Cloning And Functional Analysis Of High Expression Cullin Gene Related To Cell Cycle Under Iron-deficiency From Rice

Lack of iron is commonplace in nature, which has been a major cause for anemia, a "killer" of human health. By now, many scientists in the world who are engaged in studying iron-deficiency think that it is an effective way to solve iron-deficiency problem by promoting the iron content in food. Rice (Oryza sativa L.) is one of the most important cereal crops in the world. Researches on the response mechanism of rice in stress conditions at the cellular, physiological and molecular level will be helpful to increase the productivity potentialities of crops and improve the rice quality, which is always an important content in rice plant science.Based on the gene chip's study, we focused on the analysis of nine genes related to the cell cycle regulation. COP9 and cullin are the two major objects for the research. Cullin with the full length of cDNA 583bp and cDNA fragment of 575bp COP9 gene related to cell cycle regulation have been cloned by RT-PCR method from iron-stressed 5d rice root. Subsequently, transcriptional level analysis of cullin and COP9 was conducted with RT-PCR method and Real Time Quantitative PCR. The results indicate that there is no expression in iron-sufficiency in normal circumstances while higher expression takes place under Fe-deficiency, and the strongest expression occurs at the fifth day of Fe-deficiency treatment.What is followed is successful constructing of two new plant expression vectors pCAMBIA1302-cullin (S/A) -GFP containing fused protein CULLIN-GFP. The recombinant plasmid was then transformed into the calli of rice zhonghualO via agrobacterium EHA105. Through the tissue cultivating technology, we have gained the regenerating transgenic plants: 7 lines of over-expressed plants and 10 lines of anti-sense plants. The genome DNA of transgenic rice's leaves was extracted. Using the fragment of hygromycin gene as probe from pCAMBIA1302, it proved that cullin cDNA gene has been integrated into the genome of transformed rice plants by PCR PCR-Southern and Northern blotting analysis. By analyzing the phenotype of transgenic rice, it was found that the number of tillering in over-expressed rice increased and flowering time came earlier than wild rice and anti-sense rice, which is possible due to cullin complex involvement in the Ubiquitin pathway under iron-deficiency.At last, sub-cellular localization of CULLIN protein was analyzed by agrobacterium transformation and particle gun bombardment. The results showed that CULLIN protein was located in nucleus and plasma membrane. The research related to how cullin affect the iron's trafficking is still in progress.