热处理脱除沙梨离体植株茎尖病毒的分子机理研究

The rate of infection is high for pears trees infected by latent viruses of ACLSV and ASGV in China, which causes a severe reduction yield and quality of pear. The thermotherapy techniques have been successfully applied to obtain virus-free regeneration shoots, but the molecular mechanism of thermotherapy is still unclear, which hinders the high efficiency of elimination of viruses. In the study, The libraries of genes differentially expressed from the tips of in vitro pear shoots during different periods of heat treatment will be constructed by RNA sequencing techniques.The important candidate genes relation to high temperature elimination viruses will be selected by bioinfomatic analysis. The relatioship of the virus accumulation and differential expression patterns of these genes in the tips,central section and bases of shoots during different periods of thermotherapy will be analyzed by real-time PCR and in stiu hybridization. We will transiently express the key candidate genes by implementing the strategies of transformation overexpression vector and RNAi vector in in vitro pear and tobacco transgenic plants and investigate the effect on virus accumulation. In addition, the relationships of virus distribution,the titers of viral RNA and accumulation of siRNA characteristic of RNA silencing in the tips of shoot meristem tissue will be analyzed, meanwhile high-throughput sequencing small RNA from tip tissue during thermotherapy will be further analyzed by quantifying virus-derived siRNA and by evaluating their distribution characteristics along the respective virus genome. These results will clarify the effect of high temperature on gene silencing for RNA virus in tips of shoots. Finally, the performance of the project will reveal the molecular mechanism of thermotherapy elimination viruses in tip meristem tissue of in vitro pear shoots, which will provide theoretical foundation for high efficiency obtaining virus-free germplasm resources of fruit trees plants.

我国种植的梨普遍感染病毒，对其产量和品质有严重影响。国内外大都采用高温脱病毒处理培育无毒植株来控制病毒危害，但有关热处理脱除病毒的分子机理尚不清楚，严重影响了病毒的高效脱除。本项目拟采用RNA-seq技术及生物信息学分析，构建热处理脱除ACLSV和ASGV进程中诱导梨茎尖差异基因表达谱，筛选与病毒脱除相关的基因，推测其作用途径；分析候选基因表达的时空动态特点与病毒累积量变化的关系，通过超量表达和RNAi等策略鉴定所得基因对病毒累积量的影响。同时从另一方面比较分析热处理进程中梨茎尖病毒分布、核酸含量变化和siRNA累积量间的对应关系，测定并分析未处理和热处理条件下梨茎尖产生的来源于病毒的siRNA数量和沉默热点区域特点，明确热处理对梨寄主对茎尖病毒的基因沉默作用有何影响，解析其与茎尖病毒脱除的关系。项目的完成将揭示热处理脱除梨茎尖病毒的分子机理，为高效获得果树无病毒种质资源提供新的理论依据。

我国梨产区种植的梨树普遍感染苹果茎沟病毒（Apple stem grooving virus，ASGV），对产量和品质有严重影响。热处理结合茎尖组织培养是获取无病毒苗木的有效途径，但其分子机理尚不清楚，降低了病毒的脱除效率，制约了果树病毒病害的有效防治。. 本研究优化了提取沙梨植株不同部位核酸提取方法，测定了ASGV-Js2基因组全长。以感染ASGV组培苗沙梨“金水2号”为材料，37℃处理分别1d和5d茎尖提取的tRNA等量混合作为处理组，24℃为对照组，进行RNA-seq和小RNA测序，取得的主要结果为：.1.明确了热处理进程中梨茎尖和基部ASGV-Js2 cp和mp基因随处理时间延长，病毒累积量下降。.2.获得了与高温脱毒相关的531个基因上调表达，252个下调表达。KEGG和GO功能分析差异表达基因均参与寄主代谢途径、激素信号转导途径、植物与病原物互作途径、防御和压力反应，以及植物生长发育。采用qRT-PCR分析并明确了热处理条件下候选基因在茎尖和基部表达模式具有组织表达特异性。分析高温条件下脱毒相关候选基因表达量与病毒累积量间的关系，揭示其高温降低梨寄主体内病毒含量是高温激活了基因表达参与寄主防卫反应途径，抑制了病毒在其寄主体内的复制和移动。筛选到的与病毒脱除相关的重要候选基因，为获得抗病育种的梨基因资源提供了材料。.3.明确了热处理下梨miRNA参与调控高温降低梨病毒浓度。鉴定了37℃处理下，7个保守和77个新的miRNA差异表达基因，其预测的靶基因GO功能分析表明，主要参与代谢途径，生物和非生物压力，信号转导，生长发育等途径。qRT-PCR分析了miRNA和靶基因在茎尖和基部表达具有组织特异性，miRNA负调控的相关抗病防御和信号转导功能的靶基因在梨茎尖为上调表达，推测其靶基因在降低病毒浓度中共同起作用。.4.明确了热处理加强了沙梨寄主对茎尖病毒的基因沉默效应。分析了高温下vsiRNA 21和22nt数量增多，5'末端偏向U，在ASGV上分布不均匀；明确了高温感染ASGV的梨茎尖基因沉默途径的关键基因PpDCL2, PpRDR1和PpAGO1,2上调表达，加强抗病毒的基因沉默效应。. 研究结果揭示了梨miRNA/mRNA以及抗病相关基因，及其基因沉默参与了高温降低病毒浓度的作用，为探究高温下病毒与寄主间的互作提供理论基础。