全 文 ：Received 3 Apr. 2003 Accepted 15 Oct. 2003
Supported by the National Special Program for Research and Industrialization of Transgenic Plants (J99-A-038), the Hi-Tech Research and
Development (863) Program of China (2002AA224101), the National Natural Science Foundation of China (30270145) and the Science and
Technology Development Project of Jilin Province (20030553-1).
* Author for correspondence. Tel (Fax): +86 (0)538 8242364; E-mail: .
http://www.chineseplantscience.com
SA Induction of a Grapevine Class Ⅲ Chitinase Gene VCH3
Promoter in Transgenic Tobacco Vascular Tissue
LI Hai-Yan1, 2, LI Yu2, WEI Wei1, ZHENG Cheng-Chao3, SHU Huai-Rui4*
(1. College of Biotechnology, Jilin Agricultural University, Changchun 130118, China;
2. Mycorrhizae Institute, Jilin Agricultural University, Changchun 130118, China;
3. College of Life Sciences, Shandong Agricultural University, Taian 271018, China;
4. College of Horticulture, Shandong Agricultural University, Taian 271018, China)
Abstract: The 1 216 bp 5 upstream region of the gene encoding the class Ⅲ chitinase VCH3 was
isolated from grapevine (Vitis amurensis Rubr.) (Genbank accession number AF441123) and two inverse
salicylic acid (SA) responsive cis-acting motifs (TGACG) were found at -1 181 bp and -293 bp upstream of
the transcriptional start site, respectively. To characterize the VCH3 promoter, four chimeric constructs
varied in the length of promoter fragments from -1 187 bp, -892 bp, -589 bp and -276 bp to +7 bp relative
to the transcriptional start site were placed to the upstream of the b-glucuronidase (GUS) coding region
and transferred to Nicotiana tobacum L. cv. NC89 by Agrobacterium tumefaciens-mediated leaf discs
transformation. The functional properties of each promoter fragment were examined by fluorometric and
histochemical analysis of GUS activity in the transgenic tobacco root treated with SA. The VCH3 (-276)
GUS construct, containing only the TATA and CAAT boxes was shown to have little inducibility upon
treatment with SA. However, the similarly higher level of GUS expression was observed in the VCH3 (-589)
GUS or VCH3 (-892) GUS transgenic plants with only one cis-acting motif, while the most abundance of
GUS expression was found in the full-length promoter (-1 187 bp to +7 bp) with two cis-acting motifs.
These results indicated that the two cis-acting motifs were required for the maximal expression of the
GUS reporter gene by SA induction. In addition, the histochemical analysis of GUS activity showed that the
four VCH3 promoter fragments were more active in vascular tissue than that in outer and inner cortexes
of the transgenic tobacco roots treated by SA, suggesting that the region involved in vascular tissue-
specific expression of VCH3 promoter upon SA inducibility appears to be located between positions -276
bp and +7 bp relative to the transcriptional start site. In general, these results indicate a potential use for
the SA induction of VCH3 promoter in genetic engineering.
Key words: salicylic acid (SA); induction; VCH3 promoter; cis-acting motif; vascular tissue-specific
expression
Inducible promoters or gene-switches are used to spa-
tially and/or temporally regulate gene expression. Such regu-
lation can provide information concerning the function of a
gene in a developmental context and avoid potential harm-
ful effects due to overexpression. Thus, a range of induc-
ible promoters were isolated from various plant species and
their efficiency was characterized by directing the expres-
sion of the reporter gene encoding b-glucuronidase (GUS),
such as light- or dark-inducible promoter (Sessa et al., 1995;
Boetti et al., 1999); ABA, auxin, tetracycline, chloride or
copper-dependent promoter ( Kop et al., 1996; Sanders et
al., 1997; Boetti et al., 1999; Granger and Cyr, 2001); elici-
tor- and pathogen-inducible promoter (Yin et al., 1997). Ef-
ficient genetic engineering relies on designing appropriate
gene expression system by using those promoters to drive
gene express at the required moment and in the appropriate
tissues.
Plant resisting pathogen attack rapidly activate a wide
variety of defenses in the inoculated tissues that help pre-
vent pathogen colonization, and at slightly later times, the
infected tissues develop a hypersensitive response (HR),
in which necrotic lesions are formed at the sites of patho-
gen entry and the pathogen is restricted to these regions.
Being correlated with or slightly preceding the appearance
of a HR is because of the accumulation of salicylic acid
(SA) and the expression of several classes of pathogen-
esis-related (PR) proteins, many of which exhibit chitinase
activity (Narusaka et al., 1999). These genes are expressed
Acta Botanica Sinica
植 物 学 报 2004, 46 (2): 148-153
.Rapid Communication.
LI Hai-Yan et al.: SA Induction of a Grapevine Class Ⅲ Chitinase Gene VCH3 Promoter in Transgenic Tobacco Vascular Tissue 149
locally as well as in distant noninfected tissues (Busam et
al., 1997; Dempsey et al., 1999). Such inducible defense
mechanism, which is known as systemic acquired resis-
tance (SAR) plays a central role in disease resistance
(Dempsey et al., 1999). Many studies have reported that
chitinase provides a useful molecular and biochemical
marker for the induction of SAR (Busam et al., 1997), in
which SA serves as a mobile signal molecule to activate the
expression of chitinases gene. The functional properties of
the promoters of several of chitinase have been character-
ized (Zhu et al., 1993; Shinshi et al., 1995), and this pro-
vides the basis for dissection of the signal pathways for
induction of defense responses by elicitor or infection. In
contrast, relatively little is known about the mechanism
underlying the activation of inducible defenses in xylophyta.
In our previous work, we have isolated from grapevine
(Vitis amurensis Rubr.) the VCH3 gene encoding PR protein,
a classⅢ chitinase, and have shown that the VCH3 tran-
script accumulates in grapevine root in response to patho-
gen infection. Recently, we have firstly reported the isola-
tion of the 5 flanking region of chitinase VCH3 (Genbank
accession number AF441123) from grapevine, this region
contains two inverse TGACG sequences that have been
found to be SA-responsive cis-acting motifs in several pro-
moters (Kim et al., 1993), which are located at -1 181 bp
and -293 bp upstream of the transcriptional start site,
respectively. Here, in order to address if the SA-respon-
sive cis-motifs are of functional importance for the maxi-
mum expression of GUS induced by SA and analyze the
expression pattern of the VCH3 promoter, the properties of
the VCH3 promoter are examined by analysis of 5 deleted
fragments and GUS gene fusion in transgenic tobacco roots
in respond to SA treatment.
1 Materials and Methods
1.1 Materials
Escherichia coli strain DH5, Agrobacterium
tumefaciens strain LBA4404 , binary vector pBI121 and to-
bacco (Nicotiana tobacum L. cv. NC89) are used in this
study.
1.2 Generation of the VCH3 promoter fragments and
plasmid construction
VCH3 promoter fragments were amplified by PCR with
100 ng plasmid as template and two units Taq DNA
polymerase. Four VCH3 promoter fragments from -1 187
bp, -892 bp, -589 bp, and -276 bp to +7 bp relative to the
transcriptional start site were generated using a common 3
oligonucleotide and four different 5 oligonucleotides. To
generate VCH3 promoter — GUS chimera, various
fragments of the VCH3 promoter were cloned upstream of
b-glucuronidase coding sequence in the binary vector
pBI121. Briefly PCR products of VCH3 promoter were
blunted using klenow fragment, subsequently digested with
BamHⅠ. Plasmid pBI121 was cleaved with PstⅠ, blunted
using klenow fragment then digested with BamHⅠ. The
blunt/ BamHⅠ VCH3 promoter fragments were inserted in
blunt/ BamHⅠ pBI121.
1.3 Tobacco transformation
The GUS expression cassettes in the binary vector
pBI121 were mobilized into A. tumefaciens strain LBA4404
by one-step method. Leaf discs from N. tobacum cv. NC89
were transformed and plant were regenerated by standard
methods (Horsch et al., 1985). For each construct, the pres-
ence of the GUS gene in transformed plants was verified by
Southern blotting analysis, and 12 transformants contain-
ing one and two copies of the chimeric gene were allowed
to self-fertilize and seeds were collected and germinated on
MS agar medium with 300 mg/mL kanamycin sulfate. Kana-
mycin-resistant T2 seedlings were used as the following
induction.
1.4 Induction treatments
T2 seedlings at the four to five leaf stage were used in
SA induction treatments. The tobacco plant roots were sub-
merged to the MS medium without sucrose but supple-
mented with 1 mmol/L SA at room temperature for 24 h
before analyzing GUS activity, to control, SA was replaced
by the distilled water.
1.5 GUS activity assays and histochemical analysis
GUS activity was determined using a flurorometric as-
say as described by Jefferson et al. (1987). The roots of
different treatments were homogenized in 0.6 mL chilled
lysis buffer (0.1 mol/L sodium phosphate and 1 mmol/L
EDTA) and 10 mL was used for measuring GUS activity,
which was normalized to protein concentration for each
crude extract and calculated (pmol 4-methyl umbelliferone.
mg-1 soluble protein.min-1). Protein content was measured
by the Bradford (1976) method using BSA as a standard.
Histochemical assays were performed as follows (Stomp,
1992). One to two centimeter tobacco roots were fixed in
the fixing solution (0.1 mol/L sodium phosphate, pH 7.0;
0.1% formaldehyde; 0.1% Triton X-100; 0.1% 2-
mercaptoethano) for 30 min, and then stained in GUS stain-
ing reagent which contains 0.1 mol/L sodium phosphate,
10 mmol/L EDTA, 0.5 mmol/L pottassium ferrocyanide, 0.5
mmol/L potassium ferricyanide, 1 mmol/L X-Glucuronide
and 0.1% Triton X-100. Tissues were vacuum infiltrated
with the reaction solution in order to assure homogeneous
penetration of the substrate. After dehydrating in 100%
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004150
ethanol, the tissues were incubated in toluene for two 2-h
passages at room temperature, then embedded for section-
ing (8-10 µm in thickness).
2 Results
2.1 5 flanking region of the grape chitinase gene VCH3
contains two SA-responsive cis-acting motifs
We have recently isolated from grapevine the 1 216 bp
promoter sequence of chitinase gene VCH3 (Genbank ac-
cession number AF 441123) by adaptor-PCR (Fig.1) and the
transcriptional start site was identified by primer extension
analysis (unpublished data). The CAAT and TATA boxes
are located in -122 bp and -29 bp relative to the transcrip-
tional start site, and two inverse SA-responsive cis-acting
motifs (TGACG) are found at -1 181 bp and -293 bp up-
stream of the transcriptional start site.
Fig.1. Nucleotide sequence of Vitis amurensis chitinase VCH3 gene promoter（Genbank accession number AF441123）and the
regulatory region. VCH3 promoter sequence shown is the two inverse TGACG cis-elements which locates respectively in -1 181 bp and
-293 bp relative to the transcriptional start site. The CAAT and TATA boxes presenting respectively -122 bp and -29 bp relative to
the transcriptional start site are underlined，and the transcriptional start site is indicated by solid circle.
LI Hai-Yan et al.: SA Induction of a Grapevine Class Ⅲ Chitinase Gene VCH3 Promoter in Transgenic Tobacco Vascular Tissue 151
2.2 Construction of the VCH3 promoter-GUS chimera
and tobacco transformation
To determine if the SA-responsive cis-motifs are of func-
tional importance for the maximum expression of GUS in-
duced by SA and analyze the expression pattern of the
VCH3 promoter, the deletion fragments of VCH3 promoter
were produced by polymerase chain reactions (PCR), and
the constructed deletion fragments—GUS chimera were
transferred to N. tobacum cv. NC89 by A. tumefaciens-me-
diated leaf discs transformation. VCH3 (-276) GUS repre-
sents the minimal fragment only including TATA and CAAT
box sequences; both VCH3 (-589) GUS and VCH3 (-892)
GUS contains one TGACG element; and VCH3 (-1 187)
GUS represents the maximal fragment including two TGACG
elements (Fig.2).
2.3 Fluorometric analysis of GUS activities in transgenic
tobacco roots induced by SA
The functional properties of each promoter fragment
were examined by fluorometric analysis of GUS activity
(Fig. 3). The VCH3 (-276) GUS construct, containing the
TATA and CAAT boxes and deleting of all the cis-acting
motifs, was shown to have little inducibility upon treat-
ment by SA. And the similar level of GUS expression was
observed in the VCH3 (-589) GUS and VCH3 (-892) GUS
which contained one cis-acting motif. The highest level of
GUS expression was found in the full-length promoter (-1 187
bp to +7 bp) which contained two cis-acting motifs di-
rected the transcription of GUS (about 2.0 folds equal to
that directed by the second and third length fragments). It
indicated that all these two motifs were required for the
maximal induction of the GUS reporter gene by SA treatment.
2.4 Histochemical analysis of GUS activities in transgenic
tobacco roots induced by SA
The histochemical analysis of GUS in the transgenic
tobacco roots 24 h after treatment with SA was performed
(Fig.4). It was observed that the activities of GUS directed
Fig.2. The structure of VCH3 promoter fragments-GUS chimera. The 5 end point of each
VCH3 promoter fragment is indicated. VCH3 (-276) GUS represents the minimal fragment
(only includes TATA and CAAT box sequences. All the SA cis-acting elements are deleted); both
VCH3 (-589) GUS and VCH3 (-892) GUS are the fragments containing one cis-acting element;
and VCH3 (-1 187) GUS represents the maximal fragment containing two cis-acting elements.
Fig.3. Induction of GUS activity in transgenic tobacco roots
treated by SA. Values are means of at least 12 replicates ± SE.
by all the four promoter frag-
ments was active in vascular
tissue than that in outer and in-
ner cortexes in the cross-section
of transgenic tobacco roots
treated by SA. Furthermore,
more abundant GUS expression
was seen in VCH3 (-1 187) GUS
plants, which was consistent
with the result of fluorometric
analysis.
3 Discussion
In this study we describe a
set of four chimeric genes con-
structed by fusion of various
portion of PR protein, class Ⅲ chitinase VCH3 promoter to
the GUS reporter gene. The expression of these chimeric
genes was studied in transgenic tobacco plants to deter-
mine the regions of the promoter required for induction of
SA. Using this approach we have examined the tissue-spe-
cific expression of this gene after SA treatment.
Transformants containing a chimeric gene with 589 bp
or more of VCH3 5 flanking sequence showed substantial
increases in GUS activity after SA treatment. The highest
level of GUS expression was found in the full-length pro-
moter (-1 187 bp to +7 bp) which containing two SA-re-
sponsive cis-acting motifs directing the transcription of
GUS. SA induction of VCH3 (-1 187) GUS plants was ap-
proximately two folds equal to that in the VCH3 (-892)
GUS and VCH3 (-589) GUS plants, suggesting that all these
Acta Botanica Sinica 植物学报 Vol.46 No.2 2004152
two SA-responsive cis-acting motifs TGACG were required
for the maximal induction of the GUS reporter gene. DNA
sequences similar to the SA-responsive cis-acting motifs
were also found in other promoters (Bouchez et al., 1989;
Kim et al., 1993). These sequences are specific binding
sites for a family of trans-acting factors that are Leu-zipper
proteins (Xiang et al., 1997; Lebel et al., 1998; Strompen et
al., 1998). Whether SA treatment induces the VCH3 pro-
moter activity by increasing the level of the transcription
factor and/or by activating the protein remains to be
determined. The cDNA clones for the trans-acting factors
will be useful in solving these problems.
Tissue-specific expression was also analyzed in
transgenic plants containing the various 5 promoter dele-
tion VCH3/GUS constructs. The same patterns of expres-
sion were seen for constructs which contained 276 bp or
more of the 5 flanking region, i.e. GUS activity directed by
all the four promoter fragments was more active in vascular
tissue than that in outer and inner cortexes. Thus, the re-
gion involved in tissue-specific expression of VCH3 pro-
moter required for SA inducibility appears to be located
between positions -276 bp and +7 bp relative to the tran-
scriptional start site. In addition, many pathogens can rap-
idly spread throughout the plant if they penetrate into the
vascular system. Hence, this expression pattern of the
VCH3 promoter in root may enhance the protection afforded
to such vulnerable tissues in response to pathogen
infection. This consideration suggests that SA could serve
as a mobile signal molecule to activate the PR protein,
chitinase gene VCH3 expression in the grapevine SAR de-
fense mechanism. Generally, these results indicate that the
efficient genetic engineering will be performed to design
appropriate gene expression system by using this SA in-
duction of the VCH3 promoter to drive gene expression at
the required moment and in vascular tissues, especially, to
drive the plant disease-resistant genes.
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