Direct RT-PCR amplification of mature mRNAs in cytoplasm micropipetted from barley [Hordeum vulgare] coleoptile epidermal cell: A model system for analyzing gene expression in host cells attacked by powdery mildew [Erysiphe graminis]
Single-cell RT-PCR was used to detect gene expression in situ in single selected cells of detached barley coleoptile epidermis inoculated with Blumeria graminis f. sp. hordei. The cytoplasm was removed with a micropipette using a light microscope and directly used for RTPCR, followed by nested PCR....
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Veröffentlicht in: | Memoirs of the Faculty of Agriculture of Kinki University (Japan) 2005-03 (38) |
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Zusammenfassung: | Single-cell RT-PCR was used to detect gene expression in situ in single selected cells of detached barley coleoptile epidermis inoculated with Blumeria graminis f. sp. hordei. The cytoplasm was removed with a micropipette using a light microscope and directly used for RTPCR, followed by nested PCR. Three intron-containing genes, vacuolar ATPase B-subunit gene, cytosolic triosephosphate isomerase gene, and glycolitic glyceraldehyde-3-phosphate dehydrogenase gene were constantly expressed in this tissue and were therefore used as indicators, because shorter-size PCR-products produced by splicing are easy to detect. By simultaneously amplifying both target and indicator genes, the transcription of some stimuliinduced genes such as chitinase 2 and acidic beta-1,3-glucanase genes could be precisely detected in powdery-mildew-invaded and noninvaded, neighboring cells of the coleoptile epidermis. In addition, micropipetting only the cytoplasm without the nucleus prevented contamination with genomic DNA, which leads to miss-amplification of corresponding genomic DNA sequences of the intron-less genes during RT-PCR and subsequent nested PCR. Thus, this technique can be successfully used with coleoptile epidermal cells on which the powdery mildew fungus has attempted to invade and/or form a haustorium or with epidermal cells in the vicinity of infected cells. |
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ISSN: | 0453-8889 |