| FKBP18belongs to the family of FK506binding proteins (FKBPs), which is a class of drug receptor proteins of the immunosuppressant FK506and rapamycin (RPM). FK506, also known as tacrolimus, is a kind of macrolide antibiotics which was extracted from streptornyces tsukubaensis. Clinically, it is mainly used as immunosuppressant in liver transplantation and has also been reported in renal transplant recipients and other organ transplantations. The proliferation of cytotoxic T cells, the expression of interleukin-2recepter and the function of a variety of cytokines such as interleukin-2, y-interferon can all be inhibited by FK506.The gene FKBP18was a positive clone of an experiment made by this laboratory, which is, using the method of yeast two-hybrid, to obtain the "prey" proteins of the "bait". Kinesin like calmodulin-binding protein (KCBP), as the "bait", belongs to the family of kinesin-14, is an important protein relative to the function of flagellum. Studies had shown that KCBP involved in the normal flagella function and cell division in Chlamydomonas reinhardtii, but the mechanism remains unclear. What’s more, whether FKBP18take part in the mechanism has not been reported either.As a widespread family of proteins in prokaryotes and eukaryotes, FKBPs are involved in various activities during the normal life. For example, FKBPs regulate hormone related signal transduction pathways, in this way the seed germinated and the plant growth are under controlled. On the other hand, they probably play an important role when plants are under the pressure of abiotic, that is to say, they may strengthen their ability to resist the bad natural conditions.Flagellum/cilium, mainly composed of microtubules, is an organelle extending from cell surface. The mutation of cilia proteins can lead to developmental defects, such as multi-toe deformity, retinal degeneration and polycystic kidney disease. By far, the studies of FKBPs on animals and plants have become a hot spot, but the relationship of FKBPs and flagella/cilium remains unclear. Dunaliella salina is a kind of unicellular biflagellate green algae, which belongs to Chlorophyta, Chlorophyceae and Volcocales. It is one of the most extremely halo-tolerant eukaryotes and can live in a wide range of NaCl concentration from0.05M-5M. With all the unique advantages, Dunaliella salina can be used as a powerful model organism in the flagella study.In this study, the Dunaliella salina FKBP18cDNA5’end sequence was amplified by5’RACE method, for its3’terminal sequence has been cloned already, and the full length were obtained. In order to study the potential relationship between DsFKBP18and flagella, Dunaliella salina were treated by colchicine, heating and processing of flagella regeneration.. A total RNA of the algal cells in different states each was extracted and reversely transcripted into cDNA. By real-time PCR method, the expression level of FKBP18mRNA was analyzed.Kinesin like Calmodulin-Binding Protein (KCBP) is also an important flagella related protein. It belongs to the kinesin-14family and contains a calmodulin binding domain (CBD) near the motor domain. KCBP of D. salina (DsKCBP) has three separate domains, a homologue of myosin tail regions4(MyTH4), a like-ankle protein domain (B4.1) and a CaM-binding domain. In order to study the potential interaction sites of DsKCBP with DsFKBP18, two pairs of specific primers were designed to get the gene sequence of MyTh4and B4.1domains from DsKCBP, and the yeast two hybrid methods were used.The DsFKBP18gene open reading frame (ORF) was762bp long encoding253amino acids by the sequence analysis. The theoretical molecular weight is27.037KD and the pi value is8.6. Sequence analysis by BLAST in NCBI database conformed that the cloned sequence was Dunaliella salina FKBP18. Under colchicines’ destruction and heat treatment, as real-time Q-PCR demonstrated, the DsFKBP18mRNA expression level had both shown a rising trend. On the other hand, DsFKBPl8mRNA expression levels coincided with its growth rate during the flagella regeneration process, indicating that DsFKBPl8not only is involved in flagella disassembly, but also closely related with the flagella regeneration. The findings above-described suggest that FKBP18takes part in the signal transduction pathway of stress response from the outside environment through the flagellum.In addition, the result of yeast two-hybrid showed that DsFKBP18could interact with both MyTH4and B4.1domains of DsKCBP. It was probably that DsFKBP18function with these two domains together, or it might be false positives usually exist in yeast two-hybrid method, which remained to be further verified.In conclusion, the ORF of DsFKBP18is successfully cloned and it is not only involved in flagella disassembly, but also closely relate with the flagella regeneration. |
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