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1.
Expression Enhancement of a Rice Polyubiquitin Gene Promoter 总被引:11,自引:0,他引:11
An 808 bp promoter from a rice polyubiquitin gene, rubi3, has been isolated. The rubi3 gene contained an open reading frame of 1140 bp encoding a pentameric polyubiquitin arranged as five tandem, head-to-tail
repeats of 76 aa. The 1140 bp 5′ UTR intron of the gene enhanced its promoter activity in transient expression assays by 20-fold.
Translational fusion of the GUS reporter gene to the coding sequence of the ubiquitin monomer enhanced GUS enzyme activity in transient expression assays
by 4.3-fold over the construct containing the original rubi3 promoter (including the 5′ UTR intron) construct. The enhancing effect residing in the ubiquitin monomer coding sequence
has been narrowed down to the first 9 nt coding for the first three amino acid residues of the ubiquitin protein. Mutagenesis
at the third nucleotide of this 9 nt sequence still maintains the enhancing effect, but leads to translation of the native
GUS protein rather than a fusion protein. The resultant 5′ regulatory sequence, consisting of the rubi3 promoter, 5′ UTR exon and intron, and the mutated first 9 nt coding sequence, has an activity nearly 90-fold greater than
the rubi3 promoter only (without the 5′ UTR intron), and 2.2-fold greater than the maize Ubi1 gene promoter (including its 5′ UTR intron). The newly created expression vector is expected to enhance transgene expression
in monocot plants. Considering the high conservation of the polyubiquitin gene structure in higher plants, the observed enhancement
in gene expression may apply to 5′ regulatory sequences of other plant polyubiquitin genes. 相似文献
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Philippe Vain Kim R. Finer Dean E. Engler Richard C. Pratt John J. Finer 《Plant cell reports》1996,15(7):489-494
We report a strength comparison of a large variety of monocot and dicot intron-containing fragments inserted in the 5 untranslated leader, between the CaMV 35S promoter and the uidA gene (coding for the ß-glucuronidase: GUS). Relative strengths of the intron-containing fragments were evaluated by comparing transient GUS expression after particle bombardment in embryogenic maize and bluegrass suspension cultures. Our results confirm a dramatic dependence on the presence of an intron for chimeric gene expression in both species. On average, the maize first intron of ubi1 provided the highest enhancement of gene expression in maize and bluegrass (71- and 26-fold enhancement, respectively). Half of the introns tested affected gene expression differently in bluegrass and maize. This suggests that the intron-mediated enhancement of gene expression generally obtained with maize may not be fully applicable to all monocots. We also report enhancement of gene expression (92-fold) in a monocot species by a dicot intron (chsA intron). 相似文献
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Intron-containing fragments derived from the 5′ untranslated regions of the maize ubi1, maize adh1, rice act1 and sugarcane rbcS genes were tested for their enhancing effects on the banana bunchy top virus DNA-6 promoter (BT6.1) in banana (Musa spp. cv. Bluggoe) embryogenic cells. The rice act1 and maize ubi1 introns provided the highest levels of intron-mediated enhancement of GUS expression, increasing native BT6.1 promoter activity
by about 300-fold and 100-fold, respectively. The sugarcane rbcS intron increased expression about tenfold, whereas the adh1 intron had no significant effect. In regenerated transgenic banana plants, the ubi1 intron significantly enhanced BT6.1 promoter activity to levels similar to that of the CaMV 35 S promoter and did not appear
to affect the tissue specificity of the promoter.
Received: 28 July 2000 / Revision received: 21 August 2000 / Accepted: 4 October 2000 相似文献
6.
We describe a 5′ untranslated region (5′UTR) that dramatically increases the expression level of an exogenous gene in Aspergillus oryzae. Using a series of 5′UTR::GUS (uidA) fusion constructs, we analyzed the translation efficiency of chimeric mRNAs with different 5′UTRs at different temperatures. We found that the 5′UTR of a heat-shock protein gene, Hsp12, greatly enhanced the translation efficiency of the chimeric GUS mRNA at normal temperature (30°C). Moreover, at high temperature (37°C), the translation efficiency of the mRNA containing the Hsp12 5′UTR was far superior to that of mRNAs containing nonheat-shock 5′UTRs, resulting in much more efficient expression of GUS protein (about 20-fold higher GUS activity compared to the control construct). This 5′UTR can be used in combination with various strong promoters to enhance the expression of foreign proteins in A. oryzae. 相似文献
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Development and characterization of a chimaeric tissue-specific promoter in wheat and rice endosperm
Maria Oszvald Mark Gardonyi Cecília Tamas Imre Takacs Barnabas Jenes Laszlo Tamas 《In vitro cellular & developmental biology. Plant》2008,44(1):1-7
The recently achieved significant improvement of cereal transformation protocols provides facilities to alter the protein
composition of the endosperm, for example, to increase or decrease the quantity of one of its protein components or to express
foreign molecules. To achieve this goal, strong endosperm-specific promoters have to be available. The aim of our work was
to develop a more efficient tissue-specific promoter which is currently used. A chimaeric promoter was assembled using the
5′ UTR (1,900 bp) of the gene coding for the 1Bx17 HMW glutenin subunit protein, responsible for tissue-specific expression
and the first intron of the rice actin gene (act1). The sequence around of the translation initial codon was optimized. The effect of the intron and promoter regulatory sequences,
using different lengths of 1Bx17 HMW-GS promoter, were studied on the expression of uidA gene. The function of promoter elements, promoter length, and the first intron of the rice actin gene were tested by a transient expression assay in immature wheat endosperm and in stable transgenic rice plants. Results
showed that insertion of the rice act1 first intron increased GUS expression by four times in transient assay. The shortest 1Bx17 HMW-GS promoter fragment (173 bp) linked to the intron and GUS reporter gene provided almost the same expression level than
the intronless long 1Bx17 HMW-GS promoter. Analysis of the stable transformant plants revealed that 173 nucleotides were sufficient for endosperm-specific
expression of the uidA gene, despite 13 nucleotides missing from the HMW enhancer sequence, a relevant regulatory element in the promoter region. 相似文献
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Embryonal-suspensor tissue (EST) of Mediterranean cypress (Cupressus sempervirens L.) was tested for microprojectile-DNA delivery (by the PDS-1000/He device) for different subculture periods (9, 15, and
21 days) using the plasmid vectors pRT99GUS [containing the β-glucuronidase (GUS) and neomycin phosphotransferase (NPT II) genes, and the CaMV 35S promoter], pBI426 (with a GUS::NPT II
fusion gene under the control of a duplicated 35S RNA promoter), and pCGUδ0 (containing the GUS gene with the ubiquitin intron, under the control of the sunflower ubiquitin promoter). The relative
strengths of the promoters as determined by GUS assays were sunflower ubiquitin>35S-35S-AMVE>35S. The highest expression level
was observed when 15-day-subcultured EST was bombarded with the pCGUδ0 gene construct, which also showed high activity of the chloramphenicol acetyltransferase and NPT II genes. Green fluorescent
areas were observed on EST when bombarded with the p35S-GFP plasmid, carrying the gene for the green fluorescent protein from
the bioluminescent jellyfish Aequorea victoria.
Received: 18 November 1996/ Revision received: 19 February 1997/ Accepted: 20 November 1997 相似文献
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K. A. Torbert M. Gopalraj S. L. Medberry N. E. Olszewski D. A. Somers 《Plant cell reports》1998,17(4):284-287
The Commelina yellow mottle virus (CoYMV) infects the monocot weed Commelina diffusa. The objective of this study was to investigate the transgene expression conferred by the CoYMV promoter in a monocot species.
Friable, embryogenic oat (Avena sativa L.) tissue cultures were stably transformed with the CoYMV promoter fused to the coding region of E. coli β-glucuronidase (uidA, GUS). Developmental and tissue-specific expression of the CoYMV-GUS construct was investigated in regenerated plants and
their progeny. Histochemical GUS staining was primarily localized in the vascular tissues of shoots, leaves, floral bracts
and in roots. While ovaries stained intensely, no staining was detected in anthers or the endosperm in mature seed. The scutellum
of mature and germinating seed exhibited GUS activity.
Received: 16 April 1997 / Revision received: 23 July 1997 / Accepted: 2 August 1997 相似文献
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Wen Xian Liu Hua Liang Liu Zhi Jian Chai Xiu Ping Xu Yan Ru Song Le Qing Qu 《TAG. Theoretical and applied genetics. Theoretische und angewandte Genetik》2010,121(7):1267-1274
5′ untranslated regions (UTRs) are important sequence elements that modulate the expression of genes. Using the β-glucuronidase
(GUS) reporter gene driven by the GluC promoter for the rice-seed storage-protein glutelin, we evaluated the potential of the 5′-UTRs of six seed storage-protein
genes in enhancing the expression levels of the foreign gene in stable transgenic rice lines. All of the 5′-UTRs significantly
enhanced the expression level of the GluC promoter without altering its expression pattern. The 5′-UTRs of Glb-1 and GluA-1 increased the expression of GUS by about 3.36- and 3.11-fold, respectively. The two 5′-UTRs downstream of the Glb-1, OsAct2 and CMV35S promoters also increased GUS expression level in stable transgenic rice lines or in transient expression protoplasts. Therefore,
the enhancements were independent of the promoter sequence. Real-time quantitative RT-PCR analysis showed that the increase
in protein production was not accompanied by alteration in mRNA levels, which suggests that the enhancements were due to increasing
the translational efficiencies of the mRNA. The 5′-UTRs of Glb-1 and GluA-1, when combined with strong promoters, might be ideal candidates for high production of recombinant proteins in rice seeds. 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
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We compared the effect of the rice storage protein glutelin B-1 (GluB-1) terminator with the nopaline synthase (Nos) terminator on the accumulation of the modified house dust mite allergen mDer f 2 driven by the maize ubiquitin promoter
in transgenic rice. Accumulation of mDer f 2 in transgenic seed and leaf using the GluB-1 terminator was greater than when using the Nos terminator construct. The mDer f 2 mRNA containing the GluB-1 3′UTR was processed and polyadenylated at the same sites as the native GluB-1 mRNA in the seeds but diverged in leaves of the transgenic plants. In contrast, the poly(A) sites of mDer f 2 containing Nos 3′UTR were more divergent in both seed and leaf. These results suggest that GluB-1 3′UTR functions as a faithful terminator and that termination at the specific sites may play an important role in mRNA stability
and/or translatability, resulting in higher levels of protein accumulation. 相似文献
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The genomic upstream sequence of the rice tubulin gene OsTub6 has been cloned, sequenced and characterized. The 5′UTR sequence is interrupted by a 446 bp long leader intron. This feature
is shared with two other rice β-tubulin genes (OsTub4 and OsTub1) that, together with OsTub6, group in the same clade in the evolutionary phylogenetic tree of plant β-tubulins. Similarly to OsTub4, the leader intron of OsTub6 is capable of sustaining intron mediated enhancement (IME) of gene expression, in transient expression assays. A general
picture is drawn for three rice α-tubulin and two rice β-tubulin genes in which the first intron of the coding sequence for
the formers and the intron present in the 5′UTR for the latters, are important elements for controlling gene expression. We
used OsTua2:GUS, OsTua3:GUS, OsTub4:GUS and OsTub6:GUS chimeric constructs to investigate the in vivo pattern of beta-glucuronidase (GUS) expression in transgenic rice plants.
The influence of the regulatory introns on expression patterns was evaluated for two of them, OsTua2 and OsTub4. We have thus characterized distinct patterns of expression attributable to each tubulin isotype and we have shown that
the presence of the regulatory intron can greatly influence both the amount and the actual site of expression. We propose
the term Intron Dependent Spatial Expression (IDSE) to highlight this latter effect.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. 相似文献
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Activity of a maize ubiquitin promoter in transgenic rice 总被引:27,自引:0,他引:27
Maria-Jesús Cornejo Diane Luth Kathleen M. Blankenship Olin D. Anderson Ann E. Blechl 《Plant molecular biology》1993,23(3):567-581
We have used the maize ubiquitin 1 promoter, first exon and first intron (UBI) for rice (Oryza sativa L. cv. Taipei 309) transformation experiments and studied its expression in transgenic calli and plants. UBI directed significantly higher levels of transient gene expression than other promoter/intron combinations used for rice transformation. We exploited these high levels of expression to identify stable transformants obtained from callus-derived protoplasts co-transfected with two chimeric genes. The genes consisted of UBI fused to the coding regions of the uidA and bar marker genes (UBI:GUS and UBI:BAR). UBI:GUS expression increased in response to thermal stress in both transfected protoplasts and transgenic rice calli. Histochemical localization of GUS activity revealed that UBI was most active in rapidly dividing cells. This promoter is expressed in many, but not all, rice tissues and undergoes important changes in activity during the development of transgenic rice plants. 相似文献
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Effects of the polyubiquitin gene Ubi.U4 leader intron and first ubiquitin monomer on reporter gene expression in Nicotiana tabacum 总被引:5,自引:0,他引:5
Plesse Bertrand Criqui Marie-Claire Durr Andrée Parmentier Yves Fleck Jacqueline Genschik Pascal 《Plant molecular biology》2001,45(6):655-667
We have previously shown by RNA gel blot analyses that the tobacco polyubiquitin-encoding gene Ubi.U4 is expressed in a complex pattern during plant development (Genschik et al., 1994). In order to study its tissue-specific expression, we cloned the fragment containing the –263 bp proximal promoter of the gene, the leader intron and the first ubiquitin monomer in front of the reporter GUS gene. Histochemical analyses for GUS activity during tobacco plant development revealed that the gene is expressed at variable amounts in many plant tissues with high levels in metabolically active and/or dividing cells and in the vascular tissues of the plant. We also analysed the expression pattern of constructs in which either the intron or the intron together with the first ubiquitin monomer were deleted. Our results indicate that the ubiquitin leader intron is not only a quantitative determinant of gene expression but may also influence the tissue-specific expression pattern. 相似文献
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The Perilla (Perilla frutescens L. cv. Okdong) oleosin gene, PfOle19, produces a 19-kDa protein that is highly expressed only in seeds. The activity of the −2,015 bp 5′-upstream promoter region
of this gene was investigated in transgenic Arabidopsis plants using the fusion reporter constructs of enhanced green fluorescent protein (EGFP) and β-glucuronidase (GUS). The PfOle19 promoter directs Egfp expression in developing siliques, but not in leaves, stems or roots. In the transgenic Arabidopsis, EGFP fluorescence and histochemical GUS staining were restricted to early seedlings, indehiscent siliques and mature seeds.
Progressive 5′-deletions up to the −963 bp position of the PfOle19 promoter increases the spatial control of the gene expression in seeds, but reduces its quantitative levels of expression.
Moreover, the activity of the PfOle19 promoter in mature seeds is 4- and 5-fold greater than that of the cauliflower mosaic virus 35S promoter in terms of both
EGFP intensity and fluorometric GUS activity, respectively. 相似文献