Development of isolation procedure for the functional domains of plant chromosome

我们建立了一种聚乙二醇介导法将完整的酿酒酵母球体DNA直接转移到拟南芥原生质体中。为了监测从酵母到植物细胞的DNA转移，以插入植物内含子的b-葡萄糖醛酸酶(GUS)报告基因为报告基因。该内含子-GUS报告基因位于2 mM的载体上，在酵母转化子中不表达，而将其导入植物细胞后表达GUS活性。将含有该质粒的酿酒酵母球体与拟南芥原生质体混合后，用聚乙二醇高pH高Ca~(2+)&gt~(2+)溶液处理，培养3d后，在植物细胞提取液中检测到GUS活性。此外，GUS基因的表达在聚乙二醇处理前和处理过程中对微球核酸酶具有抗性。根据这些结果，我们得出结论，质粒DNA可以通过核酸酶抗性的过程直接从完整的酵母球形体转移到植物原生质体，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.