Functional Analysis Of Gamones Family Genes During Sexual Reproduction From Tetrahymena Thermophila

Gamone is a substance secreted by one individual and detected by other individuals of the same species through their olfactory organs,causing the latter to exhibit a change in behavior,mood,or physiological mechanism.Gamones are conserved in higher eukaryotes,but the structure and types of gamones are different in protozoan.Tetrahymena thermophila is an unicellular eukaryotic model organisms.It has asexual reproduction and sexual reproduction.In this study,gamone family genes were identified by bioinfomatic analysis.Among them,the structure,functiont,and expression regulation of GAM1 an GAM2 were analyzed.The main results were as follows:1.Identification of GAM1 and GAM2 Based on the macronuclear genome database,9 GAM genes were identified.Among them,GAM1 is 318bp,encoding 106amino acids,GAM2 is 339bp,encoding 112 amino acids.There are no intron and upregulate expression in late starvation.stage,which could induce sexual reproduction of different mating type Tetrahymena.2.Construction of GAM1 and GAM2 knockout mutants Recombinant plasmids pNeo4-GAM1 and pNeo4-GAM2 were constructed.The recombinant plasmids were transformed into different mating type T.thermophila cells,including B2086(II),Cu427(VI),and Cu428(VII)by particle bombardment.GAM1 and GAM2 knockout mutants were created by homologous recombination and resistance selection.KO-GAM1-Cu427?KO-GAM1-Cu428?KO-GAM1-B2086?KO-GAM2-Cu427?KO-GAM2-Cu428?KO-GAM2-B2086 were obtained.KO-GAM1-Cu428 and KO-GAM1-B2086 cells failed to mating,and KO-GAM2-Cu427?KO-GAM2-Cu428 also failed to mating.The results showed GAM1 and GAM2 are involved in the sexual reproduction initiation.3.Recombinant expression of GAM1 in E.coli The recombinant plasmids p SUMO-GAM1,pMBP-GAM1,p SUMO-GAM1-?N42,and pMBP-GAM1-?N42 were constructed and transformed into in E.coli.The recombinant proteins SUMO-GAM1,MBP-GAM1,SUMO-GAM1-?N42,and MBP-GAM1-?N42 were expressed with0.1mmmol/L IPTG induction at 37?.The removal of the signal peptide and MBP fusion tag improved the solubility and expression level of recombinant Gam1 protein.MBP-GAM1-?N42 were purified by Ni affinity chromotography.Gam1 improved the recognition effiency of different mating type cells.4.E.coli BL21/SUMO-GAM1 and E.coli BL21/MBP-GAM1 have high tolerance for heavy metal Under Cu²⁺and Cd²⁺stress,proliferation of E.coli BL21/SUMO-GAM1 and E.coli BL21/MBP-GAM1 is negative correlation with ion concentration.E.coli BL21/SUMO-GAM1 and E.coli BL21/MBP-GAM1 have higher tolerance for Cu²⁺and Cd²⁺than WT.The proliferation of E.coli BL21/MBP-GAM1 is faster than that of WT under 1 mmol/L Cu²⁺?400?mol/L Cd²⁺treatment.The results indicted that heterologous expression of GAM1 in E.coli improved cellular tolerance for heavy metal.5.Response of GAM1 for environmental factors and construction of mutant cells Expression level of GAM1 in Tetrahymena unregualted 73,31.5,32000,and 12000 times under Cu²⁺,Ag⁺,Ni²⁺,and Cd²⁺treatment,respectively.However,the expression of GAM1 showed less change under different temperature and p H.Recombinant plasmid pNEO4-GAM1-GFP was constructed,which contain green fluorescent protein GFP and GAM1 promoter.The pNEO4-GAM1-GFP plasmid was transformed into Tetrahymena by particle bombardment.GAM1-GFP-Cu428 and GAM1-GFP-B2086 mutants were obtained by homologous recombination and resistance selection.Green signal occurred under Cd²⁺treatment in the mutants.In sum,gamone family genes were identified in Tetrahymena.Fuction of GAM1 and GAM2 during sexual reproduction was explored.GAM1 was expressed in E.coli and purified by affinity chromotography.The expression of GAM1 was strongly induced by Ni²⁺,and Cd²⁺.Furthermore,the GAM1-GFP mutants were created,which is valuable for creating single cell biosensor in future.