Isolation And Functional Analysis Of Osk Genes In Oryza Sativa

Ubiquitin-26s proteasome is an ATP-dependent protein degradation system in nuclear and cytoplasm. It regulates manifold metabolic processes via introducing the turnover of functional proteins and degrading the unusual proteins through a complicated and accurate mechanism to recognize the target proteins. SKPl is the core component of SCF complex which is the largest enzymatic system in ubiquitin ligases (E3s) and takes limited responsibility for the specific recognition of target proteins. It has been shown that SKP1 can also associate with a large number of protein complexes other than SCFs in organism and has widespread biological functions. As the cross-point of many kinds of biological processes, SKPl regulates the normal growth and development of organisms in rate-limiting supply.In plants, Ask1(Arabidopsis Skp1-like) is the only Skp1 homologous gene that has been characterized genetically, but a little is known about Skpl families in monocot so far. Rice belongs to the monocots, is easily to be transformed and has more data on its genetic background. In order to investigate the role of ubiquitin-26s proteasome system in ovule-development of rice, we isolated the Skpl homologous genes in rice ovule and investigated their functions.Using the PCR-based strategy, we isolated three Skpl homologous genes from the rice ovule, named them as Oskl, Oskl and Osk3, respectively. Sequence analysis demonstrates the three Osks share high homology with the SKPl in other plant species. According to the analysis of phylogenetic tree, we divides the 18 rice japonica Skpl homologous genes into 8 groups. The Oskl and Osk3 fall into the first group, and Osk2 belongs to the sixth group.Northern hybridization and semi-quantitive RT-PCR were carried out to examine the expression patterns of the three genes. The Oskl gene exhibits a constitutive spectrum inall tested tissues and organs. The Osk2 gene is expressed in mature pistils and seeds at day 1 after pollination predominately. The expression levels of Osk3 gene is relatively higher in the mature tissues such as the leaf and the mature endosperm than in other tissues.Comparing with the phenotypes of wild type rice, the UBly.Oskl transgenic plants showed the etiolation phenotypes. In Arabidopsis, plants transformed with different Osks genes exhibit different phenotypes. The transgenic plants with 35s::OskJ showed the shorten-petiole and crinkled-leaf during vegetative growth, and some adult plants showed the dwarf phenotype. The transgenic plants with 35s::Osk2 exhibited an etiolation phenotype and died ahead of anthesis. The most distinct phenotype of the transgenic plants with 35s::Osk3 is that the leaf blades curved from the abaxial side to the adaxial side.Based on the expression pattern and the phenotypes of transgenic plants, we suggest that Oskl may be the housekeeping gene and play an essential role among Osks. Oskl could be the ovule-specific gene and might be involved in the process of ovule development. Osk3 may participate in the regulation of the pattern formation of abaxial-adaxial asymmetry.