Analysis Of The Seed-specific Promoters In Arabidopsis

The seeds of plants are closely related to the human life, and therefore become important target material in plant genetic engineering. Whether the exogenous gene of the conversion can be expressed correctly, efficiently and as desired in the plant recipient is of current concern. Generally, the exogenous gene is required to have a high level of expression in the particular space and time in the transgenic plants with the expected phenotype. The promoter plays a key role in regulation of gene expression. Promoter determines the position of transcription start site and the frequency with which the gene is transcribed. Structural and functional analysis of promoters is of great significance not only to elucidating the regulation mechanism of gene expression but also to the modification of crops using gene engineering. Promoters contain the core promoter and some special cis-acting elements. Those cis elements play important roles in the regulation of gene expression. Some specific elements are present in the seed-specific promoters and thus regulate the expression of these seed-specific genes.Data mining from the DNA sequences is usually separated from that of the gene expression data in the research of bioinformatics. However, gene expression is closely related to its promoter sequences, and their combination allows biologists to study the gene expression and transcriptional regulation at genome level. Given a set of upstream DNA sequences whose genes are clustered together based on their similarity in the gene expression profile, it is desirable to find the common sequence motifs that are the potential transcription factor binding sites regulating the transcription.Arabidopsis thaliana wild-type and transgenic plants used in this work were Col-0 ecotype. The expression patterns of the seed-specific genes from Arabidopsis were obtained from the chip data. To analyze the promoters of some seed genes, the promoter region was linked to the reporter gene, and the resulting chimeric promoter-GUS transcription fusion was introduced into Arabidopsis. The main results were as follows:1. The expression patterns of the seed-specific genes from Arabidopsis were obtained from Genevestigator. The information of the false positive genes in the data was deleted based on the UniGene EST patterns, and finally 63 seed genes were selected. We obtained the promoter regions of these genes from the TAIR website, and then analyzed the regulatory elements using PlantCARE Web site, indicating that most of the promoters contained some seed-specific elements.2. The promoters of the genes AT2G03520, AT3G22500, AT4G27530 were linked to the GUS reporter gene respectively, and the resulting chimeric promoter-GUS transcription fusions were introduced into Arabidopsis. The histochemical GUS staining and fluorometric assay revealed that these promoters were capable of driving the target gene to express in a seed-specific manner.3. The endogenous gene expression patterns of AT2G03520, AT3G22500, AT4G27530 were detected using the real-time quantitative PCR. The results are consistent with the microarray data and the GUS analysis, suggesting that the chip data can be used in the research of the promoter analysis.4. Deletion analysis of the promoters showed that, the AT2G03520 full-length (-444 to 1) and the deletion products 2Gâ–³1(-322 to 1)and 2Gâ–³2(-200 to 1) of the AT2G03520 promoter still drive gene activity limited to the seeds. However, the promoter activity was severely reduced in its deletion construct 2Gâ–³3(-125 to 1). AT3G22500 promoter full-length (-664 to 30) was capable of driving the target gene to express in the seeds, while the promoter activity was lost in the deletion product 3Gâ–³1(-406 to 30). The AT4G27530 full-length (-812 to -2) and the deletion fragments 4Gâ–³1(-615 to -2)conferred seed expression pattern, but 4Gâ–³2(-418 to -2)and 4Gâ–³3(-203~-2)losed seed-specific expression pattern. 5. Using the YMF, weedr, Bioprospector and Mobydick program we predicted the RY motif, B-box, G-box and other known seed-specific elements in the 1-kb upstream sequences of the 63 genes. These results indicated that the combination of the microarray data, genome sequence information and biological software can be used to analyze the regulatory elements in the promoters of the genes with the same expression patterns, and to obtain the information that is of great biological significance.