Isolation And Characterization Of An Embryo-specific Promoter From Rice Via Promoter Trapping

After completion of rice genome sequence, numerous uncharacterized genes need to be characterized for their biological functions. The mutant for particular gene is the basis for characterising gene's function. To this end, a new T-DNA multi-purpose vector pCAS04 was constrcuted in our lab, which can be used for both promoter trapping and activation tagging. A large scale of promoter trapping mutants was generated via improved Agrobacterium tumefaciens-mediated transformation. In this thesis, a line, I997 showing GUS expression specifically in the embryo of rice was identified through screening the promoter trapping mutant lines. The analysis of T-DNA flanking sequence revealed that the T-DNA was inserted at 940bp upstream of a gene encoding a putative embryo-specific protein, designated as OsESG (Oryza sativa Embryo Specific Gene). Expression analysis revealed that OsESG was preferentially expressed in embryo. Analysis of 1.1Kb OsESG promoter region demonstrated that some essential regulatory elements are probably involved in conferring the embryo specific expression pattern and stress responses. To investigate OsESG's temporal and spatial expression pattern, the 1.1 Kb upstream region of OsESG was fused to theβ-glucuronidase (GUS) reporter gene and the chimirac construct was transformed into rice. Analysis of transgenic plants indicated GUS expression was exclusively restricted to embryo, and was not detected in other tissues or organs such as endosperms, roots, stems, nodes and leaves. Futhermore, the expression of OsESG was significantly up-regulated by abscisic acid and dehydration. To analyze the biological functions of OsESG, the overexpression and RNAi vectors of OsESG were constructed. Preliminary analysis of transgenic plants suggested OsESG was invovled in certain stress responses. Through our research, people will know more about the molecular mechanisms during plant embryo development and plant stress resistance. The embryo-specific promoter cloned will be useful for specifically expressing the target gene in genetic engineering.In summary, the generation of mass promoter trapping mutant population and the following functional studies based on it will play an important role in cloning genes which possess agricultural value and identifying valuable promoters or regulatory elements. Meanwhile, it also provides methodological hints for functional genomic studies on fruits!...