拟南芥叶肉原生质体瞬时表达分析方法.pdfVIP

ATPP-Sheen-1 Transient Expression in Arabidopsis Mesophyll Protoplasts We share this protocol freely with laboratories conducting basic research. A simple registration form is required to help us keep a record of the users of this protocol and for future communication. For commercial purposes, please contact Corporate Sponsored Research Licensing Office at MGH (617-428-0200) For citing this protocol: Yoo, S.D., Cho, Y.H., and Sheen, J. 2007. Arabidopsis mesophyll protoplasts: A versatile cell system for transient gene expression analysis. Nature Protocols, 2: 1565-1575. Sheen, J. 2002, A transient expression assay using Arabidopsis mesophyll protoplasts. /sheenweb/ The protocol has been streamlined and can be applied to different types of plant materials. The growth condition of the plants seems most critical for experimental reproducibility. Each lab may need to work out the best plant growth conditions based on the available facilities and local supplies, including water, soil, air and light bulbs. Lower light (50-75 -2 -1 µmole m s ) and shorter photoperiod (13 hr) are more desirable to prolong vegetative growth. Clean air, water and soil are essential!! No need for excess nutrient or seedling transplant (sow your cold stratified seeds directly on wet soil). The quality of DNA and PEG is critical. The protocol is simple and the approach is powerful. However, it only awards success to the scientists who are willing to take the time needed to master the assay with patience and faith. Potential wounding and stress problems could be minimized during the experimental process to avoid high background. The protoplasts generated using this protocol have been used to study hormone, sugar, stress and defense responses using reporter genes that show similar responses in intact plants. Real-time RT-PCR is routinely used to confirm that the endogenous gene response is similar to that of the corresponding report