Cloning And Characterization In Vitro Of ADFs In Arabidopsis Thaliana And Physcomitrella Patens

Cytoskeleton in eukaryotic cells is to maintain cell shape, support various cellular activities. The microfilament in a dynamic structure is one of the major categories, through continuous polymerization and depolymerization to reflect its function. The realization of the function of which is through a multitude of microfilament binding protein.ADF (actin depolymerizing factor)/cofilin family is one of the ABPs(actin binding proteins),and have a crucial role in the dynamic of actin cytoskeleton.The activity of this family be regulated by different factors, such as pH,Ca2+ and the ratio of ADF/actin etc.Recent studies have shown in Arabidopsis thaliana has 12 ADF subtypes, they expressed in different development stages and organization, participate in many physiological activities of the plant.Here, we applied the method of molecular biology successful cloning including 11 members of the Arabidopsis thaliana ADFs and Physcomitrella patens ADF(one and only). And ligation to pMD18-T and expression vector, a preliminary attempt to express proteins in prokaryotic expression system, also characterized in vitro,and get the following results:1. From wild-type Arabidopsis thaliana seedlings cDNA by PCR, we successful amplification 11 members of AtADF family (in addition to AtADF12,At3g45990)and Physcomitrella patens ADF genes. Maybe in a special physiological conditions or specific expression in a certain environment conditions of a certain organ, so we temporarily failed to amplify out AtADF12.2. Cloning the gene can be expressed in prokaryotic system. Transfer the expression vector to the host cell, the target protein can be produced measured by the expression test.3. We found by co-sedimentation experiments:AtADF11 can bundle F-actin; AtADF10 and PpADF can sever F-actin, and the sever activity of PpADF can be regulated by pH and Ca2+.By the results of this experiment we revealed the differences between ADFs members, and combined with previous work, we have further understanding of the function of the ADF family, this is certain contribution to comprehensively understanding ADF family.