Screening Of A Novel Protein Interacting With FLA8from Dunaliella Salina And Preliminary Exploration Of Its Function

Background:Flagella/cilia are protruding objects from cell surface. In the process of biological evolution, they have become specialized cell devices. Flagella/cilia play important roles in cell motility, perception of environmental changes, and transmission of the signals to cell body. Ciliary abnormalities will lead to polycystic kidney disease, obesity, diabetes, cancer, and other human diseases. Formation and maintenance of flagella/cilia are crucial for vast majority of eukaryotic cells.Intraflagellar transport (IFT) is a two-way dynamic process along the axonemal microtubules, this complex process is influenced by various factors inside and outside the cells. Kinesin2is one of the kinesin superfamily members, which present in the flagellum and responsible for the forward transport of IFT. Kinesin2is a heterologous trimer, which include two homology subunit FLA8/FLA10, and a non-dynamic subunit (KAP subunit). The C-terminal of FLA8and FLA10subunit wind around each other to form a rod-like structure, their N-terminals are motor heads, which can hydrolysis ATP to generate energy. The two heads move along the microtubule to the plus end in the form of ’walking’.Dunaliella salina is a unicellular green alga with two equal length falgella. Because of its simple culture it could be used as a model organism in the study of intraflagellar transport and flagellum/cilia-related diseases.Aim:To get insight into the regulatory mechanism of the kinesin2motor protein FLA8in intraflagellar transport, we isolated proteins that interact with C-terminal region of FLA8which is the motor subunit of kinesin2from Dunaliella salina, amplified full length of FAP107cDNA and express it in E.coli BL21(DE3), and detected transcriptional level of FAP107during flagella regeneration.Methods:Yeast two-hybrid method was performed to screen the Dunaliella salina cDNA expression library using kinesin2subunit FLA8-C as bait, positive colonies were picked out by auxotroph culture and assayed for α-galactosidase activity according the manufacturer’s instructions. The cDNA fragments of prey proteins from positive colonies were detected by colony PCR and sequencing.5’RACE method was used to amplify the5’end of FAP107cDNA, pET28a(+)-FAP107prokaryotic expression vector was constructed and transformed into E.coli BL21(DE3), IPTG was added to induce the expression of the fusion protein. The interaction of FAP107and FLA8was determined by yeast cotransformation. The transcription level of FAP gene of Dunaliella salina cells during flagella regeneration was analysed by the comparative CT (2-△△CT) method using GAPDH as control.Results:Sequence analysis of positive colonies by yeast two-hybrid screening revealed that they were fragments of flagella-associated protein107(FAP107), P-loop containing Nucleoside Triphosphate Hydrolases domain(P-loop NTPase), protein-protein binding domain (PDZ domain), and cytoskeleton-associated protein Gly-rich domain(CAP-Gly domain). A cDNA sequence of1207bp was obtained as full cDNA sequence of FAP107which encoded a protein predicted to be composed of239amino acids. The deduced amino acid sequence shared homology with Chlamydomonas reinhardtii and Volvox carteri f. nagariensis(44%). The cDNA sequence and amino acid sequence has been submitted to GenBank:JN835298.1. FAP107was expressed successfully in E.coli BL21(DE3), SDS-PAGE proved that the molecular weight of HIS-FAP107was about30kD, which was identical with the predicted30.3kD. Yeast cotransformation showed that the colonies containing FAP107and FLA8were positive by auxotroph culture and assay for a-galactosidase activity. Additionally, the results of real-time quantitative PCR showed that the transcription level of FAP in Dunaliella salina was higer than the untreated group (Fgroup=344.550, P<0.001; Ftome=63.574, P<0.001; Finteraction=54.511, P<0.001) during flagella regeneration. It showed that the expression level reduced at the first60min, then increased during60-210min, declined to a normal level at300min.Conclusion:A new protein FAP107has been demonstrated to be able to interact with FLA8by yeast two-hybrid and yeast cotransformation, and it might be involved in flagellar assembly.