苹果轮纹病致病真菌Botryosphaeria dothidea中致病基因的分离和鉴定

Apple ring rot severely damaged apple production in China. But till now, the mechanism for this disease formation is still not clear. In this project, we want to isolate the pathogenesis gene which played critical role for apple ring rot development from its pathogenetic fungal Botryosphaeria dothidea. Research in plant pathology demonstrated that a tremendous gene expression change in microbe accompany their infection process. Most of them are related with pathogenesis and disease occurrence. Previous report showed that there is an obvious morphology difference of the filament between that of the B. dothidea grown in PDA medium and that isolated from the ring rot tumor in the apple shoot. Based on this finding, we hypothesis that there must be a significant gene expression change that contributes to the morphology change while related with the B. dothidea pathogenesis closely. So, at first, we are going to employ SSH (Subtractive suppression hybrid) method to identify the differentially expressed genes between the fungal grown in PDA medium and that from the apple ring tumor in the shoot.And then,we will adopt RACE-PCR to amplify the cDNA full length of that differentially expressed genes.Thirdly, based on the sequence information, we will use RNA interference strategy to suppress the expression of those differentially expressed genes identified in our research to investigated whether they played critical role for B. dothidea pathogenesis. This research will shed light on the discovering the mechanism implied in this so important apple disease.

苹果轮纹病是我国苹果产区主要病害，其发病机理一直不清楚。本研究拟从苹果轮纹病病原真菌Botryosphaeria dothidea中分离对轮纹病发生中具有重要作用的致病基因。植物病理学研究表明，病原微生物侵入植物后，病原微生物中的致病基因被大量诱导表达。前人研究从表型表明：轮纹病枝干病瘤中分离的菌丝体直径是生长于PDA培养基中的4－6倍。形态的变化暗示着基因表达的巨大差异，而且，很可能某些表达发生重大变化的基因与病原菌的致病性有关。本研究以此为依据，首先采用抑制消减杂交法研究在PDA培养基上的菌丝体和从轮纹病病瘤中分离出的菌丝体中在表达上具有重大差异的基因；然后采用RACE-PCR获得差异表达基因cDNA全长；最后，根据序列全长，在轮纹病病菌中应用RNA干扰技术抑制该基因的表达，研究该差异表达的基因在轮纹病发病中的作用。本研究将为苹果轮纹病发病分子机理研究提供基本资料。

苹果轮纹病致病真菌Botryosphaeria dothidea的致病机理一直不清楚，分离与鉴定轮纹病致病菌的致病基因，探索该病菌对苹果免疫与抗病反应的干扰机制不仅具有重要的科学意义，而且对苹果抗病品种培育具有重要应用价值。研究发现：乙烯是轮纹病致病真菌在苹果果实中发病中的关键致病因子。它在侵染过程中，被大量诱导合成，乙烯抑制了苹果果实的免疫与抗病反应，促进果实发病。.重要结果：.（1）乙烯是苹果果实轮纹病发病的关键致病因子。在轮纹病菌B. dothidea侵染苹果果实中，乙烯被大量诱导合成。乙烯信号感知抑制因子1-甲基环丙烯（1-MCP）显著阻止苹果果实轮纹病发病。.（2）MAPK-WRKY信号途径是乙烯作用于苹果免疫与抗病反应的靶点。苹果中抗病相关基因表达水平在1-MCP与轮纹病菌接菌共处理下，显著高于轮纹病菌接菌单处理。ERF转录因子家族基因表达与抗病相关基因表达在变化的趋势上不具协同性，而WRKY家族基因与抗病相关基因的表达高度协同。在进一步应用7种不同组合处理与伤害诱导中，发现 MdWRKY33-1与苹果几丁质酶编码基因MdChiB-1的表达高度协同。说明MdChiB-1与MdWRKY33二者位于同一条信号转导通路上，或二者之间存在调控关系。转基因的研究结果表明：超表达MdWRKY33-1显著提高了MdChiB-1、以及乙烯合成关键基因MdACS5a的表达，而对其他MdPR基因的表达没有影响。.（3）超表达MdMPK3、MdMPK6显著提高了苹果细胞对腐烂病菌、葡萄灰霉病病菌的抗性，而对轮纹病菌的抗性没有显著影响；超表达MdWRKY33-1没有显著提高对轮纹病菌的抗性。.因此，在轮纹病菌致病真菌中，具有促进苹果果实中乙烯大量合成的基因，是致病基因的重要候选基因。该研究为苹果轮纹病菌重要的致病候选基因的发现提供了理论依据和创新性研究成果。