Development of isolation procedure for the functional domains of plant chromosome

我们建立了一种聚乙二醇（PEG）介导的DNA从完整的酿酒酵母原生质球直接转移到拟南芥原生质体的方法。为了监测DNA从酵母到植物细胞的转移，将其中插入植物内含子的β-葡萄糖醛酸酶（GUS）报告基因用作报告基因。在基于2 mm的质粒载体上的该内含子-GUS报告基因在酵母转化体中不表达，而当质粒DNA被引入植物细胞中时，其表达GUS活性。当用PEG和高pH高Ca^2+溶液处理含有质粒的酿酒酵母原生质球和拟南芥原生质体的混合物时，在培养3天后的植物细胞提取物中检测到GUS活性。此外，GUS基因表达对PEG处理前和PEG处理期间的微球菌核酸酶处理具有抗性。从这些结果，我们得出结论，质粒DNA可以直接从完整的酵母原生质球转移到植物原生质体的核酸酶抗性过程，可能是通过细胞融合。

We developed a polyethylene glycol (PEG)-mediated direct DNA transfer method from intact Saccharomyces cerevisiae spheroplasts into Arabidopsis thaliana protoplasts. To monitor the DNA transfer from yeast to plant cells, b-glucuronidase (GUS) reporter gene in which a plant intron was inserted was used as a reporter. This intron-GUS reporter gene on a 2 mm-based plasmid vector was not expressed in yeast transformants, while it expressed GUS activity when the plasmid DNA was introduced into plant cells. When a mixture of S.cerevisiae spheroplasts harboring the plasmid and A.thaliana protoplasts was treated with PEG and high pH-high Ca^<2+> solution, GUS activity was detected in the extract of the plant cells after a three-day culture. Moreover, the GUS gene expression was resistant to micrococal nuclease treatment before and during PEG treatment. From these results, we concluded that plasmid DNA can be directly transfered from intact yeast spheroplasts to plant protoplasts by a nuclease-resistant process, possibly by the cell fusion.