Cloning And Functional Analysis Of Cyclin Dependent Kinase Inhibitor Gene OsCKI In Rice

Cell division is one of the most important biological processes of plant growth and development which contains three kinds of protein interactions of cyclin(CYC),cyclin dependent kinase(CDK)and cyclin dependent kinase inhibitor(CKI).Cyclin dependent kinase(CDK)plays a central role of the regulatory machinery controlling the progression of the cell cycle.Cyclin-CDK complex can activate CDK kinase activity and regulate the operation of the cell cycle.However CKI can inhibit the activity of CDK in the cell cycle appropriate point,negatively regulate cell cycle progression and effect the plant growth and development.In higher plants,the spatial and temporal regulation of cell division is essential to development of plant morphogenesis.Biotic and abioticl stresses are important factor in the process of regulation of the cell cycle.So far,although a large number of cell cycle regulatory genes have been identified in dicots,little is known about them in monocots model plant of rice.In order to explore new stress-tolerance genes in rice(Oryza sativa L.),expression profiles were obtained for leaf and panicle tissues at seedling,booting,heading and flowering stages of indica cultivar Pei'ai 64S plants under cold,drought and heat stresses by using the GeneChip Rice Genome Array(Affymetrix),which includes 51,279 transcripts from japonica and indica rice.A novel gene OsCKI(LOC_Os03g01740)was screened which responded to diverse stresses.By querying the NCBI database of protein,we found the gene encoding protein function is similar with setaria italica,phoenix dactylifera and brassica rapa code CKIs.Then We constructed the plant over-expression?antisense-expresses?GFP and prompter recombinant vector of OsCKI gene.In order to investegate the gene function,we have successfully transferred the above-mentioned OsCKI vectors into rice genome through agrobacterium mediation transformation system The main results were as follows:(1)Based on the bioinformatics analysis,OsCKI has no introns and cDNA was 829 bp,which contains an open read frame(ORF)of 321 bp and encodes a protein of 106 amnio acid residues with molecular weight of about 11.63KD and pI of about 9.09.(2)We analyzed OsCKI expression levels at different tissues under different stresses by gene microarray and qRT-PCR.We found that OsCKI is induced by a variety of stresses,especially in low temperature and drought treatment,indicating that the gene belongs to a multiple stress inducible gene.(3)We constructed the D-163+1300-OsCKI over-expression vector,D-163+1300-OSCKI antisense expression vector,D-163+1300-OsCKI-GFP carrier and pCAMBIA3301-OsCKI-Q prompter recombinant vector.Transgenic rice plants were obtained by transferring these recombination vectors into rice Nipponbare by agrobacterium-mediated transformation method.Then,we have carried on the detection and identification of transgenic plant.(4)Subcellular localization confirmed OsCKI gene is located in the cell nucleus which is consistent with the bioinformatics prediction.(5)By Real time PCR,we obtained the transgenic plant expression level.Compared with contrast nipponbare,expression level of three lines of transgenic plantsn is increased 12.89,17.14,12.89 fold,respectively.