【作者】 王利平；

【导师】 王国平； 洪霓；

【作者基本信息】 华中农业大学 ， 植物病理学， 2009， 博士

【摘要】 本研究对来源于中国的砂梨和加拿大温室与作物加工研究中心国家种质资源圃苹果和西洋梨的苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus,ACLSV)分离物和核果类上的樱桃绿环斑驳病毒(Cherry green ring mottle virus,CGRMV)分子变异进行了较系统的研究,明确了苹果褪绿叶斑病毒和樱桃绿环斑驳病毒的分子变异情况以及进化上的亲缘关系,为苹果、梨和核果类病毒的发生、防治奠定了基础。栽培无病毒种苗是减轻这些病毒危害的重要途径,热处理技术也广泛应用于无病毒果树种质培育中。为探索适合梨病毒的脱毒处理条件,建立更加有效的脱毒技术,本研究分析了热处理对病毒在离体植株中分布的影响,结果表明高温降低茎尖病毒浓度,对基部无影响,这种作用的不均匀性是否触发了热处理的某些抗性机制仍不清楚,进而以热处理和正常培养砂梨离体植株为研究体系,构建cDNA差减文库,筛选热处理诱导下与病毒互作的相关基因,深入分析热处理对病毒生活史及植物基因表达解析热处理脱除机制。取得的主要研究结果如下:1.本研究采用生物学法和RT-PCR对加拿大温室与作物加工研究中心国家种质资源圃核果类110个样品检测,结果表明8个样品感染CGRMV,并首次发现了CGRMV侵染李寄主。对扩增的8个CGRMV分离物的CP基因编码的氨基酸进行序列分析,结果表明CP基因变异很大,分离物之间的氨基酸同源性为90～100%,在进化亲缘关系上CGRMV分离物可以分为两个组群。2.加拿大温室与农作物加工研究中心的国家种质资源圃梨和苹果四种主要病毒的检测结果,采用DAS-ELISA对采集的438份苹果和122份梨样品进行四种主要病毒ACLSV、苹果茎沟病毒(Apple stem grooving virus,ASGV)、苹果茎痘病毒(Aple stem pitting virus,ASPV)和苹果花叶病毒(Apple mottle virus,ApMV)进行检测,结果表明,病毒复合感染现象普遍,其中ACLSV发生最普遍,其检出率达40%左右,梨样品严重感染了ASPV和ApMV,带毒率高于90%。以总RNA为模板,在检测体系中引入nad5基因作为内标的多重RT-PCR对17份苹果样品的四种主要病毒进行复检,均能扩增四种病毒的特异性目标片断,检测效果较ELISA好。3.ACLSV3′端基因的分子变异研究,以ACLSV-6860/ACLSV-7536为引物,采用改进的CTAB法提取总RNA为模板的RT-PCR对来源于加拿大22个ACLSV分离物和采用TC-RT-PCR法病毒粗提液为模板对来源于中国24个ACLSV分离物的3′端基因进行扩增、纯化、克隆和测序。序列测定表明,目标片断大小为680bp左右,该序列包含3′端CP基因(500nt,占CP核苷酸序列的85%)和3′端NCR序列。对加拿大22个ACLSV分离物的扩增片断进行了序列分析,结果表明,22个ACLSV分离物的核苷酸序列同源性为84～100%,其中11个分离物序列同源性为100%。参照报道来源于不同国家不同寄主的ACLSV分离物的序列进行进化树分析,加拿大ACLSV分离物分为2个组群,分离物Malus0422、Malus0908和Malus0422与来源日本P205位于同一组群,属于P205型,CP基因编码的氨基酸具有Ala14-Va133-Phe49-Ser104-Met158的保守序列特点,其余19个分离物位于另一个组群的两个不同分支,属于B6型,CP氨基酸具有保守序列Ser14-Leu33-Tyr49-Thr104-Leu158的特点。对来源于中国砂梨的20个分离物和来源于苹果和桃子上的4个分离物与Genbank上登录的来源于不同国家和不同寄主的分离物序列进行同源性比较和系统进化树分析,本研究的24个ACLSV分离物属于B6型,进化树分析分为两个组群,两组群之间苷酸和氨基酸同源性为86.3～87.5%和94.0～96.4%。扩增片断为686bp、来源于砂梨的的XSJ,SMJ,QX,PL,CL,QYS,GY和苹果C-AP之间同源性为95～99%的8个分离物,亲缘关系较近在进化树中归为为同一组群;扩增片断为680bp,来源于砂梨ZY,JS1,ZSZ,HL1,JQ,FS,SY BY 61-7-7,CX,CS和1个桃子C-P之间同源性为96～100%的12个分离物与来源于砂梨的XG、HL2和2个桃子(C-XA和C-HB)之间同源性为95～99%,位于另一个组群的两个不同进化分支。采用PCR-SSCP技术对中国砂梨的9个样品进行了ACLSV分子变异分析,结果表明同源性高于98%位于同一组群的的ZY、ZSZ、HL1、FS、JS1、JQ分离物的SSCP电泳为带型一致的两条弥散带型,位于另一组群同源性为99%的C—XA和HL2分离物的SSCP电泳为泳动带型一致的两条弥散带型。对分离物ZY、JQ和CL多个克隆进行序列分析,结果表明,ACLSV每个分离物由不同分子变种组成,其中有一个优势变种;优势变种和非优势变种序列间仅几个碱基差异,相似性高达99～100%,说明分离物内部存在遗传变异,ZY和JQ两个分离物的克隆序列进行多重比对,结果表明两个分离物的优势变种的序列同源性为100%。4.热处理对梨离体植株中ACLSV和ASGV分布的影响,本研究对比组织印迹杂交的两种化学显色方法检测病毒核酸产生的杂交信号强度,CDP-Star底物化学发光检测产生的杂交信号强于NBT/BCIP底物化学显色,但信号相对较弱的NBT/BCIP底物化学显色,既具有常规杂交技术的特异性,又可以直接观察病毒在梨离体植株中的分布状况,进而采用组织印迹结合化学发光显色法对热处理过程中ACLSV和ASGV在砂梨离体植株自茎尖到基部每隔0.5mm横切面的动态分布变化进行分析,结果表明,热处理50d后病毒浓度开始降低,植株茎尖无病毒核酸分布,茎干中部病毒核酸也明显下降,而茎干基部仍产生强的杂交信号,根据杂交信号的有无判断,可以观察到在茎尖2mm无杂交信号,表明获得了茎尖2mm的无毒植株,对耐热性高的ASGV病毒,在茎尖0.5mm无杂交信号,得到了茎尖0.5mm无ASGV区域,因此采用组织印迹对热处理材料进行病毒分布分析,直接提供热处理50d后切0.5mm和2mm茎尖继代离体培养可以有效脱除ASGV和ACLSV的信息。5.热处理诱导寄主基因差异表达,以37℃恒温热处理和正常培养的带有ASGV和ACLSV的黄花梨离体培养植株为研究体系,利用抑制差减杂交技术(SSH)构建了热处理诱导寄主上调和下调基因文库。通过反向Southern斑点杂交技术对两个差减文库进行筛选,经过DNA测序并在已知的核酸与蛋白质数据库中比对分析,发现两个文库中共有149个unigene差异表达,其中热处理诱导寄主上调表达的基因63个,下调表达的基因86个。在149个EST片段中,50%尚不能推断其功能,其余50%涉及抗病防御、转录、信号转导、能量、代谢、蛋白质合成等信号途径,揭示了病毒与寄主互作是一个复杂的多基因参与的过程。本研究首次利用SSH杂交技术构建了热处理诱导下热处理正、反相调节梨离体植株差异表达基因文库,筛选得到了病毒与寄主互作相关的基因片段,为认识热处理脱除病毒分子机制奠定基础。更多还原

【Abstract】 In this study we analysed Cherry green ring mottle virus(CGRMV) isolates CP gene sequences and Apple chlorotic leaf spot virus(ACLSV) isolates 3’ UTR and partial CP gene sequences of pears from Canada and China in order to know CGRMV and ACLSV molecular variability characters and the phylogenetic relationship,which established the foundation to prevent virus spread.One of most effective measures for controlling these virus diseases is utilizing certified healthy propagation materials. Thermotherapy is the most widely used technique for the production of virus-free plant materials.Higher virus elimination efficiency and the virus distribution features in in vitro pear plants during thermotherapy were analyzed in this study.The results showed that high temperature decrease the virus titers on meristerm tip,and have no effect on the base,whether it is relative to the host defence mechanism is still unknown.In vitro pear plants infected with ACLSV and Apple stem grooving virus(ASGV) were further used as materials for heat treatment and for construction of cDNA libraries and screening the genes related to host-virus interaction under thermotherapy.The virus life cycle and the host genes expression were further analyzed which will be helpful to know the mechanism of elimination of viruses from hosts.Seeking results are listed as followings:1.A survey was conducted to evaluate the sanitary status of stone fruit tree collections in the Canadian Clonal Genebank(CCG),at the Greenhouse and Processing Crops Research Center(GPCRC) in Harrow Ontario(Canada).Samples collected from clones of 110 cultivars,including 28 sweet cherry,36 sour cherry,12 hybrids and 34 plum accessions were bud-grafted onto indicator seedlings of P.serrulata ’Kwanzan’ for virus indexing,symptoms with epinasty and/or rusty necrotic fragments of midrib, suggestive of the response of Kwanzan to infection by CGRMV,were observed on indicator plants inoculated with samples from 8 clones[1 sweet cherry,1 cherry plum (P.besseyi×P.hortulana) and 6 plum].Amplicons of the expected size of 948 bp were consistently produced from 8 samples showing symptoms of CGRMV infection by One-step RT-PCR.To our knowledge,this is the first report of CGRMV infecting plum in North America.The phylogentic analyses 8 Canadian CGRMV isolates of sequences and NCBI available ones(including their origin and species when available),clustered the Canadian isolates in two groups that could be indicative for two different introduction events.2.A survey of apple(Malus domestica) and pear(Pyrus communis) viruses was carried out at the Canadian Clonal Genebank(CCG) during the fall/winter of 2007 and spring of 2008.In total,438 accessions of apple and 122 of pear were tested using randomly collected samples of leaves and/or dormant cuttings and processed using Double Antibody Sandwich - Enzyme Linked Immunosorbent Assay(DAS- ELISA). All samples were tested by ELISA for the presence of ACLSV,Apple stem pitting virus(ASPV),ASGV and Apple mosaic virus(ApMV).Specific infection rates of viruses of apples were ACLSV(48.1%),ASGV(10.0%),ASPV(6.6%) and ApMV (7.1%),and of pears were ACLSV(42.6%),ASGV(0%),AS PV(91.8%) and ApMV(90.1%).Seventeen of the apple accessions were also tested by multiplex RT-PCR and the results proved that RT-PCR is more sensitive than ELISA3.One-step RT-PCR and TC-RT-PCR using primers of CLS6860 and CLS7536 were used to amplify 22 ACLSV isolates in Canada and 24 ACLSV isolates in China.The PCR products were purified、cloned and sequences.The size of cloned fragment was 680nt corresponding to 3’ CP gene and 3’ NCR region.The nucleotide homology was 84%～100%among the 22 ACLSV isolates whereas the homology of amino acid was 91%～100%.The homology of nucleotide was 100%among the isolates of MAL0427, MAL0270,MAL0537,MAL0375,MAL0577,MAL0844,MAL0976,MAL0107, PYR0206,PYR0112,and PYR0129.The phylogenetic tree analysis at the nucleotide and amino acid level clustered the Canadian isolates in two groups.One group including of Malus0170、Malus0422 and Malus 0908 ACLSV isolates,belonged to the P205 type,the others belonged to B6 type.Meanwhile the sequence datas obtained from the 24 ACLSV isolates in China in the study were compared with previously reported sequences of ACLSV from different sources.The homology and pylogenetic tree results showed that the 24 isolates were divided into 2 groups,the homology of nucleotide and deduced amino acid sequences of 3’ gene were 86.3～87.5%and 94.0～96.4%,respectively.The similarities of nucleotide and deduced amino acid sequences among XSJ,SMJ,QX,PL,CL,QYS,GY and C-AP were 95～99%and 98～99%,respectively,which belonged to the same group. Meanwhile among the 16 isolates,12 isolates of ZY,JS1,ZSZ,HL1,JQ,FS,SY,BY, 61-7-7,CX,CS and C-P,had 99～100%homology and 4 isolates of XG、HL2、C-XA、C-HBP,had 95～99%homology,which belonged to the same group of two different branches.The molecular variability of ACLSV within 9 samples was analyzed by the PCR-SSCP.The results showed that the SSCP profile of ZY、ZSZ、HL1、FS、JS1、JQ was of the same type,the SSCP profile of C-X A and HL2 was of the another type.Sequence of JQ and ZY isolates were analyzed,it showed that ACLSV isolates were composed of a population of genetically related variants,the predominat haplotype had very tiny variation with other haplotypes within each population,only 1～10 bases variation was observed and the similarity was more than 99%among them.The sequence analysis showed that the homology of the predominant sequence is 100%between JQ and ZY isolates.4.Comparison of the results visualized by either chemical stain or X-ray film exposure showed that relatively more intense signals and less background were produced by X-ray film exposure than chemical stain for both ASGV and ACLSV.Therefore,the X-ray film exposure is more suitable for the detection of viruses with a very low titer in plant tissues.These results indicated that both ASGV and ACLSV were present in the whole plants,but the highest viral RNA titer was found in phloem parenchyma of vascular bundles in all cross sections.Tissue-printing hybridization(TPH) was used to detect ACLSV and ASGV in thermotherapy-treated in vitro cultured pears at the tip, intermediate stem and base levels,and to evaluate the effect of the tip length on the efficiency of virus elimination.It was found that both ASGV and ACLSV were present at high concentrations in the base and tip of the plants,and were present at much lower concentrations in the intermediate stem.Only tip tissues of 2mm and 0.5 mm long were ACLSV and ASGV-free,respectively,therefore,the use of tips length of 0.5mm and 2mm are recommended for improving the efficiency of virus elimination for pear meristem-tip culture.These results will be helpful for improving the efficiency for virus elimination by thermotherapy.5.Two differentially expressed genes libraries were constructed,a library of up-regulated genes and a library of down-regulated genes in virus-infected in vitro pear(pyrus proflia) were prepared by suppression subtractive hybridization(SSH). Inserts of 1200 randomly selected colonies from the forward-subtractive library and of 1000 colonies from the reverse-subtractive library were identified with the reverse southern dot-blot screening method.Sequencing results showed that 63 genes were showing up-regulated expression and 86 genes were showing down-regulated expression among 149 ESTs under heat-treatment.The function of about 52%gene fragments was unknown,while the rest 48%had putative function clarifying as disease defense,transcription,signal transduction,energy,metabolism and protein synthesis.The results indicated that the process of virus-host interactions was complex and many genes could participate in the process.In this research,we first reported the differentially expressed genes of in vitro pear under thermotherapy, which established the foundation for understanding the molecular mechanisms of virus elimination from in vitro pear plants.更多还原