苦楝丛枝植原体质粒的测定与分子特征

Complete sequence of a full-length DNA and molecular characterization of one plasmid from chinaberry(Melia azedarach Z) witches'-broom phytoplasma

【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] To clone plasmid from chinaberry witches＇-broom phytoplasma and analyse its molecular characterization.[Methods] Fragments of one plasmid（pCWBFq） in chinaberry witches＇-broom phytoplasma-Fuqing strain（CWBFq） were amplified with primer pairs which were designed according to plasmid sequences published on NCBI.Transmembrane domain and subcellular localization predictions of proteins encoded by the plasmid pCWBFq as well as phylogenetic analysis among the plasmid sequences were completed by using bioinformatic softwares.Southern blot analysis was performed to detect the plasmids existed in CWBFq and several other phytoplasmas with the pCWBFq repA probe.[Results] One complete plasmid was sequenced from CWBFq.pCWBFq comprised 4446 bp and had a nucleotide content of 73.5% A＋T and encoded six proteins.Protein P2,P3,P4 and P5 of pCWBFq contained 3,2,1 and 2 tranmembrane domains respectively,and their predicted signal peptide values were 0.989,0.505,0.918 and 0.914 respectively.Homologous comparison showed that RepA homology between pCWBFq and other phytoplasmas was between 9.6%-85.6%,however,the homology of different SSB proteins was between 74.0%-89.4%.Southern blotting with pCWBFq repA probe confirmed the existence of the plasmids in CWBFq.In addition,The hybridizations occurred with paulownia witches＇-broom phytoplasma-Nanyang strain（PaWBNy）,periwinkle virescence phytoplasma-Hainan stanin（PeVHn）,chinaberry witches＇-broom phytoplasma-Fuzhou strain（CWBFz） and mulberry dwarf phytoplasma-Puyang strain（MDPy）,whereas,no hybridizarions occurred with jujube witches＇-broom phytoplasma-Beijing strain（JWBBj）,cherry lethal yellows phytoplasma-Xichang strain（CLYXc） and Bischofia polycarpa witches＇-broom phytoplasma-Nanchang strain（BiWBNc）.[Conclusion] The plasmid encoded a replication associated protein（RepA） and a single-stranded DNA binding protein（SSB）,which were for the replication of plasmid.Four putative proteins encoded by the plasmid were predicted to contain one or more hydrophobic transmembrane domains,respectively,and presumably to be localized to the membrane.The alignment and homology analysis as well as phylogenetic analysis to the DNA and encoded protein amino acid sequences of the whole plasmids and single ORFs on the known phytoplasmal plasmids showed that the different homologous sequences have distinct variation,among which the repA gene with the largest diversity appeared in all the known plasmids while ssb with less variation were only found in 16SrI plasmids.CWBFq,PaWBNy,PeVHn,CWBFz and MDPy possessed distinct plasmids in terms of number and size,whereas there was no plasmid detected in JWBBj,CLYXc and BiWBNc,perhaps as a result of low homology among repA genes in plasmids of JWBBj,CLYXc and BiWBNc.