抗病抗体识别的赤霉病菌表面抗原基因克隆与功能鉴定

Fusarium graminearum can infect wheat, barley, corn and rice, which not only causes massive reduction of yield and quality but also produces mycotoxins that are toxic to human and domestic animals. Innate resistance germplasms to this pathogen in plants are inadequate and a recombinant antibody specific to the surface antigen of F. graminearum can enhance resistance in plants to this pathogen. However, by far the function and gene sequence that encodes the surface antigen have not been described. In this proposal 2-DE, immunoblot and MALDI-TOF/MS are used to identify the sequence coding for the antigen and subsequently to clone the gene. Then the antigen-coding gene-knock out mutants derived from different chemotypes of F. graminearum are created and subjected to the analyses of immunoproteomics and immunofluorescene labeling to verify the proper deletion of the target antigen gene, after which mycotoxin production and pathogenicity of the mutant strains and the wild type are assayed. The antigen protein is expressed and its enzymatic activity is measured. Combined comparative analyses of the transcriptome and metabolomics of different strains will be performed. qRT-PCRs together with gene-knock out are used to further identify the functions of the genes involved. These analyses will reveal the role of the surface antigen gene and the associated network in the fungal development, pathogenicity and mycotoxin biosynthesis of this fungus. Thus, completion of this proposed project will provide insight into the interaction mechanisms between the CWP2 antibody and its antigen and the resistance mechanisms of the antibody. Moreover, this study will provide the materials for further crystal analyses of the surface antigen and the improvement of the CWP2 antibody by molecular design.

赤霉病菌可侵染小麦、大麦、玉米和水稻，降低作物产量和品质，产生对人、畜有害的真菌毒素。植物抗赤霉病资源匮乏，重组抗体CWP2可特异识别赤霉病菌表面抗原，提高植物的抗赤霉病性,具有重要应用价值。但迄今为止，编码赤霉病菌表面抗原的基因及功能仍不清楚。本研究首先利用双向电泳、免疫杂交、MALDI-TOF/MS测序鉴定赤霉病菌表面抗原蛋白，克隆基因；然后创建赤霉病菌不同化学型菌株的抗原基因缺失突变体，经免疫蛋白组及荧光标记验证抗原基因缺失后，研究其产毒性和致病力；表达抗原蛋白，测定酶活性；分析、比较突变体和野生型的转录组和代谢组，整合不同组学研究结果，以qRT-PCR和基因敲除分析验证基因功能，解析表面抗原基因在赤霉病菌生长发育及致病、产毒中的作用及其调控网络。本项目的完成将为揭示CWP2抗体与抗原互作机理及抗体的抗病机理提供依据，也为进一步分析抗原晶体结构及分子设计改良抗体的亲和力提供材料。

赤霉病是我国小麦的重要病害，抗病资源匮乏。项目组曾以赤霉菌武昌菌株细胞壁蛋白为抗原，分离获得抗病抗体CWP2基因，能提高植物的赤霉病抗性。但抗体CWP2的抗病机理并不清楚。本研究旨在克隆、鉴定CWP2识别的赤霉菌抗原编码基因，揭示抗体的抗病机理。. 利用免疫蛋白组技术，经双向电泳、免疫杂交，从赤霉菌武昌菌株细胞壁蛋白中，发现CWP2特异结合的抗原为单拷贝乙二醛氧化酶（glyoxal oxidase，GLX）基因编码的蛋白质，含跨膜结构域；GLX-GFP融合蛋白细胞定位及免疫荧光标记表明，Glx蛋白位于细胞膜上。敲除GLX基因的GLX缺失突变菌株，缺少CWP2结合的蛋白质。酶活分析表明，Glx蛋白催化产生过氧化氢；GLX基因缺失突变体的酶活降低61%~68%，GLX过表达菌株的酶活提高13%~18%。CWP2可特异抑制氧化酶活性，显著降低过氧化氢产物。GLX缺失突变体的致病力降低68%~73%，DON和NIV毒素合成降低7~17倍。这些结果说明，GLX基因编码的蛋白，就是抗病抗体CWP2识别的赤霉菌表面抗原。. 序列分析表明，串珠镰刀菌、尖孢镰刀菌的GLX基因，与赤霉菌GLX基因的拷贝数和结构相同、同源性高。这两种镰刀菌GLX基因缺失突变体，在玉米和番茄上的致病力分别降低84%和68%，串珠镰刀菌的伏马菌素合成降低7倍。因此，Glx酶在镰刀菌属高度保守，是镰刀菌的致病因子，参与不同镰刀菌毒素的合成。. 利用病菌诱导及颖壳特异启动子Lem2，调控抗体CWP2融合蛋白在小麦中表达，筛选获得的小麦材料既抗赤霉菌侵入和扩展，又抗毒素积累。Lem2调控的抗体融合蛋白基因，主要在小麦颖壳和内稃中表达；赤霉菌侵染小麦颖壳24~96 h后，迅速诱导小麦颖壳中抗体融和蛋白表达量增加5倍。表明CWP2可介导植物主动免疫抗病性。. 本研究阐明了抗体CWP2的抗病机理：锚定在膜上的Glx蛋白作为受体，富集特异抗体及其融合蛋白，抗体结合抗原抑制其氧化酶活性，进而减少植物细胞壁损伤、降低病菌致病力，抗体融合蛋白的抗菌肽又破坏真菌，这种多层次的综合作用，有效阻碍病菌生长，增强植物的抗病性。这些结果为发展基于抗体的植物抗病性提供了信息和依据。