Micropipette extraction-based PCR amplification of mature mRNAs in single trichome cells of tomato leaves

摘要

Single-cell polymerase chain reaction (PCR) was conducted to detect in situ gene expression in targeted cells of tomato leaf trichomes. The cytoplasm was removed with a micropipette under a light microscope and subsequently used for reverse-transcription PCR (RT-PCR), followed by nested PCR. Two intron-containing genes, a glyceraldehyde 3-phosphate dehydrogenase gene and a plasma membrane H+-ATPase gene, were constantly expressed in these cells and therefore used as indicators of successful PCR reactions. In addition, the use of nucleus-free cellular contents for the RT-PCR and subsequent nested PCR analyses was effective for preventing contamination with the products derived from misamplification of corresponding genomic DNA sequences. Using this method, we detected the expression of certain stimuli-activated genes, following the exposure of trichome cells to volatile chemicals. Therefore, the present technique can be used to directly detect gene expression in single trichome cells of tomato leaves in response to external stimulation.