宿根矮化病菌对甘蔗侵染的蛋白质组基础及相关基因研究

Sugarcane is the most important sugar crop which produces more than 90% of the total sugar in China. Guangxi is the dominant cane sugar producer which produces more than 65% of the cane sugar in the country. Sugarcane ratoon stunting disease (RSD) is the most important disease limiting sugarcane productivity, and the infection of RSD pathogen in sugarcane plants has been limited because of the extreme difficulty in isolating and culturing the RSD pathogen Leifsonia xyli subsp. xyli (Lxx). The present proposal is going to use both the healthy and RSD infected sugarcane plants as the plant materials. The technology of 2-dimensional electrophoresis of protein and protein mass spectrometry would be employed to identify the differential proteins under RSD stress including those secreted by pathogenic bacteria cell and produced by sugarcane defense mechanisms. The functions of differential proteins would be analyzed with bioinformatics and differential protein genes related RSD will be cloned and their expressions will be analyzed by qRT-PCR and fluorescent immunohistochemistry technique. The results will reveal the basic information about proteins and related genes during infection of ratoon stunding disease into sugarcane, and provide references for further research on RSD pathogenic infection mechanism and molecular breeding of RSD resistance in sugarcane.

甘蔗是最重要的糖料作物，甘蔗糖占全国总产糖量90%以上，而广西是我国蔗糖的主导产区，每年蔗糖产量占全国65%以上。甘蔗宿根矮化病是影响甘蔗产量最重要的病害，其病原菌分离培养极为困难，因此制约了对其病原菌及其侵染致病过程的深入研究。本研究拟以甘蔗健康种苗和感染宿根矮化病的甘蔗种苗为试验材料，利用蛋白质双向电泳和质谱技术获得甘蔗在宿根矮化病胁迫下的差异蛋白，包括病原菌细胞分泌的、寄主防御机制产生的以及病原菌和寄主互作产生的蛋白质，应用生物信息学解析差异蛋白的功能；克隆与宿根矮化病相关的差异蛋白基因，利用实时荧光定量分析差异基因的表达，并应用荧光免疫组织化学技术对差异蛋白进行定性和定量分析，以期了解甘蔗宿根矮化病发生发展的蛋白质基础及相关基因，为进一步探讨甘蔗宿根矮化病的致病机理和开展抗病分子育种奠定基础。

甘蔗是最重要的糖料作物，甘蔗糖占全国总产糖量90%以上，而广西是我国蔗糖的主导产区。甘蔗宿根矮化病是影响甘蔗产量最重要的病害，其病原菌分离培养极为困难。因此，制约了对其病原菌及其侵染致病过程的深入研究。本研究成功分离培养宿根矮化病病原菌，获得其形态学特性，利用高通量测序技术（登录号：LFYU00000000）鉴定其与巴西菌株的相似性为93.61%；克隆了一推测为抗σk因子的膜蛋白基因（Lxx18460）, 制备了效价大于1:512000，能够灵敏地检测Lxx细菌总蛋白和甘蔗样品总蛋白的单克隆抗体；以甘蔗健康种苗和感染宿根矮化病的甘蔗种苗为试验材料，发现Lxx侵染可降低甘蔗出苗率、株高、茎径、节间长度、单茎重，染病蔗株的蔗糖分降低0.9%，引起抗氧化物酶的变化，染病叶片的叶绿体变形，线粒体形态异常，细胞核形态不规则，核膜破裂，染色质分布不均匀，叶片比叶重和PEPC活性降低，水势下降，膜透性与游离氨基酸含量上升，PAL， ZFP， NBS-LRR等防御抗性基因表达量上升；通过构建抑制消减杂交文库和RNA-seq高通量测序，发现染病甘蔗差异表达基因涉及能量代谢、防卫反应、信号转导、光合作用、半胱氨酸等方面，油菜素内酯合成蛋白、NBS-LRR类抗性蛋白、α-微管蛋白、ABA胁迫成熟蛋白、富含脯氨酸蛋白、翻译起始因子eif-2b α亚族等可能参与了Lxx与甘蔗的互作过程；利用蛋白质双向电泳技术分析白差异蛋白质表达情况，共找出40个差异蛋白点，其中16个上调，24个下调，涉及防卫反应、细胞生长和分裂、蛋白修饰与加工等；采用RT-PCR技术克隆了SoREMORIN、SoGSK3、SoRASP等11个差异基因，分析了这些基因在Lxx诱导下和4℃、聚乙二醇(PEG)、NaCl 非生物胁迫诱导的时空表达情况。本研究成果为进一步探讨甘蔗宿根矮化病的致病机理和开展抗病分子育种奠定基础。