Genes Expression Studies During Embryogenesis Of Rice

Embryogenesis is the first stage in the development of new life in both animals and plants. The embryogenesis of crop plants, especially rice, is mostly important for both theoretical and practical applications. In order to understand the mechanisms of modulating embryogenesis especially at its early stages, we examined expression of embryos at three stages, 48-50 hours after flowering for undifferentiated embryos, 72-74 hours after flowering for embryos just prior to differentiation and 120-122 hours after flowering for differentiated embryos using many kinds of approaches. The expression level of a number of genes showed distinct changes during different stages of rice embryogenesis.1. Using the RT-PCR technique, three rice cDNA libraries from thousands of viable embryos at above three stages were constructed. The analysis of three cDNA libraries proved to be eligible. And a differential cDNA screening was performed among three libraries. Several clones showed significant differences in signal intensity after two rounds of hybridization.2. Due to many redundant genes lies in direct cDNA libraies, we try to normalize the libraries based on reassociation kinetics that can reduce effecttively the high variation in abundance. The screening results proved it to be an effective way to enhance work efficiency.3. In our work, we attempted to obtain the first comprehensive view of global geneexpression changes and identifying genes that potentially functioning during early stages of rice embryogenesis, we use a rice high-density oligonucleotide probe microarrays representing 21,000 unique known and predicted genes, corresponding to approximately one-third of the rice genome in collaboration with Syngenta Biotechnology inc. The expression level of a number of genes showed distinct changes during different stages of rice embryogenesis.4. Through RT-PCR, we got candidate genes for later use. Furthermore, all the results were further tested by RNA in situ hybridization. Studies of expression patterns of these genes will provide new information on the molecular mechanisms about embryogenesis of rice.5. In order to study the functions of candidate genes screened from the above approaches, expression constructs were constructed for rice transformation and an agrobacterium-mediated rice transformation was used for these functional analysis. And we have got regeneration plants.In a word, studies of expression patterns of these genes will provide new information on the molecular mechanisms about embryogenesis of rice.6. Another part of this work was about Arabidopsis Ca2+-dependent protein kinase (CDPK)-related protein kinase (AtCRKl). AtCRKl contains the kinase catalytic domain and a calmodulin (CaM)-binding site. The CaM binding domain was from 412 (L) to 429 (L) (LRKSALAALAKTLTVPQL). And AtCRKl could bind CaM in a Ca2+-dependent manner. This kinase phosphorylated itself and substrate such as histone HIS and syntide-2 in a Ca2+-independent manner and the activity was stimulated by several CaM isoforms through its CaM-binding domain (CaMBD) to about ten-fold. However the stimulation amplification of the kinase activity of AtCRKl by different CaM isoforms was similar. Also apillary electrophoresis analysis demonstrating that AtCRKl was a threonine protein kinase that was consistent with predication by database analyses of AtCRKl.