Functional Study Of Arabidopsis Inositol 1,3,4-trisphosphate 5/6-kinase In Photomorphogenesis

Inositol 1,3,4-trisphosphate 5/6-kinase (5/6-kinase) is a key regulatory enzyme in the process of the inositol phosphate metabolism. In mammalian cells, the 5/6-kinase exhibits the activity of both inositol kinase and protein kinase, and is also reported to have the activity of autophosphorylation. It is conserved from plants to humans and is found even in Entamoeba histolytica. Moreover, the 5/6-kinase is also the first identified COP9 signalosome (CSN) -associated kinase in mammalian cells. But the biological function of their association still remains unclear. In plants, it is only reported that the 5/6-kinase is the ubiquitous inositol kinase. But there are not more reports on the function of this enzyme. So, the works that study if the 5/6-kinase exhibits the activity of protein kinase, and if it associates with CSN and then involves in plant photomorphogenesis will be significative for opening out the biological function of 5/6-kinase and elucidating the mechanism of plant photomorphogenesis.In the research for the dissertation, we cloned the 5/6-kinase gene AtItpk-1 fiom Arabidopsis. We also expressed the His-AtItpk-1 protein in E.coli, obtained the purified fusion protein and prepared the anti-AtItpk-1 antibody. In order to analyze the expression of AtItpk-1 under different light quality, RT-PCR and Western blot were performed. The results indicated that the expression of AtItpk-1 was strongly induced by red light, which suggested that AtItpk-1 may be involved in the response of plant to red light. In addition, the study of localization showed that AtItpk-1 is located in the nucleus in Arabidopsis.In order to study the biological function of 5/6-kinase in Arabidopsis, two T-DNA insertion mutants of AtItpk-1, designated (by us) as atitpk-1-1 and atitpk-1-2, were screened and identified. Also, the transgenic plants overexpressing AtItpk-1 were generated by transgenic technology. The phenotypic results indicated when compared with wild-type plants, the atitpk-1 mutants exhibit the phenotype that red light inhibits the elongation of hypocotyls, and the over-expression lines of AtItpk-1 show the phenotype that red light promotes the elongation of hypocotyls. In addition, when the Atltpk-l gene was transformed into atitpk-1-1 mutant, the length of hypocotyls was restored to that of the wild-type. These results suggested that the 5/6-kinase Atltpk-1 may be involved in photomorphogenesis under red light in Arabidopsis.Using the active GST-AtItpk-1 fusion protein expressed by ourselves, the autophosphorylation assay was performed. The results showed that Atltpk-l has protein kinase activity, which can provide the clues for interpreting the possible function of 5/6-kinase in the photomorphogenesis.The fus6/CSN1-3-4 was used to do the co-immunoprecipitation (co-IP) of 5/6-kinase and CSN using the anti-AtItpk-1, anti-CSN4, and anti-CSN5 antibodies. The result showed that 5/6-kinase can co-immunoprecipitated with CSN in Arabidopsis. Based on co-IP and the result that both Atltpk-1 and CSN were located in the nucleus, we think that it is possible for the association of 5/6-kinase and CSN in Arabidopsis.In our research, it was found the 5/6-kinase may be involved in photomorphogenesis under red light in Arabidopsis, possibly via interaction with CSN. The role of 5/6-kinase in photomorphogenesis is possibly based on its activity of protein kinase. But the process of plant photomorphogenesis is very complex and the detailed mechanism for 5/6-kinase involving in photomorphogenesis still needs to study further.