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时间就是金钱,效率就是生命!唯有惜时才能成功,唯有努力方可成就唯有惜时才能成功,唯有努力方可成就!小麦蓝矮植原体免疫膜蛋白(Imp) 基因的克隆和分子特性分析顾沛雯1,2,吴云锋1,王海妮1,安凤秋1(1西北农林科技大学植物保护学院与陕西省农业分子生物重点实验室/植保资源与病虫害教育部重点实验室,陕西杨凌 712100; 2宁夏大学农学院,银川 750100)摘要:【目的】探讨小麦蓝矮植原体免疫膜蛋白在介体-病原-寄主互作的分子机理。 【方法】通过植原体免疫膜蛋白基因序列两侧的保守区设计引物对 Imp 1051/Imp 2265,用 PCR 方法扩增小麦蓝矮植原体免疫膜蛋白基因;对扩增片段的最大开放阅读框和基因的同源矩阵、系统发育树分析;对克隆基因所编码蛋白进行跨膜区、亲疏水区和前导信号序列分析。 【结果】从小麦蓝矮病病株和接种长春花中均扩增到约 1.0 kb 的特异片段,其中小麦蓝矮植原体免疫膜蛋白基因长 495 bp,推导的编码蛋白含有 164 个氨基酸。与 10 种植原体的免疫膜蛋白基因进行序列同源性分析,小麦蓝矮与三叶草绿变植原体同源性最高,核苷酸和编码的氨基酸序列的同源率分别为 98.4和 95.1。蛋白质结构分析结果表明:小麦蓝矮免疫膜蛋白 N 端有一个跨膜的前导信号序列,C 端为跨膜锚定区,中间为膜外亲水区。 【结论】小麦蓝矮植原体与三叶草绿变、翠菊黄化、洋葱黄化和泡桐丛枝植原体的免疫膜蛋白为同型蛋白。关键词:小麦蓝矮植原体;免疫膜蛋白基因;同源性;蛋白结构Clone and Molecular Characterization of Immunodominant Membrane Protein Gene from Wheat Blue Dwarf PhytoplasmaGU Pei-wen1, 2, WU Yun-feng1, WANG Hai-ni1, AN Feng-qiu1 (1College of Plant Protection and Shaanxi Key Laboratory of Molecular Biology for Agriculture/Key Laboratory of Plant Protection Resources and Pest Management, Ministry of Education, Northwest Agriculture and Forest University, Yangling 712100, Shaanxi, Province; 2Agricultural College, Ningxia University, Yinchuan 750100)Abstract: 【Objective】The aim of the experiment is to get the basic molecular information of the mechanism of vector-pathogen-host plant relationships.【Method】DNA was extracted from the infected wheat and periwinkle samples and the target fragments were amplified using primers Imp1051/Imp2265 designated based on conserved regions of immundominant membrane protein gene from phytoplasm. The amplified fragment was analysed for open reading frame, homology matrix and phylogentic tree and the encoding protein was analysed for transmembrane region, hydrophilic and hydrophobic region and leader signal sequence.【Result】A 1.0 kb DNA fragment was amplified by polymerase chain reaction in DNA samples extracted from the infected wheat and periwinkle plants but not in those extracted from healthy plants. It was showed that immunodominant membrane protein gene from wheat blue dwarf (WBD) phytoplasm was 495 bp in length, encoding a predicted protein of 164 amino acids. Homology analysis of sequence from WBD and 10 phytoplasms showed that immunodominant membrane protein gene of WBD phytoplasm was closely related to those of clover phyllody (CPh) phytoplasm, with homology rate of nucleotide and amino acids sequences, at 98.4% and 95.1%. Prediction of protein structure showed that the protein from WBD phytoplasm possesses a N-terminal hydrophobic transmembrane region with export leader signal sequence, a C-terminal hydrophobic transmembrane anchor, and a central hydrophilic region, possibly external to the phytoplasm cell. 【Conclusion】Immunodominant membrane protein of WBD phytoplasm belongs to the same type with those of CPh, aster yellow (AY), onion yellow (OY) and paulownia witches broom (PaWB) phytoplasms. Key words: Wheat blue dwarf phytoplasma; Immunodominant membrane protein; Homology; Structure of protein时间就是金钱,效率就是生命!唯有惜时才能成功,唯有努力方可成就唯有惜时才能成功,唯有努力方可成就!0 引言【研究意义】植原体(phytoplasma)(原称类菌原体 mycoplasma like-organism,MLO)是一类G+C 含量比较低,不能人工培养,存在于植物筛管细胞中的类似植物病原细菌的原核生物。在自然界中,植原体通过韧皮部吸食式昆虫叶蝉、飞虱、蝽蟓等传播1,2。迄今为止,世界上报道的植物植原体病害多达 300 余种,其主要症状包括丛枝、黄化、花变叶、花器退化等2。由于植原体无细胞壁,菌体细胞膜直接与寄主植物和虫媒介体细胞接触,因此,认为其膜蛋白可能在虫媒介体植原体寄主植物的相互关系中起重要作用38。【前人研究进展】免疫膜蛋白(immunodominant membrane protein,IMP)是植原体膜蛋白中重要的蛋白之一。编码免疫膜蛋白的基因已经从甘薯丛枝3(sweet potato witches broom,SPWB,登录号 U15224)、苹果丛枝4(apple proliferation,AP,登录号 AJ011678)、桃西方 X5(western X-disease,WX,登录号AF225904)、翠菊黄化6(aster yellow,AY,登录号 AF244540)、三叶草绿变6(clover phyllody,CPh,登录号 AF244541)欧洲石果黄化7(european stone fruit yellows,ESFY,登录号AF40058)、梨衰退7(pear decline,PD,登录号AF400588)、桃黄化卷叶7(peach yellow leaf roll,PYLR,登录号 AF400589)、洋葱黄化8(onion yellow,OY,登录号 AB110271)、泡桐丛枝8(paulownia witchesbroom,PaWB,登录号AB124810)等 10 多种植原体中获得。但目前对于免疫膜蛋白在介体病 原寄主植物的互作关系中的具体功能还未见报道68。【本研究切入点】小麦蓝矮(wheat blue dwarf,WBD)植原体属于翠菊黄化组三叶草绿变亚组植原体(16Sr-C)9,15,是中国首次报道的小麦植原体病害,国外尚未报道9,12。小麦蓝矮病植原体存在于小麦筛管细胞中,在田间通过介体条沙叶蝉(Psammotettix striatus L.)在韧皮部组织吸食传播9,12,16,17。发病初期小麦心叶褪绿变淡、卷缩,后基部叶片变深绿色、增宽加厚,顶叶出现不规则的黄色宽条斑。发病后期小麦不能正常抽穗或抽塔状退化小穗,最后病株叶片全部黄化,植株死亡9,12,17。【拟解决的关键问题】本研究首次设计翠菊黄化组(16Sr )植原体10,11,15免疫膜蛋白基因的特性引物,对小麦蓝矮病植原体的免疫膜蛋白基因进行克隆与测序,并对其基因序列和表达特性进行了分析。为进一步在分子水平上研究小麦蓝矮病植原体免疫膜蛋白的功能奠定基础。1 材料与方法1.1 毒源标样小麦蓝矮病田间病株采自陕西韩城市,经介体条沙叶蝉(P. striatus L.)接种于室内小麦和长春花,具体接种方法参照顾沛雯等9,17方法进行。毒源标样均通过 16Sr RNA9,11,18,19和 tuf 基因12,13,15检测为阳性。健株对照由本实验室提供。1.2 DNA 提取毒源和健康标样按 CTAB 法提取基因组 DNA,具体方法参照 Kollar 等20的方法。1.3 PCR、产物的克隆及测序根据 AY、CPh、OY、PaWB 等植原体免疫膜蛋白基因序列两侧的保守区设计引物对Imp1051/Imp2265(上海 Sango 公司合成)进行 PCR扩增,引物对序列如下:Imp1051(5-GCCAGAAAACA ACTCAAACC-3);Imp2265(5-ACTGTTAAAGGAG GGCTTGC-3)。PCR 反应以健康植株总 DNA 和无菌双蒸水为阴性对照。PCR 产物在 1%琼脂糖凝胶(含 0.5 gml-1 EB)1TAE 缓冲体系中电泳后,取目的片段的凝胶带纯化。PCR产物纯化采用 H.Q. 1: Water control (CK); 2: Healthy wheat in greenhouse; 3 and 5: Inoculated wheat; 4 and 6: Inoculated periwinkle; 7: Healthy periwinkle in greenhouse; 8: Healthy wheat in the
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