Quantitative determination of mosaic GFP gene expression in tobacco
- 126 Downloads
A specific form of gene silencing that was observed visually as a mosaic distribution of fluorescent and non-fluorescent cells apparently dispersed at random within tissues was found in a few green fluorescent protein (GFP)-transformed tobacco lines. To characterize this event quantitatively, we studied flow cytometric measurements in GFP-expressing and -silenced cells in T1 and T2 progeny of four selected plants. The proportion of silenced cells varied considerably among the T1 lines but with notable genotype differences. Mosaic expression was inherited into the T2 generation in which the majority of progenies tested exhibited a level of silencing similar to that of their T1 parental plants. However, in some T2 progenies segregation, evident as a decrease or increase in the proportion of fluorescent cells, was observed. We discuss several factors, such as copy number, promoter activity or polyploidy, that may be the possible causes of the gene silencing, but none sufficiently explain the appearance of the mosaic distribution.
KeywordsNicotiana tabacum Mosaic gene expression Green fluorescent protein Flow cytometry
The authors are indebted to Dr. Michael J. Havey and Mark Petrashek for their help with Southern blotting analysis performed at the University of Wisconsin, Madison, Wis., USA.
- Assad-Garcia N, Ochoa-Alejo N, Garcia-Hernandez E, Herrera-Estrella L, Simpson J (1992) Agrobacterium-mediated transformation of tomatillo (Physalis ixocarpa) and tissue-specific and developmental expression of the CaMV 35S promoter in transgenic tomatillo plants. Plant Cell Rep 11:558–562Google Scholar
- Bohanec B, Luthar Z, Rudolf K (2002) A protocol for quantitative analysis of green florescent protein-transformed plants, using multiparameter flow cytometry with cluster analysis. Acta Biol Crac Ser Bot 44:145–153Google Scholar
- Feinberg A, Vogelstein B (1983) A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Ann Biochem 132:6–13Google Scholar
- Kump B, Svetek S, Javornik B (1992) Isolation of high molecular DNA from plant tissue. Res Rep Biotech Fac Univ Ljubl Agric 59:63–66Google Scholar
- Matzke MA, Matzke AJM (1996) Stable epigenetic states in differentiated plant cells: implications for somaclonal variation and gene silencing in transgenic plants. In: Russo VEA, Martienssen RA, Riggs AD (eds) Epigenetic mechanism of gene regulation. Cold Spring Harbor Laboratory Press, New York, pp 377–392Google Scholar
- Xugang L, Zhen Z, Dejiang F, Tuanjie C, Xiang L (2001) Influence of DNA methylation on transgene expression. Chin Sci Bull 46:1300–1304Google Scholar
- Xu-Gang L, Song-Biao C, Zi-Xian L, Tuan-Jie C, Qian-Chun Z, Zhen Z (2002) Impact of copy number on transgene expression in tobacco. Acta Bot Sin 44:120–123Google Scholar
- Yang NS, Christou P (1990) Cell type specific expression of a CaMV 35S-gus gene in transgenic soybean plants. Dev Genet 11:289–293Google Scholar