protein, and it differs by ca. 43 in the sequence from class I and II proteins [9].
The expression of many b-1,3-glucanases can be induced by fungal elicitors, wounding, salicylic
acid SA, ethylene, and other chemical inducers [10 – 14]. b-glucanase genes are also expressed dur-
ing the hypersensitive response HR in TMV-in- oculated leaves [10,11,15]. b-1,3-glucanases are
thought to act against pathogen directly by digest- ing b-1,3-glucans in fungal cell walls as well as act
indirectly by digesting fungal and host polysaccha- rides to produce elicitors capable of evoking the
HR [16 – 19]. Also, the b-1,3-glucanases, especially in conjunction with chitinase, are capable of hy-
drolyze fungal cell walls in vitro [20]. Since both enzymes are co-induced in response to fungal at-
tack, their function is believed to be the inhibition of fungal infection [21]. Recently, it has been
reported that class I b-1,3-glucanase deficient to- bacco plants generated by antisense transforma-
tion exhibit decreased susceptibility to necrotic virus infection [22].
So far, class I and II b-1,3-glucanases have been well studied in terms of gene regulation and func-
tion in plant. However, class III b-1,3-glucanases have been less studied in their gene regulation
except for PR-Qa and PR-Qb, the acidic and basic class III glucanase from tomato, which are
expressed upon virus infection and ethylene treat- ment, respectively [23]. In particular, cis-acting
elements in the promoters of the class III b-1,3- glucanases have not yet been characterized. In
order to study gene regulation of a class III glu- canase on the promoter level, we isolated a soy-
bean Glycine max cv Williams basic class III b-1,3-glucanase gene, designated as SGN
1
. In this paper, we report the isolation and characterization
of SGN
1
which is very closely related to the PR-Qb gene that encodes a tomato basic class III
b-1,3-glucanase gene. We analyzed the expression of the SGN
1
gene during development and in response to pathogen signals. Expression of SGN
1
was induced by pathogen signals, such as H
2
O
2
, fungal elicitor prepared from Phytophthora spp
and wounding, but not by ethephon, jasmonic acid JA and salicylic acid SA. These results indicate
that SGN
1
, a class III basic glucanase, responds differently to defense signals and is, therefore,
differently regulated in comparison class I and II plant glucanases.
2. Materials and methods
2
.
1
. Plant material and treatments Soybean G. max cv. Williams and tobacco
Nicotiana tabacum cv Xanthi were used. To ob- tain seedlings, soybean seeds were germinated and
grown in darkness for 1 – 5 days in moist vermi- culite at 28°C. To obtain tissue from mature
plants, soybean seeds were planted in sterilized soil and grown in a growth chamber Conviron E15
with a 14 h light 28°C and 10 h dark 20°C cycle for around 30 days. All plants were watered to
saturation daily and fertilized weekly with 20-20- 20 fertilizer Peters, Allentown, PA
To analyze expression of the soybean SGN
1
gene during development, hypocotyls of soybean seedlings were used. To study stress induction,
4-day old soybean seedlings were exposed to vari- ous external stresses; sterile-distilled H
2
O, 0.01 VV ethephon, 1 mM salicylate SA, 0.2 mM
jasmonate JA, 1 mM H
2
O
2
, fungal elicitor pre- pared from Phytophthora spp 50 mgml glucose
equivalents and inoculation of Pseudomonas sy- ringae pv glycinea 10
6
cellsml. The chemicals or elicitor were sprayed to the soybean seedlings in
plastic boxes and incubated for 24 h in the boxes containing 100 ml of the same treatment solutions.
For the wounding stress, the 4-day old soybean seedlings were punctured with a 20-gauge needle at
1 mm intervals along opposite sides of the entire seedling. Fungal elicitor had been isolated as de-
scribed by Simmons et al. [24] and stored at 4°C until use. For control, total RNA was isolated
from water-treated seedlings. After treatment, the tissue was frozen in liquid N
2
and stored at − 80°C until use.
Seeds of transgenic tobacco plants Ro were obtained by self pollination of the transgenic
plants. The R1 progeny were aseptically germi- nated on wet filter paper discs in petri dishes under
a 16 h-light8 h-dark cycles.
2
.
2
. Cloning of the SGN1 gene A lambda EMBL
3
soybean genomic library was screened with the tobacco b-1,3-glucanase gn
1
as a probe [25]. Approximately 300 000 recombinant
plaques were screened by plaque hybridization at 60°C in a solution containing 6 × SSC 1 × SSC is
0.15 M NaCl, 0.15 M sodium citrate, pH 7.2,
5 × Denhart’s solution [1 × Denhart’s solution is 0.02
wv Ficoll
400, 0.02
wv Polyvinylpyrrolidone, 0.02 wv BSA], 0.5 w
v SDS, 5 mM EDTA and 100 mgml sheared, denatured calf thymus DNA. The membranes
were washed twice in 2 × SSC, 0.1 SDS and for a final washed in 0.2 × SSC, 0.1 SDS for 15 min
at 60°C and then exposed to Kodak XAR-5X-ray film at − 80°C. Positive clones were mapped with
restriction
enzymes and
subcloned into
the pBluscript II vector for DNA sequencing.
2
.
3
. DNA sequencing and analysis Double-stranded DNA was sequenced by the
dideoxy chain termination method [27] with a Taq Dye primer cycle sequencing kit using an Applied
Biosystem 373A automatic DNA sequencer ABI. Nucleotide and amino acid sequence analyses were
performed with the Macintosh DNASIS program Hitachi
software Engineering
America, San
Bruno, CA.
2
.
4
. Primer extension analysis To determine the transcriptional start site on the
SGN
1
gene, a primer extension experiment was performed as described by Sambrook et al. [26]
using a synthetic 20-mer oligonucleotide 5-gatg- gaagaacttttcccac-3 complementary to nucleotides
+ 97 to + 77 of the SGN
1
gene.
2
.
5
. Genomic Southern blot analysis Southern blot analysis was carried out as de-
scribed by Hong et al. [28] using total soybean DNA isolated by a CTAB precipitation method
[29] from etiolated soybean hypocotyls. Hybridiza- tion and washing conditions were as described
above for the library screening.
2
.
6
. RNA isolation and Northern blot analysis Total RNA was isolated as described by Hong
et al. [28]. Total RNA 20 mg was denatured and separated by electrophoresis on a 1.2 agarose-
formaldehyde gel, and transferred onto a GeneS- creen Plus membrane NEN. Hybridization and
washing conditions were as described above for the library screening.
2
.
7
. Construction of a SGN1::GUS fusion gene and plant transformation
For the construction of a SGN
1::
GUS fusion chimeric gene, a 1.7 kb fragment with HincII and
HindIII restriction sites on either end containing the promoter region and part of the coding region
of the SGN1 gene was amplified by PCR using synthetic oligonucleotide primers [5-GTCGAC-
TAAGTCCATT-3
− 1618 to
− 1602
and 5-GATGGAAGAAGCTTT-TCCCAC-3 + 97 –
+ 77. The mismatches bold were introduced to
create the HincII and HindIII sites underlined]. The PCR product was then digested with HincII
and HindIII and subcloned between the SmaI and HindIII site of pBluescript SK + . The resulting
recombinant plasmid, referred to pBS-PCR, thus contained about 1.7 kb of SGN
1
promoter. A BamHIHincII fragment − 1618 – + 97 from
pBS-PCR was then inserted into the BamHISmaI site of the GUS expression vector pBI
101
.
1
to give SGN
1::
GUS construct [30]. The construct SGN
1::
GUS was introduced into Agrobacterium LBA4404 by electroporation, and
transgenic tobacco plants were generated by the leaf disc method [31,32]. Transformed plants were
selected on Murashige and Skoog MS basal medium [33] containing 200 mgml kanamycin and
500 mgml carbenicillin and grown at 25°C under a 16 h-light8 h-dark cycle.
2
.
8
. Detection of GUS acti6ity To test for expression of the SGN
1::
GUS con- struct during seed germination, transformed to-
bacco seeds were sterilized and allowed to germinate for 1 or 4 days. To test for induction of
SGN
::
GUS gene expression, leaves from the 6- or 7-week old transformed plants were used after
wounding, treatment with various stress chemicals, such as SA, JA, H
2
O
2
, and ethephon, for 18 h or inoculation with Pseudomonas syringae pv tabaci
Pst for 36 h. The tests were done by fluorometric and histochemical analysis.
Fluorometric GUS assays were carried out as described by Jefferson et al. [30]. Protein concen-
tration was determined with a Bradford assay [34], and GUS activity was expressed as nmol 4-MU
produced per mg protein per min.
Histochemical GUS assays were performed by the standard method [30]. Stained sections were
microscopically viewed and photographed with a Leitz stereomicroscope.
3. Results
