小麦蓝矮植原体染色体DNA的分离

Isolation of chromosome DNA of wheat blue dwarf phytoplasma

摘要:

【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。

Abstract:

[Objective] Isolating chromosome DNA of wheat blue dwarf (WBD) phytoplasma and establishing an effective protocol of purification for chromosome DNA. [Methods] Dif-ferential centrifugation and plus-filed gel electrophoresis (PFGE) were used to purify the chromosome DNA of WBD phytoplasma. The band observed in PFGE was confirmed by PCR and Southern blot. The effect of each step was detected by real-time quantitative PCR analysis. [Results] Using differential centrifugation and PFGE, a band about 650 kb was observed, which was confirmed as the chromosome DNA of WBD phytoplasma by Southern blot hy-bridization and PCR. Meanwhile, real-time PCR analysis showed that the relative copies of WBD phytoplasma chromosome DNA derived from differential centrifugation and PFGE was 436.5 times than that in the total DNA of infected periwinkle. [Conclusion] The size of WBD phytoplasma chromosome DNA is about 650 kb, purified WBD phytoplasma chromosome DNA can be obtained effectively using differential centrifugation and PFGE.