Construction Of CDNA Library Of Dunaliella Salina And Cloning And Characterization Of The Gene Kinesin Like Calmodulin-Binding Protein

In recent years, the methods of genetic engineering were used for algae to build high-yield, high quality new species for multiple purposes such as producing fuel, degradating pollutants and producing cheap or abundant proteins and drugs. Because Dunaliella salina, a single-cell organism without cell wall, is cultured easily, rapidly and inexpensively, the alga is able to be developed not only as a bioreactor for producing recombinant proteins or vaccines but also as a model organism for studying the gene expressions and regulations of other species. Therefore, it is urgent to clone more functional genes related to D. salina. The construction of the cDNA library of D. salina will contribute to the studies of D. salina as a bioreactor and a model organism.Cilia and flagella are evolutionarily conserved organelles, which are composed by axonemal microtubules and cilia membrane. Studies have shown that the occurrence of diseases is necessarily related to the mutations of cilia, such as polycystic kidney disease and so on. The experiments of our laboratory focus on the flagellum of D. salina as a model organelle to study the relationship between cilia and diseases.Kinesin and kinesin-like (KLPs) proteins are conservative microtubule motor proteins with the ATPase activity in eukaryotic cells. These motors play important roles in many basic developmental processes of cell. To date, many kinesins or KLPs were identified from Arabidopsis thaliana and other higher plants. Kinesin calmodulin-binding protein (KCBP) was obtained from plants as a new KLPs, which has a calmodulin-binding domain in the neighboring of its motor domain. KCBP, a member of the kinesin-14 family, is a negative point of the C-terminal microtubule motor proteins with three separate domains:a homologue of myosin tail regions 4 (MyTH4); a like-ankle protein domain (B41); a CaM-binding domain (CBD). MyTH4 and B41 have not been found in other reported kinesins. KCBP was positioned on vascular, including thepreprophase band, spindle and in film-forming body, suggesting that KCBP forms the vascular by binding the microtubules. To date, the genome sequence of KCBP homologue was only found in plants and a frew of green algae such as C. reinhardti.Kinesin was involved in assembly of flagellar microtubules. The KCBP gene in C. reinhardti is a minus-end-directed microtubule motor protein. And it played a role in the process of transportation from the end of the flagellum to the basal body. However, the KCBP gene of D.salina has not been cloned and its function in flagellar assembly has not reported so far.In this study, to clone and characterize a new kinesin like calmodulin-binding protein (KCBP) gene from Dunaliella salina, a cDNA library of Dunaliella salina was first constructed according to manufacturer's instructions of cDNA Library Construction Kit (TaKaRa). The qualification of the cDNA library was determined by measuring the number of clones, recombinant rates and length of insert fragments. The full-length cDNA sequences of the KCBP gene were screened from the cDNA library of Dunaliella salina after part cDNA sequences of KCBP gene were amplified with a pair of degenerate primers designed according to conserved homologous amino acid sequences of DIMQFG and CIFAYG. The function of KCBP in the process of flagellar disassembly induced by colchicine was identified by real-time fluorescence quantitative PCR. The high-qualified library was constructed with the titer 5.6×106 pfu/mL, the percentage of recombination about 90% and an average size of inserted fragments 1.9 kb. The full length of KCBP gene sequence from Dunaliella salina was 4514bp comprising 105bp of 5'UTR,593bp of 3'UTR and 3816bp of open reading frame (Genbank No.GU345802). The sequences analysis showed that it belonged to kinesin-14 family, which had three separate domains:a homologue of myosin tail regions 4 (MyTH4); a like-ankle protein domain (B41); a motor domain with calcium-binding sites. The function analysis of KCBP gene demonstrated that the relative mRNA abundance of KCBP gene significantly increased to 9-fold higher on 80min after flagellar disassembly with than without experiencing colchicine treatment. It is concluded that a new gene KCBP screened successfully from the cDNA library of Dunaliella salina plays an important role in the process of flagellar disassembly, and that the cDNA library constructed in this study is a good tool to the study of functional genomics of Dunaliella salina in the future.