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1.
A synthetic xylanase as a novel reporter in plants 总被引:2,自引:0,他引:2
Transient gene expression assays are often used to screen promoters before stable transformation. Current transient quantification methods have several problems, including a lack of reporter gene stability and expense. Here we report a synthetic, codon-optimised xylanase gene (sXynA) as a reporter gene for quantitative transient analyses in plants. Azurine-crosslinked xylan (AZCL-xylan) was used as a substrate for assaying xylanase activity. The enzymatic nature of the protein allows for sensitive assays at the low levels of transgene protein found in transiently transformed tissue extracts. The xylanase (XYN) protein is stable, activity slopes are linear over long time periods and assays are cost-effective. Coupled with the GUSPlus reporter gene, the XYN reporter allows sensitive and accurate quantification of gene control sequences in transient expression systems.Abbreviations Act1
Rice actin promoter
- AZCL-xylan
Azurine cross-linked xylan
- AU
absorbance units
- Blt4.9
Barley lipid transfer protein promoter
- GEB
GUS extraction buffer
- GFP
Green fluorescent protein
- GluB-1
Rice glutelin B-1 promoter
- GUS
-Glucuronidase
- LUC
Luciferase
- sXynA
Synthetic xylanase A gene
- Ubi-1
Maize ubiquitin promoter
- XAB
Xylanase assay buffer
- XYN
Xylanase
Communicated by P. Lakshmanan 相似文献
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Bertazzon N Raiola A Castiglioni C Gardiman M Angelini E Borgo M Ferrari S 《Plant cell reports》2012,31(1):133-143
Grapevine is an economically important crop, and the recent completion of its genome makes it possible to study the function
of specific genes through reverse genetics. However, the analysis of gene function by RNA interference (RNAi) in grapevine
is difficult, because the generation of stable transgenic plants has low efficiency and is time consuming. Recently, transient
expression of genes in grapevine leaves has been obtained by Agrobacterium tumefaciens infiltration (agroinfiltration). We therefore tested the possibility to silence grapevine genes by agroinfiltration of RNAi
constructs. A construct to express a double strand RNA (dsRNA) corresponding to the defense-related gene VvPGIP1, encoding a polygalacturonase-inhibiting protein (PGIP), was obtained and transiently expressed by agroinfiltration in leaves
of grapevine plants grown in vitro. Expression of VvPGIP1 and accumulation of PGIP activity were strongly induced by infiltration with control bacteria, but not with bacteria carrying
the dsRNA construct, indicating that the gene was efficiently silenced. In contrast, expression of another defense-related
gene, VST1, encoding a stilbene synthase, was unaffected by the dsRNA construct. We have therefore demonstrated the possibility of transient
down-regulation of grapevine genes by agroinfiltration of constructs for the expression of dsRNA. This system can be employed
to evaluate the effectiveness of constructs that can be subsequently used to generate stable RNAi transgenic plants. 相似文献
4.
Identification of a potent MAR element from the human genome and assessment of its activity in stably transfected CHO cells
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Tian‐Yun Wang Xiao‐Yin Wang Hong‐Yan Xu Chun‐Peng Zhao Guang‐Hua Xu 《Journal of cellular and molecular medicine》2018,22(2):1095-1102
Low‐level and unstable transgene expression are common issues using the CHO cell expression system. Matrix attachment regions (MARs) enhance transgene expression levels, but additional research is needed to improve their function and to determine their mechanism of action. MAR‐6 from CHO chromosomes actively mediates high and consistent gene expression. In this study, we compared the effects of two new MARs and MAR‐6 on transgene expression in recombinant CHO cells and found one potent MAR element that can significantly increase transgene expression. Two MARs, including the human CSP‐B MAR element and DHFR intron MAR element from CHO cells, were cloned and inserted downstream of the poly(A) site in a eukaryotic vector. The constructs were transfected into CHO cells, and the expression levels and stability of eGFP were detected by flow cytometry. The three MAR sequences can be ranked in terms of overall eGFP expression, in decreasing order, as follows: human CSP‐B, DHFR intron MAR element and MAR‐6. Additionally, as expected, the three MAR‐containing vectors showed higher transfection efficiencies and transient transgene expression in comparison with those of the non‐MAR‐containing vector. Bioinformatics analysis indicated that the NFAT and VIBP elements within MAR sequences may contribute to the enhancement of eGFP expression. In conclusion, the human CSP‐B MAR element can improve transgene expression and its effects may be related to the NFAT and VIBP elements. 相似文献
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Raffaele Lombardi Patrizia Circelli Maria Elena Villani Giampaolo Buriani Luca Nardi Valentina Coppola Linda Bianco Eugenio Benvenuto Marcello Donini Carla Marusic 《BMC biotechnology》2009,9(1):96
Background
In recent years, different HIV antigens have been successfully expressed in plants by either stable transformation or transient expression systems. Among HIV proteins, Nef is considered a promising target for the formulation of a multi-component vaccine due to its implication in the first steps of viral infection. Attempts to express Nef as a single protein product (not fused to a stabilizing protein) in transgenic plants resulted in disappointingly low yields (about 0.5% of total soluble protein). In this work we describe a transient expression system based on co-agroinfiltration of plant virus gene silencing suppressor proteins in Nicotiana benthamiana, followed by a two-step affinity purification protocol of plant-derived Nef. 相似文献6.
Wheat line CSP44, a selection from an Australian bread wheat cultivar Condor, has shown resistance to stripe rust in India
since the last twenty years. Seedlings and adult plants of CSP44 showed susceptible infection types against stripe rust race
46S119 but displayed average terminal disease severity of 2.67 on adult plants against this race as compared to 70.33 of susceptible
Indian cultivar, WL711. This suggests the presence of nonhypersensitive adult plant stripe rust resistance in the line CSP44.
The evaluation of F1, F2 and F3 generations and F6 SSD families from the cross of CSP44 with susceptible wheat cultivar WL711 for stripe rust severity indicated that the resistance
in CSP44 is based on two genes showing additive effect. One of these two genes isYr18 and the second gene is not yet described. 相似文献
7.
Nunzia Scotti Fiammetta Alagna Enrico Ferraiolo Gelsomina Formisano Lorenza Sannino Luigi Buonaguro Angelo De Stradis Alessandro Vitale Luigi Monti Stefania Grillo Franco M. Buonaguro Teodoro Cardi 《Planta》2009,229(5):1109-1122
Plants have been recognized as a promising production platform for recombinant pharmaceutical proteins. The human immunodeficiency
virus Gag (Pr55gag) structural polyprotein precursor is a prime candidate for developing a HIV-1 vaccine, but, so far, has been expressed at
very low level in plants. The aim of this study was to investigate factors potentially involved in Pr55gag expression and increase protein yield in plant cells. In transient expression experiments in various subcellular compartments,
the native Pr55gag sequence could be expressed only in the chloroplast. Experiments with truncated subunits suggested a negative role of the
5′-end on the expression of the full gene in the cytosol. Stable transgenic plants were produced in tobacco by Agrobacterium-mediated nuclear transformation with protein targeted to plastids, and biolistic-mediated plastid transformation. Compared
to the nuclear genome, the integration and expression of the gag transgene in the plastome resulted in significantly higher protein accumulation levels (up to 7–8% TSP, equivalent to 312–363 mg/kg
FW). In transplastomic plants, a 25-fold higher protein accumulation was obtained by translationally fusing the Pr55gag polyprotein to the N-terminus of the plastid photosynthetic RbcL protein. In chloroplasts, the Pr55gag polyprotein was processed in a pattern similar to that achieved by the viral protease, the processing being more extended
in older leaves of mature plants. The Gag proteins produced in transgenic plastids were able to assemble into particles resembling
VLPs produced in baculovirus/insect cells and E. coli systems. These results indicate that plastid transformation is a promising tool for HIV antigen manufacturing in plant cells.
Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.
IGV publication no. 330 相似文献
8.
Jean-Jacques M. Van Oosten Alain Gerbaud Casper Huijser Paul P. Dijkwel Nam-Hai Chua Sjef C.M. Smeekens 《The Plant journal : for cell and molecular biology》1997,12(5):1011-1020
Many plant genes are responsive to sugars but the mechanisms used by plants to sense sugars are unknown. A genetic approach has been used in Arabidopsis to identify genes involved in perception and transduction of sugar signals. For this purpose, an in vivo reporter system was established consisting of the light- and sugar-regulated plastocyanin promoter, fused to the luciferase coding sequence (PC—LUC construct). At the seedling stage, expression of the PC—LUC gene is repressed by sucrose, and a number of sucrose-uncoupled (sun) mutants were selected in which sucrose is unable to repress the activity of the PC promoter. Three mutants have been characterized in more detail. The sugar analog 2-deoxy-d -glucose (2DG) was used to repress whole plant photosynthesis, PC—LUC gene expression and total ribulose-1,5-bisphosphate activity. It was found that the sun6 mutation makes plants unresponsive to these 2DG-induced effects. Moreover, unlike wild-type plants, sun6 mutants are insensitive to elevated levels of glucose in the growth medium. These findings suggest that the SUN6 gene is active in a hexose-activated signal transduction pathway. 相似文献
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解析基因的剪接加工机制是了解植物形态建成、生长发育和逆境胁迫应答的重要环节.与动物相比,植物中相应的研究进展较为缓慢.利用农杆菌介导的烟草瞬时表达系统,分别对单子叶植物水稻BADH2和双子叶植物拟南芥GR7基因片段在烟草叶片中的转录后剪接加工进行分析.结果表明,一些重要剪接调控元件在植物中保守存在,而烟草瞬时表达系统可以作为研究高等植物剪接调控的重要工具,快捷灵敏地检测基因的剪接加工方式. 相似文献
11.
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
12.
Transient expression of green fluorescent protein in various plastid types following microprojectile bombardment 总被引:6,自引:0,他引:6
Julian M. Hibberd Philip J. Linley Muhammad S. Khan John C. Gray 《The Plant journal : for cell and molecular biology》1998,16(5):627-632
The green fluorescent protein gene ( gfp ) is a widely used reporter in both animals and plants. Fusions between the plastid rrn promoter or the Escherichia coli trc promoter and the gfp coding region have been delivered to chloroplasts using gold or tungsten microprojectiles, and fluorescence from GFP was visible in individual tobacco chloroplasts and in the abnormally large chloroplasts of the arc 6 mutant of Arabidopsis thaliana 2–4 days after bombardment. The fusion of the gfp coding region to the bacterial trc promoter demonstrated that a bacterial promoter is active in chloroplasts in vivo . GFP was also detectable in amyloplasts of potato tubers and in chromoplasts of marigold petals, carrot roots and pepper fruits 4 days after bombardment. This demonstrates that GFP can be used as a reporter for transient gene expression in chloroplasts and in non-photosynthetic plastids in a range of higher plants. 相似文献
13.
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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Kazuo Nakashima Tomohiro Kiyosue Kazuko Yamaguchi-Shinozaki Kazuo Shinozaki 《The Plant journal : for cell and molecular biology》1997,12(4):851-861
A cDNA, ERD1, isolated from one-hour-dehydrated plants of Arabidopsis thaliana L. encodes a putative protein that is similar to the regulatory ATPase subunit (ClpA) of the Clp protease and contains a putative chloroplast-targeting transit-peptide at the N-terminus. A chimeric gene with the putative plastid-targeting sequence of the erd1 gene fused to the synthetic green-fluorescent protein (sGFP) gene was constructed and introduced into Arabidopsis protoplasts. The N-terminal region of the ERD1 protein directed the sGFP protein into the plastids of the protoplasts, and functioned as a transit peptide. Northern blot analysis indicated that expression of the erd1 gene was induced not only by water stress, such as dehydration and high salinity, but also by natural senescence and dark-induced etiolation. The erd1 gene was not strongly induced by exogenous abscisic acid. A chimeric gene with the 0.9 kb promoter region of the erd1 gene fused to the β-glucuronidase (GUS) reporter gene was constructed, and tobacco plants transformed with the construct. The GUS reporter gene driven by the erd1 promoter was induced by dehydration and high salt stress at significant levels in the transgenic plants. The GUS gene was strongly expressed in older leaves without dehydration, and was induced by dark-induced etiolation. Furthermore, GUS activity was reduced by cytokinin treatment during dark-induced etiolation. These results indicate that expression of the erd1 gene is developmentally up-regulated by senescence as well as by water stress. 相似文献
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Felicity Z. Watts Neil Butt Philip Layfield Jesse Machuka Julian F. Burke Anthony L. Moore 《Plant molecular biology》1994,26(1):445-451
An Arabidopsis thaliana gene (UBC6) encoding a homologue to ubiquitin-conjugating enzymes has been isolated which is capable of encoding a protein of 183 amino acids of ca. 21 kDa. Northern analysis indicates that the gene is expressed in flowers, seeds and, to a somewhat lesser extent, in 10-day seedlings but not in mature leaves, callus and pre-flowering plants. This pattern of expression is confirmed using transgenic Arabidopsis plants containing a UBC6 promoter-GUS gene fusion construct. These plants displey GUS activity in mature anthers prior to dehiscence, in developing embryos, sepals and the style after pollination. 相似文献
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Surendar Reddy Dhadi Aparna Deshpande Wusirika Ramakrishna 《Plant Molecular Biology Reporter》2012,30(1):36-45
A novel Agrobacterium tumefaciens-mediated transient expression assay (AmTEA) was developed for young plants of different cereal species and the model dicot
Arabidopsis thaliana. AmTEA was evaluated using five promoters (six constructs) and two reporter genes, gus and egfp. The constitutive 35S promoter and the promoter of the rice glutaredoxin gene showed gus and egfp expression in the cereals analyzed in the present study. A promoter for the DEAD-box RNA helicase family protein gene from
Arabidopsis showed similar expression patterns of reporter genes in stable transgenic lines as well as in transient expression lines
of Arabidopsis. Agrobacterium tumefaciens co-cultivation and plant incubation times were optimized using 35S and the rice expressed protein gene promoter (R2-273).
The possibility of non-specific expression of the reporter genes was ruled out by using the antibiotic carbenicillin and the
comparison of expression of the reporter genes driven by full-length and truncated R2-273 promoters. AmTEA considerably reduced
time, space, labor, and cost requirements. Ease of use with stress treatments is another major advantage of this method. AmTEA
can be automated and used for large-scale studies to decipher promoter and gene functions with the ultimate goal to enhance
the performance of cereal crops against biotic and abiotic stresses. 相似文献