Isolation And Alignment Analysis Of PHK1 Gene Of Chinese Cabbage

In plant growth and development, they will be frequently affected by a variety of osmatic stresses, such as drought, salinification or freezing injury, etc. Plants have established molecular mechanisms to adapt these stresses in the long term of evolvement. The process of plants responding to stresses is a process of signal perception, transmitting and cellular reaction. Great progress has been made in the study on the osmotic stresses respond signal pathway. The achievement mainly focused on the signal transduction, cellular reaction and the expression of the relative genes. Protein factors (such as DREB, MAPK and phospholipase) that regulate signal transduction and stress inducible gene expression, and some protective proteins (such as proline synthetases, freezing-resistance proteins, and cysteine proteinases) have been characterized. It is not clear about how plants percept stress signals until recently, a break throughout was made with the rapid progress in the Arabidopsis genomic researches, and the isolation of AHK1 (Arabidopsis histidine kinase 1). AHK1 is a member of HK family of Arabidopsis. It is a kind of hybrid histidine kinases and has three conserved domains including a signal input domain, a transmitter domain and a receiver domain. In Arabidopsis, AHK1 involves in respond to osmotic stresses, and functions in a similar manner as SLN1, an osmotic sensor in yeast.This dissertation work was aimed at isolating an AHK1 homologous gene, named PHK1 (pekinensis histidine kinase 1) gene by the author, from Chinese cabbage.A DNA sequence of 7598 bp in length containing the PHK1 gene, and a cDNA sequence of 2848 bp in length were segregated using molecular biological techniques such as inverse PCR, conventional PCR and Genome Walking, and informatics methods.Alignment analysis of PHK1 gene was conducted based on the bioinformatics online programs, such as GENSCAN, CDD, SWISS-MODEL, Clustal X and RNAdraw. The ORF of PHK1 was predicted to be 2589 bp, and the deduced peptide was 862 amino acids in length. In comparison with the responding sequences of AHK1, the homology of CDS and the deduced amino acid sequence of PHK1 are 85.68 % and 88.57 % respectively. Conserved domain analysis showed that PHK1 contained a transmitter domain and a receiver domain, and unrooted relationship tree analysis showed that the evolutional relationship between PHK1 and AHK1 was closest. The 3'UTR region of PHK1 is 208 bp in length, and the homology of the 3'UTR sequence of PHK1 was 66.86 % with AHK1, which was much lower than that of the coding sequence. The secondary structure of 3'UTR region of PHK1 and AHK1 was similar based on the prediction of RNAdraw program.The experimental results and the analysis presented in this dissertation demonstrated that an unknown PHK1 gene which is homologous to AHK1 was isolated from Chinese cabbage. It is presumed that PHK1 may act as an osmosensor to take the changes of environmental signals, involve in the signal transduction and induce the respond to reduce the osmostatic stress injury. Further verification and analysis are under the way.Chinese cabbage is an important vegetable. The research on its molecular mechanism of osmotic stress response is an applied basic one. The results of this dissertation work will provide a basis for further characterization of PHK1, for study on the molecular mechanism of Chinese cabbage in response to osmotic stress, and will promote the relative researches on other cruciferae crops.