Function Analysis Of The Promoter Of Wheat Pollen Specific Gene Tapsg719

The tissue-specific expression identification of the gene regulated by wheat pollen-specific gene TaPSG719's promoter was carried out in this paper, and some progress in the promoter study in plant gene regulation theory was obtained, which is a base for male sterile line construction.This paper is based on the cloned promoter of the gene TaPSG719 (Genbank ID: FJ497025) by our laboratory, and two fragments of the promoter were amplified by PCR, which are about 1.0 kb?and?1.7 kb in length, respectively. Both fragments were digested by restriction endonuclease Bam HI first and then Hind III after they were introduced into T-vector. Meanwhile, vector pBI121 was treated exactly the same way with those enzymes. These two fragments then were linked to the longer fragment of digested vector pBI121. As the vector pBI121 has a GUS gene, the vectors obtained successfully were named p1.0GUS and p1.7GUS, respectively. Two vectors were transformed into agrobacterium, and the transformed agrobacterium was used to bring the fusion gene of promoter fragment/reporter gene GUS into tobacco. Transgenic plants were gained by tissue culture. The gDNA were extracted from the regenerated plants'leaf. PCR with different primers were carried out to select positive ones. Then GUS histochemistry stain was done, the material included positives ones, negative ones, and transgenic ones with the fusion gene of promoter fragment/ GUS, and the tissue stained were root, caulis, leaf, sepal, immature pollen, mature pollen and stigma.Experiment result:①Identification of p1.0GUS and p1.7GUS by PCR and later sequencing showed that the vectors were constructed successfully;②Identification of regenerated plants by PCR proved that the foreign gene had inserted into the genome of tobacco;③After GUS histochemistry stain, no tissue of the negative tobacco turned blue and positive ones gave a completely opposite result, and in the transgenic ones with the fusion gene only pollen turned blue;④In the transgenic ones with the fusion gene,immature pollen turned deep blue after stain, while only very few of the mature pollen turned blue. That gene GUS displayed tissue-specific expression is obviously the result of the regulation of the fragments of the promoter. The expression activity of GUS gene was distinctly different in the different developing stages of pollen from the transgenic tobacco with the fusion gene.