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A-to-I editing challenger or ally to the microRNA process   总被引:4,自引:0,他引:4  
Ohman M 《Biochimie》2007,89(10):1171-1176
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采用免疫亲和层析法分离纯化含次黄嘌呤核苷的mRNA(I-mRNA)。将Poly-I与BSA交联,并用Poly-I-BSA交联体免疫家兔,获取抗次黄呤核苷抗血清,斑点印迹法检测到该抗体滴度高、选择性强,将抗次黄嘌呤核苷抗体与蛋白A Sapharose交联,并制备抗体亲和层析柱。将小鼠肺mRNA上样于层析柱,淋洗层析柱后,用洗脱液洗脱I-mRNA,并以斑点印迹法在洗脱液中检测到很强的I-mRNA阳性信号;淋洗液中I-mRNA阳性信号的强度则随淋洗液体积的增加而减弱。以上结果表明,应用本方法可以有效分离得到含次黄嘌呤核苷的mRNA。  相似文献   

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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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Pentatricopeptide repeat (PPR) proteins belong to a family of approximately 450 members in Arabidopsis, of which few have been characterized. We identified loss of function alleles of SLO2, defective in a PPR protein belonging to the E+ subclass of the P-L-S subfamily. slo2 mutants are characterized by retarded leaf emergence, restricted root growth, and late flowering. This phenotype is enhanced in the absence of sucrose, suggesting a defect in energy metabolism. The slo2 growth retardation phenotypes are largely suppressed by supplying sugars or increasing light dosage or the concentration of CO(2) . The SLO2 protein is localized in mitochondria. We identified four RNA editing defects and reduced editing at three sites in slo2 mutants. The resulting amino acid changes occur in four mitochondrial proteins belonging to complex I of the electron transport chain. Both the abundance and activity of complex I are highly reduced in the slo2 mutants, as well as the abundance of complexes III and IV. Moreover, ATP, NAD+, and sugar contents were much lower in the mutants. In contrast, the abundance of alternative oxidase was significantly enhanced. We propose that SLO2 is required for carbon energy balance in Arabidopsis by maintaining the abundance and/or activity of complexes I, III, and IV of the mitochondrial electron transport chain.  相似文献   

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转RNAi-SPS5.1拟南芥的获得及表型分析   总被引:1,自引:0,他引:1  
在克隆了拟南芥蔗糖磷酸合成酶AtSPS5.1的基础上,选择其中特异性的188 bp片段,以正反两个方向插入克隆载体pKANNIBAL中,再将该克隆载体连接入具有NPTⅡ筛选标记的表达载体pART-27中,构建成RNAi-SPS5.1干涉载体.采用农杆菌介导的真空渗透法转化拟南芥,得到T0代的种子,筛选阳性植株,并进行RT-PCR检测,结果表明:RNAi技术能够有效抑制SPS5.1基因的表达,且SPS5.1被干涉后植株的生长发育明显较野生型差,说明该基因的表达对拟南芥生长发育具有重要作用.  相似文献   

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在RNA代谢过程中,需要许多蛋白和核酸的参与,其中一类蛋白就是RNA解旋酶。RNA解旋酶通过水解ATP获得能量来参与RNA代谢的多个方面,包括核内转录、pre-mRNA的剪切、核糖体发生、核质运输、蛋白质翻译、RNA降解、细胞器内基因的表达。DEAD-box蛋白家族是RNA解旋酶中最大的亚家族,它具有9个保守结构域,因motifyⅡ的保守氨基酸序列Asp-Glu-Ala-Asp(DEAD)而命名。该家族在酵母、拟南芥(Arabidopsis thaliana Heynh.)和人类基因组中都有较多的家庭成员。近年来,研究者对拟南芥DEAD-box蛋白家族的结构和功能进行了一些研究,本文着重总结DEAD-box基因家族对拟南芥生长发育的影响。  相似文献   

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李磊  刘彤  刘斌  刘忠权  司朗明  张荣 《生物多样性》2010,18(5):497-1177
拟南芥(Arabidopsis thaliana)自然居群的表型特征代表其在自然环境下的适应状况, 不同居群间特征的对比可以为了解拟南芥表型变化规律, 进而分析其形成过程和机制提供重要线索。本研究以分布于新疆北部天山、塔尔巴哈台山和阿尔泰山的10个种群的9个表型性状为基础, 对比分析了小尺度、局域尺度和区域尺度环境下原生境拟南芥种群表型性状的变化。结果发现, 不同性状对环境变化的反应不同, 其中株高、株重、根重、根长、单个果实重、果实开裂力度在3种环境尺度下种群间的差异均达到极显著水平, 而分枝数、果实长度的种群间变化不显著, 种群间的表型分化系数较低。不同环境尺度下株重、根重、单株果数均表现出一致的协变格局, 反映了生理功能性状之间整合对拟南芥适应环境的重要性。同时, 各种群间整体的性状协变差异性明显, 根长、单个果实重、分枝数、果实长度、果实开裂力度等特征与其他特征协变具有明显的局部性, 局域尺度和区域尺度环境之间的变化较大。聚类分析发现区域尺度上的不同种群聚合在一起的现象非常突出, 进一步表明拟南芥的表型特征受微环境的强烈影响。Mantel检验表明, 小尺度上10个种群株高、株重、根重、单个果实重、果实长度、果实开裂力度6个性状变化存在显著的空间相关性, 而分枝数、根长的相关性却不显著。因此, 我们认为拟南芥表型变化受小尺度环境的影响强烈, 但在表型层面并非所有性状都与原生境气候存在遗传关联。  相似文献   

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天山北部拟南芥生存群落特征及其与环境的关系   总被引:3,自引:1,他引:3  
为了解拟南芥在天山北部的分布状况及环境依赖特点, 分析拟南芥的自然选择特征, 本文对天山北部分布的13个拟南芥(Arabidopsis thaliana)生存群落结构、组成及其与环境关系进行了研究, 并分析了拟南芥与群落主要物种的种间联结性。结果表明: 拟南芥生存的群落结构简单, 其中天山北坡中段的石河子、一四三团、沙湾、独山子地区的8个群落均为草本类型, 优势种相似, 而与伊犁果子沟、额敏和阿勒泰的5个群落差别较大。属的区系成分分析表明世界分布、北温带分布以及地中海、西亚至中亚分布型成分占大多数, 具有典型的地中海旱生植物区系分布特征, 体现了本地拟南芥分布及演化的干旱、半干旱的地理环境特点。采用双向指示种分析(TWINSPAN)将13个群落分为新疆绢蒿–猪毛菜–角果毛茛(Seriphidium kaschgaricum–Salsola collina–Ceratocephalus testiculatus)、新疆绢蒿–猪毛菜(S. kaschgaricum–S. collina)、新疆绢蒿–狭果鹤虱(S. kaschgaricum–Lappula semiglabra)、新疆绢蒿–旱麦草(S. kaschgaricum–Eremopyrum triticeum)、勿忘草–草原苔草(Myosotis sylvatica–Carex liparocarpos)5个群落类型。去势典范对应分析(DCCA)表明纬度、坡向、土壤有机质及pH值是决定天山北部拟南芥种群分布的主导因子。拟南芥分布与群落内其他物种有极强的依赖关系, 与13个群落62个主要物种的种间联结性分析表明, 共有119个正关联性种对, 明显高于72个负关联性种对, 与各群落优势种呈显著正关联。拟南芥种群分布数量在群落间差异较大, 分布于降雨较少的天山中部浅山地带拟南芥种群数量均高于降雨较丰富的天山西部伊犁果子沟地区, 是否发生适应性分化需要深入研究。  相似文献   

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蛋白质的亚细胞定位信息对于深入了解该蛋白质的功能具有重要意义。本文对一个预测的拟南芥叶绿体未知功能基因At4g22890 编码蛋白进行了叶绿体定位研究。我们克隆了该基因5′端长208 bp 的DNA 片段, 与绿色荧光蛋白(GFP) 基因构建重组表达载体pMON530-cTP-GFP, 经农杆菌介导转化拟南芥。转基因植株经激光共聚焦显微镜观察, GFP 荧光仅在叶绿体中观察到, 表明所克隆的DNA 序列编码的多肽能够将At4g22890 编码蛋白质引导进入叶绿体, 由此推测该蛋白质为叶绿体蛋白质。  相似文献   

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Lee WH  Kim YK  Nam KH  Priyadarshi A  Lee EH  Kim EE  Jeon YH  Cheong C  Hwang KY 《Proteins》2007,68(4):1016-1019
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Three distinct editosomes are required for the uridine insertion/deletion editing that creates translatable mitochondrial mRNAs in Trypanosoma brucei. They contain KREPB6, KREPB7, or KREPB8 proteins and their respective endonucleases KREN3, KREN2, or KREN1. RNAi knockdowns of KREPB6, KREPB7, and KREPB8 variably affect growth and RNA editing. KREPB6 and KREPB7 knockdowns substantially reduced in vitro insertion site cleavage activity of their respective editosomes, while KREPB8 knockdown did not affect its editosome deletion site cleavage activity despite inhibition of growth and editing. KREPB6, KREPB7, and KREPB8 knockdowns disrupted tagged KREN3, KREN2, or KREN1 editosomes, respectively, to varying degrees, and in the case of KREN1 editosomes, the deletion editing site cleavage activity shifted to a smaller S value. The varying effects correlate with a combination of the relative abundances of the KREPB6-8 proteins and of the different insertion and deletion sites. Tagged KREPB6-8 were physically associated with deletion subcomplexes upon knockdown of the centrally interactive KREPA3 protein, while KREN1-3 endonucleases were associated with insertion subcomplexes. The results indicate that KREPB6-8 occupy similar positions in editosomes and are important for the activity and specificity of their respective endonucleases. This suggests that they contribute to the accurate recognition of the numerous similar but diverse editing site substrates.  相似文献   

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RNA silencing is a broadly conserved machinery and is involved in many biological events. Small RNAs are key molecules in RNA silencing pathway that guide sequence-specific gene regulations and chromatin modifications. The silencing machinery works as an anti-viral defense in virus-infected plants. It is generally accepted that virus-specific small interfering (si) RNAs bind to the viral genome and trigger its cleavage. Previously, we have cloned and obtained sequences of small RNAs from Arabidopsis thaliana infected or uninfected with crucifer Tobacco mosaic virus. MicroRNAs (miRNAs) accumulated to a higher percentage of total small RNAs in the virus-infected plants. This was partly because the viral replication protein binds to the miRNA/miRNA* duplexes. In the present study, we mapped the sequences of small RNAs other than virus-derived siRNAs to the Arabidopsis genome and assigned each small RNA. It was demonstrated that only miRNAs increased as a result of viral infection. Furthermore, some newly identified miRNAs and miRNA candidates were found from the virus-infected plants despite a limited number of examined sequences. We propose that it is advantageous to use virus-infected plants as a source for cloning and identifying new miRNAs.  相似文献   

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Plastids (chloroplasts) of higher plants exhibit two types of conversional RNA editing: cytidine-to-uridine editing in mRNAs and adenosine-to-inosine editing in at least one plastid genome-encoded tRNA, the tRNA-Arg(ACG). The enzymes catalyzing RNA editing reactions in plastids are unknown. Here we report the identification of the A-to-I tRNA editing enzyme from chloroplasts of the model plant Arabidopsis thaliana. The protein (AtTadA) has an unusual structure in that it harbors a large N-terminal domain of >1000 amino acids, which is not required for catalytic activity. The C-terminal region of the protein displays sequence similarity to tadA, the tRNA adenosine deaminase from Escherichia coli. We show that AtTadA is imported into chloroplasts in vivo and demonstrate that the in vitro translated protein triggers A-to-I editing in the anticodon of the plastid tRNA-Arg(ACG). Suppression of AtTadA gene expression in transgenic Arabidopsis plants by RNAi results in reduced A-to-I editing in the chloroplast tRNA-Arg(ACG). The RNAi lines display a mild growth phenotype, presumably due to reduced chloroplast translational efficiency upon limited availability of edited tRNA-Arg(ACG).  相似文献   

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As in many other organisms, tRNA-derived RNAs (tDRs) exist in plants and likely have multiple functions. We previously showed that tDRs are present in Arabidopsis under normal growth conditions, and that the ones originating from alanine tRNAs are the most abundant in leaves. We also showed that tDRs Ala of 20 nt produced from mature tRNAAla (AGC) can block in vitro protein translation. Here, we report that first, these tDRs Ala (AGC) can be found within peculiar foci in the cell that are neither P-bodies nor stress granules and, second, that they assemble into intermolecular RNA G-quadruplex (rG4) structures. Such tDR Ala rG4 structures can specifically interact with an Arabidopsis DEA(D/H) RNA helicase, the DExH1 protein, and unwind them. The rG4-DExH1 protein interaction relies on a glycine-arginine domain with RGG/RG/GR/GRR motifs present at the N-terminal extremity of the protein. Mutations on the four guanine residues located at the 5′ extremity of the tDR Ala abolish its rG4 structure assembly, association with the DExH1 protein, and foci formation, but they do not prevent protein translation inhibition in vitro. Our data suggest that the sequestration of tDRs Ala into rG4 complexes might represent a way to modulate accessible and functional tDRs for translation inhibition within the plant cell via the activity of a specific RNA helicase, DExH1.  相似文献   

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