叶蝉传播小麦蓝矮植原体专化性的分子机制研究

Wheat Blue Dwarf phytoplasma (WBDp) is one kind of important crop pathogens in agriculture. It is specifically transmitted by the leafhopper, Psammotettix striatus and leads to wheat blue dwarf diseases. WBD disease occurs periodically in Northewestern China and causes large loss. Currently, this interaction mechanism such as how Psammotettix striatus specifically transmits WBDp remains unclear. The previous study of our lab found immunodominant membrane protein, Imp, was a key protein which may play an important role in specific transmission. This project will cover several following aspects: 1. studying WBDp cycling transmission pathway in vector body and determining the difference in this pathway between insect host and non-host; 2. Using qRT-PCR and immuno-electron microscope to study the location of Psammotettix striatus body involved in WBDp reproduction and the WBDp movement between cells; 3. Screening the key factors about specific transimission;4. Using RNAi to reduce the expression of these key insect factors and analyse the the insect host ability about WBDp acquisition and WBDp spread and identifying the function of these insect host gene candidates. This project will determine the molecular mechanism of WBDp specific transmission and explore a new stragety to control WBD diseases.

小麦蓝矮植原体是一种重要的农业有害微生物。由条沙叶蝉(Psammotettix striatus)传播危害，引起小麦蓝矮病(WBD)。在我国西北麦区周期性发生，造成严重的损失。目前，植原体如何与叶蝉互作，及条沙叶蝉传播植原体的机理都不清楚。本实验室前期研究发现植原体免疫膜蛋白（Imp）在植原体昆虫传播中发挥重要作用。本项目拟开展以下几个方面的工作：1.研究WBD植原体在介体条沙叶蝉体内的偱回传播途径，明确介体条沙叶蝉和非介体间的偱回差异；2.利用实时荧光定量PCR技术和超微切片免疫电镜技术研究WBD植原体在介体条沙叶蝉体内的繁殖场所与细胞间扩散移动方式；3.筛选与介体传播相关的关键因子；4.用RNAi 技术体系，沉默抑制关键因子功能表达后，分析获毒和传毒能力被抑制的程度，鉴定侯选关键因子的功能。明确条沙叶蝉传播小麦蓝矮植原体专化性分子机制，为植原体病害的防治探索新的途径。

小麦蓝矮植原体是一种重要的农业有害微生物。由条沙叶蝉(P. striatus)传播危害小麦，引起小麦蓝矮病(WBD)。在我国西北麦区造成严重损失。目前，对条沙叶蝉传播植原体的机理不清楚，制约病害防控。本项目研究内容：1.研究WBD植原体在介体条沙叶蝉体内的偱回传播途径；2.利用实时荧光定量PCR技术和超微切片免疫电镜技术研究WBD植原体在介体条沙叶蝉体内的繁殖场所与细胞间扩散移动方式；3.筛选与介体传播相关的关键因子；4.用RNAi 技术体系，沉默抑制关键因子功能表达后，鉴定侯选关键因子的功能。明确条沙叶蝉传播小麦蓝矮植原体专化性分子机制，为植原体病害的防治探索新的途径。. 取得的重要结果：① 从植原体基因组中克隆出体免疫膜蛋白（Imp）基因424bp，插入原核表达载体pMAL-c2x转入E.coli 原核表达WBD Imp，纯化Imp作为抗原，免疫注射雄性大白兔肌肉组织制备抗血清。经琼脂双扩散测定抗血清效价为1:1000，将Imp抗体与荧光素FITC结合。②转移介体条沙叶蝉饲取WBD植原体不同时间采样，解剖肠道组织和唾液腺，用植原体Imp-FITC免疫荧光抗体标记观察，明确了植原体在叶蝉体内的循回过程：植原体快速进入滤室和中肠肠腔内，12-24h进入中肠和后肠上皮细胞， 36h后进入唾液腺中。③构建了条沙叶蝉泛素化膜蛋白酵母文库，用Imp杂交筛选到10种互作蛋白，分别是Actin、细胞分裂周期蛋白（Cdc42）、α微管蛋白（α-Tubulin）、ATP合成酶（ATP synthase）等；分别从叶蝉基因组中克隆到Actin等10种互作蛋白的基因全长。其中Actin基因长为1131bp, , α-Tubulin基因长为1458bp， ATP合成酶基因长为1563bp。④ 进一步用WBD膜蛋白Imp从叶蝉cDNA酵母文库中筛选出10种互作蛋白，利用BiFC、Co-IP方法验证确定α-Tubulin、Cdc42为互作靶标蛋白。⑤用RNAi技术验证叶蝉体内靶标蛋白Tubulin、Cdc42的传毒功能，研究发现叶蝉微丝蛋白Tubulin调控叶蝉介体专化性传播机制，Cdc42控制叶蝉唾液腺中植原体的繁殖能力。.