Plant Molecular Biology 36: 407–415, 1998.
1998 Kluwer Academic Publishers. Printed in Belgium.
A class III acidic endochitinase is speciﬁcally expressed in the developing
seeds of soybean (Glycine max [L.] Merr.)
Nana Asare Yeboah, Masaomi Arahira, Van Hai Nong
, Deyu Zhang, Kazunari Kadokura,
Akira Watanabe & Chikafusa Fukazawa
Genetic Engineering Laboratory, National Food Research Institute, Ministry of Agriculture Forestry and
Fisheries, Kannondai, Tsukuba Science City, Ibaraki 305, Japan (
author for correspondence);
National Centre for Natural Science and Technology, Institute of Biotechnology, Nghiado, Tuliem, Hanoi, Vietnam
Received 19 December 1996; accepted in revised form 26 September 1997
Key words: cDNA cloning, class III acidic endochitinase, C-terminal extension, Glycine max, protein sequence,
A soybean chitinase which has an apparent molecular mass of 28 kDa by SDS-PAGE, and has chitinase speciﬁc
activity of 133 units per mg protein at pH 5.2 and an apparent pI of 5.7, was puriﬁed from mature dry seeds.
Based upon the selected part (the residue positions 10–17) of the determined N-terminal 38 amino acid sequence,
a 23-mer degenerate oligonucleotide was synthesized and used for the PCR cloning of the chitinase cDNA. The
resulting 1340 bp cDNA was comprisedof a 5
-untranslated region of 39 bases, a coding region correspondingto a
25 amino acid signal sequence, followed by a mature 308 amino acid sequence (calculated molecular mass 34269,
calculated pI 4.7), and a 235 nucleotide 3
-terminal untranslated region including 24 bases of the poly(A) tail. By
comparing the deduced primary sequence with those of plant chitinases known to date, this enzyme was more than
50% identical to every class III acidic chitinase, but has no signiﬁcant similarity to other families of chitinases. The
comparison also showed that the C-termininal region of this chitinase is markedly extended, by at least 31 residues.
Northern blot analysis demonstrated that this mRNA species is remarkably transcribed from the early stage until
the late middle stage of seed development, whilst it is hardly expressed in the leaves and the stems of soybean.
Spatial and temporal expression of this single gene imply that this class III chitinase is mainly devoted to the seed
defense, not only in development but also in dormancy of soybean seed. This is the ﬁrst reported isolation and
cDNA cloning of a class III acidic endochitinase from seeds. According to the chitinase nomenclature we propose
that this enzyme wouldbe classiﬁed into a new class of chitinase PR-8 family, together with a Sesbaniahomologue.
Chitinase [EC 220.127.116.11]is an enzyme which catalyzes
the hydrolysis of
-1,4 linkages of the N-acetyl-D-
glucosamine polymer chitin, a component of fungal
cell walls and exoskeleton of insects, which is not
known to occur naturally in plants. It is believed,
therefore, that the plant enzyme functions as a defense
against pathogenic attacks.
The nucleotide sequence data reported will appear in the DDBJ,
EMBL and GenBank Nucleotide Sequence Databases under the
accession number AB 000097.
al stimuli, including pathogenic attacks, has largely
been exploited to characterize a number of chitinases
from various sources. According to a revised nomen-
clature , there are four chitinase families, which
are further divided into classes. Signiﬁcant similarity
exists between class I (Chia1), class II (Chia2)and
class IV (Chia4) enzymes constituting now PR-3 fam-
ily (family 19 of glycosyl hydrolases),but not between
the class III (Chib1) belonging to PR-8 family (fam-
ily 18 of glycosyl hydrolases) and any of the classes
mentioned (see  for review). Furthermore, several
classes of chitinases may occur in the same plant spe-