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

 【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。

Clone and Molecular Characterization of Immunodominant Membrane Protein Gene from Wheat Blue Dwarf Phytoplasma

Abstract：【Objective】The aim of the experiment is to elucidate the molecular mechanism of vector-pathogen-host plant relationships.【Method】Using primers Imp1051/Imp2265 designated by conserved regions of immundominant membrane protein gene from phytoplasmas amplify DNA samples extracted from infected wheats and periwinkles; The amplified fragment is analysed for open reading frame,homology matrix and phylogentic tree; Encoding protein is anlysised for transmembrane region, hydrophilic and hydrophobic region and leader signal sequence.【Result】A 1.0kb DNA fragment was amplified by polymerase chain reaction in DNA samples extracted from infected wheats and periwinkles but not in those extranted from healthy plants. It was showed that immundominant membrane protein gene from wheat blue dwarf(WBD) phytoplasma was 495 bp in length, encoding a predicted protein of 164 amino acids. Homology analysis of sequence from WBD and 10 phytoplasmas showed that immundominant membrane protein gene of WBD phytoplasma was closely related to those of clover phyllody(CP) phytoplasma, with homology rate of nucleotide and amino acids sequences, 98.5% and 95.1%. Prediction of protein structure showed the immunodominant membrane proteins from WBD phytoplasma possess a N-terminal hydrophobic transmembrane regions with export leader signal sequence, a C-terminal hydrophobic transmembrane anchor, and a central hydrophilic region, possibly external to the phytoplasma cell.【Conclusion】Immundominant membrane protein of WBD phytoplasma belong to the same type with those of CP, aster yellow(AY), onion yellow(OY) and paulownia witches’-broom(PaWB) phytoplasmas.