共查询到20条相似文献,搜索用时 31 毫秒
1.
Zhen Wang Changbin Chen Yunyuan Xu Rongxi Jiang Ye Han Zhihong Xu Kang Chong 《Plant Molecular Biology Reporter》2004,22(4):409-417
In the last decade, RNA interferences (RNAi) has proven to be an effective strategy to knock out homologous genes in a wide
range of species. Based on its principle, a new generation of vectors containing an inverted target sequence separated by
an intron as a loop, developing simplifications to the procedure of RNAi construction are required to improve the efficiency
of gene inactivation techniques. Here, a novel polymerase chain reaction (PCR)—based RNAi vector pTCK303 with a maize ubiquitin
promoter, 2 specific multiple enzyme sites, and a rice intron was constructed for monocot gene silencing. With this vector,
only 1 PCR product amplified by a single pair of primers and 2 ligation reactions were needed to create an RNAi construct,
which shortened the time span before being transformed into the plant. To test the efficiency of vector pTCK303, a rice geneOsGAS1 was used, and its RNAi construct was introduced into rice calli. Southern blot analysis of the transgenic rice confirmed
the presence of theOsGAS1 RNAi structure. The decrease inOsGAS1 level in the transgenic rice was detected by Northern blot probed with anOsGAS1-specific sequence. Moreover, the rate of inhibition of the RNA expression level in RNAi transgenic rice was approximately
85% according to our real-time PCR. Therefore, the RNAi vector pTCK303 based on the homology-dependent gene-silencing mechanisms
facilitated the inhibition of endogenous genes in a monocot and was proven to be a practical and efficient platform for silencing
a rice gene.
These authors contributed equally to this work. 相似文献
2.
ERA1是控制植物气孔开闭的一个重要基因,根据其保守域构建RNA干扰(RNAi)载体并转化拟南芥,考察转基因植株的生长、气孔导度、离体叶片失水率以及ERA1和相关基因表达,探讨siRNA介导的ERA1表达下调对拟南芥抗旱性的影响。结果表明:转基因拟南芥株系中ERA1的表达受到明显抑制,其离体叶片失水率低于野生型,但并未出现ERA1缺失突变体的负面生长表型;转基因株系对ABA处理比野生型更敏感,其ABA处理株的根长显著变短,气孔孔径更小;转基因株ABI1、ABI2、ATHB6的表达量降低,而RAB18、RD29B、ADH1的表达量升高,siRNA介导的ERA1表达下调可能会激活RAB18、RD29B等逆境响应元件。研究发现,采用RNAi技术可以有效下调ERA1表达,在没有过多负面生长表型的前提下提高拟南芥的抗旱性,且ERA1表达下调可能通过ABA途径正面影响拟南芥的抗旱性。 相似文献
3.
4.
Hui-Juan Li Ai-Fang Yang Xue-Cheng Zhang Feng Gao Ju-Ren Zhang 《Plant Cell, Tissue and Organ Culture》2007,89(1):37-48
Sucrose: sucrose 1-fructosyltransferase (1-SST) cDNA from Lactuca sativa, coding the enzyme responsible for lower degree polymers fructan biosynthesis, was cloned by RT-PCR and RACE methods. The
1-SST cDNA under the control of CaMV 35S promoter was introduced into tobacco by Agrobacterium-mediated leaf disc transformation protocol. Fructan synthesis in vitro and carbohydrate analysis showed that sense transgenic
tobacco plant displayed sucrose: sucrose 1-fructosyltransferse activity. After freezing stress, significant increases in electrolyte
leakage and malondialdehyde were found in the wild type and anti-sense transgenic plants, while no apparent differences were
observed in sense transgenic plants. Meanwhile, water soluble carbohydrate, fructan and fructose of sense transgenic plants
remarkably increased, compared with those of wild type and anti-sense plants. No significant difference was detected in superoxide
dismutase activity between transgenic and wild type plants. The above results demonstrated that the expression of 1-SST gene improved the freezing resistance of transgenic tobacco plants. 相似文献
5.
Aluminum (Al) toxicity is one of the major factors that limit plant growth in acid soils. Al-induced release of organic acids
into rhizosphere from the root apex has been identified as a major Al-tolerance mechanism in many plant species. In this study,
Al tolerance of Yuzu (Citrus Junos Sieb. ex Tanaka) was tested on the basis of root elongation and the results demonstrated that Yuzu was Al tolerant compared
with other plant species. Exposure to Al triggered the exudation of citrate from the Yuzu root. Thus, the mechanism of Al
tolerance in Yuzu involved an Al-inducible increase in citrate release. Aluminum also elicited an increase of citrate content
and increased the expression level of mitochondrial citrate synthase (CjCS) gene and enzyme activity in Yuzu. The CjCS gene was cloned from Yuzu and overexpressed in Nicotiana benthamiana using Agrobacterium tumefaciens-mediated methods. Increased expression level of the CjCS gene and enhanced enzyme activity were observed in transgenic plants compared with the wild-type plants. Root growth experiments
showed that transgenic plants have enhanced levels of Al tolerance. The transgenic Nicotiana plants showed increased levels of citrate in roots compared to wild-type plants. The exudation of citrate from roots of the
transgenic plants significantly increased when exposed to Al. The results with transgenic plants suggest that overexpression
of mitochondrial CS can be a useful tool to achieve Al tolerance. 相似文献
6.
Dorothee Wegener Peter Steinecke Thomas Herget Iris Petereit Christina Philipp Peter H. Schreier 《Molecular & general genetics : MGG》1994,245(4):465-470
A chimeric gene encoding a ribozyme under the control of the cauliflower mosaic virus (CaMV) 35S promoter was introduced into transgenic tobacco plants. In vivo activity of this ribozyme, which was designed to cleave npt mRNA, was previously demonstrated by transient expression assays in plant protoplasts. The ribozyme gene was transferred into transgenic tobacco plants expressing an rbcS-npt chimeric gene as an indicator. Five double transformants out of sixteen exhibited a reduction in the amount of active NPT enzyme. To measure the amount of ribozyme produced, in the absence of its target, the ribozyme and target genes were separated by genetic segregation. The steady-state concentrations of ribozyme and target RNA were shown to be similar in the resulting single transformants. Direct evidence for a correlation between reduced npt gene expression and ribozyme expression was provided by crossing a plant containing only the ribozyme gene with a transgenic plant expressing the npt gene under control of the 35S promoter, i.e. the same promoter used to direct ribozyme expression. The expression of npt was reduced in all progeny containing both transgenes. Both steady-state levels of npt mRNA and amounts of active NPT enzyme are decreased. In addition, our data indicate that, at least in stable transformants, a large excess of ribozyme over target is not a prerequisite for achieving a significant reduction in target gene expression. 相似文献
7.
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
8.
A study of an enzyme that reacts with the sulfenic acid produced by the alliinase in Petiveria alliacea L. (Phytolaccaceae) to yield the P. alliacea lachrymator (phenylmethanethial S-oxide) showed the protein to be a dehydrogenase. It functions by abstracting hydride from sulfenic acids of appropriate structure to form their corresponding sulfines. Successful hydride abstraction is dependent upon the presence of a benzyl group on the sulfur to stabilize the intermediate formed on abstraction of hydride. This dehydrogenase activity contrasts with that of the lachrymatory factor synthase (LFS) found in onion, which catalyzes the rearrangement of 1-propenesulfenic acid to (Z)-propanethial S-oxide, the onion lachrymator. Based on the type of reaction it catalyzes, the onion LFS should be classified as an isomerase and would be called a “sulfenic acid isomerase”, whereas the P. alliacea LFS would be termed a “sulfenic acid dehydrogenase”. 相似文献
9.
Tang Wei Newton Ronald J. 《Molecular breeding : new strategies in plant improvement》2005,16(3):235-246
Mature zygotic embryos of recalcitrant Christmas tree species Fraser fir [Abies fraseri (Pursh) Poir], and Nordmann fir (Abies nordmanniana L.k.), and Virginia pine (Pinus virginiana Mill.) were used as explants for Agrobacterium tumefaciens strain GV3850-mediated transformation using the gfp (green fluorescent protein) gene as a reporter. Factors including media used for inoculation and co-cultivation, concentrations
of acetosyringone, and antibiotics in tissue culture media have been evaluated. A high transformation frequency was obtained
on TE medium containing 50μM acetosyringone and using 500 mg/l timentin to eliminate bacteria. Transient gene expression was
observed in all three Christmas tree species, but transgenic plants were only produced from Virginia pine. Stable integration
and expression of transgenes in the plant genome of Virginia pine was confirmed by polymerase chain reaction (PCR), Southern
and northern blot analyses. These results demonstrated that a stable transformation system has been established in Virginia
pine and this system would provide an opportunity to transfer economically important genes into Christmas tree species. 相似文献
10.
11.
Yasumasa Kuwahara Hidetoshi Hara Shoziro Ishii Hiroshi Fukami 《Bioscience, biotechnology, and biochemistry》2013,77(3):447-448
Lachrymatory factor synthase (LFS), an enzyme essential for the synthesis of the onion lachrymatory factor (propanethial S-oxide), was identified in 2002. This was the first reported enzyme involved in the production of thioaldehyde S-oxides via an intra-molecular H+ substitution reaction, and we therefore attempted to identify the catalytic amino acid residues of LFS as the first step in elucidating the unique catalytic reaction mechanism of this enzyme. A comparison of the LFS cDNA sequences among lachrymatory Allium plants, a deletion analysis and site-directed mutagenesis enabled us to identify two amino acids (Arg71 and Glu88) that were indispensable to the LFS activity. Homology modeling was performed for LFS/23–169 on the basis of the template structure of a pyrabactin resistance 1-like protein (PYL) which had been selected from a BLASTP search on SWISS-MODEL against LFS/23–169. We identified in the modeled structure of LFS a pocket corresponding to the ligand-binding site in PYL, and Arg71 and Glu88 were located in this pocket. 相似文献
12.
Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants 总被引:1,自引:0,他引:1
A novel, constitutively expressed gene, designated MtHP, was isolated from the model legume species Medicago truncatula. Sequence analysis indicates that MtHP most likely belongs to the PR10 multi-gene family. The MtHP promoter was fused to a -glucuronidase gene to characterize its expression in different plant species. Transient assay by microprojectile bombardment and hairy root transformation by Agrobacterium rhizogenes revealed GUS expression in leaf, stem, radicle and root in M. truncatula. Detailed analysis in transgenic Arabidopsis plants demonstrated that the promoter could direct transgene expression in different tissues and organs at various developmental stages; its expression pattern was similar to that of CaMV35S promoter, and the level of expression was higher than the reporter gene driven by CaMV35S promoter. Deletion analysis revealed that even a 107 bp fragment of the promoter could still lead to a moderate level of expression. The promoter was further characterized in white clover (Trifolium repens), a widely grown forage legume species. Strong constitutive expression was observed in transgenic white clover plants. Compared with CaMV35S promoter, the level of GUS activity in transgenic white clover was higher when the transgene was driven by MtHP promoter. Thus, the promoter provides a useful alternative to the CaMV35S promoter in plant transformation for high levels of constitutive expression. 相似文献
13.
Cytokinins play important roles in regulating plant growth and development. A new genetic construct for regulating cytokinin content in plant cells was cloned and tested. The gene coding for isopentenyl transferase (ipt) was placed under the control of a 0.821 kb fragment of the 1-aminocyclopropane-1-carboxylic acid (ACC) oxidase gene promoter from Lycopersicon esculentum (LEACO1) and introduced into Nicotiana tabacum (cv. Havana). Some LEACO10.821 kb-ipt transgenic plant lines displayed normal shoot morphology but with a dramatic increase in the number of flower buds compared to nontransgenic plants. Other transgenic lines produced excessive lateral branch development but no change in flower bud number. Isolated leaves of transgenic tobacco plants showed a significantly prolonged retention of chlorophyll under dark incubation (25°C for 20 days). Leaves of nontransformed plants senesced gradually under the same conditions. Experiments with LEACO10.821 kb-gus transgenic tobacco plants suggested auxin and ethylene involvement in induction of LEACO10.821 kb promoter activity. Multiple copies of nucleotide base sequences associated with either ethylene or auxin response elements were identified in the LEACO10.821 kb promoter fragment. The LEACO10.821 kb-ipt fusion gene appears to have potential utility for improving certain ornamental and agricultural crop species by increasing flower bud initiation and altering branching habit. 相似文献
14.
Transgenic hairy root system is important in several recalcitrant plants, where Agrobacterium tumefaciens-mediated plant transformation and generation of transgenic plants are problematic. Jute (Corchorus spp.), the major fibre crop in Indian subcontinent, is one of those recalcitrant plants where in vitro tissue culture has
provided a little success, and hence, Agrobacterium-mediated genetic transformation remains to be a challenging proposition in this crop. In the present work, a system of transgenic
hairy roots in Corchorus capsularis L. has been developed through genetic transformation by Agrobacterium rhizogenes harbouring two plasmids, i.e. the natural Ri plasmid and a recombinant binary vector derived from the disarmed Ti plasmid
of A. tumefaciens. Our findings indicate that the system is relatively easy to establish and reproducible. Molecular analysis of the independent
lines of transgenic hairy roots revealed the transfer of relevant transgenes from both the T-DNA parts into the plant genome,
indicating the co-transformation nature of the event. High level expression and activity of the gusA reporter gene advocate that the transgenic hairy root system, thus developed, could be applicable as gene expression system
in general and for root functional genomics in particular. Furthermore, these transgenic hairy roots can be used in future
as explants for plantlet regeneration to obtain stable transgenic jute plants. 相似文献
15.
16.
Hemavathi Chandrama Prakash Upadhyaya Ko Eun Young Nookaraju Akula Hyun soon Kim Jeon Jae Heung Oh Man Oh Chenna Reddy Aswath Se Chul Chun Doo Hwan Kim Se Won Park 《Plant science》2009,177(6):659-667
Vitamin C (ascorbic acid) is an essential component for collagen biosynthesis and also for the proper functioning of the cardiovascular system in humans. Unlike most of the animals, humans lack the ability to synthesize ascorbic acid on their own due to a mutation in the gene encoding the last enzyme of ascorbate biosynthesis. As a result, vitamin C must be obtained from dietary sources like plants. In this study, we have developed transgenic potato plants (Solanum tuberosum L. cv. Taedong Valley) over-expressing strawberry GalUR gene under the control of CaMV 35S promoter with increased ascorbic acid levels. Integration of the GalUR gene in the plant genome was confirmed by PCR and Southern blotting. Ascorbic acid (AsA) levels in transgenic tubers were determined by high-performance liquid chromatography (HPLC). The over-expression of GalUR resulted in 1.6–2-fold increase in AsA in transgenic potato and the levels of AsA were positively correlated with increased GalUR activity. The transgenic lines with enhanced vitamin C content showed enhanced tolerance to abiotic stresses induced by methyl viologen (MV), NaCl or mannitol as compared to untransformed control plants. The leaf disc senescence assay showed better tolerance in transgenic lines by retaining higher chlorophyll as compared to the untransformed control plants. Present study demonstrated that the over-expression of GalUR gene enhanced the level of AsA in potato tubers and these transgenics performed better under different abiotic stresses as compared to untransformed control. 相似文献
17.
18.
Shunhua Ding Qingtao Lu Yan Zhang Zhipan Yang Xiaogang Wen Lixin Zhang Congming Lu 《Plant molecular biology》2009,69(5):577-592
To investigate the possible mechanisms of glutathione reductase (GR) in protecting against oxidative stress, we obtained transgenic
tobacco (Nicotiana tabacum) plants with 30–70% decreased GR activity by using a gene encoding tobacco chloroplastic GR for the RNAi construct. We investigated
the responses of wild type and transgenic plants to oxidative stress induced by application of methyl viologen in vivo. Analyses
of CO2 assimilation, maximal efficiency of photosystem II photochemistry, leaf bleaching, and oxidative damage to lipids demonstrated
that transgenic plants exhibited enhanced sensitivity to oxidative stress. Under oxidative stress, there was a greater decrease
in reduced to oxidized glutathione ratio but a greater increase in reduced glutathione in transgenic plants than in wild type
plants. In addition, transgenic plants showed a greater decrease in reduced ascorbate and reduced to oxidized ascorbate ratio
than wild type plants. However, there were neither differences in the levels of NADP and NADPH and in the total foliar activities
of monodehydroascorbate reductase and dehydroascorbate reductase between wild type and transgenic plant. MV treatment induced
an increase in the activities of GR, ascorbate peroxidase, superoxide dismutase, and catalase. Furthermore, accumulation of
H2O2 in chloroplasts was observed in transgenic plants but not in wild type plants. Our results suggest that capacity for regeneration
of glutathione by GR plays an important role in protecting against oxidative stress by maintaining ascorbate pool and ascorbate
redox state. 相似文献
19.
Increased Septoria musiva resistance in transgenic hybrid poplar leaves expressing a wheat oxalate oxidase gene 总被引:8,自引:0,他引:8
Liang Haiying Maynard Charles A. Allen Randy D. Powell William A. 《Plant molecular biology》2001,45(6):619-629
A cDNA clone of a wheat germin-like oxalate oxidase (OxO) gene regulated by the constitutive CaMV 35S promoter was expressed in a hybrid poplar clone, Populus × euramericana (`Ogy'). Previous studies showed that OxO is likely to play an important role in several aspects of plant development, stress response, and defense against pathogens. In order to study this wheat oxalate oxidase gene in woody plants, the expression of this gene and the functions of the encoded enzyme were examined in vitro and in vivo in transgenic `Ogy'. The enzyme activity in the transformed `Ogy' was visualized by histochemical assays and in SDS-polyacrylamide gels. It was found that the wheat OxO gene is expressed in leaves, stems, and roots of the transgenic `Ogy' plants and the encoded enzyme is able to break down oxalic acid. Transgenic `Ogy' leaves were more tolerant to oxalic acid as well as more effective in increasing the pH in an oxalic acid solution when compared to untransformed controls. In addition, when leaf disks from `Ogy' plants were inoculated with conidia of the poplar pathogenic fungus Septoria musiva, which produces oxalic acid, the OxO-transformed plants were more resistant than the untransformed controls. 相似文献
20.
Promoters for pregenomic RNA of banana streak badnavirus are active for transgene expression in monocot and dicot plants 总被引:4,自引:0,他引:4
Schenk PM Remans T Sági L Elliott AR Dietzgen RG Swennen R Ebert PR Grof CP Manners JM 《Plant molecular biology》2001,47(3):399-412
Two putative promoters from Australian banana streak badnavirus (BSV) isolates were analysed for activity in different plant species. In transient expression systems the My (2105 bp) and Cv (1322 bp) fragments were both shown to have promoter activity in a wide range of plant species including monocots (maize, barley, banana, millet, wheat, sorghum), dicots (tobacco, canola, sunflower, Nicotiana benthamiana, tipu tree), gymnosperm (Pinus radiata) and fern (Nephrolepis cordifolia). Evaluation of the My and Cv promoters in transgenic sugarcane, banana and tobacco plants demonstrated that these promoters could drive high-level expression of either the green fluorescent protein (GFP) or the -glucuronidase (GUS) reporter gene (uidA) in vegetative plant cells. In transgenic sugarcane plants harbouring the Cv promoter, GFP expression levels were comparable or higher (up to 1.06% of total soluble leaf protein as GFP) than those of plants containing the maize ubiquitin promoter (up to 0.34% of total soluble leaf protein). GUS activities in transgenic in vitro-grown banana plants containing the My promoter were up to seven-fold stronger in leaf tissue and up to four-fold stronger in root and corm tissue than in plants harbouring the maize ubiquitin promoter. The Cv promoter showed activities that were similar to the maize ubiquitin promoter in in vitro-grown banana plants, but was significantly reduced in larger glasshouse-grown plants. In transgenic in vitro-grown tobacco plants, the My promoter reached activities close to those of the 35S promoter of cauliflower mosaic virus (CaMV), while the Cv promoter was about half as active as the CaMV 35S promoter. The BSV promoters for pregenomic RNA represent useful tools for the high-level expression of foreign genes in transgenic monocots. 相似文献