In situ hybridizations

A part of a proof of concept, we constructed our first cDNA libraries in collaboration with Mohan B. Singh's lab at the University of Melbourne. In particular, we were interested in finding examples of cell specific gene expression. Using RT-PCR screening, we were able to isolate one clone that was very strongly expressed in the Svn, and one clone that was very strongly expressed in the Sua. To confirm these results, we conducted in situ hybridization on pollen sections using riboprobe constructs from these two clones. The first clone is found principally in the Svn and represents a polyubiquitin (Figure 1). The second clone is found principally in the Sua and possesses high sequence homology to a potassium transporter of Arabidopsis.

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Figure 2. In situ hybridization of polyubiquitin (clone 37). This is particularly highly expressed in the Svn, as confirmed by RT-PCR. New libraries have polyubiquitin highly represented among the clones. Interestingly, this polyubiquitin differs in the 5'-UTR, which may be related to its selective pattern of expression (see Singh et al. 2002). Two preparations are shown in three different lighting regimes: differential interference contrast DIC (left), mixed epifluorescence EF and DIC (middle), and EF (right). Anti-sense (left): Anti-sense probe labels the Svn (arrowhead), while the Sua (arrow) remain essentially unlabeled. The vegetative nucleus is located below the sperm cells. Sense (right): Sense probe did not label any observed preparations.

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Figure 3. In situ hybridization of clone 34, which is similar to NP_186854.1, a potassium transporter, putative (KUP3/KT4/POT4) of Arabidopsis thaliana. Based on our partial sequence, there was reasonably strong homology. Two preparations are shown in three different lighting regimes: differential interference contrast DIC (left), mixed epifluorescence EF and DIC (middle), and EF (right). Anti-sense (left): Anti-sense probe labels the Sua (arrowhead) and vegetative nucleus (arrow), while Svn, is visible only on DIC/EF and EF views. Identity of Sua was confirmed by plastid DNA nucleoids (data not shown). Sense (right): No label observed.

Figure 4. In situ hybridizations of clone A7 in pollen of Plumbago zeylanica. This is a surface protein associated with the sperm cells. This discriminates between a wide variety off(1) A7 antisense probe. The Sua is visible following detection by digoxigenin-labeled RNA. The arrow shows the Sua. (2) Same pollen grain as in 1 observed using mixed epifluorescence and bright field microscopy. (3) A7 sense probe. (4) Same pollen grain as in 3 observed using mixed epifluorescence and bright field microscopy.

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This file was last modified on Wednesday, 19-Aug-2009 16:45:30 CDT