【作者】 阴筱；

【导师】 李向东；

【作者基本信息】 山东农业大学 ， 植物病理学， 2013， 博士

【摘要】 玉米是我国重要的粮食作物、饲料作物和工业原料。病害的发生给我国玉米生产造成了严重损失，其中粗缩病可导致玉米严重减产，甚至绝产，是玉米上危害最严重的病害之一。引起我国玉米粗缩病的主要是呼肠孤病毒科（Reoviridae）斐济病毒属（Fijivirus）的水稻黑条矮缩病毒（Rice black-streaked dwarf virus，RBSDV）。RBSDV的病原特征、传播途径等生物学特性和基因结构以及功能解析已有所研究，但其分子变异、进化机制和致病机理还不十分清楚。由于田间寄主植物和传毒昆虫的发生数量、带毒率与玉米粗缩病的发生密切相关，了解RBSDV田间寄主，及时检测传毒介体灰飞虱的带毒率，对于制定玉米粗缩病的防控措施具有重要意义；明确影响RBSDV进化的因素，了解病毒在自然条件下的进化趋势，研究病毒与寄主间的互作，可为抗病品种选育及病毒病的可持续控制提供理论指导。本研究建立并优化了RBSDV的检测体系，明确了RBSDV的田间寄主，测定了RBSDV重排体SDZZ10的全基因组序列，从山东玉米上检测到南方水稻黑条矮缩病毒（Southern rice black-streaked dwarf virus，SRBSDV），分析了RBSDV的群体遗传结构，并鉴定了玉米中与RBSDV P8互作的蛋白。具体结果如下：1、针对RBSDV的S10片段设计了4对引物，通过比较不同的Mg2+浓度、Taq DNA聚合酶用量、退火温度、检测引物建立了最佳RT-PCR检测体系：10×PCR buffer2.5μL，25mM MgCl21.5μL，dNTP (each2.5mM)2.0μL，检测引物F4（5’-AGY GAA GAA TTTGTA GGT GTG-3’）和R4（5’-GTT TCA ACA AAT GAC GCT AC-3’）(10μM)各1.0μL，5U/μL Taq DNA聚合酶0.2μL,模板RNA5.0μL，加11.8μL水将体系补至25μL。利用这一体系可以从单头灰飞虱和30ng玉米样品提取的总RNA中检测到病毒的存在，同时从禾本科的牛筋草（Eleusine indica）、野燕麦（Avena fatua）、马唐（Digitariasanguinalis）、稗草（Echinochloa crusgalli）、狗尾草（Setaria viridis）、狗牙根（Cynodondactylon），菊科苣荬菜（Sonchus brachyotus）、醴肠（Eclipta prostrata），苋科的反枝苋（Amaranthus retroflexus）等均检测到RBSDV，首次发现双子叶植物苣荬菜、反枝苋也是RBSDV的自然寄主。2、测定了RBSDV分离物SDZZ10所有可读框（ORF）的序列，与已知的2个RBSDV分离物Hbm和Zjr全基因组序列比较，SDZZ10的大多数ORF与Hbm相应ORF的核苷酸序列一致率更高，其蛋白与Hbm相应蛋白的氨基酸一致率也更高，但SDZZ10的ORF3，ORF4，ORF9-2和ORF10与Zjr相应ORF的核苷酸一致率更高，P4，P9-1和P9-2与Zjr相应蛋白的氨基酸一致率更高。在ORF8和ORF10的系统进化树中，SDZZ10分别属于不同的组，说明SDZZ10是一个自然发生的重排体。3、测定了南方水稻黑条矮缩病毒（SRBSDV）分离物JNi4的S7-S10基因序列。JNi4的S7到S10和RBSDV相应片段的核苷酸一致率分别为72.6-73.1%，72.3-73%，73.9-74.5%和77.3-79%，与SRBSDV HN和GD分离物相应片段的一致率为99.7%，99.1%-99.7%，98.9%-99.5%和98.6%-99.2%。JNi4在根据S7到S10基因组序列构建的系统发生树中和GD、HN形成一个独立的分枝。这些结果证实了SRBSDV作为斐济病毒属一个独立种的观点，证明JNi4是SRBSDV的一个分离物。山东是目前为止发生SRBSDV的最北地区。4、测定了来自山东、江苏和安徽等地水稻和玉米101个RBSDV分离物的S8（S8包含一个开放阅读框ORF8，编码次要衣壳蛋白）和103个分离物的S10（ORF10编码主要衣壳蛋白）的序列。RBSDV的S8和S10基因处于负选择。RBSDV三个分离物的S8和两个分离物的S10存在明确的重组现象。中国RBSDV种群根据S8基因可以分为3个组，而根据S10基因可以分为2个组，分组与分离物的地理和寄主来源之间没有相关性。在同时获得S8和S10序列的85个分离物中有17个是组间重排体，30个是亚组间重排体。不同地区和不同寄主的RBSDV亚种群内和亚种群间基因交流频繁，来自中国玉米的种群处在扩张趋势。未发现RBSDV新谱系。这些结果表明重组、重排、负向选择压力及基因交流是影响中国RBSDV进化的重要因素。5、根据SDZZ10S8序列构建了诱饵载体质粒pGBKT7-S8，利用酵母双杂系统从玉米cDNA文库中筛选与RBSDV P8互作的玉米蛋白。经假阳性排除、序列测定以及在GenBank数据库中blast检索，初步筛选到26种可能互作的蛋白，并进一步证实玉米40S核糖体蛋白S13可以和RBSDV P8互作。为了确定与P8互作的40S核糖体蛋白S13的区域，将其基因平均分为三段（N端、M段、C端）。按照N、M、C、N+M和M+C进行PCR扩增，并将片段正确连入相应载体中构建了5个缺失突变体，分别进行酵母验证和荧光双分子互补验证，结果表明40S核糖体蛋白S13与RBSDV P8互作的区域为N端和C端。更多还原

【Abstract】 Maize is one of the most important food and forage crops and industrial raw materials.Diseases lead to severe losses of maize production in China. Maize rough dwarf disease(MRDD) is devastating and caused significant losses to maize production. As a species of thegenus Fijivirus in the family Reoviridae, Rice black-streaked dwarf virus (RBSDV) can causeMRDD in maize. The biological characteristics and spread of MRDD, genome structure andgene function of RBSDV have been well documented, but its molecular diversity andevolution mechanism of RBSDV remain largely unknown. Because natural hosts in the field,incidence and percentage of viruferous small brown planthopper are closely related tooccurrence of MRDD, quick and efficient detection of RBSDV field hosts and viruferousvector are critical for providing prevention and control measures. Elucidating the factorsaffecting RBSDV evolutio and understanding the interaction between host and viral proteinswill provide theoretical guide for the breeding of resistant maize cultivar to RBSDV and thesustainable control of MRDD.In the study, we established and optimized the detection system for RBSDV, anddetected hosts in the field, sequenced the genome of a RBSDV reassortant, detected SRBSDVfrom Shandong maize, analyzed the genetic structure of RBSDV populations, and identifiedmaize protein interacting with P8. The results were as follows:Firstly, four pairs of primers were designed according to the nucleotide sequences ofRBSDV S10. RT-PCR detection system for RBSDV was optimized after comparing primerpair, concentrations of Mg2+and Taq polymerase and times of RNA dilution. The optimalRT-PCR system was10×PCR buffer2.5μL,25mM MgCl21.5μL, dNTP (each2.5mM)2.0μL, primers F4and R4(10μM) each1.0μL,5U/μL Taq DNA polymerse0.2μL, templateRNA5.0μL, sterilized water11.8μL to make a total volume of25μL. With the methodestablished, RBSDV can be detected from RNA extracted from single Laodelphax striatellusor30ng infected maize leaves. Besides graminceous plants, Sonchus brachyotus andEclipta prostrate from family Asteraceae and Amaranthus retroflexus fromAmaranthaceae are also natural hosts of RBSDV. Secondly, we reported the complete genomic sequences of all the open reading frames(ORFs) in SDZZ10. Comparing with two RBSDV isolates whose complete genomicsequences were available, the most ORFs and corresponding proteins of SDZZ10sharedhigher nucleotide (nt) or amino acid (aa) identities with RBSDV-Hbm; While ORFs3,4,9-2and10of SDZZ10shared higher nt identities with RBSDV-Zjr, P4, P9-1and P9-2sharedhigher aa identities with RBSDV-Zjr. Phylogenetic analyses of ORF8and ORF10showedthat SDZZ10was clustered into different groups, indicating that SDZZ10is a naturalreassortant.Thirdly, we sequencing S7-S10of SRBSDV isolate, JNi4. The S7to S10of JNi4sharenucleotide identities of72.6-73.1%，72.3-73%，73.9-74.5%and77.3-79%, respectively, withcorresponding segments of Rice black-streaked dwarf virus isolates, and identities of99.7%,99.1%-99.7%,98.9%-99.5%, and98.6%-99.2%with those of SRBSDV isolates HN and GD.JNi4forms a separate branch with GD and HN in the phylogenetic trees constructed withgenomic sequences of S7to S10. These results confirm the proposed taxonomic status ofSRBSDV as a distinct species of the genus Fijivirus and indicate that JNi4is an isolate ofSRBSDV. Shandong is so far the norther nmost region where SRBSDV is established.Fourly, we obtained the sequences of101segment8(S8; carries ORF8encoding theminor core capsid protein) and103S10(ORF10encoding the major capsid protein). BothORF8and ORF10are under negative selection. The S8sequences of3isolates and S10sequences of two isolates were ‘clear’ recombinants. The RBSDV population of China can beclassified into three groups according to the S8and two according to the S10sequences,irrespective of their hosts or geographical origins. Among85RBSDV isolates with both S8and S10sequences available,17are between-group reassortants,30are between-subgroupreassortants. The RBSDV subpopulations from different geographical regions and hosts showfrequent gene flow within or between subpopulations. The RBSDV population from maize inChina is in a state of expanding. In this study no new emergent population was detected.Taken together, our results indicate that recombination, reassortment, negative selection andgene flow are important factors that drive evolution of RBSDV in China.Finally, to identify the maize protein interacting with RBSDV P8, a cDNA library wasscreened by yeast two-hybrid system with pGBKT7-S8as bait plasmid which carried theORF8of RBSDV isolate SDZZ10. Totally26maize proteins were obtained as potentialinteracting proteins. We further confirmed that the40S ribosomal protein S13can interactwith RBSDV P8by YTHS and BiFC. To determine the specific interaction region, we dividedthe sequence of the40S ribosomal protein S13to three regions (N, M and C), and constructed five deletion mutants. According to the results of YTHS and BiFC, we concluded that the40Sribosomal protein S13interacted with P8via its N-and C-termini.更多还原