| Calcium as a universal second messenger regulates a range of cellular and physiological processes in plants and animals. It is believed that changes in cytosolic Ca2+ are sensed by a group of Ca2+-binding proteins including CaM-like domain protein kinases (CDPKs) and calmodulin (CaM). CDPKs, well-characterized kinases, have been showed to be modulated by binding of Ca2+ to a carboxyl-terminal CaM-like sequence with EF hands and mediate many of the effects of Ca2+ in plant cells.CaM is a ubiquitous intracellular Ca2+ receptor involved in transducing a variety of extracellular signals. Whereas mammalian cells, such as those of humans, contain only one CaM protein encoded by at least three differentially regulated CaM genes, plant cells have multiple CaM genes which encode numerous CaM isoforms. The existence of multiple divergent CaM isoforms in plants poses the question whether or not they allow differential regulation of targets and can confer different Ca2+ sensitivity to CaM-binding enzymes or proteins. One group of CaM-binding proteins is composed of Ca2+/CaM-dependent protein kinases. In animals, a large number of Ca2+/CaM-dependent protein kinases (CaMKs) have been identified, including CaMKs I, II and IV. Among them, multifunctional CaMK II, a well-characterized kinase that can phosphorylate a broad range of proteins upon binding to Ca2+/CaM, is thought to play important roles in a variety of cellular events in mammals.Although typical Ca2+/CaM-dependent protein kinases with activities similar toCaMK in animals have not been identified in plants, several protein kinases that share sequence homology with CaMK have been identified in plants. While the CaM-binding abilities of these kinases have been demonstrated, the effects of CaM on their activities has not been established. A chimeric Ca2+/CaM-dependent protein kinase (CCaMK) with a visinin-like Ca2+-binding domain has been characterized from lily and tobacco. Whereas CaM is required for substrate phosphorylation by this CCaMK, CaM suppresses its autophosphorylation, indicating that CCaMK has different enzymatic and structural characteristics from classic animal CaMKs. Recently, a CaM-binding protein kinase, OsCBK, was isolated from rice. Though OsCBK can bind to CaM with high affinity, both autophosphorylation and substrate phosphorylation of this kinase is CaM-independent.A current topic in plant molecular biology is how calmodulin-binding protein kinase (CBK) and CaM-mediated signal transduction pathway is involved in regulation of plant growth and development The biochemical characterization of Arabidopsis calmodulin-regulated CDPK-related protein kinase (AtCRK5) and tobacco calcium/calmodulin-dependent protein kinase (NtCaMKl) were analyzed. We also studied the function of ACRK5 during Arabidopsis development by using an Arabidopsis thaliana genome DNA library. The main results are summarized as follows:This project is composed of two major parts. One is using an Arabidopsis thaliana genome DNA library, which based on binary vector pMD-1 (which is binary vector pBI121 by origin, the genome DNA fragments which were inserted into binary vector, under the control of CaMV 35S), to transform of Arabidopsis thaliana by Vacuum infiltration. There may be three kinds of mutant when scanning the mutant-libraty, overexpressed, antisense and knockout mutants. By optimized the methods of growth, transfromation and selection, we have set up a stable system. The timing of flowering is important for the reproductive success of plants. We devised a method way that alters the balance of a gene product by over-expression or repression of the gene randomly, then the phenotypes of the mutants can be observed for gene family. A novel gene that regulates the timing of flowering was identified in Arabidopsis and characterized as a Arabidopsis calmodulin- regulated CRK-AtCRK5, which is invoved in the transition from vegetativeto reproductive development. The amino-terminal region of AtCRK5 contains all 11 conserved subdomains characteristic of the catalytic dom...
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