棣棠丛枝病相关植原体的分子鉴定.docVIP

 n 棣棠丛枝病相关植原体的分子鉴定# 常文程，李向东** （山东农业大学植保学院植物病理系，山东 泰安 271018） 5 10 15 20 25 30 35 40 摘要：用植原体 16S rRNA 基因和 tuf 基因通用引物，对表现丛枝症状的棣棠 DNA 进行巢 式 PCR 扩增，分别得到了约 1.2kb 和 800bp 的特异性片段。序列分析表明：与棣棠丛枝相 关植原体（KWB）的 16S rRNA 基因和 tuf 基因均与翠菊黄化植原体组（16SrI）D 亚组泡 桐丛枝株系一致率最高，为 99.4%和 99.5%。在系统进化树中，KWB 也与 16Sr I-D 亚组分 离物聚类到一起。利用虚拟 RFLP 分析发现，KWB 的 16S rRNA 基因 Alu I、Hha I、Mse I、 Taq 教育部博士学科点专项基金限制性酶切图谱与 16S rI-D 亚组植原体一致。以上结果表明， 与棣棠丛枝病相关的植原体属于 16Sr I-D 亚组。 关键词：棣棠；植原体；16S rRNA 基因；巢式 PCR；tuf 基因 中图分类号：S432.42 Molecular Identification of the Phytoplasma Associated with Kerria Witches’-Broom CHANG Wencheng, LI Xiangdong (Depratment of Plant Pathology, College of Plant Protection, Shandong Agricultural University, Shandong Taian 271018) Abstract: By using universal primers for phytoplasmal 16S rRNA and tuf genes, two specific fragments of ca.1.2kb and 800 bp in length were amplified with nested-PCR from the DNA of kerria showing witches’-broom symptom. The fragments were sequenced and subjected to identity and phylogenetic analysis. The results indicated that the 16S rRNA and tuf gene of phytoplasma isolate associated with kerria witches’-broom (KWB) shared highest identities of 99.4% and 99.5%, respectively, with paulownia witches’-broom phytoplasma (PaWB) which belongs to subgroup D of aster yellows group (16SrI-D). In the phylogenetic trees constructed with 16S rRNA and tuf genes, KWB was clustered with phytoplasmas of 16Sr I-D. The 16S rRNA gene of KWB had identical patterns with a 16Sr I-D phytoplasma for Alu I、 Hha I、 Mse I、 Taq I in the virtual RFLP analysis. All these results indicated that the phytoplsama associated with kerria witches -broom belonged to the subgroup D of aster yellows group (16SrI-D). Key words: kerria witches’-broom; phytoplasma; 16S rRNA; nested-PCR; tuf gene 植原体（Candidatus Phytoplasma）是一种没有细胞壁的原核微生物，主要由取食韧皮部 的昆虫（如叶蝉、飞虱等）传播，也可由 菟丝子寄生和嫁接等途径传播，常常引起植株黄 化、丛枝、花器变态、萎缩等症状。迄今为止，世界上报道的植物植原体病害有 1000 余种， 仅我国就有 100 多种，造成了巨大的损失。 由于植原体尚不能进行体外培养，对其生物学特性了解较少。近年来，分子生物学的迅 速发展极大地推动了植原体