Characterization Of Expression Of D-type Cyclin Gene Poptr;CYCD1;1and Poptr;CYCD3;3from Poplar

Plant D-type cyclin controls the process of cell cycle which plays an important role in cell division. To research the characteristics of Poptr;CYCD1;1,we utilized the D-type cyclin Poptr;CYCD1;1as the research object and constructed the vector p1301-D1;1-RNAi and then transformed it into the tobacco,which is the over-expression of Poptr;CYCD1;1and Populus simoniixP.nigra, which is wild type. We also utilized the Poptr;CYCD3;3as the research object, cloned the promoter and demonstrated its characteristics and studied its expression pattern through the Agrobacterium mediated Arabidopsis thaliana flowers. We also constructed the vector expression the fused protein with GFP and imported it into the onion skin using the particle gun method to detect the Poptr;CYCD3;3subcellular localization.We over-express it in the tobacco to research its effect, the results show as the following statements.(1) We transformed the gene inhibition-expressing Poptr;CYCD1;1into transgenic tobacco which is the over-expression of Poptr;CYCD1;1and get20positive resistance buds, and obtained20transgenic lines by the PCR detection. Contrasted to the same development period tobacco over-expressioning Poptr;CYCD1;1gene, the expression of quantity of tobacco which is inhibition-expression was decreased distinctly by RT-PCR and Northern blot. Its characteristics was almost recovered to the WT tobacco. The results indicated that the vector of RNAi is effective.(2) We generated transgenic Populus which is inhibition-expression Poptr;CYCD1;1gene and obtained8transgenic lines by the PCR detection. Its expression quantity was generally lower than WT by RT-PCR and Northern blot detection, whereas the tansgenic line did not display much difference in the differentiation medium and remains to be further study.(3) The Poptr;CYCD3;3open reading frame (ORF) is1161bp in length, encoding a predicted polypeptide of386amino acids with a molecular weight of97634.3Da and a PI of5.04. We found the Poptr;CYCD3;3amino acid sequence has the most homology with castor-oil plant.(4) The fluorescence of the fused protein CYCD3;3-GFP concentrates to the nuclear compared with the control, the result showed that the Poptr;CYCD3;3is localized in the nucleus.(5) We get8lines of transgenetic Arabidopsis thaliana through the hygromycin screen, the Poptr;CYCD3;3promoter are fused into the Arabidopsis thaliana genome. GUS activity detection showed that the gene expresses in the growing point after germination7days.(6) We generated transgenic tobacco over-expressing Poptr; CYCD3;3gene and obtain10transgenic lines by the PCR detection, while compared with the wild type tobacco, transgenic Poptr;CYCD3;3line did not display much difference from the seeding of tissue culture to planting seeding and the reason remains to be further study.