【作者】 刘文文；

【导师】 王锡锋；

【作者基本信息】 中国农业科学院 ， 植物病理学， 2013， 博士

【摘要】 水稻条纹病毒（Rice stripe virus，RSV）是纤细病毒属（Tenuivirus）的代表种，引起的水稻条纹叶枯病是温带和亚热带稻区最重要的水稻病害之。在自然条件下，RSV主要是依靠介体灰飞虱以持久增殖的方式在寄主植物间进行传播，介体灰飞虱的传播是水稻条纹叶枯病暴发成灾的主要原因，因此对介体灰飞虱传播RSV机制的研究具有重要意义。病毒在介体昆虫内的运动，复制和扩散除了需要自身的外壳蛋白（CP）外还需要借助于介体蛋白，通过与介体蛋白的互作从而克服介体昆虫体内的传播障碍，最终到达唾液腺，并随唾液的分泌到达寄主植物，来完成自身的传播。本研究利用酵母双杂交核系统和分离泛素膜系统对RSV与灰飞虱之间的互作进行了研究。先后构建了两个用于筛选蛋白质相互作用发生在细胞核内和细胞质内的灰飞虱cDNA文库。首先以RSV CP和SP为诱饵对酵母双杂核系统cDNA文库筛选并得到3个介体蛋白，其中肌动蛋白与RSV CP互作，假设蛋白与RSV SP互作，还有种属于RR2家族的表皮蛋白与RSV CP和SP都互作。随后以RSV CP为诱饵对酵母双杂膜系统cDNA文库进行了筛选，获得114个介体蛋白，进步利用该系统对其中17个蛋白与RSV CP的互作进行验证，明确了12个与其互作的介体蛋白，分别为核糖体相关膜蛋白，含有NAC domain的蛋白（NAC），钙转运ATP酶，突触囊泡蛋白，硒蛋白T，Jagunal，G蛋白受体，核糖体蛋白L13，卵黄蛋白原（Vg），40S核糖体蛋白S28，Atlastin，RR1家族的表皮蛋白（NCuP）。最后通过化学发光免疫共沉淀技术明确了与RSV CP互作的5个介体蛋白从强到弱依次为NCuP，Atlastin，Jagunal，Vg和NAC。以酵母双杂得到的NCuP和Vg序列为基础，通过5’ RACE方法得到了两个基因的全长，分别登录到NCBI，登录号为KC485263和KC469581。利用免疫荧光共聚焦显微镜技术明确了NCuP不仅与RSV CP可以在Sf9细胞内结合，还可以与RSV病毒粒子在灰飞虱细胞系和血细胞结合。通过对灰飞虱不同组织NCuP的ELISA检测，表明该蛋白在血淋巴中含量最高。随后本研究运用了RNAi技术沉默了NCuP的表达，发现灰飞虱RSV含量下降了65%并且传毒率也下降了40%，这表明NCuP与RSV的结合保护了病毒不被免疫系统降解，说明未与NCuP结合的RSV在血淋巴中被降解或不能复制，导致病毒量下降而影响了传毒效果。同时还利用免疫荧光共聚焦显微镜技术明确了Vg与RSV病毒粒子可在灰飞虱卵巢的生殖区内完全共定位，而在卵母细胞内几乎没有共定位，由于灰飞虱的卵巢是端滋型营养方式，推测RSV是通过与Vg在卵巢外结合被Vg的受体带入到了卵巢的生殖区，然后病毒与Vg分开，经由营养丝直接进入到了卵母细胞内，随着卵母细胞进而传递给后代。更多还原

【Abstract】 Rice stripe virus (RSV), a typical member of the genus Tenuivirus, causes rice stripe disease,which is one of the most serious rice diseases in subtropical and temperate regions of Asia. RSV ismainly transmitted by the small brown planthopper (SBPH)(Laodelphax striatellus) in apersisent-propagative manner. Transmission is a critical step in the infection cycle of every virusbecause it controls dispersal in space and time, thus directly influences its epidemiology. SBPH,especially in high density, can cause damage to rice plants when sucking the sap. Even at a much lowerdensity, it can lead to more significant disease epidemics and yield losses because of virus transmission.Therefore, it is important to study the transmission mechanism of RSV by SBPH. The viral componentsinvolved in these interactions are relatively well established. Virus coat protein (CP) plays a major rolefor the invasion of various insect vector tissues and hence for the successful infection of the salivaryglands and subsequent introduction of viruses into plants.In this study, two cDNA library of SBPH were constructed using yeast two hybrid GAL4systemand split-ubiquitin yeast membrane system. These two systems are used to studying the proteininteractions occured in the nucleus and cytoplasm respectively. Firstly, a SBPH cDNA library of yeasttwo hybrid GAL4system was screened by RSV CP and SP as bait proteins. Three proteins of SBPH,actin, one hypothetical protein of unknown function and a cuticular protein which belongs to RR2family, were obtained. Out of three proteins, actin interacted with RSV CP, hypothetical proteininteracted with RSV SP and a cuticular protein interacted with both RSV CP and SP respectively.Seondly, a SBPH cDNA library of split-ubiquitin yeast membrane system was screened by RSV CP as abait protein. One hundred and fourteen proteins were obtained as putatives interactors and17proteinswere chosen to confirm the interaction with RSV CP using yeast membrane system. Twelve proteins ofSPBH were confirmed and they are ribosome associated membrane protein, protein with NAC domain(NAC), Calcium-transporting ATPase, Synaptic vesicle protein, Selenoprotein T, Jagunal, G-proteincoupled receptor, Ribosomal protein L13, Vitellogenin (Vg),40S ribosomal protein S28, Atlastin andNovel cuticular protein (NCuP) which belong to RR1family respectively. Finally, five proteins ofSBPH were identified the interactions with RSV CP by CO-IP assay as an independent confirmationanalysis. Different efficiency of protein interactions from strong to weak were NCuP，Atlastin，Jagunal，Vg and NAC.Based on the screening sequences from yeast two hybrid，the full-length cDNA of NCuP and Vgwere acquired by Rapid Amplification of cDNA Ends (RACE). Two cDNA sequences of NCuP and Vghad been deposited in GenBank under accession no. KC485263and no. KC469581. NCuP couldcolocalized not only with RSV CP in Sf9cells, but also with ribonucleoprotein particles (RNPs) of RSVin the cell line and hemocytes of SBPH by fluorescent confocal microscopy. This demonstrated thatNCuP was involved in specific interactions that formed virus-vector combinations in the insect vectors.To learn more about NCuP, we investigated its expression levels among different organs of L. striatellusby ELISA. The results showed that the quantity of NCuP expression was higher in the hemolymph. To reveal the function of NCuP in virus transmission in the vector, we utilized a powerful strategy offunctional study, insect RNAi. The introduction of NCuP dsRNA into L. striatellus by microinjectionwas performed. The quantity of RSV in the SBPH silenced the expression of NCuP by RNAitechnology was65%lower than that of control groups and the virus transmission efficiency wasreduced40%. The declining quantity of virus demonstrated that NCuP could protect the virus fromdegradation in the hemolymph. Some persistent propagative virus could move from the intestine tohemolymph and be transported to the salivary glands. Colocalization of Vg and RNPs of RSV wasobserved in the germarium of SBPH ovarioles. Thus, VgR mediated endocytosis might haveoccasionally incorporated RSV encapsulated in Vg into the germarium of SBPH ovarioles and the RSVparticles then spread into the oocytes through nutritive cord.更多还原