中华苜蓿根瘤菌噬菌体的分离及主要壳蛋白基因g23的系统进化分析

【目的】揭示苜蓿根瘤菌噬菌体的形态学特征及主要壳蛋白g23基因的分布地位,为根瘤菌噬菌体的生态学研究提供数据支持。【方法】以中华苜蓿根瘤菌(Sinorhizobium meliloti USDA1002T)为宿主,采用双层平板法分离土壤环境中的苜蓿根瘤菌噬菌体,利用电子显微镜观察纯化的噬菌体形态特征;提取噬菌体DNA,PCR扩增编码噬菌体壳蛋白的g23基因,构建系统进化树,以形态学鉴定和分子生物学相结合的方法,明确分离获得的苜蓿根瘤菌噬菌体g23基因的系统进化地位。【结果】分离获得了3株噬菌体,头部均呈二十面体,直径大小为81–86 nm,尾部有收缩尾鞘,长度大约为54–70 nm。克隆测序结果显示,获得的3株噬菌体g23基因株间相似度较高,但与可培养的Exo T-、Schizo T-、T-、Pseudo T-evens相似度较低。系统进化分析表明,获得的3株噬菌体不隶属于目前已划分的不同环境噬菌体g23基因的分类类群中。【结论】3株噬菌体均属于肌尾噬菌体科的裂性噬菌体,与目前获得的所有噬菌体g23基因相似性较低,属于新的侵染中华苜蓿根瘤菌的噬菌体株。

Isolation and phylogenetic analysis of major capsid gene (g23) of bacteriophages infecting Sinorhizobium meliloti

Objective] In order to provide scientific data for studying the ecology of phage infecting Sinorhizobium meliloti, we examined morphological characteristics of rhizobiophages and their phylogenetic status of the major captain protein g23.Methods] Rhizobiophages were isolated by the double-layer plate method with host Sinorhizobium meliloti USDA1002^T. The morphological characteristic of rhizobiophages were studied by transmission electron microscope. Meanwhile, rhizobiophage DNA was extracted, and the g23 that encodes the major capsid protein of bacteriophages was chosen as objective gene in PCR amplification.Results] Three rhizobiophages were isolated, all had an icosahedral head with approximately 81 to 86 nm in diameter and a long contractile tail with 54 to 70 nm in length. Basic local alignment search tool searches in website of national center for biotechnology information (NCBI) revealed that the g23 amino acid sequences obtained in this study had high identity with each other, but had very lower identity with those from T-evens, PseudoT-evens, SchizoT-evens and ExoT-evens. Phylogenetic analysis showed that the isolated g23 sequences formed a unique clade with those clones obtained from different ecosystem.Conclusion] All results indicated that the isolated rhizobiophages belong to family Myoviridae, a new group of T4 phages, which had lower identity with the g23 clones obtained in different environment.