苹果树腐烂病菌致病木聚糖酶的基因功能及其抑制蛋白的鉴定

Apple tree canker, caused by Valsa ceratosperma, is one of the most devastating and economically important diseases of apple prcduction in China. At present, measures to control losses caused by V. ceratosperma include using agriculture management cultures, chemical protection and treatment diseased scars. However, these measures can not control the disease effectively because the pathogenic factors and pathogenicity mechanisms of V. ceratosperma is still not clear. Our research group had confirmed that V. ceratosperma could produce a series of cell wall-degrading enzymes (CWDEs) including xylanase, polygalacturonase (PG), polymethylgalacturonase ( PMG), cellulose (Cx) and β-glucosidase. In all the CWDEs, the maximum activity of xylanase was the highest and it showed the most powerful macerating ability. The enzyme activities of xylanase were closely correlated with the virulence of different strains. In additon, it was showed that the disease resistance of plant and it's the inhibiting ability to xylanase activity was positive correlation, which provides a new idea for the control of apple valsa canker. This proposed project will investigate three aspects of the problem starting from xylanase produced by V. ceratosperma in the infection process. The first aspect is to isolate and clone the genes of xylanase by constructing genome library and RACE technology.The second aspect is to analyze gene funtion using gene expression characteristics and gene knochout methods. The last one is to screen the inhibition protein of pathogenic xylanase from apple germ plasm resources, to study the role of xylanase inhibitor in the disease defense of plant, and to detect it's affect on the growth, sporulation and infection of V. ceratosperma. After the program finished, we expecte to reveal xylanse is a pivotal pathogenic factor and it's role in the infection process of V. ceratosperma, to isolate and purify the apple xylanase inhibitor and understand it's the biocontrol effect on apple tree canker. The results of the project will provide essential informantion and references for completely revealing the pathogenicity mechanisms of V. ceratosperma and controlling the plant diseases using inhibition protein.

黑腐皮壳菌引起的苹果树腐烂病是对苹果产业威胁最大的一种毁灭性病害，但迄今对腐烂病菌的致病因子及其致病机理尚不明确，导致现有防治措施不能有效控制病害的发生和流行。本课题组研究证实腐烂病菌侵染致病过程中产生活性极强的木聚糖酶，且酶活性与菌株致病力存在密切相关性；在研究中还发现寄主的抗病性与其抑制木聚糖酶活性的能力呈正相关。本项目拟从苹果树腐烂病菌分泌的木聚糖酶入手，通过构建基因组文库和RACE技术，分离克隆木聚糖酶同源基因的全长序列；利用基因表达分析和基因敲除技术，解析木聚糖酶基因的功能；以此为基础，筛选致病木聚糖酶的抑制蛋白，分析其在植物抗病防御反应中的作用，进而研究抑制蛋白对腐烂病菌生长、繁殖及侵染的影响。研究结果将明确腐烂病菌的致病因子及木聚糖酶基因在病原菌致病性中的作用，明确苹果木聚糖酶抑制蛋白对腐烂病的生防效果，为全面揭示腐烂病菌的致病机理及应用酶抑制蛋白防治植物病害奠定基础。

由黑腐皮壳菌(Valsa mali var. mali)引起的腐烂病是苹果树上的一种毁灭性病害，严重制约了我国苹果产业的发展，由于缺乏对病原菌致病机理的了解，导致该病尚无法得到有效控制。本项目对苹果树腐烂病菌木聚糖酶基因进行了克隆，解析了木聚糖酶基因的功能，获得了木聚糖酶抑制蛋白并测定了其对腐烂病菌的抑制作用。.以苹果树腐烂病菌强致病力菌株的基因组DNA为模板，进行了De novo测序，其全基因组约为41 Mb，共有9225个基因，这为全面解析苹果树腐烂病菌的致病机制及其与寄主的互作研究奠定了基础。对Xylanase I和Xylanase II进行了克隆，开放阅读框分别为1320 bp和681 bp，属于糖基水解酶10家族和11家族。构建了两个基因的重组表达载体pET-Xylanase，并制备了木聚糖酶的多克隆抗体，其效价高达8000倍且特异性强。融合蛋白具有极高的木聚糖酶活性，接种苹果愈伤组织后可见明显的组织浸解症状。.离体培养条件下，温度和碳源均可影响腐烂病菌木聚糖酶基因的表达；病菌侵染致病过程中，基因的表达量均在接种后1 d开始显著升高，接种后2~3 d到达峰值，但Xylanase I的表达量显著高于Xylanase II。利用PEG介导的原生质体转化法，获得了包括Xylanase I和Xylanase II在内的9个木聚糖酶基因的敲除突变体，但仅Xylanase I的缺失突变体致病力显著降低。离体枝条接种后7 d和11 d，突变体所致病斑面积相比野生型菌株分别下降了71.9%和53.4%，木聚糖酶活性下降了24.9%和34.2%，但基因回复突变体的致病力和木聚糖酶活性均回复到野生型菌株水平。.对苹果基因组中检索到的4个木聚糖酶抑制剂基因进行了克隆和原核表达，发现1320 bp和1317 bp两个基因的融合蛋白具有可溶性。腐烂病菌侵染过程中，两个木聚糖酶抑制剂基因在感病材料中的最大表达量分别升高了24.3和13.7倍，而在抗病材料（1号砧木）中升高了42和59倍。两个基因的融合蛋白均可抑制腐烂病菌菌丝的生长和孢子萌发，并对腐烂病菌的侵染和扩展有一定的抑制作用，但1320基因融合蛋白的效果更显著，暗示木聚糖酶抑制蛋白对腐烂病有明显的防效和应用潜能，为腐烂病的安全控制提供了新思路。