Real time RT-PCR methodology: Real time RT-PCR will be used to examine the expressional profiles of multiple genes in parallel and to provide information on differential expression patterns. The figure below represents typical results:
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| Histone 3.3 was used as a control (1: Sua, 2: Svn). Clone A7 is shown as 3 (Sua) and 4 (Svn). For each sperm cell type, the reactions were repeated at least once (data analyzed using ABI Prism 7000. In the Sua, clone A7 displays expression levels that are 10.7-fold higher in Sua than Svn. Clone A7 is shows significant sequence homobology with At AGP20 and is presumably a membrane bound transmembrane protein |
About 2000 Sua and Svn sperm cells are used to extract total RNA. RNase-free DNase I is used to eliminate genomic DNA contamination in the column. Total RNA is reverse transcribed by oligo(dT). About 50 cells are required for SYBR Green real time PCR. As a control, a housekeeping gene, such as histone 3.3, which is expressed in both Sua and Svn sperm cells, can be used to monitor the expression profiles of other genes. (Other housekeeping genes will also be selected and used as controls as well, which will serve as an additional precaution in case there is a subtle differential in any given housekeeping gene. The following tissues will be tested: microspores, bicellular pollen, seedlings, root, stem, leaf, mature pollen, sepal, petal, mature ovule, fertilized ovule, Sua, and Svn, as indicated in the following examples.
Some illustrations of observed gene expression patterns: The following images are illustrative of different patterns that have been observed to date, but are not exhaustive. Current results are still being analyzed and extended. Also, in situs are not yet available for these to confirm patterns of expression during development.
The illustration below shows a sequence that is expressed in microspores, Sua and Svn. Interestingly, it does not appear to be expressed in the bicellular pollen or in the pollen in general, although this conflicts with its presence in the Sua and Svn, which are present within the pollen. The sequence is known to be in whole mature pollen, yet the relative concentration of transcripts appears to be too low to be detected. It is not possible to discriminate whether the mRNA is "sequestered" in the male reproductive cells, as expression in the generative cell has not been confirmed.
| Svn Seq A3J |
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 | Apparently the pattern of gene expression of this sequence is related to generative and sperm cell development. This clone was isolated from microarray screening. |
Next is an example of a sequence that is expressed principally in the sporophytes. Low expression is detected in microspores and bicellular pollen, high expression in all vegetative sporophyte tissues, with expression also noted in reproductive sporophyte tissues. The Sua and Svn, however, have very low levels of expression. Microarray data suggested that it displayed over four times more expression in the Svn.
| Svn Seq A6B |
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 | Apparently this gene is principally expressed in sporophytic cells. Isolated from microarray screening, this was expressed in trace amounts in the Svn. |
Here are some other patterns of gene expression. This page is still under active development. More will appear soon.
This file was last modified on Wednesday, 28-Jan-2004 00:48:52 CST