Development of isolation procedure for the functional domains of plant chromosome

植物染色体功能域分离方法的开发

• 批准号: 04454068
• 负责人: KAWAMOTO Shinichi
• 金额: $ 4.22万
• 依托单位: Hokkaido University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (B)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04454068/
• 关键词: Arabidopsis thaliana; Reporter gene; Cell fusion; DNA transfer; Saccharomyces cerevisiae; 酵母; YACライブラリー; 染色体機能部位

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.

我们建立了一种聚乙二醇（PEG）介导的DNA直接从完整的酿酒酵母（Saccharomyces cerevisiae）球质体转入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，采用插入植物内含子的b-葡萄糖醛酸酶（GUS）报告基因作为报告基因。该内含子-GUS报告基因在2 mm的质粒载体上在酵母转化子中不表达，而当质粒DNA导入植物细胞时，其表达GUS活性。用PEG和高ph -高Ca^<2+>溶液处理含有质粒的酿酒酵母球质体和拟蓝藻原生质体的混合物，培养3天后，在植物细胞提取物中检测到GUS活性。此外，在PEG处理前和处理过程中，GUS基因表达对微局部核酸酶具有抗性。根据这些结果，我们得出结论，质粒DNA可以通过抗核酸酶的过程直接从完整的酵母球质体转移到植物原生质体中，可能是通过细胞融合。

项目成果

Yoshimichi Hatsuyama: "Direct transfer of plasmid DNA from intact yeast spheroplasts into plant protoplasts." Plant Cell Physiol.35. 93-98 (1994)

Yoshimichi Hatsuyama：“将质粒 DNA 从完整酵母原生质体直接转移到植物原生质体中。”

Y.Hatsuyama, N.Sunaga, Y.Habu, M.Ishikawa, S.Kawamoto, S.Naito and Y.Ohno: "Direct transfer of plasmid DNA from intact yeast spheroplasts into plant protoplasts" Plant Cell Physiol.35. 93-98 (1994)

Y.Hatsuyama、N.Sunaga、Y.Habu、M.Ishikawa、S.Kawamoto、S.Naito 和 Y.Ohno：“将质粒 DNA 从完整酵母原生质体直接转移到植物原生质体中”Plant Cell Physiol.35。