Transformation And Expression Of The Gene Encoding CrAC3 In Chlamydomonas Reinhardtii

Cilia are important organ in cellular activities;primary cilia can regulate various signal transduction induced by the external physical and biochemical changes.Defects in cilia can cause many related diseases,such as retinitis pigmentosa,polycystic kidney disease,neural tube defects,etc.Therefore,it has a very important significance to study the cilia structure,movement,physiological process and function.Biflagellated Chlamydomonas reinhardtii is an important model organism to study cilia.Chlamydomonas reinhardtii fertilization process needs to be completed through the complex signal transduction cascade.In this cascade reaction process,agglutinin proteins on the gametic flagellar surface would induce the increasing in intracellular levels of cAMP,leading to a series of cell changes and gamete activation prepared for cell-cell fusion.Among them,an adenylyl cyclase?AC?can convert ATP to cAMP,which plays a key role in the signal transduction pathway induced by gamete adhesion.Therefore,this paper studies on type ? adenylyl cyclase CrAC3,detecting and analyzing the expression and function of its protein in Chlamydomonas reinhardtii.The main results are as follows:1.Screen out the type ? adenylyl cyclase CrAC3 using JGI genome database?Chlamydomonas version 4?.CrAC3 encoding 1391 aa and its transcripts possess gamete-specific property.Through bio informatics analysis,it shows that CrAC3 is a hydrophobic membrane protein containing six transmembrane helix,including phosphorylation and glycosylation sites,and the protein prediction size of 180 KD.CrAC3 are most similar to other ciliated ACs such as Paramecium,Tetrahymena and Plasmodium reported to contain an ion channel domain.These results provide the basis for the detection and analysis of CrAC3 protein.2.Transfer the CrAC3 gene driven by exogenous promoter RBCS-HSP70 containing a selective marker APHVIII with 3x HA tag in the C-terminal into Chlamydomonas reinhardtii wild type cells using transgenic technique,then screen out the transformant of S11?mt-?.By using the methods of immunofluorescence and western blotting binding with anti-HA antibody detection,it showed that the expression of protein CrAC3-HA-RBCS was exclusively in the S11?mt-?resting gametes,the actived gametes,and were all expressed in the flagella.But the expressed protein size is 75 KD,which means the gene might be trimmed in the cell.The results provide a foundation for studying and comparing the expression of endogenous protein CrAC3 between the transformed protein CrAC3-HA-RBCS and their related functions in Chlamydomonas reinhardtii.3.Chlamydomonas reinhardtii fla10 cell encoding protein CrAC3-HA-RBCS was obtained by cells hybridization,the hybrid cell named C-16?mt+?.Using Western blotting with anti-HA antibody detection,which revealed that protein CrAC3-HA-RBCS expressed extremely in C-16?mt+?gametic flagella is similar to that of S11?mt-?.It indicated that the protein CrAC3-HA-RBCS in S11?mt-?cells can be inherited through cells hybridization.The results provide bases on the genetic study of protein CrAC3-HA-RBCS,the protein movement in cells and its function.4.Cultured Chlamydomonas reinhardtii mutant fla10?mt+?and hybrid cell C-16?mt+?in impermissible temperature,using western blotting and Rat anti-HA antibody detection,it found that the protein CrAC3-HA-RBCS shifted from C-16?mt+?gametic flagella to its cell body.And the IFT protein complex A and B detected by IFT antibody have the same trend as protein CrAC3-HA-RBCS detected by anti-HA antibody.Therefore,suggesting that protein CrAC3,HA-RBCS expression in C-16?mt+?gametes is associated with IFT system,namely the movement of protein CrAC3-HA-RBCS depending on the IFT system.5.Depending on the monoclonal antibody technique,combined with the detection of western blot and candidate monoclonal antibodies detection,a monoclonal antibody 1J11₁10824 against Chlamydomonas reinhardtii CrAC3 peptides was screened out.The target protein size is 180 KD in 6145C?mt-?and S11?mt-?gametes stained by 1J11₁10824 antibody,and it consistent with the prediction size of CrAC3.Further extraction the flagella from the mixture of S11?mt-?and 21gr?mt+?in different mating times,the 1J11₁10824 antibody detection results are same as using the Rat anti-HA antibody detection.This result not only clears the antibodies of CrAC3 and CrAC3-HA-RBCS protein,but also provides a tool for the next step studies on the two proteins.6.The construct DNA,named CrAC3-HA-endo,encoding Chlamydomonas reinhardtii CrAC3 driven the endogenous promoter obtained from BAC clone was transferred into Chlamydomonas reinhardtii mutant fla10,and obtained the transformed cells was screened out.This result will offer the basis of next studies in combination the monoclonal antibody 1J11₁10824 against CrAC3 peptides with CrAC3-HA-endo detection,also,in the expression of protein CrAC3-HA-endo and its functions in Chlamydomonas reinhardtii gametes activation and zygotes formation.