Rangasamy D, Ratledge C, Woolston C J
Department of Applied Biology, University of Hull, Hull, HU6 7RX, UK Fax no.: +44-1482-465458 E-mail:
Plant Cell Rep. 1997 Jul;16(10):700-704. doi: 10.1007/s002990050305.
Protoplasts isolated from pea leaves (Pisum sativum L. cv. Hurst Greenshaft) were electroporated in the presence of plasmid pDR#1, which contains the rat liver ATP:citrate lyase gene fused to a duplex 35S cauliflower mosaic virus promoter with a transit peptide sequence of the Rubisco small subunit. The level of enzyme expression and viability of protoplasts were both influenced by polyethylene glycol treatment before electroporation. Under the optimised electroporation conditions, an average increase of ATP:citrate lyase activity of 14% was observed in the transfected cells after 24 h, with a similar magnitude of change in the abundance of the corresponding mRNA. Immunoblot analysis confirmed the correct expression and targeting of ATP:citrate lyase protein in the chloroplasts of pea protoplasts. These results provide a basis for the establishment of a procedure for targeting heterologous protein into pea plastids in the presence of a transit peptide.
从豌豆叶片（豌豆品种Hurst Greenshaft）分离得到的原生质体，在含有质粒pDR#1的情况下进行电穿孔处理。该质粒包含与双链35S花椰菜花叶病毒启动子融合的大鼠肝脏ATP：柠檬酸裂解酶基因，并带有核酮糖-1,5-二磷酸羧化酶小亚基的转运肽序列。电穿孔前的聚乙二醇处理会影响原生质体的酶表达水平和活力。在优化的电穿孔条件下，转染细胞在24小时后ATP：柠檬酸裂解酶活性平均增加了14%，相应mRNA丰度的变化幅度相似。免疫印迹分析证实了ATP：柠檬酸裂解酶蛋白在豌豆原生质体叶绿体中的正确表达和靶向定位。这些结果为在转运肽存在的情况下将异源蛋白靶向导入豌豆质体的方法建立提供了基础。
