Cloning And Functional Analysis On Flagellar Regeneration Of Kinesin Family Protein KIF4from Dunaliella Salina

In mammals, most cell types have cilia, protruding structures involved in sensing mechanical and chemical signals from the extracellular environment, that act as major communication hubs for signaling controlling cell differentiation and polarity. It has been proposed that cancer cells with cilia disorder reduce or alter their response to extracellular cues that regulate growth and differentiation. But the machanism of cilia assembly regulation and its function in tumorigenesis are still unclear. Since the intraflagellar transport (IFT) was discovered in the biflagellate green alga Chlamydomonas, the mechanisms of flagellar/ciliary assembly and disassembly have aroused widespread concerns. Flagellar/ciliary assembly and disassembly, which determine the length of the flagella/cilia, require the IFT system composed of the molecular motors, kinesin-2and dynein and IFT particles to supply the necessary substances to the tip of flagella/cilia and to bring flagellar/ciliary turnover products back to the base. It is well known that kinesin-2plays a key role in flagellar/ciliary assembly, however, the role of other kinesin family protein members are still unclear.Dunaliella salina (D. salina) is a unicellular biflagellate eukaryotic alga without cell wall, which has a pair of same length flagella of13μm. The flagella of D. salina were employed as model organelle to investigate the cilia assemly mechanism. In this study, a kinesin family protein gene KIF4cDNA of D.salina was cloned and its effect on flagellar regeneration was investigated. Methods(1) A pair of degenerate primers was designed to clone the kinesin family protein dsKIF4by reverse transcription PCR.(2)3’and5’RACE nested PCR was employed to obtain the full length of dsKIF4.(3) The relative abundance of dsKIF4mRNA was detected during flagellar regeneration by real-time PCR.(4) To investigate the effect of dsKIF4in flagellar regeneration at protein level a prokaryotic expression vector was constructed and the fusion protein was induced by IPTG and it antibody was being prepared after purification of the fusion protein.Results(1) A720bp cDNA fragment of D. salina was obtained by reverse transcription PCR using a pair of degenerate primers.(2) A1134bp and a960bp cDNA fragment were obtained by3’and5’RACE nested PCR respectively and then a2214bp cDNA was obtained after splice, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species such as InvA Kinesin from Volvox carteri (56%)> kinesin family protein from Chlamydomonas reinhardtii (58%) and KIF4C from human (42%) and was named dsKIF4as it contained a KIF4motor domain.(3) The relative abundance of dsKIF4mRNA was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration.(4) The fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.ConlusionA cDNA fragment of720bp was obtained by reverse transcription PCR using a pair of degenerate primers. And then a2214bp cDNA fragement was cloned by3’ and5’ RACE nested PCR, which encoded736amino acids. The induced protein shared high homology with kinesin family protein from other species and was named dsKIF4as it contained a KIF4motor domain. The relative abundance of dsKIF4mRNA was detected by real-time PCR and the results showed that it was increased during flagellar regeneration, demonstrating that dsKIF4might be involved in flagellar regeneration. In addition, in order to investigate the effect of dsKIF4in flagellar regeneration at protein level, we the fused protein of dsKIF4was heterogenous expressed in E. coli and the His-dsKIF4protein was expressed with molecular weight of～50kDa. The anti-dsKIF4has been preparing after his-dsKIF4was purified.