Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection...Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.展开更多
Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragmen...Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.展开更多
Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and...Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.展开更多
植物病毒病是一类严重危害农作物生产的重要病害。已报道的植物抗病毒基因主要在抑制病毒增殖和阻止病毒扩散中起作用。病毒的复制涉及自身的编码蛋白及其与寄主蛋白间的互作,参与病毒复制的寄主蛋白很多,如真核翻译起始因子eIF4E和eIF...植物病毒病是一类严重危害农作物生产的重要病害。已报道的植物抗病毒基因主要在抑制病毒增殖和阻止病毒扩散中起作用。病毒的复制涉及自身的编码蛋白及其与寄主蛋白间的互作,参与病毒复制的寄主蛋白很多,如真核翻译起始因子eIF4E和eIF4G,植物的内膜系统等,相关蛋白的功能丧失或构型改变可阻滞病毒的复制;此外,植物细胞内的硫氧还蛋白可调节细胞的氧化还原状态,进而阻断病毒的增殖。病毒在植物体内的扩散包括胞间移动和长距离迁移,植物抗病蛋白(R蛋白)通过识别病毒的无毒因子(Avr)促发防御反应,诱导过敏性坏死,限制病毒在细胞间的扩散,编码这类抗病蛋白的基因主要为TIR-NBS-LRR和CC-NBS-LRR。病毒的长距离迁移涉及的因素很多,目前仅发现韧皮部的RTM蛋白可能以多聚蛋白的形式抵制病毒的长距离移动。另外,RNA沉默也是植物抵制病毒侵染的免疫反应机制。本文旨在综述植物的各种抗病毒机制和相关的抗病基因,并探讨分子标记辅助选择(marker-assisted selection,MAS)、定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)和转基因等生物技术在抗病改良中的应用前景。展开更多
文摘Odontoglossum ringspot virus (ORSV) infects perennial orchids (Phalaenopsis amabilis) and causes a widespread viral disease. RNA-silencing of viral genes is a promising and effective way of controlling viral infection in plants. An inverted repeat (IR) fragment of the ORSV coat protein gene, cp, was inserted into the pXGY1 vector to generate the silencing construct, pXGY1-ORSV, which was introduced into Nicotiana benthamiana via Agrobacterium-mediated infiltration. A total of 15 homozygous pXGY1-ORSV transgenic N. benthamiana T1 plants were obtained from five transgenic lines, and ORSV cp gene multiplication was reduced by at least 75% - 95% in 12 T2 plants, demonstrating their increased resistance to ORSV. An infectious ORSV clone, pCAMBIA2300-ORSV, was generated to facilitate rigorous analyses of plant viral resistance. Semi-quantitative RT-PCR (sqRT-PCR) and northern-blot analyses revealed that levels of ORSV multiplication and ORSV coat protein were significantly reduced in pXGY1-ORSV transgenic N. benthamiana. Western-blot from pXGY1-ORSV inoculated leaves of ORSV infected P. amabilis also revealed the significant decrease and even degradation of ORSV-CP protein. Disease symptoms were not observed in transgenic plants. These results indicate a high level of ORSV-resistance in pXGY1-ORSV transgenic N. benthamiana.
基金This work was funded by the East Carolina Christmas Tree Program (2002).
文摘Short interfering RNA (siRNA) is widely used for studyingpost-transcriptional gene silencing and holds great promise as a tool for both identifying functionof novel genes and validating drug targets. Two siRNA fragments (siRNA-a and -b), which weredesigned against different specific areas of coding region of the same target green fluorescentprotein (GFP) gene, were used to silence GFP expression in cultured gfp transgenic cells of rice(Oryza sativa L.; OS), cotton (Gossypium hirsutum L.; GH), Eraser fir [Abies fraseri (Pursh) Poir;AF], and Virginia pine (Pinus virginiana Mill.; PV). Differential gene silencing was observed in thebombarded transgenic cells between two siRNAs, and these results were consistent with theinactivation of GFP confirmed by laser scanning microscopy, Northern blot, and siRNA analysis intested transgenic cell cultures. These data suggest that siRNA-mediated gene inactivation can be thesiRNA specific in different plant species. These results indicate that siRNA is a highly specifictool for targeted gene knockdown and for establishing siRNA-mediated gene silencing, which could bea reliable approach for large-scale screening of gene function and drug target validation.
基金co-financed by the European Union(European Social Fund–ESF)Greek national funds through the Operational Program"Education and Lifelong Learning"of the National Strategic Reference Framework(NSRF)–Research Funding Program:Heracleitus Ⅱ+1 种基金the European Social Fund(G.M.)Postdoctoral Grant LS1-1190(F.V)
文摘Multicellular organisms, like higher plants, need to coordinate their growth and development and to cope with environmental cues. To achieve this, various signal molecules are transported between neighboring cells and distant organs to control the fate of the recipient cells and organs. RNA silencing produces cell non-autonomous signal molecules that can move over short or long distances leading to the sequence specific silencing of a target gene in a well defined area of cells or throughout the entire plant,respectively. The nature of these signal molecules, the route of silencing spread, and the genes involved in their production, movement and reception are discussed in this review. Additionally, a short section on features of silencing spread in animal models is presented at the end of this review.
文摘植物病毒病是一类严重危害农作物生产的重要病害。已报道的植物抗病毒基因主要在抑制病毒增殖和阻止病毒扩散中起作用。病毒的复制涉及自身的编码蛋白及其与寄主蛋白间的互作,参与病毒复制的寄主蛋白很多,如真核翻译起始因子eIF4E和eIF4G,植物的内膜系统等,相关蛋白的功能丧失或构型改变可阻滞病毒的复制;此外,植物细胞内的硫氧还蛋白可调节细胞的氧化还原状态,进而阻断病毒的增殖。病毒在植物体内的扩散包括胞间移动和长距离迁移,植物抗病蛋白(R蛋白)通过识别病毒的无毒因子(Avr)促发防御反应,诱导过敏性坏死,限制病毒在细胞间的扩散,编码这类抗病蛋白的基因主要为TIR-NBS-LRR和CC-NBS-LRR。病毒的长距离迁移涉及的因素很多,目前仅发现韧皮部的RTM蛋白可能以多聚蛋白的形式抵制病毒的长距离移动。另外,RNA沉默也是植物抵制病毒侵染的免疫反应机制。本文旨在综述植物的各种抗病毒机制和相关的抗病基因,并探讨分子标记辅助选择(marker-assisted selection,MAS)、定向诱导基因组局部突变(targeting induced local lesions in genomes,TILLING)和转基因等生物技术在抗病改良中的应用前景。
