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【目的】测定苦楝丛枝植原体(CWB植原体)质粒并分析其分子特征。【方法】扩增苦楝丛枝植原体质粒片段并进行系统进化分析,预测质粒编码蛋白的跨膜区、亚细胞定位;以质粒pCWBFq repA为模板制备探针,利用Southern blot方法检测苦楝丛枝植原体及其他几种植原体的质粒。【结果】测定了苦楝丛枝植原体福清株系的一个质粒pCWBFq,该质粒全长4446 bp,A+T含量为73.5%,编码6个蛋白,其中pCWBFq P2-P5五个编码蛋白分别含有3、2、1和2个跨膜区,其信号肽(Singnal Peptide,SP)信号值分别为0.989、0.505、0.918和0.914。氨基酸序列相似性比较表明:pCWBFq RepA蛋白与其他植原体质粒RepA的同源性在9.6%-85.6%之间,而pCWBFq SSB蛋白与其他植原体质粒SSB的同源性在74.0%-89.4%之间。用pCWBFq repA探针检测到苦楝丛枝植原体中质粒的存在,同时也能够检测到16SrI组的泡桐丛枝植原体(PaWBNy),海南长春花绿变植原体(PeVHn),苦楝丛枝植原体福州株系(CWBFz)和桑树萎缩植原体濮阳株系(MDPy)中的质粒,但16SrV组的枣疯植原体北京株系(JWBBj)、樱桃致死黄化西昌株系(CLYXc)和重阳木丛枝南昌株系(BiWBNc)中未能检测到任何杂交信号。【结论】苦楝丛枝植原体质粒pCWBFq编码的6个蛋白中,除与质粒复制有关的RepA和SSB外,另外4个均为含有疏水结构的分泌蛋白或膜蛋白。植原体质粒的同源基因间存在程度不同的变异,其中repA基因在所有植原体质粒上均存在,但变异性相对较大,而ssb基因仅存在于16SrI组植原体质粒中,且变异相对较小。16SrI组的CWBFq、PaWBNy、PeVHn、CWBFz和MDPy中均存在数量和大小不同的质粒,而16SrV组的JWBBj、CLYXc和BiWBNc中或含有的质粒因与pCWBFq repA探针的同源性较低而不能被检测到。
【Objective】 The objective of this study was to determine the molecular mass of CWB phytoplasma (CWB) and to analyze its molecular characteristics. 【Method】 Plasmid fragments of Melia azedarach were amplified and phylogenetically analyzed to predict the transmembrane and subcellular localization of plasmid-encoded proteins. The plasmid pCWBFq repA was used as a probe to prepare the probe. Southern blot was used to detect the phytoplankton Body and several other plastid plasmids. 【Result】 A plasmid pCWBFq was isolated from Fusarium graminearum Fuqing strain, which was 4446 bp in length and 73.5% in A + T, encoding six proteins, of which pCWBFq P2-P5 contained 3 , 2, 1 and 2 transmembrane regions. The signal values of Singnal Peptide (SP) signals were 0.989, 0.505, 0.918 and 0.914, respectively. The comparison of amino acid sequence similarity showed that the homology of pCWBFq RepA with other phytoplasma plasmids was between 9.6% -85.6%, while that of pCWBFq SSB was 74.0% -89.4% between. The plasmid pCWBFq repA was used to detect the presence of plasmids in M. meliloti and PaWBNy, PeVHn, (CWBFz) and mulberry atrophy phytoplasma Puyang strain (MDPy). However, in the 16SrV group, JWBBj, CLYXc and Chongyangmu No hybridization signal was detected in Zhixianchang line (BiWBNc). 【Conclusion】 Among the six proteins encoded by pCWBFq, the other four were secreted proteins or membrane proteins with hydrophobic structure, except RepA and SSB which are related to plasmid replication. The phytoplasma plasmids had different degrees of homology. The repA gene was found in all the phytoplasma plasmids, but the variability was relatively high. However, the ssb gene only existed in the 16SrI phytoplasma plasmid, and the variation was relatively small. Plasmids of different sizes and sizes existed in CWBFq, PaWBNy, PeVHn, CWBFz and MDPy of 16SrI group, while the plasmids contained in JWBBj, CLYXc and BiWBNc of 16SrV group were inferior in homology to pCWBFq repA probe Detected.