英文 原生质体制备.pdf

ATPP-Sheen-1 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. For commercial purposes, please contact Corporate Sponsored Research Licensing Office at MGH (617-428-0200) For citing this protocol: 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. 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 time to master the system 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. RT-PCR is routinely used to confirm that the endogenous gene response is similar to that of the corresponding reporter gene. Good luck with your experiments! Jen Sheen, Dept of Molecular Biology, Wellman 11, MGH, Boston, MA 02114 Updated on 9/26/97, 2/28/98, 9/11/00, 12/16/00, 4/6/01, 9/1/01, 9/29/01, 10/3/01, 4/21/02, 5/27/02, 10/10/03, 12/23/03, 3/22/04 References: Yanagisawa et al., 2003, Nature 425: 521-525 Asai et al., 2002, Nature 415: 977-983 Sheen, 2001, Plant Physiol. 127:1466-1475 Hwang Sheen, 2001, Nature 413: 383-389 Kovtun et al., 2000, PNAS 97: 2940-2945 Abel Theologis, 1994, Plant J. 5: 421-427 Masson Paszkowski, 1992, Plant J. 2: 829-833