| Summary: | 碩士 === 國立中興大學 === 農業生物科技學研究所 === 88 === Aims to isolate genes specifically expressed before pollination in rice, we used differential screening and obtained several cDNA fragments. Using sequence homology search analysis, BF171 clone sharing high sequence similarity with a pollen-specific CDPK (calcium-dependent calmodulin-independent protein kinase) gene in maize, was identified. A rice gene homolog of maize CDPK, designated as OSCK1 (Oryza sativa calcium-dependent calmodulin-independent protein kinase 1), was isolated using BF171 as probe in screening of rice cDNA and genomic library. OSCK1 encodes a polypeptide which contains 541 amino acids (predicted molecular mass is 59 kDa). OSCK1 protein includes one N-terminal protein kinase domain, on junction domain with inhibitory role, and one C-terminal calmodulin domain with four putative EF-hand calcium-binding loops. Northern blot analysis indicated that a single signal, roughly estimated as ~2.3 kb, can be detected by OSCK1 probe. The OSCK1 transcripts were highly accumulated in maturing panicles, but were barely detectable in young or booting panicles, and were drastically disappeared in grains after pollination. In a more detailed dissection, the OSCK1 transcripts were found to be very abundant in mature pollen. Such results strongly suggest that the expression of OSCK1 is expressed restricted in mature pollen. High stringency hybridization analysis of genomic DNA revealed that OSCK1 was constituted by at least two closely related members in rice. To further investigate the expression pattern of OSCK1 gene, the upstream promoter was isolated and its transcriptional start site was determined by high resolution S1 nuclease mapping. The gene expression and physiological roles of OSCK1 are evaluated by promoter study and transgenic plant analysis respectively. Preliminary results showed that the OSCK1 promoter, instead of confer pollen-specificity, drive the downstream GUS expression ubiquitously in stems, leaves, and flowers of Arabidopsis.
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