| Actin, the major component of the dynamic microfilaments system, exists in nearly all eukaryotic cells, and plays an essential role in living activities. In plants, actins are encoded by multi-gene families. There are many kinds of actin isoforms expressed in tissue-specificity and different phases during plant development for different functions. Actin isoforms have complicated interactions with actin-binding proteins. So it is important to study the physicochemical properties of plant actin isoforms in vitro for further understanding their functions and dynamic regulation in vivo. Although fluorescence labeling method was widely used in the observation of microfilaments' dynamic distributions in vivo, all of the traditional methods have their limits. The use of GFP fusion and the preparation of new type of algae fluorescent probe will facilitate our further study on plant actins.By gene fusion and prokaryotic expression, we purified a pea actin isoform (PEAc1), His-tagged PEAc1, His-tagged GFP and His-tagged PEAc1-GFP from inclusion body. After filtrating a series of induction condition, we expressed and purified His-tagged PEAc1 with soluble form in a large amount. The secondary structure of fusion proteins was determined by circular dichroism spectrum (CD). Compared with the results predicted with biology software, His-tagged PEAc1-GFP expressed in soluble form has more reasonable secondary structure, indicating that the fusion of His-GFP-tag facilitates PEAcl's soluble expression and correct folding. This may be a good method for obtaining soluble and abundant plant actin isoforms and other proteins which could not fold exactly and exist in inclusion body by prokaryotic expression.The polymerization ability of the fusion actins above was studied in detail. The his-tagged PEAcl-GFP purified from the supernatants could polymerize into green fluorescent filamentous structures with diameter, length and shape being identical to that of muscle F-actins, which could be labeled by TRITC-phalloidin (a specific agent for staining actin microfilaments), and were identified as having a 9 nm diameter by negative staining, corresponding with that of the muscle F-actins (7-10 nm). Under polymerization conditions, His-tagged PEAcl-GFP polymerized with kinetics similar to those of skeleton muscle actin, that is, an obvious lag nucleation period at the beginning of polymerization and an S-like typical polymerization curve could be obtained. The critical concentration is 0.75 umol/L near to that of chicken muscle actin (0.56 umol/L) under the same condition. All above showed that PEAcl preserved the polymerization activities of actin, and GFP fusion gave facilities for the study of microfilaments properties in vitro.Monomer His-tagged PEAc1-GFP could notably inhibit DNase I activities. Polymer His-tagged PEAc1-GFP efficiently activated myosin Mg-ATPase activity, which indicated that PEAcl might take part in correlative living activities in vivo. Moreover, this result provided experimental proof in vitro for fusing GFP to actin isoform directly to study the dynamics of microfilaments and its regulation in vivo.We prepared rabbit anti-pea actins polyclonal antibodies using PEAcl as antigen which being expressed and purified from prokaryotic cells, and the antibodies possessed better immunity activity to pea actins. .From the direct mutant of Spirulina platensis(SP-D), we got high purity and activityphycobiliprotein which could grow crystals. The algae fluorescent probe prepared by coupling the above polyclonal antibody to phycobilipotein not only keeps the property of stronger anti-fluorescence quenching but also has the lower fluorescent background when it was used for labeling stoma cells of pea tendril. The data above showed that algae phycobiliprotein fluorescent probes not only facilitate the study to pea actins but also provide a doable method to label other plant proteins. It has a good foreground for algae fluorescence probe to be used in the immunity fluorescent detection of plant cells. The gene clon...
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