Gene Sequense Analysis Of IFT25 And IFT27 Of Chlamydomonas Reinhardtii And Their Interplay Study In Vivo And In Vitro

Intraflagellar Transport (IFT) is the bi-directional movement of cilia transportation particles from the cell and the cilia tip in the distance. The cell cilia transportation particles are composed of compound A and B. IFT-A is made up by at least 8 transported proteins, while IFT-B 12 transported proteins. Cilia is a kind of cell antenna, responsible for passing information from extracellular to intracellular, and IFT particles play an important role in the transportation and assemblies of cilia in eukaryotic cells. The missing and disfunction would cause human bodies to suffer diabetes, nutritional deficiencies, obesity, and other cilia disease, however, the molecular mechanism of how the transportation in cell cilia being regulated is still poorly understood.IFT25 is a kind of small phosphorylated protein in the Complex B in cilia, while IFT27 is a kind of small G protein. They are encoded by ift25 and ift27.In the first part, bioinformatics analysis of IFT25 and IFT27 in Chlamydomonas reinhardtii show that both of them are conserved sequences. IFT25 is hydrophilic protein, however, IFT27 is hydrophobic protein.Transmembrane helices and signal peptide are not detected so that they belong to the non-secretory intracellular protein.In the second part, we studied the interaction of IFT25 and IFT27 in vitro.First of all,we inserted ift25 and ift27 into expression vectors pGEX-2T and pMALC2H, separately. Then,we import the constructed vectors pGEX-2TrIFT25 and pMAL-C2H-IFT27 into BL21. With the analysis of western blotting,we find that IFT25 is water-soluble, and IFT27 is water-insoluble. When IFT27 was co-expressed with IFT25, the protein were also soluble.In the third part, we studied the interaction of IFT25 and IFT27 in vivo.The gene silencing methods was adopted to do this research. Taking FLA 10 as internal reference, Western blotting was used to detect the expression level of a target protein. It was found that, contrasting with wild-type (CC-125) in the transcription level, when the gene ift25 was silenced, the transcription level of ift25 gene was reduced, but if(27 was increased. When the ift27 gene silenced, the transcription level of ift27 was reduced,and ift25 was unchanged. By the analysis of expression level in protein, we could find that when there was a decrease in IFT25 protein levels, the level of IFT27 accompanied it to reduce; and when IFT27 protein reduced, the level of IFT25 remained the same as before.We have known that the defect of IFT27 leads to cilia disease. IFT25 and IFT27 may have a combined effect on ciliary assembly and deeply influence its physiological function as a result of the work I did.