【作者】 章松柏；

【导师】 谢联辉； 吴祖建；

【作者基本信息】 福建农林大学 ， 植物病理学， 2013， 博士

【摘要】 水稻（Oryza sativa）是我国的主要粮食作物，病毒病一直是影响我国粮食稳定生产的一个重要问题。近十年来，多种水稻病毒病相继爆发流行，给水稻生产造成了巨大的经济损失，其中由一种新的植物呼肠孤病毒---南方水稻黑条矮缩病毒（Southern Rice black streaked dwarf virus，SRBSDV）引起的南方水稻黑条矮缩病尤为重要。国内外学者对这种新病毒的生物学和分子生物学进行了许多研究，明确了南方水稻黑条矮缩病的地理分布、症状、病原性质、传播途径、品种抗性等生物学特性，并解析了病毒的基因组结构和少数编码蛋白的功能，但是对于病毒在寄主组织的分布和动态变化、多数编码蛋白的功能、病毒的致病机制、寄主抗感病作用机制等仍不清楚。因此，保持对水稻病毒病的监测，了解病毒在寄主体内的分布和动态变化，以及研究病毒与寄主间的互作关系，对于病毒病的防控以及阐明病毒的致病机制等方面具有重要的意义。首先，于2009-2012年间，在湖北、福建、江西、湖南、海南等省进行了水稻病毒病田间流行调查，并承担了这些省份水稻病毒病的诊断和检测任务，在此基础上，分析了水稻病毒病的种类、分布和流行特点。结果如下：首次报道了南方水稻黑条矮缩病毒在湖北水稻和玉米上的发生，以及验证了水稻条纹病毒在湖北水稻产区的存在；福建水稻病毒的种类较多，有SRBSDV、RBSDV(Rice blackstreaked dwarf virus)、RSV(Rice stripe virus)、RRSV(Rice ragged stunt virus)、RDV(Rice dwarf virus)、RGSV(Rice grassy stunt virus)，国内流行的水稻病毒病在福建均有分布；湖南和江西两省是近几年我国水稻病毒病发生的重灾区，病原以SRBSDV为主，RRSV次之；海南省是我国南方水稻黑条矮缩病和水稻锯齿叶矮缩病的重要初侵染来源地。其次，测定了SRBSDV湖北分离物的全基因组序列，并以此序列为基础扩增了病毒13个基因的全长，在本氏烟细胞中分别表达这些基因，观察所表达蛋白的亚细胞定位。结果显示：SRBSDV湖北分离物全长29122bp，基因组核酸S10-S1的登录号分别为HM585270-HM585879；P1、P4、Pns52、P91这4个蛋白主要定位于细胞质，在细胞质中形成聚集体，Pns52、Pns91能形成较大颗粒状聚集体，而P1、P4则形成点状的聚集体；P2、P8、P3、P10这4个蛋白的定位相似，即可定位于细胞核，也可以定位于细胞质，在细胞质中都能够形成一些点状聚集体；P51、Pns6、Pns71、Pns72和Pns92定位相似，分散于整个细胞，定位于细胞质中或膜上，沿细胞骨架等组分形成丝网状结构，Pns6和P51在细胞质中还能够形成颗粒状的聚集体。再次，通过电镜观察、RT-PCR和实时定量PCR技术研究了病毒在水稻组织中的分布和动态变化以及种子各组分的带毒情况。结果显示：SRBSDV只存在于韧皮部细胞中，是一个韧皮部组织限制的病毒；不同生育期的病株种子各组分都可以带毒，成熟种子虽可以携带SRBSDV，但是不能够经过种子传播；在水稻幼苗接种病毒后的30天内，根组织的病毒含量一直大于地上部分组织，病毒含量在12天或14天内增长比较缓慢，26d左右时可达到比较明显的水平；幼苗期根组织内的病毒含量远大于地上部分的叶片和叶鞘，而分蘖期根、叶、叶鞘、茎中的病毒含量差别不大，至扬花灌浆期后茎中的病毒含量远大于其它部位，进一步说明病毒在不同生育期、不同组织中的含量是不同的。最后，以SRBSDV Pns6为诱饵，利用酵母双杂交系统筛选水稻cDNA文库，钓取了24个可能与之互作的水稻蛋白；从中选取8个蛋白，经过RT-PCR获得了其完整阅读框，并将它们重组到酵母载体和BiFC植物表达载体上，通过酵母菌回复互作验证和BiFC体系的荧光互补验证，8个水稻蛋白中有5个能够与Pns6互作，这5个水稻蛋白是40S ribosomal protein S9-2、ubiquitin-conjugating enzyme、AP2domain containing protein、zinc finger protein、eukaryotic translation elongationfactor1A。在此基础上，进一步研究了OseEF1A(eukaryotic translation elongationfactor1A from Oryza sativa)与SRBSDV Pns6互作的意义以及OseEF1A与水稻病毒互作的普遍性，取得了如下结果：（1）SRBSDV Pns6除了与水稻eEF1A互作外，还与自身的基质蛋白Pns91互作，共定位实验表明Pns6可能招募Pns91和OseEF1A形成复合体，从而在病毒基因组复制和增殖中起某种作用；（2）RRSV的Pns10和Pns6都能够与OseEF1A在酵母中互作以及在本氏烟细胞中共定位，说明和SRBSDV一样，RRSV能够利用OseEF1A为病毒基因组复制和增殖服务；（3）除上述两种病毒外，OseEF1A还能够与RGDV的Pns11，RSV的NS3，RGSV的NS1、NS3和PC3等病毒蛋白在酵母中互作，并且还能够与作为DNA病毒的双生病毒相关蛋白在酵母中互作，从而证明了植物病毒利用eEF1A的普遍性。更多还原

【Abstract】 Rice is one of the most important crop plants in China. Viral diseases are a majorthreat to rice production. In the last decade, several rice virus diseases had broken outand caused great harm to agricultural production in China. In these virus diseases,southern rice black streaked dwarf disease, caused by Southern Rice black streakeddwarf virus(SRBSDV), a newly identified species in the genus Fijivirus, becameparticularly important for it had brought huge loss to people in recent years. Thebiology and molecular biology about it were studied by many domestic and overseasscholars. Some biological or molecular biological characteristics of SRBSDV, such asgeographical distribution, symptom, property of pathogen, cytopathology and varietyresistance, genome structure, virus-encoded proteins, were made clear, while thefunctions of most virus-encoded proteins, distribution and change of the virus in thehost, pathogenic mechanism of the virus, and molecular mechanisms of plantresistance and susceptibility to SRBSDV have not been known. Therefore, the studies,which on the surveillance of rice virus diseases, the interaction between viral proteinsand host proteins, the distribution and change of the virus in the host, have importantsignificance for elucidating these questiones.Firstly, in2009-2012, the occurrence and epidemiology of rice virus diseases inFujian, Hubei, Jiangxi, Hunan, Hainan provinces were investigated and the diagnostictests of rice virus diseases were taken for these provinces. Based on these, the virusvarieties, distributive characteristics and epidemiology of rice virus diseases in theseprovinces were analysed.The results were as below: in Hubei province, theoccurrence of southern rice black streaked dwarf disease on rice an maize was firstreported and the existence of rice stripe disease was confirmed; The rice virusdiseases outbreak in domestic had almost all occurred in Fujian province, and thevirus varieties were SRBSDV, RBSDV(Rice black streaked dwarf virus), RSV(Ricestripe virus), RRSV(Rice ragged stunt virus), RDV(Rice dwarf virus), RGSV(Ricegrassy stunt virus); Hunan and Jiangxi provinces were the most seriously regionswhere rice virus diseases, caused by SRBSDV or/and RRSV, were pandemic in recent years; the overwintering virus sources in Hainan were the important primary source ofSRBSDV and RRSV in southern China.Secondly, the genome of SRBSDV Hubei isolate was determined by randomprimer amplification method, and based on the genome, the full-length of13viralgenes were amplified and expressed in cells of Nicotiana benthamiana, respectively.And then the subcellular localizations of virus-encoded proteins were observed byconfocal fluorescent microscopy. The results showed: the genome of SRBSDV Hubeiisolate was29122bp and the accession numbers of segment10to1wereHM585270to HM585879; P1, P4, Pns52, P91could localize in the cytoplasm andform polymers as small mobile particles(P1, P4) or large mobile particles(Pns52, P91);P2, P8, P3and P10could localize both in the cell nucleus and in the cytoplasm asmobile particles; P51, Pns6, Pns71, Pns72and Pns92could localize in the cellcytoplasm and/or on the cell membrane and all form network structures attached oncytoskeletons, Pns6and P51could also form small granular polymers in the cellcytoplasm.Thirdly, the distribution and change of the virus in host were analysed byRT-PCR, electronic microscopy and Real-time PCR. The results showed: SRBSDVonly existed in the phloem cells of rice plants and was a phloem-limited viruse;SRBSDV could exist in every part of rice seeds from infected rice plants at differentstages, but it was not a seed-transmitted virus; The virus volume in roots was morethan leaf sheaths and leaves at young seedling stage, but at tillering stage the virusvolume were a roughly similar in roots, leaf sheaths, leaves and stems, while atflowering and filling stage the virus volumes in different parts was various and that ofmidstems was the most.The results further illustrate that the virus volume was indynamic changes in different part of infected rice plants at different growth stages.Finally, in order to study the interaction between the virus and host plant, Pns6ofSRBSDV was used as bait protein to screen rice Library by yeast two-hybridsystem(YTHS). And24rice proteins were obtained for their possibly interacting wtihPns6.8proteins were selected for further experiments. Constructed YTHS and BiFCvectors of the8proteins and identified their interactions with Pns6by YTHS and BiFC. The results showed that Pns6could interact with5proteins of them as40Sribosomal protein S9-2, ubiquitin-conjugating enzyme, AP2domain containingprotein, zinc finger protein, eukaryotic translation elongation factor1A. Then on thisbasis, eukaryotic translation elongation factor1A from Oryza sativa (OseEF1A) wasselected to study the significance of its interaction with Pns6for virus infection and totest the universality of the interactions between OseEF1A and rice viruses. The resultsshowed:1, SRBSDV Pns6could interact with itslf and with Pns91in yeast, OseEF1Acould co-localize with Pns6, but not Pns91when the two corresponding proteins wereexpressed together in epidermal cells of N. benthamiana. However, when the threeproteins were expressed together, co-localization of OseEF1A and Pns91could beobserved;2, RRSV Pns10and Pns6could also interact with OseEF1A in yeast and inepidermal cells of N. Benthamiana. When OseEF1A and Pns6or Pns10were expressedtogether, co-localization could be observed. It is interesting to note that both RRSV andSRBSDV use viroplasm-associated proteins to interact with OseEF1A. Therefore, theinteraction of viroplasm-associated proteins with OseEF-1A may sequester the riceprotein to sites not accessible by its cellular partners;3, In yeasts, OseEF1A also couldinteract with proteins from other rice viruses such as Pns11of RGDV, NS3of RSV,NS1, NS3, PC3of RGSV and AC4proteins from Clerodendrum yellow mosaic Chinavirus and Tomato yellow leaf curl Guangdong virus. These results confirmed theuniversality of plant virus utilizing eEF1A.更多还原