Molecular Cloning of a Genomic DNA Encoding Yam Class IV Chitinase

Abstract

Genomic DNA for a class IV chitinase was cloned from yam (Dioscorea opposita Thunb) leaves and sequenced. The deduced amino acid sequence shows 50 to 59% identity to class IV chitinases from other plants. The yam chitinase, however, has an additional sequence of 8 amino acids (a C-terminal extension) following the cysteine that was reported as the last amino acid for other class IV chitinases; this extension is perhaps involved in subcellular localization. A homology model based on the structure of a class II chitinase from barley was used as an aid to interpreting the available data. The analysis suggests that the class IV enzyme recognizes an even shorter segment of the substrate than class I or II enzymes. This observation might help to explain why class IV enzymes are better suited to attack against pathogen cell walls.

Cloning and Characterization of a Balsam Pear Class I Chitinase Gene (Mcchit1) and Its Ectopic Expression Enhances Fungal Resistance in Transgenic Plants

“…To analyze the expression pattern, the balsam pear seedlings were transplanted in pots and grown at 28-33 C during the day and 20-25 C at night in a greenhouse. For wounding treatment, the mature leaves were cut into strips (around 2 cm in width), and kept moist for 24 h on filter paper at [28][29][30] C. N. benthamiana was employed to express the McChit1 gene ectopically. The regenerated plants were transplanted in pots and maintained at [28][29][30][31][32][33] C during the day and 20-25 C at night in the greenhouse.…”

“…The treated seedlings were grown at [25][26][27][28][29][30] C for 4 d in a growth chamber, and the infected seedlings were scored. Each treatment was replicated three times, and each replicate contained 30 seedlings.…”

“…[23][24][25][26][27][28][29][30] On the basis of the primary structures, plant chitinases are classified into seven classes (I-VII). [23][24][25] Classes I, II, and IV belong to family 19 glycosyl hydrolases, and share a homologous catalytic domain.…”

Intracellular Localization of a Class IV Chitinase from Yam

“…Furthermore, this yam chitinase has an additional 8 amino acids (the C-terminal extension) following the last cysteine at the C-terminal, compared with other class IV chitinases. 9) With respect to the Cterminal extension, some class I chitinases have been found in bean, 23) potato, 24) tobacco, 25) rice 26,27) and grape 28) to have a C-terminal extension like this yam class IV chitinase. As a general rule, chitinases carrying such a C-terminal extension are localized in the vacuole and those lacking it are secreted out of the cell.…”

“…6) In order to investigate the structure of chitinase E, therefore, its genomic DNA was cloned from yam leaves in the previous study. 9) The nucleotide sequence (YC4-v) has been deposited in the GenBank data base (under accession no AB102714). The deduced amino acid sequence showed 50 to 59% identity to class IV chitinases from other plants.…”