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【目的】分离小麦蓝矮(WBD)植原体染色体DNA,并建立WBD植原体染色体分离纯化体系。【方法】采用差速离心和脉冲电泳(PFGE)方法富集纯化WBD植原体染色体DNA,并通过PCR和Southern blot进行检测验证,实时荧光定量PCR方法对分离纯化效果进行定量检测。【结果】脉冲电泳凝胶中出现一条大小约为650 kb的条带,经PCR检测和Southern blot分析表明该条带为WBD植原体的染色体DNA。实时荧光定量PCR检测结果表明采用差速离心与脉冲电泳结合的方法可以将WBD植原体基因组的相对拷贝数提高436.5倍。【结论】采用差速离心与脉冲电泳法结合可以有效地从感染WBD长春花中分离到纯的WBD植原体染色体DNA,WBD植原体染色体DNA大小约为650 kb。
【Objective】 The purpose of this study was to isolate the chromosomal DNA of wheat blue-dwarf (WBD) phytoplasma and to establish a phytoplankton-isolating and purifying system for WBD. 【Method】 Chromosomal DNA of purified WBD was enriched by differential centrifugation and pulse electrophoresis (PFGE). The DNA was purified by PCR and Southern blot. The effect of isolation and purification was quantified by real-time fluorescence quantitative PCR. 【Result】 A band of about 650 kb appeared in the gel electrophoresis. The result of PCR and Southern blot showed that the band was the chromosomal DNA of WBD phytoplasma. Real-time PCR results showed that the relative copy number of the WBD phytoplasma genome could be increased by 436.5 folds by differential centrifugation and pulse electrophoresis. 【Conclusion】 Chromosomal DNA of pure WBD phytoplasma can be effectively isolated from WBD-infected cats by differential centrifugation and pulse electrophoresis, and the chromosome DNA of WBD is about 650 kb.