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Functional Analysis of the Arabidopsis Transcription Factor DYT1

Author: YuYu
Tutor: YangZhongNan
School: Shanghai Normal University
Course: Biochemistry and Molecular Biology
Keywords: Arabidopsis transcription factor DYT1
CLC: Q943.2
Type: Master's thesis
Year: 2011
Downloads: 81
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Abstract


DYSFUNCTIONAL TAPETUM1 (DYT1) is a vital component of anther development and tapetal function in Arabidopsis. In this study, we carried out DYT1 promoter segmented deletion to search the region that contain regulatory element and spatiotemporal expression of DYT1 and prepared its polyclonal antibodies by prokaryotic expression. To identify the promoter region essential for regulating developmental expression of DYT1, series truncated promoter fragments of DYT1 were cloned to drive the GUS (β-glucuronidase) gene expression. Identified full-length promoter -632 and -550 bp(the translational start orientation) demonstrated normal expression of GUS in anthers. However, deletion of the promoter region containing the ACGT and GTGA motifs drastically reduced GUS expression and no histochemical GUS signal in further truncation. Additionally, the GUS expression pattern driven by the -550 promoter is correspond with the previous analysis of DYT1 expression in the tapetum from stage 3 to 5 under the Ler background.The region with specified motifs binding sites and genetic information of DYT1 promoter is responsible for early tapetal development.In addition, the 624bp cDNA of DYT1 ORF sequence was amplified with a specific primer pair and cloned into the pET-32a(+). The recombinant plasmid was then transformed into E. coli BL21. A soluble protein was induced with IPTG, and SDS-PAGE analysis of the protein showed a molecular weight of around 50KD, corresponding with the putative molecular mass of DYT1. Therefore, as the antigen, the protein was used to immune the rabbits to obtain polyclonal antibody, the titer of which was over 1:5120 demonstrated by ELISA assay. Additionally, the immunological specificity of polyclonal antibody was validated by Western Blot. These results lay a solid foundation for further investigating the regulatory mechanism of DYT1 in anther development.

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CLC: > Biological Sciences > Botany > Plant Cell Genetics > Plant Genetic Engineering
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