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Garlic ( Allium sativum ) chitinases: characterization and molecular cloning
Author(s) -
Damme Els J. M.,
Willems Pieter,
Torrekens Sophie,
Leuven Fred,
Peumans Willy J.
Publication year - 1993
Publication title -
physiologia plantarum
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.351
H-Index - 146
eISSN - 1399-3054
pISSN - 0031-9317
DOI - 10.1111/j.1399-3054.1993.tb00140.x
Subject(s) - chitinase , complementary dna , allium sativum , biology , cdna library , signal peptide , biochemistry , peptide sequence , microbiology and biotechnology , amino acid , molecular cloning , nucleic acid sequence , gene , botany
Leaves and bulbs of garlic ( Allium sativum L.) contain a chitinase which can be separated into three different isoforms with similar molecular structure and N‐ terminal amino acid sequence. SDS‐PAGE of the alkylated chitinase revealed two distinct polypeptides of 32 and 33 kDa. Induction studies of the chitinase in leaves of garlic plants indicated that not only treatment with ethephon or salicylate and wounding but also a temperature shock strongly increased the enzyme level. cDNA libraries constructed from poly(A)‐rich RNA isolated from young garlic shoots and bulbs were screened for chitinase clones using the cDNA clone CCH4 encoding a basic potato chitinase as a probe. Two different cDNA clones (designated CHITAS 1 and CHITAS 2)of ca 1 000 bp were isolated and their sequences analyzed. The amino acid sequences deduced from both cDNA clones were homologous though not identical to the N‐terminal sequences of the mature chitinases. Although both clones encode highly homologous chitinases their sequences definitely differ in that they have different signal peptides and one of them contains a glycine‐rich domain. The garlic chitinases are apparently translated from an mRNA of 1200 nucleotides which encodes a proprotein of approximately 32 or 33 kDa for CHITAS 1 and CHITAS 2, respectively. Co‐translational removal of the signal peptide will result in a 30 (for CHITAS 1) or 31 kDa (for CHITAS 2) protein with an isoelectric point of 4. 94 (for CHITAS 1) or 6. 12 (for CHITAS 2). Garlic chitinases are encoded by a small gene family as shown by Southern blot analysis of genomic DNA isolated from garlic. The garlic chitinases show a high degree of sequence homology to the previously isolated chitinases from dicotyledonous as well as monocotyledonous species, indicating that these proteins have been conserved from an evolutionary point of view.