| In higher plants, cytokinins involve in the regulation of plant growth and development by regulating division and differentiation of plant cells. They participate in a wide variety of physiological processes, such as involving in gamete cell and embryo development, and cell division; promoting bud differentiation; regulating apical dominance; inhibiting root elongation; promoting the formation of vascular bundles, the development of flowers and fruits, and the production of anthocyanin; participating in the biosynthesis of chloroplast, stress response and pathogen resistance, as well as delaying blossom time and leaf senescence and so on. Recent researches have shown that cytokinin involved in Dictyostelium spore formation.In prokaryotic and eukaryotic cells, two-component signaling system is an important way to sense and transmit the environmental signals. It is currently a hot issue to identify the components and the functions of the system. Studies on the two-component signal system of arabidopsis were listed as those of the U.S. NSF 2010 projects and had made significant progresses. Researches show that two-component signaling system involves in transduction and transmission of plant hormone (ethylene, cytokinin), osmosis and light signals. The cytokinin receptors belong to the family of histidine protein kinases, they are two-component signaling system components. Three membrane-bound cytokinin receptors, AHK2, AHK3 and AHK4, were found in Arabidopsis thaliana. They can combine with the extracellular cytokinins and transduct the signals into the cells. AHK2 and AHK3 express in plant roots, stems, leaves and flowers, while AHK4 mainly express in roots. If AHK4 gene mutated, the plant would not exhibit the cytokinin inhibition to root elongation.Chinese cabbage (Brassica pekinensis Rupr.) is an important vegetable crop. The published genome sequences of Chinese cabbage are not complete. There are no genome-wide or individual characterizations of the components of this signaling system in Chinese cabbage.In this dissertation work, we carried out a range of molecular biological experiments, such as RT-PCR and RACE, to isolate the gene which is homologous with AHK4 (named PHK4) gene. The isolated cDNA sequence of PHK4 is 3156 bp. Alignment analysis of PHK4 gene was conducted based on the bioinformatics online programs, such as NCBI, Bioedit, CDD, SWISS-MODEL, Clustal X and RNAdraw. The ORF of PHK4 was predicted to be 2985 bp, and the deduced peptide was 994 amino acids in length. In comparison with the responding sequences of AHK4, the homology of cDNA and the deduced amino acid sequence of PHK4 are 86.65 % and 92.07 % respectively. The analysis based on Bioedit showed that the physicochemical properties between PHK4 and AHK4 are similar. Conserved domain and tertiary structure prediction analysis based on NCBI and SWISS-MODEL showed that PHK4 contained a trans-membrane domain, a transmitter domain and a receiver domain, and unrooted relationship tree analysis based on Clustal X showed that the evolutional relationship between PHK4 and AHK4 was closest. The 3'UTR region of PHK4 is 171 bp in length, and the homology of the 3'UTR sequence of PHK4 with AHK4 was 39.86 %, which was much lower than that of the coding sequence. The secondary structure prediction based on RNAdraw program showed that the 3'UTR region of PHK4 and AHK4 was similar.The experimental results and the analyses presented in this dissertation demonstrated that an unknown PHK4 gene which is homologous to AHK4 was isolated from Chinese cabbage. It is presumed that PHK4 may be a histidine kinase and may act as a receptor which involve in the cytokinin signal transduction. Further verification and analysis are under the way. The function of PHK4 can be validated by yeast functional complementary experiments.We plan to restor the yeast mutation by transfering PHK4 gene into the yeast mutant. In this dissertation, the mutant yeast (TM182) was cultured, and the eukaryotic expression vectors (pCUY315 and pCUY90)of yeast were cloned. These works provide basises for the functional verification of PHK4. |
