利用酵母双杂交膜系统筛选与小麦蓝矮植原体免疫膜蛋白互作的异沙叶蝉蛋白

正小麦蓝矮病(Wheat blue dwarf,WBD)是我国西北麦区一种重要植原体病害,在我国西部地区危害严重。该病害由异沙叶蝉(Psammotettix alienus L.)专化性传播,介体传毒成为病害流行的重要中心环节[1]。本实验室前期通过免疫荧光标记研究发现WBD植原体免疫膜蛋白(immunodominant membrane protein, IMP)与介体异沙叶蝉肌动蛋白互作,说明IMP在植原体传播和致病过程中起关键作用。     

利用酵母双杂交膜系统筛选与小麦蓝矮植原体免疫膜蛋白互作的异沙叶蝉蛋白

In order to investigate interactive proteins in leafhopper (Psammotettix alienus L.) with WBD phytoplasma, the protein interaction analysis was performed by using WBD phytoplasma IMP (immunodominant membrane protein) as bait protein to screen a cDNA library of leafhopper using a split-ubiquitin yeast membrane system. Through the screening test, 30 clones were obtained from the cDNA library and 8 proteins were identified by searching against NCBI database, such as tubulin, peptidyl-prolyl cis-trans isomerase, Cdc42 protein, ribosomal proteins and ATP-F0 subunit protein, etc. The interactions between IMP and 8 putative proteins were further confirmed by co-transformation and β- Galactosidase assays. This study could be useful for understanding the molecular interaction mechanism between WBD phytoplasma and insect vector and the specificity of transmission.     

双重RT-PCR法同步检测单头异沙叶蝉携带的两种小麦病毒

异沙叶蝉可传播小麦矮缩病毒Wheat dwarf virus(WDV)和小麦黄条纹病毒Wheat yellow striate virus(WYSV)两种病毒。本文根据WDV和WYSV的基因序列分别设计两种病毒的特异性引物对,以含有上述两种病毒的异沙叶蝉样品总RNA为模板,以随机引物为3′端通用引物反转录获得cDNA,然后在同一个PCR反应体系加入两对引物,分别得到与预期相符的773 bp和322 bp扩增产物条带。通过对引物浓度、dNTP和rTaq用量以及退火温度等条件进行优化,建立了能在同一异沙叶蝉体内检测WDV与WYSV两种小麦病毒的双重RT-PCR方法。该双重PCR方法特异性强、敏感性高,可以快速准确地在一个体系里同步检测介体昆虫体内的两种病毒,有效地检测虫体内病毒带毒率,这些结果可为病毒预测预报和病害防治提供参考。     

介体条沙叶蝉传播小麦蓝矮病植原体特性研究

小麦蓝矮病植原体(wheat blue dwarf,WBD)属于翠菊黄化组三叶草绿变亚组植原体(16SrⅠ-C),由介体条沙叶蝉(Psammotettix striatus L.)专化性传播。通过电镜超微结构观察,在接种小麦、长春花和带毒条沙叶蝉体内有大量植原体,而在健康植物组织、无毒条沙叶蝉和带毒条沙叶蝉所产卵中未见植原体的存在。通过介体传毒试验和PCR检测发现,条沙叶蝉最适获毒期为7 d,植原体在虫体内的潜育期为1 5～1 7 d,接毒期为2～3 d。条沙叶蝉一旦获毒可终生持毒和传毒。不同虫态的条沙叶蝉带毒率没有明显的差异,但寄生植物的种类影响其带毒率。     

条沙叶蝉触角形态及其感器类型

【目的】观察比较条沙叶蝉(Psammotettix striatus)成若虫、雌雄两性的触角及其感器的形态、数量和分布,为条沙叶蝉的行为生物学、化学生态学和电生理学的研究提供参考。【方法】应用电镜扫描技术,系统观察条沙叶蝉的触角及其感器的类型、数量和分布。【结果】条沙叶蝉成虫两性间无差异,触角呈刚毛状,包括柄节、梗节和鞭节3部分,柄节及梗节较粗短,其上具众多耳状突起,梗节近端部着生有3～5根毛形感器;鞭节细长,分为多亚节,第1、2、4亚节上各有1个锥形感器,第3亚节上着生1毛形感器,其余亚节上均无感器存在。【结论】条沙叶蝉的触角感器类型及数量在雌雄个体间无差异,若虫触角上感器的分布、数量及种类与成虫一致。     

利用酵母双杂交系统筛选介体异沙叶蝉中与小麦矮缩病毒外壳蛋白互作的蛋白质

【目的】利用分离泛素酵母双杂交膜系统（split-ubiquitin yeast membrane system）,以小麦矮缩病毒（Wheat dwarf virus,WDV）的外壳蛋白（CP）基因为诱饵对异沙叶蝉（Psammotettix alienus L.）cDNA文库进行筛选,研究异沙叶蝉传播WDV的分子机制。【方法】以笔者实验室饲养的异沙叶蝉为材料,提取其总RNA后取100 ng进行纯化,利用SMART法反转录合成ds cDNA,经过Sfi I酶切纯化,连接到pPR3-N文库载体上,构建得到以pPR3-N为载体的异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库。同时,构建带有Sfi I酶切位点的诱饵载体pDHB1-WDV CP,经功能检测后用诱饵载体初步筛选pPR3-N空文库,寻找适合筛库的条件和确定His基因产物抑制剂3-氨基-1,2,4-三唑（3-AT）的使用浓度。然后用诱饵载体筛选异沙叶蝉cDNA文库,对筛选结果进行分析,再通过共转验证和β-半乳糖苷酶检测进一步验证是否发生互作。利用Uniprot和KEGG在线网站,对筛到的蛋白进行gene ontology（GO）注释和Pathway分析。【结果】初级文库库容量超过2.0×10⁶ cfu,文库实际扩增数量大于1.3×10⁶ cfu,文库重组率大于97%,扩增文库插入片段平均长度大于1 000 bp,表明异沙叶蝉cDNA文库的质量较高。酶切验证显示诱饵载体pDHB1-WDV-CP中CP的插入完整而准确。功能检测表明融合蛋白能够正确表达。在3-AT浓度为5 mmol?L^-1的筛选条件下,诱饵载体筛选异沙叶蝉cDNA文库得到280个克隆,经测序和Blast比对分析最终得到12个可能与WDV的CP发生互作的异沙叶蝉蛋白质。将这12个蛋白质再次进行共转验证和β-半乳糖苷酶检测,最终得到9个蛋白质与WDV CP互作。GO注释显示,9个蛋白参与的生物过程包括蛋白去磷酸化、碳水化合物代谢过程、先天性免疫应答、模式识别受体的信号通路、运输、同向运输和乙醇氧化等;分子功能包括金属离子结合活性、蛋白磷酸酶活性、信号模式识别受体的活性、水解酶活性、磷酸离子载体活性和叶酸运输活性等。参考KEGG数据库,这些蛋白参与的代谢途径有泛素介导的蛋白水解途径、内吞作用、花生四烯酸代谢途径、cAMP信号通路和模式识别受体的信号通路等。【结论】异沙叶蝉分离泛素酵母双杂交膜系统cDNA文库的成功构建与筛选,为研究异沙叶蝉与小麦矮缩病毒的互作机制研究奠定了基础。     

条沙叶蝉生物学与生态学研究

作者依据成虫产卵、若虫存活与发育等指标测定了条沙叶蝉对19种供试禾本科作物及杂草的寄主适应性,明确寄主植物14种,食料植物4种,非寄主食料植物1种。同时发现,不同小麦品种对条沙叶蝉存在不同程度抗虫性。通过室内试验和田间调查,明确条沙叶蝉各虫态生活习性及在关中年生活史和发生特点,在关中,条沙叶蝉1年发生4代,以卵越冬,全年各世代主要在杂草地完成周年发育循环,麦田发生条沙叶蝉主要是在小麦秋苗期。在20、25、30、35℃4种恒温与狗尾草、小麦、玉米3种食料条件下初步明确温度、食料对条沙叶蝉若虫发育、存活与成虫寿命及产卵的影响。     

Sequence analyses of Wheat dwarf virus isolates from different hosts reveal low genetic diversity within the wheat strain

Wheat dwarf virus (WDV) causes disease in wheat (Triticum aestivum) and barley (Hordeum vulgare) in many parts of Europe. The host range also includes many species of the family Poaceae. WDV is only transmitted by the leafhopper Psammotettix alienus. During a five‐year period (2001–2005), grass samples were collected in central Sweden in the vicinity of fields with WDV‐infected winter wheat. Screening with ELISA and PCR identified WDV in a low number of samples (8/1098) from only three grass species: Apera spica‐venti, Avena fatua and Poa pratensis. In addition, triticale was found to be positive. Fourteen WDV isolates from Avena fatua, Apera spica‐venti, Triticum aestivum, Lolium multiflorum, Poa pratensis, triticale and the insect vector Psammotettix alienus, were partially sequenced (ca. 1200 nucleotides), providing the first published WDV sequences from the insect vector. All isolates belonged to the wheat strain of WDV and the genetic diversity was low. Phylogenetic analyses showed no clear grouping according to geographical location or host species. The results suggest that the same WDV genotypes are infecting both wheat and grasses in Sweden. Interestingly, one group of isolates (subtype B) formed a distinct clade in the phylogenetic tree. Subtype B was always found in mixed infection with the main genotype. Complete sequencing of a subtype B isolate showed that it was 98·6% identical to a typical wheat isolate from the same plant.     

Partial resistance to Wheat dwarf virus in winter wheat cultivars

Four Hungarian winter wheat cultivars were investigated for their susceptibility to the geminivirus Wheat dwarf virus (WDV). Previously, two cultivars (Mv Regiment and Mv Emese) were assessed by breeders to exhibit virus symptoms in the field, whereas Mv Dalma and Mv Vekni showed few symptoms. Two inoculation techniques for WDV, vector transmission with the leafhopper Psammotettix alienus and agroinoculation, were used. Leafhopper transmission was more efficient than agroinoculation. However, irrespective of the technique used, no Mv Dalma or Mv Vekni plants showed clear WDV symptoms. In contrast, 3/30 Mv Emese and 4/36 Mv Regiment plants showed dwarfing and chlorosis after agroinoculation and 13/17 and 14/15 plants, respectively, had clear WDV symptoms after vector transmission. WDV‐specific PCR showed that Mv Vekni and Mv Dalma plants could be infected, especially following vector transmission (approximately 50% infection), but at significantly lower frequency than Mv Emese or Mv Regiment plants (100% infection). Furthermore, real‐time PCR showed that WDV DNA accumulated to much lower levels in infected Mv Vekni and Mv Dalma plants than in infected Mv Regiment and Mv Emese plants. The data strongly suggest that Mv Vekni and Mv Dalma are partially resistant to WDV infection. As WDV resistance has not previously been identified in wheat, and because WDV can cause significant yield losses, the resistance of Mv Vekni or Mv Dalma will provide a valuable breeding resource.