我国部分地区柑橘衰退病毒基因型研究

Study on Genotypes of Citrus Tristeza Virus from Some Citrus Cultivation Regions in China

作者: 专业:植物病理学 导师:洪霓 年度:2010 学位:硕士  院校: 华中农业大学

Keywords

Citrus tristeza virus, RT-PCR, strains, genotype, sequence analysis

        柑橘衰退病为柑橘上发生危害最严重的病毒病,在世界主要柑橘产区均有发生。引起该病害的病原柑橘衰退病毒(Citrus tristeza virus,CTV),属于线形病毒科(Closteroviridae),长线形病毒属(Closterovirus)。根据其在寄主植物上所引起的不同症状,可分为速衰株系、茎陷点株系和弱毒株系。根据CTV基因组序列特点,可将其分为T36,VT,T3和T30基因型,其中T30为弱毒株系,其它为强毒株系。为明确我国发生的CTV基因型特点及其混合发生状况,本研究对来自我国部分地区的CTV进行了分析,结果如下。1.比较分析了以总RNA为模板的常规RT-PCR、免疫捕捉RT-PCR(IC-RT-PCR)和试管捕捉RT-PCR(TC-RT-PCR)3种方法进行CTV检测及株系区分的效果,结果显示,3种方法检测CTV均具较好的检测效果,但采用总RNA为模板时在进行常规检测及株系鉴定时,扩增产物的电泳条带均相对更清晰些;其它两种方法的扩增产物电泳条带虽相对较弱,不适合于用Bi-RT-PCR方法进行CTV株系的鉴定,但可用于大量样品的CTV检测。2.采用Bi-RT-PCR技术,对来自不同地区的141份柑橘样品进行了CTV强毒和弱毒株系的鉴定。结果显示,有107份柑橘样品的CTV检测结果为阳性,带毒率为75.8%,且所检测到的CTV均为强毒株系。3.选取10份柑橘样品,对其CP进行了扩增及扩增产物的HinfⅠ/RFLP分析,根据Gillings(1993)限道的分类规则,初步确定来自样品S45的CTV(CTV-S45)为HinfⅠ/RFLP类群V,LR1、S36、S37、S38和S39存在类群Ⅲ和Ⅴ的CTV混合感染,而样品S46为我国新鉴定出的类群Ⅷ与类群Ⅴ的混合侵染。4.选取以上8个CTV感染的柑橘样品及本研究室前期鉴定获得的2个CTV单一分离物感染的柑橘样品,采用10对基因型鉴定引物对其进行基因型分析,结果显示,除N21为VT基因型、N4为T30基因型CTV单一感染外,5个样品B15、B16、S36、S39和LRl存在基因型为VT、T36和T3的CTV混合感染;BX与S40存在4种基因型VT、T36、T3和T30的CTV混合感染;CTV-S45可能带有T3和T30基因型。这些结果表明,CTV强致病性株系VT和T3型在我国发生普遍。5.对采用引物VT-K17扩增产物进行了克隆及序列分析,结果显示,所测定分离物可较为明显地分为VT基因型与T30基因型两大类群。其中来自样品S45的2个分离物CTV-S45-2和CTV-S45-4与弱毒株系T30和T385亲缘关系较近,其扩增片段的核苷酸和氨基酸同源性分别在96.6%和94.9%。其它分离物与VT基因型聚为一类,而在该基因型类群中存在较大的分子变异,这些分离物可进一步分为3个组群,不同组群间存在特异性的核苷酸和氨基酸位点差异。6.对CTV-S45的CP、K17和Pol基因及5’UTR作进一步测序和序列分析,发现其CP基因编码氨基酸序列与T30同源性较高为97.3%,但在强毒和弱毒株系的标志性氨基酸位点(第63、122和124个氨基酸位点),CTV-S45与强毒株系相一致,分别为T、V、F。而在K17和Pol基因及5’UTR区域,则表现强毒和弱毒株系混合侵染的特点,推测可能存在不同株系间的基因重组或为一较特殊的分离物。
    Citrus tristeza is found in worldwide citrus production areas, which is among the most servere virus disease. The pathogen Citrus tristeza virus (CTV) is a member of genus Closterovirus, family Closteroviridae.Now, majorly three CTV strains, including quick decline, stem pitting and mild strains have been identified based on the virus-induced symptoms on host citrus plants. Four CTV genotyps T36, VT, T3 and T30 also can be identified based on the divergence of their genome sequence. Among these genotypes, T30 is a mild strain and others belong to servere strains. In order to understand the strain and genotype constitution of CTV population from China, CTV infected samples were collected from some citrus production areas in China and analysed. The results are as followings.The efficiency of three RT-PCR methods, including a universal RT-PCR using total RNA as temple, imuno-capture RT-PCR (IC-RT-PCR) and tuber-capture RT-PCR (TC-RT-PCR), were comparied for the detection and strain differentiation of CTV.Results showed that the virus could be efficiently detected by all these three RT-PCR methods. However, the amplified products of RT-PCR using total RNA as temple had more intensive electrophoresis bands. Both IC-RT-PCR and TC-RT-PCR were not good for the strain differentiation by Bi-RT-PCR.Totally 141 citrus samples were collected and Bi-RT-PCR was used for the identificaion of CTV strains.Result showed that 107 citus samples were positive for CTV, among 75.8%.All detected CTV isolates belonged to the severe strains.The CTV CP genes were amplifed and the obtained products were subjected to HinfⅠ/RFLP. Based on the grouping rules from Gillings(1993), CTV-S45 was identifed as HinfⅠ/RFLP groupⅤ. Samples LR1,S36, S37, S38 and S39 were mixed infected by CTV isolates belonging to HinfⅠ/RFLP groupsⅢandⅤ. The CTV-S46 was a mixture of CTV isolates belonging to HinfⅠ/RFLP groupsⅧandⅤ.The genotypes of the CTV isolates from the above samples and 2 previously reported isolates were analysed by RT-PCR using 10 sets of primers targeted to the specific gene regions of different CTV genotypes.Results showed that only N21 and N4 were belonged a single VT genotype and T30 genotype, respectively. All others were mix-infected by CTV isolates belonging to more than two genotypes.Among these samples, B15、B16、S36、S39 and LR1 were mix-infected by CTV isolates belonging to VT、T36 and T3 genotypes, BX and S40 could be mix-infected by CTV isolates belonging to four genotypes VT, T36, T3 and T30.The isolate CTV-S45 showed the genotypes of T3 and T30. The obtained results indicates that the infection of CTV genotypes VT and T3,which are servere strains, are common in cultivted citrus in China.The amplified products usinng the primer set VT-K17 were cloned and sequenced. The phylogenectic tree constructed based on their nucleotide sequences showed that all these CTV isolates were clustered into two groups, belonging to VT and T30 genotype. Two isolates CTV-S45-2 and CTV-S45-4 from sample S45 were clustered into a branch with mild strain T30 and T385,which shared 96.6%(nt) and 94.9%(aa) identities. All other isolates were clustered into large branch with the sereve strain VT and three sub-groups were visualized in the brach due the high divergencities among these isolates. Sequence comparison also illustrated some sub-group specific nucleotide acid and amino acid sits.Forthermore, the genes CP, K17 and Pol and 5’UTR from the CTV-S45 were sequenced. Sequence analysis results showed that the deduced amino acid sequnce of CP gene had a high similarity with T30, the CP marked amino acid sites (T63, V122 and F124), which were used for the indentificatiom between CTV mild and sereve strains, were, the same as those from sereve strains. However, its K17 and Pol and 5’UTR regions showed the types from both mild and sereve strains. These results indicated that gene recombination between different genotypes might occourred or it was an isolate with specific molecular characteristics.
        

我国部分地区柑橘衰退病毒基因型研究

摘要6-8
Abstract8-9
缩略语表10-11
1.文献综述11-19
    1.1 CTV的国内外研究进展11-17
        1.1.1 CTV的寄主范围11
        1.1.2 CTV的传播方式11
        1.1.3 CTV的基因组分子生物学特性11-13
        1.1.4 CTV的株系分化研究13-15
            1.1.4.1 不同CTV株系在指示植物上产生的症状差异13
            1.1.4.2 CTV不同株系的CP基因特点13-14
            1.1.4.3 CTV不同株系的基因组分化特点14-15
        1.1.5 CTV的防治研究15-17
            1.1.5.1 MSCP防治16-17
            1.1.5.2 抗CTV基因工程17
    1.2 研究目的与意义17-19
2.材料与方法19-25
    2.1 研究材料19-20
        2.1.1 样品来源19
        2.1.2 试剂19
        2.1.3 引物合成19-20
    2.2 研究方法20-25
        2.2.1 RNA提取20-22
            2.2.1.1 总RNA提取20-21
            2.2.1.2 dsRNA提取21-22
        2.2.2 PCR扩增22-23
            2.2.2.1 RT-PCR22-23
            2.2.2.2 免疫捕捉RT-PCR23
            2.2.2.3 试管捕捉RT-PCR23
        2.2.3 CP基因RFLP分析23
        2.2.4 PCR产物回收、转化及测序23-25
            2.2.4.1 PCR产物回收23-24
            2.2.4.2 载体连接24
            2.2.4.3 热激转化24
            2.2.4.4 PCR法筛选阳性克隆24-25
        2.2.5 序列分析25
3. 结果与分析25-48
    3.1 三种方法检测CTV效果比较25-26
    3.2 CTV强毒和弱毒株系的鉴定26-28
        3.2.1 Bi-RT-PCR鉴定CTV强毒和弱毒株系结果26-27
        3.2.2 CTV CP基因扩增产物的HinfⅠ/RFLP分析结果27-28
    3.3 不同CTV基因型分析28-48
        3.3.1 多重分子标记分析(MMM)28-32
        3.3.2 VTK17扩增片段的核苷酸及氨基酸序列分析32-41
        3.3.3 CTV-S45样品的基因型分析41-48
4. 总结与讨论48-50
参考文献50-60
附录60-62
致谢62
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