烟草叶背面注射-Protocol.pdf

PROTOCOL Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants Imogen A Sparkes, John Runions, Anne Kearns Chris Hawes School of Life Sciences, Oxford Brookes University, Oxford OX3 0BP, UK. Correspondence should be addressed to I.A.S. (isparkes@brookes.ac.uk). Published online 30 November 2006; doi:10.1038/nprot.2006.286 s l Expression and tracking of fluorescent fusion proteins has revolutionized our understanding of basic concepts in cell biology. The o c o protocol presented here has underpinned much of the in vivo results highlighting the dynamic nature of the plant secretory pathway. t o r Transient transformation of tobacco leaf epidermal cells is a relatively fast technique to assess expression of genes of interest. These p e r cells can be used to generate stable plant lines using a more time-consuming, cell culture technique. Transient expression takes from u t a 2 to 4 days whereas stable lines are generated after approximately 2 to 4 months. n / m o c . e INTRODUCTION r u t Expression of fluorescent proteins (FPs) has been used in many express the transgene construct. Transient expression of FPs in a n . systems to investigate protein interactions, trafficking, turnover, tobacco epidermal cells is a relatively fast process requiring only w w organelle biogenesis, movement and inheritance. During the last 2–4 days from infiltration to expression. Subsequent generation w / / decade we have seen the development of GFP variants allowing the of stable transgenic plants takes from 2 to 4 months using a tissue : p t im