| Summary: | 碩士 === 國立中興大學 === 生物科技學研究所 === 103 === DAF-Like gene 1(DAFL1) is a gene that encodes a putative RING-finger E3 ligase protein and GUS activity was specifically detected in the ovule of the DAF1::GUS carpel. Dominant-negative mutation of DAFL1 causes ovule defects in Arabidopsis plants. To identify and investigate the interacting proteins for DAFL1, yeast two-hybrid assay was applied. AT5G65180 (ENTH1), the potential target substrate protein of the DAFL1 was further characterized in Arabidopsis. ENTH family proteins are characterized by a conserved 150 amino acid sequence at the N-terminus of their proteins and have been found to act as adaptor proteins, which are involved in the clathrin-mediated endocytosis and have an essential role for growth, development and hormonal signaling. In this study, promoter assay by transforming constructs fusing the promoter of ENTH1 with GUS reporter gene (ENTH1::GUS) in Arabidopsis was performed. The result indicated that GUS activity was detected in most of the tissues but not in the cauline leaf, rosette leaf and stem. Strong GUS signals was especially detected in the carpel. Furthermore, 2X35S::ENTH1 was transformed into Arabidopsis for functional analysis. 2X35S::ENTH1 showed a similar phenotype to DAFL1 mutant plants by producing short silique with about half of seeds to that in wild-type plants. These results indicated that ENTH1 may be the target substrate protein for DAFL1. Further analysis of the protein interaction between full length ENTH1 and DAFL1 by yeast two-hybrid system will be performed. Moreover, the detailed analysis of the ovule defects should provide a deeper understanding of how ovule formation was regulated by DAFL1 and its interacting protein ENTH1.
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