【作者】 吴官维；

【导师】 洪霓；

【作者基本信息】 华中农业大学 ， 植物病理学， 2014， 博士

【摘要】 由柑橘衰退病毒(Citrus tristeza virus, CTV)引起的柑橘衰退病是影响柑橘生长和产业发展的重要病害。CTV为长线形病毒科(Closteroviridae)长线形病毒属(Closterovirus)的成员,已知存在明显CTV株系分化和多个基因型,目前有关基因型鉴定方法和我国CTV分离物的基因型组成信息仍亟待完善。该病毒具有两个衣壳蛋白组分,即CP和CPm,二者除为结构蛋白外,还具有多种重要的生物学功能。深入研究CP和CPm的遗传变异和抗原特性可为探究CTV的系统进化特点及解析其生物学特性等提供重要信息。本研究对来源于我国部分地区CTV分离物的基因型组成、群体遗传结构以及CP的抗原表位特性进行了深入研究,取得的主要研究结果如下：1.我国CTV分离物的基因型研究以采自湖北和江西两省的10个CTV分离物为对象,采用已报道的11个基因型多重分子标记引物(VTK17、VTPOL、VT-5’、T3K17、T30K17、T30POL、T30-5’、 T36K17、T36POL、T36-5’和B165-LProⅡ)对其进行多重分子标记(MMM) RT-PCR分析,结合扩增产物测序和系统进化分析。结果表明,我国CTV种群中,除了已知的4种基因型(VT、T36、T30和T3),首次鉴定出B165和RB基因型。而且在我国CTV分离物中多种基因型混合侵染的现象较普遍,仅发现三个分离物N4、S4和BX为单一基因型侵染。同时发现,基因组5‘端的分子标记引物特异性较高,可较好区分相应的基因型,而位于POL和K17区域的分子标记特异性较低,不能特异区分VT和T3、VT和T30以及T30和RB基因型。对K17、POL和LPro II区域进行氨基酸多重比对分析的结果表明,存在基因型特异性氨基酸位点。S45-VTPOL序列虽与T36聚为一大支,但与T36相似性仅89.7%。类似地,B15-LProⅡ和B16-LProⅡ序列与B165聚为一支,但与参考分离物B165相似性很低,核苷酸和氨基酸水平分别介于70.0-81.8%和67.1-76.6%,重组分析未从这些序列中检测到重组事件,表明我国CTV种群可能存在新的基因型。此外,发现MMM与标记序列系统进化分析结果存在不一致情况,因此基因型的鉴定需采用MMM-RT-PCR与序列分析相结合。2.CP和CPm基因的遗传多样性和进化特点研究从我国4个主要柑橘产区(湖南、四川、江西和湖北省)收集柑橘叶片或枝条样品共计85份,其中6份湖北样品为已知阳性样品,四川样品均表现衰退症状。RT-PCR检测结果表明,CTV总体检出率为32.8%(26/79),而四川样品CTV检出率达100%(11/11)。采用引物CPm (F)和T36-CP (R)对32份阳性CTV样品进行扩增,从29份样品克隆到了含有部分p61基因、完整CPm和CP基因以及两者中间非编码间隔区的大片段(～1540bp)。序列分析表明各分离物之间和内部均表现较高的遗传多样性。基于1540bp片段核苷酸序列进行系统进化分析的结果表明,我国CTV分离物可以划分为6组,除已报道的5个组群(RB、T30、T36、HA和VT),新鉴定出仅含中国分离物的组群Ⅵ。来自湖南样品的CTV分离物多为RB(克服枳壳抗性)组群,而在VT组群内存在一以中国蚜传分离物AT-1为代表的中国特有亚群,该亚群分离物相比其他所有分离物,P61蛋白C端均特异插入了一个氨基酸位点,将该亚群命名为AT-1。分别采用CP和CPm基因核苷酸序列构建的系统发育树具有类似拓扑结构,但有的组群或分离物在两系统进化树中位置有变化。在CPm系统进化树中,T30基因型组群的分离物聚在RB组群,形成单一亚组RB-d,与其他3个RB亚组的遗传距离较近,介于0.024±0.005-0.037±0.005。对1540bp片段序列进行重组分析,结果表明CPm基因是CTV基因组的重组热点区域之一,在该区域共计检测到25个重组事件,而CP基因内仅检测到2个重组事件。基因漂移分析表明我国与其他国家或地区的CTV分离物存在明显的基因交流,且全球CTV分离物CP群体处于明显扩张趋势；自然选择分析表明CP和CPm基因均处于负向选择压力下；此外,从两个基因内部分别发现新的正向选择密码子,包括位于CPm基因的第9位密码子和位于CP基因的第31、41和68位密码子。以上结果表明基因漂移、基因重组和负向选择压力是我国CTV复杂种群形成的主要原因。3. CTV-AT-1分离物基因组全长的克隆和序列分析AT-1为通过蚜传获得的亚分离物,对其全基因组进行了测序,大小为19,252bp(GenBank Accession No. JQ061137)。 CP Hinf I/RFLP结合系统发育进化分析表明,该分离物属于VT基因型和RFLP第Ⅲ组群,为我国优势组群,且为单一侵染。基因组序列分析表明,AT-1与VT组群的T318A分离物相似性最高,为96.8%,并聚为一支,与属于T36组群的Qaha和Mexico两个分离物相似性最低,均为80.9%。各开放阅读框和两端非编码区相似性分析表明,在5’-UTR和3’-UTR区分别与参考分离物VT和B165的相似性最高,为98.1%和98.9%；在12个开放阅读框中,5’端ORFla和RdRp的核苷酸序列均与参考分离物VT相似性最高,而其氨基酸序列分别与参考分离物T30和T3具有最高相似性；3’端除p6外的9个基因在核苷酸和氨基酸水平上均与B165分离物相似性最高。重组分析进一步确认AT-1为VT和B165的重组子,且重组位点位于RdRp和p33之间。4. CTV CP的抗原表位作图和定点突变分析随机选取10株CTV特异性单克隆抗体,以CTV-S4分离物CP为对象,采用蛋白截短法结合Western blotting分析,鉴定出7株单克隆抗体识别的3个抗原表位区域。其中MAb5和6识别表位区域I (48LGTQQNAALNRDLFLT63), MAb3和7识别表位区域III (114LSDKLWTDVVFN125), MAb1、4和10识别表位区域Ⅱ(97DDDSTGIT104),该区域为新鉴定的抗原表位区域。采用合成多肽(97DDDSTGIT104、97DDDSTGI103和97DDDSTG102)和ELISA分析,进一步确认表位区域Ⅱ为MAb1、4和10的识别区域,且C端氨基酸T104和I103的缺失对表位识别具有显著影响。不同CTV分离物CP基因编码氨基酸多重比对分析表明,该区域高度保守。定点突变分析表明,该区域的突变会一定程度上影响抗体对表位Ⅱ的识别,其中I103M和S100T突变会显著降低MAb1、4和10对表位Ⅱ的识别能力。5. CTV-HB1CP与单克隆抗体互作的关键氨基酸位点的鉴定和表位结构分析采用上述10株单克隆抗体对CTV-HB1病毒粗提液和CP基因原核表达蛋白进行Western blotting分析,结果表明5株单抗(MAb1、4、5、6和10)不能识别。CP基因编码氨基酸序列的多重比对分析表明,位于表位区域Ⅱ的第98位点在除HB1外的所有CTV分离物中高度保守,均为天冬氨酸(D98),而HB1为甘氨酸(G98)。将S4-CP的D98突变为G98, MAb1、4和10不能识别表位Ⅱ,表明D98为这3个单抗识别大多数CTV分离物的重要位点。进一步回复突变(将HB1-CP的G98突变为D98)可以导致这3个单抗重新识别HB1CP,表明G98是HB1CP与这3个单抗互作的关键氨基酸位点。CTV-S4和CTV-HB1两个分离物CP之间的结构预测和比较分析表明,氨基酸位点G98暴露的表面积区域过小可能是不能识别的主要原因。CTV-HB1和CTV-S4在MAb5和6识别的表位区域Ⅰ(aa48-63)的序列相同,结合结构分析,可能构象变化是CTV-HB1CP不能被这两个单抗识别的主要原因。更多还原

【Abstract】 Citrus tristeza virus is the causal agent of citrus tristeza disease, which has been one of the most important diseases affecting citrus growth and industry. CTV, a member of the genus Closterovirus in the family Closteroviridae, possesses a number of distinct and characterized strains and genotypes. Up to now, there is still incomplete understanding of the characterization method of CTV genotype, and the CTV genotype composition in China. CTV has two main structural proteins, major coat protein (CP) and minor coat protein (CPm), and both of them participate in multiple biological functions. Further study on the genetic variation and antigenic characteristics of CPm and CP will provide important information on the CTV evolution history and possible important structural or functional domains in two structural proteins. In this study, the genotype composition of CTV isolates from some regions in China, genetic structure of CTV population in China, and epitopes in CP are studied in depth. The main results are listed as follows:1. Genotype study of CTV isolates from ChinaTotally10CTV-infected citrus samples collected from Hubei and Jiangxi provinces were used for genotype study. By using RT-PCR method with11multiple molecular markers (MMMs)(VTK17, VTPOL, VT-5’, T3K17, T30K17, T30POL, T30-5’, T36K17, T36POL, T36-5’, and B165-LPro II), combined with sequencing of the products and phylogenetic analysis. Our results revealed four known genotypes (VT, T36, T30and T3) in Chinese CTV population, and two genotypes RB and B165were identified for the first time in China. In addition, only three isolates N4, S4and BX were singly infected, while other seven isolates were infected with mixtures of two or more genotypes. The results indicated that the MMMs in5’region were much more specific with the corresponding genotype, while MMMs in POL and K17regions were nonspecific, and could not discriminate between VT and T3, VT and T30, and T30and RB. Multiple alignments of deduced amino acid sequences in K17, POL, and LPro II regions showed genotype-specific amino acids in CTV isolates. Although the sequences of S45-VTPOL clustered with reference isolate T36, the similarity of nucleotide sequence between them was only89.7%. Similarly, the sequences of B15-LPro Ⅱ and B16-LProⅡ clustering with B165showed only70.0-81.8%nt and67.1-76.6%aa identity with the reference isolate B165. Recombination analysis showed no recombinant events in these sequences, suggesting novel genotypes existing in CTV population in China. Our results also found that that the genotype identification results showed somewhat inconsistency when comparing the RT-PCR and sequence analysis results, thus, genotype assignment of CTV cannot be based solely on the amplification profiles of MMMs, and sequencing of MMMs is required.2. Genetic diversity and evolution characteristics of CP and CPm genesTotally85citrus leaf or branch samples (including six CTV positive samples from Hubei province) collected from four main citrus growing provinces (Hunan, Sichuan, Jiangxi, and Hubei) were used in this study, and most of samples from Sichuan showed decline symptoms. RT-PCR results showed that the total detection rate of CTV is32.8%(26/79), while that of CTV from samples in Sichuan was100%(11/11). By using the primer sets CPm (F) and T36-CP (R) with RT-PCR,32CTV-infected samples were further used for the cloning of an approximately1540-bp fragment consisting of partial P61, complete CPm, CP and the internal non-coding region. The fragment sequences from29isolates were obtained. Sequence analysis revealed intra-and inter-isolate sequence diversity. Phylogenetic analysis of the whole fragment nucleotide sequences revealed five known groups (RB, T30, T36, HA, and VT), and one new group VI solely consist of Chinese CTV isolates. Most of isolates from Hunan province belonged to group RB, and one popular and unique CTV subgroup representated by aphid transmissible isolate AT-1in the group VT was found in China, and this subgroup had unique aa insertion in the C-terminus end of P61, here designated as AT-1. The CPm-and CP-based phylograms shared similar tree topologies with fragment-based phylogram except for some groups. In CPm-based phylogram, isolates represented by the mild strain T30formed a subgroup RB-d, which has low genetic distance ranging from0.024±0.005to0.037±0.005with other three subgroups in the group RB. Recombination analyses of the1540-bp sequences revealed CPm gene as the new recombination hot regions across CTV genome, and25recombination events occurring in CPm were detected, while only two recombination events occurred in the CP gene. Our results revealed frequent gene flow between subpopulations from China and other countries, and the CTV subpopulations of CP gene worldwide were in a state of increasing. Analysis of synonymous and nonsynonymous substitutions indicated that both CPm and CP genes are under strong negative selections, and positively selected sites at the position9in CPm, and the positions of31,41, and68in CP were identified in CTV for the first time. Our results suggested that gene flow, recombination, and negative selection could contribute to the complex CTV population in China.3. Cloning and sequence analysis of the complete genomic sequence of AT-1Isolate CTV-AT-1was obtained by aphid transmission, and the complete genomic sequence of AT-1was cloned, sequenced, and deposited in the GenBank with an accession number JQ061137. The AT-1had19,252nt. Hinf I/RFLP analysis of CP gene combined with sequence analysis showed that AT-1was single infection and belonged to the prevalent Hinf/RFLP group III and VT genotype. Complete genome sequence analysis showed that the AT-1had the highest identity to the aphid-transmissible sub-isolate T318A (96.8%) and clustered together in whole genome-based phylogram, while the lowest identity to the isolates Qaha and Mexico (80.91%) belonging to the group T36. Sequence analysis of each gene and two UTRs revealed that AT-1had highest identity to representative isolates VT (98.1%) and B165(98.9%) in5’-UTR and3’-UTR, respectively. In addition, AT-1showed highest identity irrespective of nt and aa level with isolate B165in continuous nine genes except for p6in3’terminal, while in ORF1a and RdRp regions in5’terminal, AT-1had highest nucleotide identity with VT, and amino acid identity with T30and T3, respectively. Recombination analysis further demonstrates that AT-1was a recombinant from two parents VT and B165, and the breaking point was located in the region between RdRp and p33.4. Epitope mapping and site-directed mutagenesis of coat protein of CTVTen monoclonal antibodies were selected randomly to map the epitopes in the coat protein of isolate CTV-S4. By using truncated coat proteins and Western blotting method, we identified three epitope regions recognized by seven monoclonal antibodies. Of them, MAb5and6recognized the epitope I region (48LGTQQNAALNRDLFLT63), MAb3and 7recognized the epitope III region (114LSDKLWTDVVFN125), and MAb1,4, and10recognized the epitope II region (97DDDSTGIT104), which was a novel epitope. The epitope was further confirmed by using synthesized oligopeptides and ELISA method, and the deletion of two amino acids (T104and1103) in the C-terminus of epitope II has significant effect on its reaction with these three MAbs. Multiple alignments of deduced amino acid sequences of CP gene showed that the epitope II region is much conserved in CTV isolates belonging to different groups. Site-directed mutagenesis analysis showed that the natural variations in this region could not abolish the reaction between MAbs and the epitope II, while I103M and S100T variation reduced the reaction signals significantly.5. Identification of the critical amino acids in the interaction between CTV-HB1coat protein and monoclonal antibodies and epitope structure analysisThe reactivity of those10monoclonal antibodies with the crude extracts and expressed coat protein of isolate CTV-HB1was evaluated by Western blotting method. Results showed that five monoclonal antibodies (MAb1,4,5,6and10) could not recognize the CP of CTV-HB1. Multiple alignments of deduced aa sequences of CP gene showed conserved and the same aa site D98in all CTV isolates except for HB1(G98). When the D98in S4-CP was replaced with G98, MAbs1,4, and10could not recognize the mutated epitope II, suggesting the important role of D98in the recognization of CP by these three MAbs. In addition, the interaction between HB1-CP and these three MAbs was recovered when G98in HB1-CP was replaced with D98, indicating that the presence of G98in CP of HB1is crucial for its interaction with them. Further comparative analysis of predicted epitope and structure of coat protein between isolates CTV-S4and CTV-HB1suggested that the less exposed surface area of G98in HB1CP might result in the negative reaction with these MAbs. Our results also suggested that the conformational differences in the epitope I (48LGTQQNAALNRDLFLT63) between the CPs of isolates S4and HB1might contribute to the different reactions of two isolates to MAbs5and6, although the amino acid sequences in the epitope I region was the same between them.更多还原