【作者】 杜振国；

【导师】 谢联辉； 吴祖建；

【作者基本信息】 福建农林大学 ， 微生物学， 2011， 博士

【摘要】 在植物及多种真核生物中,基因沉默是一种保守的抗病毒机制。为对付这种防御机制,大多数植物病毒及一些动物病毒编码一或多个基因沉默抑制子。水稻是我国最重要的粮食作物之一。病毒病一直是威胁水稻生产的一个重要因素。近年来,水稻锯齿叶矮缩病毒(Rice ragged stunt virus, RRSV)和南方黑条矮缩病毒(Southern rice black-streaked dwarf virus, SRBSDV)的流行给我国水稻生产造成了严重损失。本实验克隆了RRSV和SRBSDV两种病毒非结构蛋白全长ORF,并分别将它们连接到pPZP212或pEarleyGate 100载体,将所得质粒分别转化到农杆菌(Agrobacterium tumefaciens ) EHA105,然后分别与携带有35S-GFP的农杆菌混合,注射本氏烟16c。在注射后第3天,所有注射区域均出现了较强的绿色荧光。但只有在共注射35S-RRSVS6加35S-GFP或35S-SRBSDVS6加35S-GFP的区域中,荧光强度一直持续到第7天。另外,在共表达RRSV S6突变体加35S-GFP的区域中,荧光强度从第3天开始出现下降,到第7天基本消失。在注射后第7天,GFP的mRNA在共表达RRSV的Pns6和35S-GFP的区域中大量积累,而此时,在这些区域中,GFP对应的siRNA基本检测不到。把RRSV的Pns6连接到马铃薯X病毒(Potato virus X,PVX)上后,该蛋白能显著增强PVX的致病性。从这些结果中,本实验推断RRSV和SRBSDV的Pns6具有基因沉默抑制子活性。进一步的分析表明RRSV的Pns6不能抑制由双链GFP诱导的沉默。并且,在缺失一段对核酸结合必须的区域后,Pns6不再具有抑制子活性。在本氏烟(Nicotiana benthamiana)叶片表皮细胞中表达时,RRSV的Pns6主要定位于细胞周围,并形成一些可区分的小点。本实验室已有研究结果表明RRSV的Pns6可能为一运动蛋白。Pns6的多功能性促使我们以其为诱饵,运用酵母双杂交实验进行了水稻cDNA文库的筛选。对经过筛选而得到的阳性克隆,本实验进行了测序,序列分析表明,至少有44个水稻蛋白可能与RRSV的Pns6存在着互作。本实验对这些蛋白按GO注释进行了分类,结果表明这些蛋白在功能上具有着很大的多样性。本实验室先前的研究表明,水稻条纹病毒(Rice stripe virus, RSV)的p2可以与水稻的SGS3 (OsSGS3)互作。并且在RSV侵染的水稻中,5个可以被TAS3产生的反式作用小干扰RNA识别的生长素响应因子基因在表达上出现了上调,这表明RSV的侵染可能影响了水稻的反式作用小干扰RNA途径。本实验开展了一些旨在进一步了解p2与OsSGS3互作的生物学意义的研究,并得到以下结果:1)RSV的p2是一基因沉默抑制子,它能抑制由单链GFP诱导的基因沉默并能显著增强PVX的致病性。2)在RSV侵染的水稻中,生长素相关基因并没有发生一致的上调,这表明在本实验室先前的研究中,反式作用小RNA靶标基因上调是特异的。3)p2缺失N端的40个氨基酸后,仍能和OsSGS3互作,但缺失了C端20个氨基酸的p2不能和OsSGS3互作。4)本实验制备了OsSGS3的多克隆抗体。5)本实验构建了过表达OsSGS3的转基因水稻植株,单管单苗接种实验表明,与野生型水稻比,过表达OsSGS3的转基因水稻对RSV表现出较强的抗性。这些结果对我们进一步认识RSV与水稻的互作有着重要的意义。更多还原

【Abstract】 RNA silencing is an evolutionally conserved antiviral mechanism in plants and some other eukaryotes. As a counter defense, most plant and some animal viruses encode one or multiple silencing suppressors. Rice is one of the most important crop plants in China. Viral disease constitutes one major threat to rice production. Rice ragged stunt virus (RRSV), which is the type species of Orayzavirus, and Southern rice black-streaked dwarf virus (SRBSDV), a newly proposed species of Fijivirus, are two rice viruses that have caused great yield losses to rice production in China in recent years.In this study, the ORFs of the non-structural proteins of RRSV and SRBSDV were cloned and ligated into the vectors pPZP212 or pEarleyGate 100 respectively. The plasmids, which were named 35S-RRSVS6 , 35S-RRSVS7, 35S-RRSVS10, 35S-SRBSDVS6 , 35S-SRBSDVS7 and 35S-SRBSDVS9 respectively, were individually transformed into the Agrobacterium tumefaciens EH105. The transformants were each mixed with a strain of Agrobacterium carrying the construct 35S-GFP and the mixtures were inoculated into leaves of Nicotiana benthamiana 16c. All innoculated leaf patches showed strong green fluorescence at 3 days post inoculation (dpi), but only in patches co-infiltrated by 35S-RRSVS6 plus 35S-GFP or 35S-SRBSDVS6 plus 35S-GFP did the fluorescence intensity maintained until 7dpi. However, the green fluorescence declined in leaf patches coexpressing GFP and mutant forms of the ORF of RRSV or SRBSDV S6 at 3 dpi and disappeared at 7dpi. In addition, in leaf patches co-infiltrated with 35S-RRSVS6, but not 35S-RRSV△S6 plus 35S-GFP, the accumulation of GFP mRNA was high and that of GFP siRNA was barely detectable at 7 dpi. When cloned into PVX, pns6 of RRSV could greatly enhance the pathogenicity of the chimeric virus. We concluded from these results that the pns6 of RRSV and SRBSDV have silencing suppressor activities. Further analyses showed that RRSV pns6 could not inhibit the silencing induced by dsGFP and its silencing suppressor activities were abolished when a region involved in nucleic acid binding in this protein was deleted.Previously, we have found that the pns6 of RRSV might be a movement protein. The multi-functional nature of this protein promoted us to search possible host proteins that could interact with it. To this end, a rice cDNA library was screened using yeast two hybrid experiments with RRSV pns6 as bait. A number of positive clones were obtained and sequence analysis of the cDNA inserts resulted in the identification of 44 candidate rice proteins that might interact with RRSV pns6. These proteins were classified according to their GO annotations. The results revealed that they might be involved in a wide range of cellular processes. In addition, the cellular localization patterns of the pns6 of RRSV were observed by transiently expressing it in leaf cells of Nicotiana benthamiana. The results showed that the Pns6 of RRSV mainly accumulated in the cell periphery, forming distinct and punctate small dots there.Previous studies of this lab showed that p2 of RSV could interact with SGS3 of rice. In RSV infected rice, the expression of five ARFs that were targets of TAS3 derived tas-siRNAs was upregulated, indicating that RSV infection resulted in the alteration of the tas-siRNA pathway in rice. Further experiments were conducted in this study to explore the biological implications of the interaction between RSV p2 with OsSGS3 and the following results were obtained: 1) p2 of RSV is a silencing suppressor. It could suppress the silencing induced by sense GFP in N. benthamiana 16c and could greatly enhance the pathogenicity as well as accumulation of PVX in N. benthamiana. 2) Several auxin related genes exhibited downregulation in RSV infected rice. This indicated that the increasing in expression of auxin related genes was not a general response of rice to RSV infection, confirming the specificity of the elevated expression of the ARFs targeted by tas-siRNAs detectd by our lab previously. 3) It was shown that the region encompassing the N-terminal amino acids 1-40 is dispensable for the interaction between p2 and OsSGS3. However, deletion of the 20 amino acids located on the C terminus of p2 abolished its interaction with OsSGS3. 4) Polyclonal antibodies to OsSGS3 were prepared. 5) Transgenic rice overexpressing OsSGS3 was obtained. The inoculation experiments in this study using one tube-one seedling method indicated that the transformants showed enhanced resistance to RSV compared with wild type rice.更多还原