共查询到20条相似文献,搜索用时 31 毫秒
1.
RNA interference (RNAi) has been used to suppress gene expression in various eukaryotic organisms. In plants, RNAi can be
induced by introduction of an RNAi vector that transcribes a self-complementary hairpin RNA. Most basic RNAi constructs have
an inverted repeat interrupted with a spacer sequence. To test silencing capability of RNAi constructs, we developed an in
vivo assay that is based on the RNAi-mediated changes of the α-linolenic acid content in hairy roots. A tobacco endoplasmic
reticulum ω-3 fatty acid desaturase (NtFAD3) is the main enzyme for production of α-linolenic acid of root membrane lipids.
Tobacco hairy roots transformed with the RNAi vectors against the NtFAD3 gene showed a decrease in α-linolenic acid content. The frequency of RNA silencing was more affected by spacer sequence than
by spacer length, at least between 100 and 1800 bp. Since significant amounts of hairpin RNA against the NtFAD3 gene remained in the transgenic plants displaying a weak silencing phenotype, low degree of silencing was attributed to low
efficiency of hairpin RNA processing mediated by Dicer-like proteins. Our results show the possibility of producing a broad
range of the RNAi-induced silencing phenotypes by replacing the spacer sequence of RNAi construct. 相似文献
2.
The superior performance of F1 hybrids has a significant impact on agricultural productivity. For commercial application, the availability of an efficient
system for obtaining male-sterile lines of crops is an essential prerequisite. Here we have investigated the use of RNA interference
(RNAi) technology to silence a male-specific gene in the model host tobacco. TA29 is expressed exclusively in anthers at the time of microspore development. About 10 out of 13 tobacco lines transformed with
a hairpin RNAi construct containing TA29 sequences were male sterile. Transgenic plants were phenotypically indistinguishable from non-transgenic plants. At the anthesis
stage, pollen grains from transgenic, male-sterile plants were aborted and lysed in comparison to the round and fully developed
pollen in non-transgenic plants. Microscopic analysis of anthers showed selective degradation of tapetum in transgenic plants
with no microspore development. One week after self-pollination, the ovules of non-transgenic plants were double the size
of those in transgenic plants, due to successful self-fertilization. Male sterile transgenic plants set seed normally, when
cross-pollinated with pollen from non-transgenic plants, confirming no adverse effect on the female parts of the flower. These
results show that silencing of male-specific genes by RNAi is potentially a useful tool for generating male-sterile lines
for producing hybrid seed. 相似文献
3.
Shin-ichiro Oka Sayaka Hirai Kwan Sam Choi Hiroaki Kodama 《Journal of Plant Biology》2008,51(3):227-232
The amplification mechanism of short interfering RNAs (siRNAs) along the transgene sequence exists in RNA interference (RNAi).
The RNA-dependent RNA polymerase synthesizes complementary RNAs by using the transgene mRNA as a template, and the secondary
siRNAs are generated from the outside of primary RNAi target. Four independent RNAi vectors which produced primary siRNAs
against distinct regions of the tobacco endoplasmic reticulum ω-3 fatty acid desaturase gene (NtFAD3) were transiently expressed in leaves of theNtF4D3-overexpressed transgenic plants. Regardless of the RNAi vector used, the secondaryNtFAD3 siRNAs were generated preferentially from the 3’ downstream region of the transgene. Secondary siRNAs from the 5’ upstream
region adjacent to the annealing site of primary siRNAs accumulated under the detection level. Our results suggest that different
regulatory mechanisms are involved in the spreading of RNA silencing into 5’ upstream and 3’ downstream regions of the target
sequence, respectively. 相似文献
4.
Hong J Wei N Chalk A Wang J Song Y Yi F Qiao RP Sonnhammer EL Wahlestedt C Liang Z Du Q 《Biochemical and biophysical research communications》2008,368(3):703-708
RISC (RNA-induced silencing complex) is a central protein complex in RNAi, into which a siRNA strand is assembled to become effective in gene silencing. By using an in vitro RNAi reaction based on Drosophila embryo extract, an asymmetric model was recently proposed for RISC assembly of siRNA strands, suggesting that the strand that is more loosely paired at its 5′ end is selectively assembled into RISC and results in target gene silencing. However, in the present study, we were unable to establish such a correlation in cell-based RNAi assays, as well as in large-scale RNAi data analyses. This suggests that the thermodynamic stability of siRNA is not a major determinant of gene silencing in mammalian cells. Further studies on fork siRNAs showed that mismatch at the 5′ end of the siRNA sense strand decreased RISC assembly of the antisense strand, but surprisingly did not increase RISC assembly of the sense strand. More interestingly, measurements of melting temperature showed that the terminal stability of fork siRNAs correlated with the positions of the mismatches, but not gene silencing efficacy. In summary, our data demonstrate that there is no definite correlation between siRNA stability and gene silencing in mammalian cells, which suggests that instead of thermodynamic stability, other features of the siRNA duplex contribute to RISC assembly in RNAi. 相似文献
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6.
RNA interference (RNAi) efficiently induces sequence-specific gene silencing in mammalian cells through short interfering
RNA (siRNA) of 21–23 nucleotides synthesized in vitro or expressed by DNA-based vector. However, introduction of siRNA into
mammalian cells by transfection limits the application of RNAi, especially when it is necessary to generate long-term gene
silencing in vivo. Virus vector-mediated RNAi provides an alternative to transfection. In the present study, we investigated
such transduction system and showed that retrovirus vector-mediated RNAi can substantially down-regulate expression of mouse
adult β-globin gene in MEL cells. The results suggest that retrovirus vector-delivered RNAi may find its use in functional
genomics and in gene therapy. 相似文献
7.
Molecular and biochemical characterization of a novel xylanase from the symbiotic <Emphasis Type="Italic">Sphingobacterium</Emphasis> sp. TN19 总被引:1,自引:0,他引:1
Junpei Zhou Huoqing Huang Kun Meng Pengjun Shi Yaru Wang Huiying Luo Peilong Yang Yingguo Bai Zhigang Zhou Bin Yao 《Applied microbiology and biotechnology》2009,84(2):323-333
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RNA interference in crop plants 总被引:13,自引:0,他引:13
Kusaba M 《Current opinion in biotechnology》2004,15(2):139-143
10.
《Bioscience, biotechnology, and biochemistry》2013,77(12):2674-2677
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana. 相似文献
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13.
An CI Sawada A Fukusaki E Kobayashi A 《Bioscience, biotechnology, and biochemistry》2003,67(12):2674-2677
Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana. 相似文献
14.
Noncoding Flavivirus RNA Displays RNA Interference Suppressor Activity in Insect and Mammalian Cells
Esther Schnettler Mark G. Sterken Jason Y. Leung Stefan W. Metz Corinne Geertsema Rob W. Goldbach Just M. Vlak Alain Kohl Alexander A. Khromykh Gorben P. Pijlman 《Journal of virology》2012,86(24):13486-13500
West Nile virus (WNV) and dengue virus (DENV) are highly pathogenic, mosquito-borne flaviviruses (family Flaviviridae) that cause severe disease and death in humans. WNV and DENV actively replicate in mosquitoes and human hosts and thus encounter different host immune responses. RNA interference (RNAi) is the predominant antiviral response against invading RNA viruses in insects and plants. As a countermeasure, plant and insect RNA viruses encode RNA silencing suppressor (RSS) proteins to block the generation/activity of small interfering RNA (siRNA). Enhanced flavivirus replication in mosquitoes depleted for RNAi factors suggests an important biological role for RNAi in restricting virus replication, but it has remained unclear whether or not flaviviruses counteract RNAi via expression of an RSS. First, we established that flaviviral RNA replication suppressed siRNA-induced gene silencing in WNV and DENV replicon-expressing cells. Next, we showed that none of the WNV encoded proteins displayed RSS activity in mammalian and insect cells and in plants by using robust RNAi suppressor assays. In contrast, we found that the 3′-untranslated region-derived RNA molecule known as subgenomic flavivirus RNA (sfRNA) efficiently suppressed siRNA- and miRNA-induced RNAi pathways in both mammalian and insect cells. We also showed that WNV sfRNA inhibits in vitro cleavage of double-stranded RNA by Dicer. The results of the present study suggest a novel role for sfRNA, i.e., as a nucleic acid-based regulator of RNAi pathways, a strategy that may be conserved among flaviviruses. 相似文献
15.
A chemical-regulated inducible RNAi system in plants 总被引:21,自引:0,他引:21
Constitutive expression of an intron-containing self-complementary 'hairpin' RNA (ihpRNA) has recently been shown to efficiently silence target genes in transgenic plants. However, this technique cannot be applied to genes whose silencing may block plant regeneration or result in embryo lethality. To obviate these potential problems, we have used a chemical-inducible Cre/loxP (CLX) recombination system to trigger the expression of an intron-containing inverted-repeat RNA (RNAi) in plants. A detailed characterization of the inducible RNAi system in transgenic Arabidopsis thaliana and Nicotiana benthamiana plants demonstrated that this system is stringently controlled. Moreover, it can be used to induce silencing of both transgenes and endogenous genes at different developmental stages and at high efficiency and without any detectable secondary affects. In addition to inducing complete silencing, the RNAi can be produced at various times after germination to initiate and obtain different degrees of gene silencing. Upon induction, transgenic plants with genetic chimera were obtained as demonstrated by PCR analysis. Such chimeric plants may provide a useful system to study signaling mechanisms of gene silencing in Arabidopsis as well as other cases of long-distance signaling without grafting. The merits of using the inducible CLX system for RNAi expression are discussed. 相似文献
16.
Nomenclature and functions of RNA-directed RNA polymerases 总被引:13,自引:0,他引:13
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DNA interference: a simple and efficient gene-silencing system for high-throughput functional analysis in the fern adiantum 总被引:1,自引:0,他引:1
Kawai-Toyooka H Kuramoto C Orui K Motoyama K Kikuchi K Kanegae T Wada M 《Plant & cell physiology》2004,45(11):1648-1657
RNA interference (RNAi) has become a powerful tool for determining gene function and is used in a wide variety of organisms. Since it is necessary to generate double-stranded RNA (dsRNA) as an inducer for RNAi, preparation of RNAi-inducing constructs is somewhat cumbersome and time consuming, especially for the thousands of genes used in a genome-wide analysis. To overcome these problems, we have developed a more convenient gene-silencing method in the fern Adiantum using double-stranded DNA (dsDNA) as a model system for functional analysis in plants. Delivery of dsDNA fragments homologous to an endogenous gene into gametophytic cells can induce sequence-specific gene silencing. As it only requires dsDNA fragments homologous to a target gene, PCR-amplified fragments are enough to trigger gene silencing. Maximum gene silencing efficiencies of >90% have been achieved for transformed plants. In addition, simultaneous transfer of dsDNA fragments corresponding to multiple genes still has a silencing effect for individual genes. We term this approach 'DNA interference'. 相似文献
19.
Isabel Weinheimer Yaming Jiu Minna-Liisa Rajam?ki Olli Matilainen Jukka Kallij?rvi Wilmer J. Cuellar Rui Lu Mart Saarma Carina I. Holmberg Jussi J?ntti Jari P. T. Valkonen 《PLoS pathogens》2015,11(3)
Certain RNA and DNA viruses that infect plants, insects, fish or poikilothermic animals encode Class 1 RNaseIII endoribonuclease-like proteins. dsRNA-specific endoribonuclease activity of the RNaseIII of rock bream iridovirus infecting fish and Sweet potato chlorotic stunt crinivirus (SPCSV) infecting plants has been shown. Suppression of the host antiviral RNA interference (RNAi) pathway has been documented with the RNaseIII of SPCSV and Heliothis virescens ascovirus infecting insects. Suppression of RNAi by the viral RNaseIIIs in non-host organisms of different kingdoms is not known. Here we expressed PPR3, the RNaseIII of Pike-perch iridovirus, in the non-hosts Nicotiana benthamiana (plant) and Caenorhabditis elegans (nematode) and found that it cleaves double-stranded small interfering RNA (ds-siRNA) molecules that are pivotal in the host RNA interference (RNAi) pathway and thereby suppresses RNAi in non-host tissues. In N. benthamiana, PPR3 enhanced accumulation of Tobacco rattle tobravirus RNA1 replicon lacking the 16K RNAi suppressor. Furthermore, PPR3 suppressed single-stranded RNA (ssRNA)—mediated RNAi and rescued replication of Flock House virus RNA1 replicon lacking the B2 RNAi suppressor in C. elegans. Suppression of RNAi was debilitated with the catalytically compromised mutant PPR3-Ala. However, the RNaseIII (CSR3) produced by SPCSV, which cleaves ds-siRNA and counteracts antiviral RNAi in plants, failed to suppress ssRNA-mediated RNAi in C. elegans. In leaves of N. benthamiana, PPR3 suppressed RNAi induced by ssRNA and dsRNA and reversed silencing; CSR3, however, suppressed only RNAi induced by ssRNA and was unable to reverse silencing. Neither PPR3 nor CSR3 suppressed antisense-mediated RNAi in Drosophila melanogaster. These results show that the RNaseIII enzymes of RNA and DNA viruses suppress RNAi, which requires catalytic activities of RNaseIII. In contrast to other viral silencing suppression proteins, the RNaseIII enzymes are homologous in unrelated RNA and DNA viruses and can be detected in viral genomes using gene modeling and protein structure prediction programs. 相似文献