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

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

【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Imp1051/Imp2265,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1.0kb的特异片段,其中小麦蓝矮植原体免疫膜蛋白基因长495bp,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。

【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 acentral 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.