黄瓜离体叶绿体转化方法

An In Vitro Cucumber Chloroplast Transformation Method

叶绿体是植物细胞内执行光合作用的半自主性细胞器,叶绿体转基因是研究叶绿体基因表达调控机制的重要技术。通常在细胞和组织水平进行转化时需要叶绿体同质化,因此实验周期较长。该文以无菌培养的黄瓜绿色子叶为材料,通过差速离心分离叶绿体,以0.33 mol/L山梨醇为叶绿体洗涤和悬液,在13 k V/cm电击电压条件下进行转化。经PCR、RT-PCR鉴定和荧光显微镜观察,证明外源基因能导入离体叶绿体并可进行表达。该方法有望为包括鉴定叶绿体表达载体功能等基础性研究工作提供快捷途径。

Chloroplasts are semi-autonomous organelles in which photosynthesis takes place and chloroplast transformation is an important technique for investigating regulation mechanism of gene expression in the chloroplast. Transformation of chloroplasts by cells or tissues normally requires a long experimental period for achieving homoplasmic lines. In this paper, a chloroplast transformation method is reported, by which chloroplasts are isolated by differential centrifugation from green cotyledons of cucumber cultivated under sterile conditions, washed and suspended in 0.33 mol/L sorbitol, and then transformed by electroporation at 13 kV/cm voltage. The presence of exogenous gene and its expression in chloroplasts after transformation were confirmed by PCR, RT-PCR and observation of GFP fluorescence by fluorescence microscopy. It is hopeful that this method can provide a quick way for identification of efficient chloroplast expression vectors and other basic research.