Cloning, Expressing And Characterization Of Centrin In Ceratopteris Thalictroides

Centrin (caltractin) is a member of the calmodulin (CaM) superfamily ofEF-hand calcium-binding proteins. These proteins contain four helix-loop-helixdomains,the so-called EF-hands, which may each bind one Ca2+. It is an essentialcomponent of centrosomal structures(basal body or spindle pole body) in a wide rangeof organisms,ranging from alga land yeast and protozoa to higher plants andhumans.Centrin is required for proper cell division,and plays important roles incontraction of centrin-based fiber systems in eukaryotic cells as well.During spermatogenesis in ferns, organelles such as blepharoplast, basal body,flagella, are formed de novo in cells. It is unclear whether centrin is involved in theformation of motile apparatus in ferns. In this study, Ceratopteris thalictroides istaken as experimental material.We clone the complete DNA sequence of centrinand construct prokaryotic expression vector to make it express. This study hasimportant implications for the follow-up study of the role of centrin gene in theformation of motile apparatus in ferns during spermatogenesis.Using Ceratopteris thalictroides as experiment material, the total RNA wasextracted from gametophyte.Then the cDNA sequence of centrin was cloned byRACE. The full-length of centrin gene sequence of Ceratopteris thalictroides is1077bp, the open reading frame is512bp encoding127amino acids. Phylogenicanalyzing by the Blastp on NCBI showed that the identity to other plant is veryhigh. Phylogenetic tree constructed by the software of Clustalx2.0andMEGE5.0. The result show that the centrin genes of C.thalictroides, C. rainhandtiiand P. patens gather as a class. That reveal they have close evolutionary relationshipto each other. Furthermore, the recombinant pET32-centrin plasmids were transformed into host E. coli BL21for expression. The recombinant proteinabout60kDa was induced by IPTG, in line with the expected size. Therecombinant protein was used for western blot detection, and the hybridizationbands were obviously. It indicated that the centrin gene was an encoding gene inCeratopteris thalictroides, which encodes a protein in the Ceratopteristhalictroides male gametophyte, and which has an important effect onCeratopteris thalictroides sperms. This study lays a foundation for elucidatingthe molecular mechanism of spermatogenesis in ferns.