other unknown proteins isolated from the embryonic axis or the radicle tissue.

gene expression, muskmelon seeds, activity, chitinase, isoforms, regulation, cloning