【作者】 林林；

【导师】 吴云锋； 陈剑平；

【作者基本信息】 西北农林科技大学 ， 植物病理学， 2009， 博士

【摘要】 葱属植物上病毒病发生普遍,严重影响产量和品质。开展病毒分子生物学研究是了解病害发生过程、制定有效防治策略的基础。本文中,我们首先对引起陕西洋葱黄条症状的病毒进行了鉴定,采用马铃薯Y病毒属(genus Potyvirus)简并引物结合该病毒特异引物扩增并测定了其基因组cDNA全序列(AM267479);在了解其基因组结构特点的基础上,利用酵母双杂交系统对该病毒及其同属病毒大豆花叶病毒半夏分离物(SMV-P)各编码产物间相互作用关系进行了细致分析;同时,对该病毒P3蛋白与寄主蛋白组分的互作关系也作了较为深入地研究。结果表明,该病毒基因组全序列由10427个核苷酸组成,含有一个编码3, 340氨基酸的开放阅读框。序列分析表明,该病毒与胡葱黄条病毒ZQ2分离物(SYSV-ZQ2)高度相似,核苷酸同一性为93.2%,多聚蛋白的同一性为97.5%,由此推测该病毒为SYSV的洋葱分离物,命名为SYSV-O。这是SYSV侵染洋葱的首次报道。蛋白裂解位点分析发现,除NIb/CP裂解位点外,SYSV-O与SYSV-ZQ2的裂解位点完全一致(NIb/CP裂解位点分别为VSYQ/A和VSYQ/V)。对SYSV分离物基因组3’-末端部分核苷酸序列的系统进化树分析表明,SYSV分离物形成4个明显的群体:印度尼西亚胡葱分离物成簇为一个群体,日本大葱、薤、分葱分离物和一个印度尼西亚大葱分离物成簇为一个群体,中国杭州分葱分离物和中国章丘大葱分离物(ZQ2)成簇为一个群体,中国杭州大葱分离物、章丘大葱分离物(ZQ1)和陕西洋葱分离物成簇为一个群体。分离物间的进化与其寄主植物及地理分布有显著相关性。酵母双杂交技术是研究蛋白质互作的有效工具,在目前Potyvirus属病毒的研究中,人们已经利用该技术发现了一些病毒编码蛋白的互作,然而对于Potyvirus属病毒编码的全部蛋白之间存在怎样的互作关系还不明确。本文对此开展了部分工作,绘制了两种Potyvirus属病毒(SYSV-O和SMV-P)编码蛋白间的互作图谱。结果表明,SYSV-O P1蛋白可以自身互作,同时与P3、6K1、CI、VPg、NIa-pro、CP存在互作;Hc-Pro可以自身互作,同时与P3、NIa-pro、VPg、CP互作;P3蛋白可以自身互作,还可与除CP之外的所有病毒蛋白互作;6K1和6K2都可以和P3、VPg、NIa-pro互作;CI存在自身互作,还与P1、P3、6K2、VPg、NIa-pro互作;VPg可以自身互作,同时与除NIa-pro和CP之外的其他蛋白都互作;NIa-pro可以自身互作,也和除了VPg之外的其他蛋白互作;NIb也可自身互作,同时还与VPg、NIa-pro和CP互作;CP蛋白可自身互作,还可与P1、Hc-Pro、CI、VPg、NIa-pro、NIb互作。SMV-P编码蛋白的互作与SYSV-O有相同之处,但也有其特有的互作。这些互作中很多都和以前在Potyvirus属成员中的研究结果相一致,但是6K1、6K2与其他蛋白的互作以及P1、P3、NIb的自身互作均为首次发现。病毒编码蛋白之间的互作有方向性,这种互作的方向性可能是不同载体在酵母细胞中有各自喜好的折叠方式或暴露的结合位点不同而造成的。其中两个病毒中都存在而且在不同的方向都能检测到的互作只有6K1/NIa-pro组合。检测到3组两个病毒的HC-Pro和对方病毒编码蛋白的互作:SMV-P-HC-Pro/ SYSV-O-NIa-pro, SYSV-O-HC-Pro/ SMV-P-NIa-pro, SYSV-O-HC-Pro/SMV-P-NIb,认为这种不可能的互作是由于两个病毒同类编码蛋白之间的同源性而产生的。SYSV-O与同属洋葱黄矮病毒(OYDV)基因组结构相似,也编码一个大的P3蛋白,然而目前对于其功能尚不清楚。本文利用酵母双杂交技术筛选到P3的寄主互作蛋白,初步分析了P3可能的功能及作用机制。结果表明,SYSV-O P3与洋葱1,5-二磷酸核酮糖羧化酶/加氧酶(Rubisco)大亚基(rbcL)具有强烈的互作反应,Rubisco是植物光合作用中的关键酶,说明P3蛋白可能通过影响寄主植物的光合作用参与症状表现过程。克隆洋葱rbcL全长基因后,利用酵母双杂交系统和免疫共沉淀试验进一步证实了两者间的互作。缺失突变体分析发现,rbcL N端结构域是与P3互作的关键区域,而P3的互作区域可能位于膜外部分的N端。在其他编码蛋白和其他病毒上的试验表明,OYDV-P3和SMV-P-P3也可与rbcL互作,并且SYSV-O的8个蛋白(除了6K1和CP)和SMV-P的7个蛋白(除了6K1, CI和CP)与rbcL也存在互作,表明potyvirus属成员的P3蛋白与rbcL的互作关系是普遍存在的;potyvirus属病毒的多种编码蛋白可能都不同程度地参与了寄主症状的表达。更多还原

【Abstract】 Virus diseases of Allium are common and seriously affect both quality and yield. Molecular biology techniques are essential to detect and identify the pathogens. In this work, the complete nucleotide sequence of a potyvirus infecting onion in Shaanxi Province was determined (AM267479) using universal primers for members of the family Potyviridae and further specific primers based on the sequence determined. Using these data the interactions between the 10 proteins encoded by the virus were investigated using the yeast two hybrid system (YTHS). For comparison, the same study was also carried out on another potyvirus, an isolate of Soybean mosaic virus from Pinellia ternata (SMV-P). Moreover, the interaction between the P3 protein and the host proteins has been also studied. The complete cDNA of this potyvirus was 10,427 nucleotides in length excluding the poly(A) tail. It contains a single large open reading frame and was predicted to encode a polyprotein of 3,340 amino acids. BLAST showed that the sequence of Shallot yellow stripe virus SYSV-ZQ2 was related to it mostly. They had 93.2% nucleotide identity and 97.5% amino acid identity. Obviously, the virus isolated from onion in Shaanxi is an isolate of SYSV, named SYSV-O, and this provides the first report that SYSV can infect the onion. The proteolytic cleavages sites of the SYSV-O ten mature proteins were the same as those of SYSV-ZQ2 except NIb/CP, which are VSYQ/A and VSYQ/V, respectively. Phylogenetic analysis of the 3′-terminal nucleotide sequences of SYSV isolates confirmed the existence of four distinct groups. One is the isolate from Indonesia shallot, and the other includes the isolates from Japan Welsh onion, rakkyo bunching onion and Indonesian Welsh onion. China Hangzhou bunching onion isolates and Zhangqiu isolates make up the third group. The last group includes Chinese Hangzhou welsh onion isolates, Zhangqiu welsh onion ZQ1 isolate and the new Shaanxi onion isolate. The evolutionary relationship between different isolates was significantly correlated with their geographical origin and their host plant species.YTHS has been an effective and powerful tool for studying potyvirus protein functions, and some interesting interactions between the viral proteins have previously been found. However, it is still not clear what interactions occur amongst all ten of the potyvirus proteins. In these studies we have mapped the complete set of interactions between the proteins of SYSV-O and SMVP. The results showed that: P1 protein can self-interact and interact with P3, 6K1, CI, VPg, NIa-pro, CP; HC-Pro can self-interact and interact with P3, NIa-pro, VPg, CP; P3 can self-interact and interact with all of the other viral proteins except CP; the two 6K proteins can interact with P3, NIa-pro, VPg; CI can self-interact and interact with P1, P3, 6K2, NIa-pro, VPg; VPg can self-interact and interact with all of the other viral proteins except NIa-pro and CP; NIa-pro can self-interact and interact with all of the other viral proteins except VPg; NIb can self-interact and interact with VPg, NIa-pro, CP; CP can self-interact and interact with P1, HC-Pro, CI, VPg, NIa-pro, NIb. The interaction of SMV-P proteins is similar to those of SYSV-O in many aspects, but some differences occurred. Although many of these interactions are identical to those in previous reports, there are some new findings including the 6K interactions with the other viral proteins and the self-interactions of P1, P3 and NIb. Many interactions showed directionality, suggesting that proteins fused in one direction may have more favourable protein folding or exposure of binding sites than those in the other direction. A potentially significant and novel finding in our experiments was that the interaction between 6K1 and NIa-Pro are consistently strong in both directions for both viruses. This activity was retained in a slightly truncated form of the SMV-P protein 6K1. We also detected interaction of three (AD/BD) combinations SMV-HC-Pro/SYSV-NIa-Pro, SYSV-HC-Pro/SMV-NIa-Pro and SYSV-HC-Pro/ SMV-NIb from the 38 combinations.The genomic structure of SYSV-O is similar to that of another potyvirus, Onion yellow dwarf virus (OYDV). Both contain a large P3 protein, whose function is not yet clearly understood. Using SYSV-O P3 as bait, we screened for interacting proteins of onion. The results showed that the Ribulose-1, 5-bisphosphate carboxylase/ oxygenase (RuBisCO) large subunit (rbcL) interacts with P3 of SYSV-O strongly. Such interaction was further confirmed by co-immunoprecipitation assay in vitro. RuBisCO is the key protein of photosynthesis and its carboxylase/oxygenase activity center is located on the rbcL. This implies that the P3 protein is involved in symptom expression through interfering with its host plant’s photosynthesis. Studies using deletion mutants revealed that the N-terminal of SYSV-P3 and OYDV-P3 was involved in the interaction with the N-terminal of rbcL (137aa). It is predicted P3 is a trans-membrane protein and the outer-membrane part consists of the interaction domain. The P3/rbcL interaction also occurs in OYDV and SMV-P, and rbcL can interact with eight proteins of SYSV-O (except 6K1 and CP) and seven proteins of SMV-P (except 6K1, CI, CP). These results showed that the interaction between P3 of Potyvirus and rbcL is common, and that many other potyvirus proteins may also be involved in symptom development.更多还原