The Research Of The Correlation Between Enlargement Of Chinese Cabbage Clubroot And Expression Of BrHK4?BrRR15 And BrRR16 Genes

External environment signals are transfered across the membrane into cells with the multi-step protein phosphorylation in the bicomponent signal system of the eukaryotic cells,which regulates gene expression and further causes the physiological and biochemical reactions of the cells.In recent years,two-component signaling system,confirmed its participation in the cytokinin signaling,has been identified in Arabidopsis,tomato,rice and other higher plants.Plant cytokinin receptors,elements of the plant two-component signaling system,belong to the histidine protein kinase family.AHK2,AHK3 and AHK4/CRE1/WOL were found to exist as cytokinin receptors in the current research of Arabidopsis,and AHK4mainly expresses in the roots.There are 32 response regulators in the cytokinin signal transduction network of Arabidopsis.The typical response regulators are divided into Type A and Type B;The ARRs of type A play a negative regulatory role in the cytokinin signal transduction.The ARRs of type B,which have transcriptional activation activity,regulate expression of the downstream genes as positive response regulators.Further studies indicate that ARR15 and ARR16 are involved in the response of cytokinins in the roots,and they response to the AHK4 in Arabidopsis.Both Chinese cabbage(Brassica rapa spp.Pekinensis)and Arabidopsis are cruciferous plants.In our lab gene sequences homologous with AHK4 have been amplified from Chinese cabbage self-line AB-81,including the DNA,CDS,3~?-UTR and 5~?-UTR sequences,and the gene is named BrHK4,which we formerly named PHK4.Meanwhile,a series of experiments about BrHK4 gene has been conducted:The cytokinin receptor function of BrHK4 gene has been validated preliminarily by eukaryotic expression system of yeast and functional complementation experiments of Arabidopsis mutants;It has been demonstrated that the relative molecular weight of prokaryotic expressed BrHK4 is about 116 KD;A plant expression vector of GUS gene drived by BrHK4 gene promoter has been constructed.The reseach results about BrHK4 gene in this work are as follows:(1)Explore the key period of root fungus infection of Chinese cabbage.The results showed that 14 days after germination was the optimum inoculation time,with the highest incidence rate of 90%and the shortest average onset time of 16 days after inoculation.(2)The relative quantitative data results of the real-time PCR analysis of BrHK4,BrRR15,BrRR16 genes expression in different periods of incidence when chinese cabbages infected with clubroot pathogen revealed that in different periods of incidence the expression level of the three genes of the swollen roots were higher than that of the control roots.It can be inferred that the enlargement of cabbage roots caused by Plasmodiophora brassicae Woronin parasitism was closely related to the expression of BrHK4,BrRR15,BrRR16 genes.(3)GUS gene drived by BrHK4 promoter was transformed into Arabidopsis genome by Agrobacterium capullo transformation method,and the positive plants have been obtained by screening.PCR amplification of positive plants DNA has got specific fragment of the objective genes,laying a foundation for further research about the expression of BrHK4 gene.