Cloning And Functional Analysis Of Intraflagellar Transport Protein Ift88Gene From Dunaliella Salina

Dunaliella salina is a highly salt-tolerant unicellular eukaryotic cells with about13μm long biflagellate, which has a typical"9+2" structure. Using some chemical or physical methods can regulate the flagellar’s regeneration and disassembling. Compared with another model organism-Chlamydomonas reinhardtii, D. salina has no cytoderm, which is closer to mammalian in the cell structure, and D. salina is very suitable modal organism to investigate flagella length regulation mechanism as it has the characteristic of simple cell structure, short growth cycle, easy to culture and to observe the the flagellum morphology and structure.The flagellumand cilium are an organelle projecting from the surface of various cells, their structures are basically the same. Intraflagellar transport (IFT) is a microtubule-based motility located between the flagellar membrane and axoneme, in which groups of protein particles are transported from the base to the tip of the flagellum (anterograde) by kinesin II and from the tip to the base (retrograde) by dynein. IFT is required for assembly and maintenance of eukaryotic flagella and cilia. The IFT particles are composed of at least20protein subunits,which form two complexes, called complex A and B. IFT88is one of the components of the IFT-B, which is encoded by IFT88gene. IFT88gene is capable of encoding a tetratricopeptide repeat sequence (TPR) family, which is very important for the transport function because it can mediating protein-protein interactions. IFT88has been shown to interact with IFT52, IFT46, Hsp40in Chlamydomonas reinhardtii.. Mutations of Tg737,a similar gene of IFT88, in mouse can cause polycystic kidney disease, and it also have a certain relationship with mice visual and olfactory dysfunction.IFT88plays an important role in the flagellum assembly, the IFT protein in are very conservative in both green algae, nematode and vertebrates, and the function of IFT88are not reported in D. salina.So in this study, The IFT88cDNA was cloned using designed3’and5’inner/outer primers based on the591bp nucleotides fragments according to the flagella regeneration transcriptome sequencing.Remove flagella by using mechanical polishing method, D. Salina flagella regenerated after returned to normal culture conditions. Then the D. salina flagella were observed by both silver staining and iodine staining method. The total RNA of cells at different times after deflagellation was extracted and reversely transcripted. Then the transcriptional expression of the IFT88was observed by real-time fluorescence quantitative PCR. The open reading frame was predicted using ORF Finder and then cloned. After induced by1mM IPTG for4h at37℃, protein of the IFT88was expressed in E. coli BL21(DE3). SDS-PAGE method was using to detect the IFT88protein expression. Using D. Salina as a model organism to investigate the function of IFT88will provide the foundation of further study of the IFT88functions within the cell.Method1. Cloning of IFT88cDNA from D. salinaThe IFT88cDNA was cloned using designed3’and5’inner/outer primers based on the591bp nucleotides fragments according to the flagella regeneration transcriptome sequencing. The sequence was spliced by using DNAMAN,and then validated by PCR the open reading frame of the IFT88sequence.2.Functional analysis of IFT88in D.SalinaRemove flagella by using mechanical polishing method, D. Salina flagella regenerated after returned to normal culture conditions. Then the D. salina flagella were observed by both silver staining and iodine staining method. The total RNA of cells at part different times after deflagellation was extracted and reversely transcripted. Designed a pair of primers based on the primer design requirements of real-time fluorescence quantitative PCR. Real time-PCR was used to investigate the variation at relative transcriptional level during flagella regeneration in D. Salina.3. Heterologous expression of IFT88in D. SalinaThe the open reading frame of IFT88sequence in D. Salina was connected to the prokaryotic expression vector pET28a (+). And the vector of pET28a-IFT88was transformed into E. coli BL21(DE3). After induced by1mM IPTG for4h at37℃, protein of the IFT88was expressed in E. coli BL21(DE3). SDS-PAGE method was using to detect the IFT88protein expression.Results1. Cloning of IFT88cDNA from D. SalinaA3054bp of cDNA products was obtained, including316bp5’UTR,428bp3’UTR and2400bp open reading frame.The predicted ORF encoded799amino acids. Protein blast showed this amino acid sequence had highly homology with the IFT88of other species, such as Chlamydomonas reinhardtii (83%).2.Functional analysis of IFT88in D.SalinaD. salina cells was separated with its flagella by choosing mechanical grinding method. The variation of flagella regeneration in D. Salina was observed by both silver staining and iodine staining method. During flagella regeneration, the expression of IFT88mRNA level was increased in30min, and afterwards decreased rapidly. The change of IFT88at the transcriptional level was consistent with the flagellum regeneration rate. The results show that IFT88may plays an crucial role during flagellar regeneration in D. Salina. 2. Heterologous expression of IFT88in D. SalinaCompared with control group, the result of SDS-PAGE proved that IFT88of D.salina was expressed successfully, and there was a specificity band of-90kDa, it’s just the same size as the predicted IFT88protein.Conclusion1. A3054bp of IFT88cDNA from D. salina has been obtained, which contains316bp5’UTR,428bp3’UTR and2400bp open reading frame, and encods799amino acids.2. The expression of IFT88mRNA is increased at30min during flagellar regeneration, suggesting that IFT88may play a crucial role during flagellar regeneration in D. Salina.3. An expression of IFT88protein with88kDa has been induced in E. coli, which lays a foundation for the further preparation of anti-IFT88antibodies.