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1.
Sasaki K Ito H Mitsuhara I Hiraga S Seo S Matsui H Ohashi Y 《Plant molecular biology》2006,62(4-5):753-768
The wound-induced expression of tpoxN1, encoding a tobacco peroxidase, is unique because of its vascular system-specific expression and insensitivity to known wound-signal compounds such as jasmonic acid, ethylene, and plant hormones [Sasaki et al. (2002) Plant Cell Physiol 43:108–117]. To study the mechanism of expression, the 2-kbp tpoxN1 promoter region and successive 5′-deletion of the promoter were introduced as GUS fusion genes into tobacco plants. Analysis of GUS activity in transgenic plants indicated that a vascular system-specific and wound-responsive cis-element (VWRE) is present at the −239/−200 region of the promoter. Gel mobility shift assays suggested that a nuclear factor(s) prepared from wounded tobacco stems binds a 14-bp sequence (−229/−215) in the −239/−200 region in a sequence-specific manner. A mutation in this 14-bp region of the −239 promoter fragment resulted in a considerable decrease in wound-responsive GUS activity in transgenic plants. An 11-bp sequence, which completely overlaps with the 14-bp sequence, was found in the 5′ distal region (−420/−410) and is thought to contribute to the wound-induced expression together with the 14-bp. The −114-bp core promoter of the tpoxN1 gene was indispensable for wound-induced expression, indicating that the 14-bp region is a novel wound-responsive cis-element VWRE, which may work cooperatively with other factors in the promoter. 相似文献
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Functional characterization of a cotton late embryogenesis-abundant D113 gene promoter in transgenic tobacco 总被引:4,自引:0,他引:4
Previous studies have shown that mRNA and protein encoded by late embryogenesis-abundant (LEA) gene D113 from Gossypium
hirsutum L. accumulate at high levels in mature seeds and also in response to abscisic acid (ABA) in young embryo. In this study,
we studied the expression of four promoter 5′ deletion constructs (−1383, −974, −578 and −158) of the LEA D113 gene fused to beta-glucuronidase (GUS). GUS activity analysis revealed that the −578 promoter fragment was necessary to direct
seed-specific GUS expression in transgenic tobacco plants (Nicotiana tabacum L.). To further investigate the expression pattern of LEA D113 promoter under environmental stresses, 2-week-old transgenic tobacco seedlings were exposed to ABA, dehydration, high salinity
and cold treatments. GUS activity in the seedlings was quantified fluorimetrically, and expression was also observed by histochemical
staining. An apparent increase in GUS activity was found in plants harboring constructs −1383, −974 and −578 after 24 h of
ABA or high-salinity treatments, as well as after 10 days of dehydration. By contrast, only a slight increase was observed
in all the three lines after cold treatment. Virtually no change in expression was found in construct −158 in response to
dehydration, salinity and cold, but there was a moderate response to ABA, suggesting that the region between −574 and −158
was necessary for dehydration- and salinity-dependent expression, whereas ABA-responsive cis-acting elements might be located in the −158 region of the promoter. 相似文献
5.
Vahid Omidvar Siti Nor Akmar Abdullah Amir Izadfard Chai Ling Ho Maziah Mahmood 《Planta》2010,232(4):925-936
The 1,053-bp promoter of the oil palm metallothionein gene (so-called MSP1) and its 5′ deletions were fused to the GUS reporter
gene, and analysed in transiently transformed oil palm tissues. The full length promoter showed sevenfold higher activity
in the mesocarp than in leaves and 1.5-fold more activity than the CaMV35S promoter in the mesocarp. The 1,053-bp region containing
the 5′ untranslated region (UTR) gave the highest activity in the mesocarp, while the 148-bp region was required for minimal
promoter activity. Two positive regulatory regions were identified at nucleotides (nt) −953 to −619 and −420 to −256 regions.
Fine-tune deletion of the −619 to −420 nt region led to the identification of a 21-bp negative regulatory sequence in the
−598 to −577 nt region, which is involved in mesocarp-specific expression. Gel mobility shift assay revealed a strong interaction
of the leaf nuclear extract with the 21-bp region. An AGTTAGG core-sequence within this region was identified as a novel negative
regulatory element controlling fruit-specificity of the MSP1 promoter. Abscisic acid (ABA) and copper (Cu2+) induced the activity of the promoter and its 5′ deletions more effectively than methyl jasmonate (MeJa) and ethylene. In
the mesocarp, the full length promoter showed stronger inducibility in response to ABA and Cu2+ than its 5′ deletions, while in leaves, the −420 nt fragment was the most inducible by ABA and Cu2+. These results suggest that the MSP1 promoter and its regulatory regions are potentially useful for engineering fruit-specific
and inducible gene expression in oil palm. 相似文献
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The promoter of Brassica campestris Male Fertile 5 (BcMF5), a pollen coat protein member, class A (PCP-A) gene family, was isolated from Brassica rapa L. ssp. chinensis Makino (Chinese cabbage-pak-choi) by Thermal Asymmetric Interlaced Polymerase Chain Reaction (TAIL-PCR). Sequence analysis
suggested that the 605-bp promoter of BcMF5 appears to be a pollen promoter. In an attempt to confirm the promoter activity of BcMF5 promoter, −609 to +3 bp and −377 to +3 bp fragments of the upstream sequence of BcMF5 were inserted at the site upstream of the coding region of the uidA gene in the sense orientation to construct two deletion expression vectors. Transient expression analysis in onion epidermal
cells by particle bombardment showed that both −609 to +3 bp and −377 to +3 bp fragments of BcMF5 promoter were capable of driving β-glucuronidase gene expression. Furthermore, by Agrobacterium-mediated genetic transformation
method, Arabidopsis transgenic KanR plants were obtained. GUS assay analysis revealed that the promoter of BcMF5 induced gene expression at the early stage of anther development and drove high levels of GUS expression in anther walls,
upper regions of petals, pollen, and pollen tubes in the middle and late stage of anther development, but did not drive any
expression in sepals and pistils. 相似文献
9.
Li Ang Chen LiangLiang Ren HaiYun Wang XueChen Zhang HaiWen Huang Rong-Feng 《中国科学:生命科学英文版》2008,51(3):280-285
In rice, the characterization of OsEBP-89 is inducible by various stress-or hormone-stimuli, including ethylene, abscisic acid (ABA), jasmonate acid (JA), drought
and cold. Here, we report the investigation of essential DNA region within OsEBP-89 promoter for methyl jasmonic acid (MeJA) induction. PLACE analysis indicates that this promoter sequence contains multiple
potential elements in response to various stimuli. First, we fused this promoter with GUS gene and analyzed its expression
under MeJA treatment through Agrobacterium infiltration mediating transient expression in tobacco leaves. Our results revealed that this chimeric gene could be inducible
by MeJA in tobacco leaves. To further determine the crucial sequences responsible for MeJA induction, we generated a series
of deletion promoters which were fused with GUS reporter gene respectively. The results of transient expression of GUS gene
driven by these mutant promoters show that the essential region for MeJA induction is positioned in the region between −1200
and −800 in OsEBP-89 promoter containing a G-box (−1127), which is distinct from the essential region containing ERE (−562) for ACC induction.
In all, our finding is helpful in understanding the molecular mechanism of OsEBP-89 expression under different stimuli. OsEBP-89, essential DNA region, methyl jasmonic acid, transient assay, promoter, tobacco leaves
Contributed equally to this work
Supported by the National Basic Research Program of China (Grant No. 2006CB101700) and the National Natural Science Foundation
of China (Grant Nos. 30671135, 30525034 and 30730060) 相似文献
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In our previous study, we identified a Rosa chinensis heat shock protein (HSP) gene, RcHSP17.8, which was induced by abiotic stresses, such as high temperature and osmotic stress. To analyze the expression of RcHSP17.8 and the function of cis-acting elements in the promoter region, a 1,910 bp fragment of the upstream sequence of the RcHSP17.8 translation initiation codon and five promoter deletion fragments were fused to a β-glucuronidase (GUS) report gene. These
plasmids were transferred to Arabidopsis thaliana via Agrobacterium. GUS staining was seen in all the organs, especially in the vascular tissues after heat treatment. In transgenic Arabidopsis, GUS expression driven by the full length promoter was significantly higher under heat shock, but no GUS activity was detected
under other abiotic stresses. Deletion analysis indicated that the region from −178 to −771 was essential for the promoter’s
response to high temperature. 相似文献
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SBgLR (Solanum tuberosum genomic lysine-rich) gene was isolated from a potato genomic library using SB401 (S.
berthaultii 401) cDNA as probe. RT-PCR analysis of SBgLR gene expression profile and microscopic analysis of green fluorescent protein (GFP) expression in tobacco plants transformed
with SBgLR promoter-GFP reporters indicate that SBgLR is a pollen-specific gene. A series of 5′deletions of SBgLR promoter were fused to the β-glucuronidase (GUS) gene and stably introduced into tobacco plants. Histochemical and quantitative assays of GUS expression in transgenic
plants allowed us to localize an enhancer of SBgLR promoter to the region −345 to −269 relative to the translation start site. This 76 bp (−345 to −269) fragment enhanced GUS
expression in leaves, stems and roots when fused to −90/+6 CaMV 35S minimal promoter. Deletion analysis showed that a cis-element, which can repress gene expression in root hairs, was located in the region −345 to −311. Further study indicated
that the −269 to −9 region was sufficient to confer pollen-specific expression of GFP when fused to CaMV 35S enhancer.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
Authors Zhihong Lang and Peng Zhou contributed equally to this work. 相似文献
14.
Chen X Wang Z Gu R Fu J Wang J Zhang Y Wang M Zhang J Jia J Wang G 《Plant cell reports》2007,26(9):1555-1565
By screening a genomic library of maize, a 2.2 kb 5′ flanking fragment of Zpu1 gene, encoding the pullulanase-type starch debranching enzyme, was isolated. Promoter fragments of various lengths, including
the full 5′ flanking sequence (−2267 to −1) (Z1), a 3′ deletion (−2267 to −513) (Z5) and three 5′ deletions extending to −1943
(Z2), −1143 (Z3) and −516 (Z4) upstream of the translational initiation codon (ATG), were fused to the GUS reporter gene and
introduced into tobacco. When these constructs were tested in transgenic tobacco plants, seed-preferred GUS activity was observed
in pZ1-transgenic lines. In pZ2-transgenic lines, the GUS activity was not only restricted to seeds, but was also detected
in calyxes, petals, stamens and mature leaves. At the same time, negligible GUS activity was detected in roots, stems, young
leaves, stigmas and ovaries from the transgenic tobacco plants, which had integrated the full isolated sequence of Zpu1 promoter or its deletions. Deletion analysis indicated that the promoter contained a putative positive cis-regulatory element and the proximal region (−516 to −1) was essential for directing the expression of gus reporter gene. Analysis of GUS activity during the fruit development and seed germination suggested that Zpu1 promoter is active both in starch anabolism and in starch catabolism, which is consistent with the function of the endogenous
gene in maize. GUS activity in leaves under light and darkness confirmed that Zpu1 promoter functions in the starch degradation of photosynthetic tissues in the dark phase of the diurnal cycle. 相似文献
15.
We previously cloned and analyzed the 1,893-bp promoter region (−1,915 to −23) of the tomato (Lycopersicon esculentum) Lehsp23.8 gene, whose expression is induced by treatment with high or low temperatures, heavy metal, or abscisic acid (ABA). In our
present work, we examined how this expression is regulated. A comprehensive quantitative promoter deletion and base-substitution
analysis was conducted under various environmental conditions. The proximal region (−565 to −23 bp) of the Lehsp23.8 promoter harbors cis-regulatory elements that conferred high levels of heat-induced expression in transgenic tobacco. Mutation of the five proximal
HSEs (HSE1 to 5) of that promoter led to an absence of heat inducibility. The AT-rich regions between −255 bp and −565 bp
(AT-rich1 to 4) in the promoter might serve as enhancers for such heat-induced expression. Deletion and HSE mutation analysis
indicated that other cis-acting elements also function in response to low temperature, heavy metal, and ABA and that HSE1 to 5 act at least as cis-acting elements in multiple-stress responses of Lehsp23.8. These results reveal that those five proximal HSEs and AT-rich regions function interdependently in the expression of Lehsp23.8 in response to non-heat stresses. Furthermore, the putative elements CRT/DRE, AP-1, and ABRE in that promoter are not required
for multiple-stress induction. 相似文献
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In a study of the 5′-flanking sequence of the Zea mays L. (maize) Glb1 gene in vitro, serial promoter deletions were generated and linked with the β-glucuronidase (GUS) reporter gene. The promoter deletion-GUS fusions were introduced into the maize P3377 cell line by particle
bombardment. GUS assays indicated that treatment of the maize cultured cells with abscisic acid (ABA) was required for Glb1-driven GUS transient expression, and that the –272-bp sequence of the Glb1 promoter was sufficient for ABA-regulated expression of GUS. The longest undeleted sequence used, –1391 GUS, showed relatively
low expression which could be indicative of an upstream silencer element in the Glb1 promoter between –1391 and –805. Further studies show that the Glb1-driven GUS activity of bombarded maize P3377 cells increases with increasing ABA concentration (up to 100–300 μm). Site-directed mutagenesis of a putative ABA response element, Em1a, abolished GUS expression in P3377 cells. This observation
indicated that the Em1a sequence in the Glb1 5′ regulatory region is responsible for the positive ABA regulation of gene expression.
Received: 9 May 1997 / Revision received: 9 November 1997 / Accepted: 8 December 1997 相似文献
18.
Earlier, a pollen-specific Oryza sativa indica pollen allergen gene (OSIPA), coding for expansins/pollen allergens, was isolated from rice, and its promoter—upon expression in tobacco and Arabidopsis—was found active during the late stages of pollen development. In this investigation, to analyze the effects of different
putative regulatory motifs of OSIPA promoter, a series of 5′ deletions were fused to β-glucuronidase gene (GUS) which were stably introduced into rice and Arabidopsis. Histochemical GUS analysis of the transgenic plants revealed that a 1631 bp promoter fragment mediates maximum GUS expression
at different stages of anther/pollen development. Promoter deletions to −1272, −966, −617, and −199 bp did not change the
expression profile of the pollen specificity. However, the activity of promoter was reduced as the length of promoter decreased.
The region between −1567 and −199 bp was found adequate to confer pollen-specific expression in both rice and Arabidopsis systems. An approximate 4-fold increase in the GUS activity was observed in the pollen of rice when compared to that of Arabidopsis. As such, the OSIPA promoter seems promising for generation of stable male-sterile lines required for the production of hybrids in rice and other
crop plants. 相似文献
19.
The first enzyme in the flavonoid pathway, chalcone synthase, is encoded by a gene (CHS) whose expression is normally under developmental control. In our previous studies, an 896-bp promoter region of a flower-specific
CHS gene was isolated from Lilium orential ‘Sorbonne’, and designated as PLoCHS. Here, the PLoCHS promoter was fused to the β-glucuronidase (GUS) gene to characterize its spatial and temporal expression in Petunia
hybrida ‘Dreams Midnight’ using an Agrobacterium-mediated leaf disc transformation method. Our results demonstrated that GUS expression was present in flowers, but reduced or absent in the other tissues (leaf and stem) examined. In petals, GUS activity reached its peak at flower developmental stage 4, and decreased at later stages. Deletion analysis indicated that
even a 307-bp fragment of the PLoCHS promoter could still direct flower-specific expression. Further deletion of the region from −261 to −72 bp resulted in weak
expression in different organs, including flowers, leaves and stems. This evidence combined with prediction of cis-acting elements in the PLoCHS promoter suggests that the TACPyAT box located in this promoter plays a key role in the regulation of organ-specific expression. 相似文献
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In this study, 900-bp (signed as p including nucleotides –1 to –886) and partly deleted (signed as dp including nucleotides
–1 to –414) COMT (caffeate/5-hydroxyferulate O-methyltransferase) promoters from Populus tremuloides Michx. were fused to the GUS reporter gene, and the tissue-specific expression patterns of the promoters were determined in Betula pendula Roth along the growing season, and as a response to mechanical bending and wounding. The main activity of the PtCOMTp- and
PtCOMTdp-promoters, determined by the histochemical GUS assay, was found in the developing xylem of stems during the 8th–13th
week and in the developing xylem of roots in the 13th week of the growing season. The GUS expression patterns did not differ
among the xylem cell types. The PtCOMT promoter-induced GUS expression observed in phloem fibres suggests a need for PtCOMT
expression and thus syringyl (S) lignin synthesis in fibre lignification. However, the PtCOMTdp-promoter induced GUS expression
in stem trichomes, which may contribute to the biosynthesis of phenylpropanoid pathway-derived compounds other than lignin.
Finally, a strong GUS expression was induced by the PtCOMT promoters in response to mechanical stem bending but not to wounding.
The lack of major differences between the PtCOMTp- and PtCOMTdp-promoters suggests that the deleted promoter sequence (including
nucleotides −415 to −886) did not contain a significant regulatory element contributing to the GUS expression in young B. pendula trees. 相似文献