【作者】 吴建国；

【导师】 谢联辉； 李毅；

【作者基本信息】 福建农林大学 ， 植物病理学， 2010， 博士

【摘要】 水稻矮缩病毒(Rice Dwarf Virus,RDV),属于呼肠孤病毒科(Reoviridae)、植物呼肠孤病毒属(Phytoreovirus),是水稻普通矮缩病的病原,广泛分布于日本、朝鲜、菲律宾、尼泊尔等东南亚水稻种植区,并且在我国南方水稻产区普遍发生,并有不同程度的流行,给我国水稻生产造成严重损失。利用Affymetrix水稻全基因组芯片检测RDV侵染抗病水稻品种宜香2292(YX229)后基因表达的差异,其结果显示:水稻被RDV侵染后22 d,其基因变化倍数大于1.5倍,p-value小于0.05的表达差异显著的基因有856个,其中上调的基因为838个,下调的为18个;其中,大量的核仁和核糖体相关基因被诱导。对具有明确的基因功能注释的581个差异表达基因,按其基因功能划分为14类,其中胁迫防御相关基因占13.77%,转录因子占9.98%,代谢相关基因占9.29%,信号转导相关基因占9.29%,蛋白质降解相关基因占8.43%,蛋白质生物合成相关基因占6.20%,转座及反转座子蛋白/RNA沉默路径中的相关基因占5.51%,细胞壁相关基因占5.16%,内源激素相关基因占2.41%,分泌路径相关基因占2.07%,运输相关基因占1.38%,细胞周期和细胞骨架相关基因分别占1.03%,而没有明确功能分类的基因居多,为24.44%。通过融合GFP表达实验,证实RDV编码的运动蛋白Pns6是一个膜定位蛋白,主要定位于细胞膜以及核膜和胞间连丝上;Pns10编码的RNA沉默抑制因子,其定位形式和Free eGFP相似,主要定位于细胞质;RDV Pns11是一个双定位信号分子,主要定位于细胞核和叶绿体。同时以烟草核仁基因Fibrillarin为Marker基因,根据RDV Pns11氨基酸序列结构特点,对Pns11的定位形式进行了进一步研究,结果表明:RDV Pns11是一个核仁定位信号分子(Nucleolus localization signals, NoLS),能够与Fibrillarin共定位,且RDV Pns11的核定位信号(Nuclear location signal, NLS)是Pns11定位细胞核和核仁所必需的。对RDV Pns11NLS的缺失突变体的定位研究揭示,Pns11 NLS中的碱性氨基酸对核仁定位是至关重要的,其中NLS中132-144位8个碱性氨基酸“-RK----------KKGGKK-”和Pns11 NLS的C-端的6个碱性氨基酸“-RKSKRK-”是Pns11的核仁定位信号(NoLS),即是RDV Pns11定位核仁所必需的。为了深入研究RDV Pns11与核仁或核仁相关基因之间的关系,以水稻和烟草核仁基因所编码的Fibrillarin蛋白作为RDV Pns11潜在的靶蛋白,通过酵母双杂交和双分子荧光互补试验揭示,RDV Pns11可能与Fibrillarin蛋白存在弱的相互作用;TRV病毒诱导Fibrillarin基因沉默试验表明:Fibrillarin基因沉默后烟草植株发育迟缓,顶端优势基本散失,植株矮化,新叶扭曲畸形和白化,这与RDV侵染水稻后所产生的病害症状存在一定的相似性,这暗示RDV侵染寄主后,可能通过改变核仁相关基因的表达或与其互作,从而诱发一系列病毒病害症状的产生。以Fibrillarin基因沉默后的烟草植株为材料,研究揭示Fibrillarin基因的沉默不会影响RDV Pns11的定位;然而,RDV Pns11的RNA沉默抑制活性可能依赖于核仁蛋白-Fibrillarin,推测Pns11可能与Fibrillarin或Fibrillarin基因下游的某些基因存在互作。水稻矮缩病毒和水稻瘤矮病毒(Rice gall dwarf virus,RGDV)同属植物呼肠孤病毒属成员,且RDV Pns11与RGDV Pns12在氨基酸序列结构上存在22%的相似性。利用农杆菌共浸润的方法发现RGDV Pns12蛋白是该病毒的第二个RNA沉默抑制因子。Pns12能够抑制正义链RNA诱发的局部沉默,但不能抑制由双链RNA诱发的局部沉默,在异源病毒表达载体PVX中,Pns12能够增强PVX对本氏烟草的致病性。其结果表明,Pns12蛋白为RNA沉默抑制因子,可能作用于RNA沉默路径中dsRNA形成的上游,这与RDV Pns11具有RNA沉默抑制功能的研究结果是一致的。此外对RGDV Pns12亚细胞定位结果也显示,RGDV Pns12和RDV Pns11的定位形式也是相似的,主要定位于烟草表皮细胞的细胞核和核仁中,推测该基因可能在细胞核或核仁中发挥功能。更多还原

【Abstract】 Rice dwarf virus (RDV), which belongs to the genus Phytoreovirus in the family Reoviridae, is one of the most widespread and disastrous rice-infecting viruses causing great yield reduction in southeast Asian countries, such as Japan, Korea, Philippines and Nepal. Rice seedlings of the moderately resistant variety YX-2292 were collected at 22 days post inoculation (dpi) for transcriptome analysis (using the GeneChip Rice Genome Array of Affymetrix). The results showed that a total of 856 genes were RDV-responsive, in which 838 genes were upregulated and 18 downregulated, by applying SAM (Significant Analysis of Microarray) software with the criteria of fold changes>1.5; false discovery rate (q-value) <0.05 to screen significantly differentially expressed genes. Interestingly, these studies showed that a great number of nucleolar and ribosomal genes was affected. Among the 856 RDV-responsive genes, only 581 have gene annotations. To gain further insight into the functions of RDV-responsive genes, these genes were classified into 14 categories: including unclassified for genes with no annotated function (24.44%), stress/defense related genes (13.77%), transcription factor (9.98%), signal transduction (9.29%), metabolism (9.29%), protein fate (8.43%), protein synthesis (6.20%), transposon/ retrotransposon protein / RNA silencing pathway genes/possible genome stress induced genes (5.51%), cell wall function (5.16%), phytohormone related(2.41%), secretory pathway (2.07%), transporter/chanel (1.38%), cytoskeleton or cytosjeleton associated (1.03%) and cell cycle related (1.03%).Tansient expression after fusion with eGFP showed that Pns6, the movement protein of RDV localized mainly in cell membrane and formed punctate sites reminiscent of plasmodesma. Pns10, a silencing suppressor of RDV, has a diffuse pattern of localization, which resembles free eGFP to localize in cytoplasmic. Pns11 was shown to be a dual targeted protein, which could be found in both the chloroplasts and the nuclei. The nuclear localization of Pns11 was further studied. The results showed that Pns11 colocalized with fibrillarin, which is often usd as a marker gene of the nucleolus. This suggested that Pns11 was a nucleolus localized protein. Futher studies showed that a nuclear location signal located in Pns11 was required for the nucleolar localization of Pns11. Deletion mutants of Pns11 were created and their localization characterized. The results indicated that the basic amino acids of Pns11 NLS play an essential role in Pns11 location. The 8 basic amino acids“RK----------KKGGKK”, 132-144 site, and the C-terminal six basic amino acids “-RKSKRK”in the Pns11 NLS are the Pns11 nucleolus localization signals (NoLS), which is necessary for Pns11 nucleolus location.To further study the relationships between Pns11 and the nucleolus or nucleolus related genes. Possible interactions between the rice and tobacco fibrillarin and RDV Pns11 were explored by using yeast two hybrid experiments and BiFC.The results of BiFC suggested that Pns11 might weakly interact with fibrillarin. Fibrillarin of Nicotiana benthamiana was silenced by using VIGS. The fibrillarin-silenced N. benthamiana showed phenotypes including loss of apical dominance reduced plant size and dwarfism, delayed development distortion and albinism of newly developed leaves. These, to some extent, resemble symptoms caused by RDV, suggesting that RDV might induce the development of symptomes through the alteration of nucleolar genes expression or through interaction with nucleolar components. The fibrillarin-silenced N. benthamiana was used to further characterize Pns11. The results showed that the absence of fibrillarin had no effects on the nucleular localization of Pns11. However, the silencing suppressor acctivities of Pns11 might depend on the presence of fibrillarin. It is likely that Pns11 may interact with fibrillarin or some other genes downstream of fibrillarin.Rice gall dwarf virus (RGDV) and RDV both belong to the genus Phytoreovirus. RGDV Pns12 has 22% sequence identities with RDV Pns11. In this study, we showed that Pns12 of RGDV exhibited silencing suppressor activity in coinfiltration assays with the reporter green fluorescent protein (GFP) in transgenic Nicotiana benthamiana line 16c. Pns12 of RGDV suppressed local silencing induced by sense RNA but had no effect on that induced by dsRNA. Expression of Pns12 also enhanced Potato virus X pathogenicity in N. benthamiana. Collectively, these results suggested that RGDV Pns12 might function as a virus suppressor of RNA silencing (VSR) that target an upstream step of the dsRNA formation in the RNA silencing pathway. Further, we showed that Pns12 localized mainly in nucleus and nucleolus in tobacco epidermal cells, which is similar to RDV Pns11.更多还原