Part:BBa_K3890001
pLAT52 pollen specific promoter
Pollen specific promoter from tomato (Solanum lycopersicum). It has almost no expression in other floral or vegetative organs. It has been expressed in other model plants, such as Arabidopsis thaliana and Nicotiana tabacum, with similar expression profile, besides having homologues in other species’ genomes [1,2].
Usage and Biology
LAT52 is described as a very specific anther promoter expressed during microsporogenesis and can be used for guided pollen specific expression with minimal off-targets, having been successfully tested in other plant species besides tomato [1]. Studies with pollen expression have been very important especially for plant reproduction and development studies [2]. In the USP-Brazil 2020/2021’s team, it was used for isolating a protein expression to pollen in plants treated with pesticides as means to make it safer for bees.
Characterization
Through our whole project’s circuit (BBa_K3890000), we could confirm the efficiency of the pLAT52 local expression: we found no leaks to important plant parts such as leaves.
Figure 1.Scheme of steps for RT-qPCR. RNA from leaf and pollen are extracted and purified, then the samples are used as template for cDNA synthesis. Next, the standardized cDNA was used to perform the qRT-PCR to prove the difference of transcripts rate.
RT-qPCR assays were successfully carried out. Significative expression at transcript level for CYP2 (median Ct value of 21.4) and GUS (median Ct value of 20.5) genes were detected only for pollen of transgenic plants (median Ct values were above 31.9 for both genes in cDNA samples from pollen and leaves of control plants and from leaves of transgenic plants, as well as undetermined for both genes in negative control). These findings confirm the specific expression of CYP6G1 and GUS genes, at least at the transcriptional level, in the pollen of transgenic tomato plants and its functionality to produce in vivo transcripts.
GUS histochemical assays were also performed to check for the local expression of the promoter and staining was detected in the structure, through microscopy, confirming the efficiency of the promoter for an optimal local expression.
Figure 2. Schematic representation for β-glucuronidase (GUS) assay on pollen. The catalysis of X-Gluc produces a chromophore that can be easy to visualize and shows the traduction of transcript.
References
1. Twell D, Yamaguchi J, McCormick S. Pollen-specific gene expression in transgenic plants: coordinate regulation of two different tomato gene promoters during microsporogenesis. Development. 1990 Jul;109(3):705-13. PMID: 2401221.
2. Twell D, Wing R, Yamaguchi J, McCormick S. Isolation and expression of an anther-specific gene from tomato. Mol Gen Genet. 1989 Jun;217(2-3):240-5. doi: 10.1007/BF02464887. PMID: 2770694.
Sequence and Features
- 10COMPATIBLE WITH RFC[10]
- 12COMPATIBLE WITH RFC[12]
- 21COMPATIBLE WITH RFC[21]
- 23COMPATIBLE WITH RFC[23]
- 25COMPATIBLE WITH RFC[25]
- 1000COMPATIBLE WITH RFC[1000]
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