| The biochemical characterization of two tobacco calcium/calmodulin-binding protein kinases (NtCBKl/2) were analyzed; On the basis of optimization of in situ RNA hybridization technique, we studied the temporal and spatial expression of NtCBKl in shoot apical meristem during the transition to flowering. We also studied the function of NtCBKl during tobacco development by using transgenic analysis. The main results are summarized as follows:1. The define of calmodulin-binding domain: NtCBKs were expressed in prokaryotic cells as recombinant proteins. Using biotinylated calmodulin as probe, NtCBKs could bind calcium/calmodulin when calcium was added to the reaction buffer, while NtCBKs could not in present of EGTA. This result showed that the expressed proteins had calmodulin-binding ability in a calcium-dependent manner. Based on the analysis of software, the calmodulin-binding domains of NtCBKs are speculated. Furthermore, truncated NtCBKs were expressed in E.coli, and the calmodulin-binding domains were identified in the respectively positions.2. The activity analysis of NtCBKs: NtCBKs were expressed in BAC-To-BAC system, and purified by Ni-NTA and CaM-sepharose. The autophosphorylation and substrate phosphorylation of NtCBKs were calcium/calmodulin-independent, and the phosphorylated animo acids were serine/thronine. These data show that NtCBKs are serine/threonine protein kinases that bind Ca2+/CaM, but whose enzymatic activity is independent of Ca2+/CaM. The effects of NtCaMs on activity of NtCBKs were analyzed, finding Ca2+/CaM could stimulate the activity of NtCBKs. For NtCBKl, three tobacco CaM isoforms (NtCaMl/3/13) have been shown to bind to NtCBKl with the same dissociation constants (KD) and have the same effect in activating of NtCBKl. For NtCBK2, NtCaMl/3/13 have been shown to bind to NtCBK2, but with different dissociation constants as follows: 55.7 nM for NtCaM1, 25.4 nM for NtCaM3 and 19.8 nM for NtCaMl3 respectively, indicating that NtCaM3 and NtCaM13 had higher affinity to NtCBK2 than NtCaMl. The enzymatic activity of NtCBK2 was also differently modulated by various CaM isoforms. While the phosphorylation activity of NtCBK2 was assayed to be enhanced by only about two-three folds by the presence ofNtCaM1, the activity could be amplified up to eight-nine folds by NtCaM3 or ten-eleven folds by NtCaMl3, suggesting that NtCaM3 and NtCaMl 3 were better activators than NtCaMl for NtCBK2.3. The expression pattern of NtCBKl: The expression of NtCBKl had distinct temporal and spatial differences in shoot- apical meristem (SAM) during flower development of tobacco. In vegetative phase, NtCBKl mRNA distributed widely in actively young leaf primordium and SAM. Secondly NtCBKl gene expression was undetectable in the SAM of floral determination phase and the succedent formed young floral primordium, while the expression of NtCBK1 reverted to high level in floral organ primordia which were coming into being from floral primordium. In addition, the NtCBK1 promoter was fused to the GFP reporter gene to give the construct pNtCBKl:GFP, which was introduced into wild-type tobacco by Agrobacterium tumifaciens strain LBA4404 mediated transformation. Using the transgenic tobacco with pNtCBKl:GFP, we speculated the expression of NtCBK1 promoter by studying the distributing of GFP. We also detected the change of GFP in tobacco SAM during flowering. There was much expression of GFP in the meristem which was going to form floral primordium, and little expression signal of GFP was obtained in young floral primordium, but in succedent floral organ primordia, the expression of NtCBK1 rise to the before. Lots of GFP signal was detected in all meristems of 35S:GFP transgenic tobacco including floral primordium and floral organ primordia. These results imply the possible role of NtCBK1 during flowering transition.4. Function study of NtCBK1 by transgenic analyses: To further explore the possible role of NtCBK1 in flower transition, the transgenic analyses were carried out with several different expression co...
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