Studies On Embryo-Specific Gene OsESG1 And Its Promoter In Rice

Receptor-like protein kinase (RLKs) belongs to a subfamily of protein kinase. The S-RLKs which has the S-domain of RLKs is an important group of receptor-like protein kinase family. They contain a cytoplasmic protein kinase domain that is activated by ligand binding to the extracellular receptor domain, and regulating the signal transduction pathway through a reversible phosphorylation system by phosphorylation/dephosphorylation. As the receptor in signal transduction, S-RLKs play some key roles in developmental and signal transduction processes in higher plants. In this research, a mumber of S-RLK family gene named OsESGl (Oryzo Sativa Embryo Specific Gene 1) and it’s upstream promoter which is a 1.4 kb fragement named OsESP1 were isolated using genomic DNA of rice (Oryza sativa L. cv. Zhonghua 11) as the template. OsESG1 and OsESPl were cloned into the plant expression vector and transformed into rice callus through Agrobacterium-mediated transformation, and then the hygromycin resistant transgenic lines were harvested. The expression profiles and function of OsESG1, and the activity and the key cis-elements of OsESP1, were analysed and investigated in detail. The main results were summerized and listed as follows:1. Using the OsESP1 full-length promoter and the series deleted promoters such as OsESPQ1, OsESPQ2, OsESPQ3, OsESPQ4 and OsESPQ5, the series promoter-GUS expression vectors were constructed, and the transient and stable expression systems were used to investigated the expression patterns and transcriptional activation abilities of the promoters in rice seeds. The results showed that the full-length and series deleted promoters all owned embryo specific expression patterns, and different transcriptional activities of them were observed and investigated.2. GUS activities of different promoter fragments were further analyzed using the fluorometric method. The results demonstrated that the GUS activity of OsESP1 transgenic plants were higher than that of transgenic plants transformed with OsESPQ4 or OsESPQ5, however, there was slightly difference of GUS activity between OsESPQ4 and OsESPQ5 transgenic plants. But the GUS activity of the OsESPQ4 and OsESPQ5 transgenic plants were higher than that of the OsESPQ1, OsESPQ2 or OsESPQ3 transgenic plants. Considering OsESPQ4 and OsESPQ5 all contain fragment of OsESPQ2, it seems that OsESPQ2 comprises tissue specific cis-acting elements and fragment, and OsESPQ2 is necessary in regulating promoter to express specifically and efficiently. Meantime, the deleted promoters of OsESPQ1, OsESPQ2 and OsESPQ3 had weak GUS activity seperatly, but when OsESPQ1 or OsESPQ3 combaintion with fragment OsESPQ2 respectively, the higher GUS activity could be obtained, suggesting that the interaction and cooperation of multiple cis-acting elements from different regions of promoter were necessary for efficient expression of OsESP1.3. The expression patterns of OsESG1 were analyzed by RT-PCR in different rice tissues, such as root, shoot, leaf, flower, seed coat, endosperm and embryo, and the embryo specific pattern was found. Further OsESG1 expression pattern analysis of embryo in different developmental stages showed that the expression level of OsESG1 kept increasing with increasing of seed maturity. The results suggested that OsESG1 is an embryo specific gene and it may play roles during the seed maturation process.4. In order to identify the function of OsESG1, the sense and antisense expression vectors of OsESG1 were constructed and transformed into callus of rice (Oryza sativa L. cv. Zhonghua 11) through Agrobacterium-mediated transformation. Comparing the phenotypic characterization of the transgenic plants with wild types indicated that the seeds of over expression transgenics germinated later than that of wild-type, and the radicle or plumule of over expression transgenic plants were shorter than those of wild-type plants. The root development of over expression transgenics delayed during the vegetative growth stage, and the number of adventitious root or even root hairs of over expression transgenics were less than those of wild-type plants. Therefore, these results indicated that OsESG1 may involve in the plant development process by inhibiting the seed germination, seedling and root development.5. The OsESG1 over expression Arabidopsis were also harvested through Agrobacterium-mediated transformation. The phenotype observation showed that the growth of transgenic seedlings were inhibited, and seed germination rate of transgenics also infected and delayed by Nacl or ABA treatment. The results above further confirmed that OsESG1 may regulate and participate in growth and development process of plant.In brief, an embryo-specific gene OsESGl and embryo-specific promoter OsESP1 were isolated from rice, and the expression patterns and biological functions were investigated and analyzed. The results showed that OsESPl is an embryo-specific promoter, and the specifically and efficiently expression of OsESPl required the interaction and cooperation of multiple cis-acting elements from different regions of promoter, especially the elements of OsESPQ2. By harvesting the transgenic plants of rice and Arabidopsis, the function of embryo-specific gene OsESGl was analyzed, and the results indicated that OsESGl may play roles in growth and development process of plants by influencing the seed germination, seedling and root development.