Immunopurification of polyribosomal complexes of arabidopsis for global analysis of gene expression

被引:187
作者
Zanetti, ME
Chang, IF
Gong, FC
Galbraith, DW
Bailey-Serres, J [1 ]
机构
[1] Univ Calif Riverside, Ctr Plant Cell Biol, Dept Bot & Plant Sci, Riverside, CA 92521 USA
[2] Univ Arizona, Dept Plant Sci, Tucson, AZ 85721 USA
关键词
D O I
10.1104/pp.105.059477
中图分类号
Q94 [植物学];
学科分类号
071001 ;
摘要
Immunoaffinity purification of polyribosomes ( polysomes) from crude leaf extracts of Arabidopsis ( Arabidopsis thaliana) was achieved with transgenic genotypes that overexpress a translational fusion of a ribosomal protein ( RP) with a His(6)- FLAG dual epitope tag. In plants with a cauliflower mosaic virus 35S: HF- RPL18 transgene immunopurification with anti- FLAG agarose beads yielded 60- Svedberg ribosomal subunits, intact 80- Svedberg monosomes and polysomes. Sucrose density gradient fractionation of the purified complexes demonstrated that the distribution of polysome size was similar in crude cell extracts and the purified complexes. The immunopurified complexes included putative cytosolic RPs of Arabidopsis and ribosome-associated proteins, as well as full- length transcripts of high and low abundance. Whole- genome profiling using long DNA oligonucleotide- based microarrays provided a high level of reproducibility between polysomal mRNA samples immunopurified from two independent biological replicates ( r approximately 0.90). Comparison of immunopurified and total cellular RNA samples revealed that for most of the genes, the mRNAs were associated with the epitope- tagged polysomal complexes, with an average relative level of association of 62.06% +/- 6 4.39%. The results demonstrate that the immunopurification of polysomes can be a valuable tool for the quantification of mRNAs present in translation complexes in plant cells. This technology can be extended to evaluation of mRNA populations at the cell- or tissue- specific level by regulation of the tagged RP with distinct promoters.
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页码:624 / 635
页数:12
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