Cloning And Functional Analysis Of FLA8 Gene Of Kinesin-2 Subunit From Dunaliella Salina

Flagella are evolutionarily conserved organelles with special functions playing important roles in cell motility and the process of signal perception and transmission from external environment to intracellular. Flagella defect and motility changes are associated with many abnormalities, such as polycystic kidney disease, obesity, diabetes, cancer. Material transport within flagella and flagellar assembly are regulated by intraflagellar transport (IFT) which is composed of anterograde transport from basal body along the axoneme to flagella tip and retrograde transport. The anterograde transport is carried out by kinesin-2 and the retrograde transport by dynein. Kinesin-2 is the only class of kinesin with heteromeric motor subunits composed of three subunits:two different motor subunits including FLA 8 and FLA10, and a non-motor binding subunit. Studies have shown that Chlamydomonas FLA10 is localized in mitotic spindle during mitosis and between the outer microtuble of flagellar axoneme and flagellar membrane. Flagellar resorption is blocked due to FLA10 gene mutation. Chlamydomcnas FLA8 is associated with chromosomal loss but the influence of FLA8 on the process of flagellar regeneration has not been reported.Dunaliella salina (D. salina) is a unicellular and biflagellate eukaryotic alga without cell wall. The pair of flagella has the same length about 13μm with typical 9 +2 structure. The change of flagellar length and motility can be observed under microscope. In this study, D. Salina was used as a model organism and its flagella was for investigating the role of FLA8 in the process of flagellar regeneration in order to elaborate the function of FLA8 in flagellar assembly and IFT mechanism. A pair of degenerate primers was designed according to the conserved amino acids sequence GYNGTIF and NEDPKDA from Volvox, Chlamydomonas, and Chlorella. The full-length cDNA of the FLA8 gene was obtained by RT-PCR,3' and 5' RACE technology. Subsequently, flagella were collected from cell bodies using mechanical method. During the process of flagellar regeneration, total cellular RNA was extracted at different stages and then reversely transcripted to cDNA. A pair of specific primers designed by Primer Premier 6.0 software was used in real-time fluorescence quantitative PCR to analyse the function of the FLA8 gene in the process of flagellar regeneration.The results showed that the full-length cDNA sequence of the cloned FLA8 gene was 2612 bp possessing a 90 bp 5'UTR, a 167 bp 3'UTR and a 2355 bp open reading frame encoding 784 amino acids. The theoretical pI value of FLA8 was 6.65 and the molecular weight was 85.097 kDa. Amino acid sequence homologous analysis indicated that FLA8 of D. salina shared high homology with other organisms, such as Volvox (73%), Chlamydomonas reinhardtii (72%), Chlorella (53%), suggesting that the cloned sequence is D. salina FLA8 amino acid sequences. Real-time quantitative PCR results showed that the expression of the D. salina FLA8 remained high at mRNA level during flagellar regeneration. Within the first 3 h, flagella grew quickly at the initial stage and the expression of FLA8 was significantly higher than other times. It is concluded that the high expression of the FLA8 gene plays an important role in the process of flagellar regeneration of D. salina.