嗜热四膜虫有性后代的分离纯化
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摘要
在实验条件下诱导四膜虫种群交配进行有性生殖时,交配后的种群中除有性后代外,还会混杂一定比例的亲本细胞,而高纯度的有性后代是研究四膜虫一些基本生物学问题的重要基础。本研究利用四膜虫交配时不能摄食并且新产生的有性后代几小时后才恢复摄食能力的特性,以嗜热四膜虫(Tetrahymenathermophila)交配种群为对象,在其有性生殖阶段后期向交配体系中加入Fe3O4磁性纳米颗粒,未交配的亲本细胞由于能够摄入磁性颗粒而具有磁性;再利用有性后代和未交配细胞的磁性差异,在磁场作用下将有性后代分离纯化出来。通过优化过柱分离的时间点、分离柱的口径、分离柱中铁粉/石英粉的质量等分离条件,有性后代的分离纯度及收率均能达到98%以上。该分离方法亦可为其他种四膜虫或纤毛虫有性后代的分离纯化提供借鉴。
When Tetrahymena cells are induced to mate for sexual reproduction in the lab,the resulting population always contains a certain proportion of unmated parental cells besides sexual progeny.However,highly purified sexual progeny is an important basis for studying some basic biological questions of Tetrahymena.In this study,based on the characteristics that mating Tetrahymena loses the ability to ingest particles and the newly produced sexual progeny does not recover this ability within several hours,we added the Fe3 O4 magnetic nanoparticles to the mating population of T.thermophila at the late stage of sexual reproduction and only the unmated parent cells would become magnetic because they still had the ability to ingest the magnetic particles,which made possible an efficient isolation of sexual progeny under the action of magnetic field.By optimizing the time point at which cell population was passed through the separation column,the diameter of the column and the quality of iron/quartz powder in the column,we could make the purity and yield of the sexual progeny both more than 98%.The established method can also be used for the separation and purification of the sexual progeny of other Tetrahymena or ciliate species.
When Tetrahymena cells are induced to mate for sexual reproduction in the lab,the resulting population always contains a certain proportion of unmated parental cells besides sexual progeny.However,highly purified sexual progeny is an important basis for studying some basic biological questions of Tetrahymena.In this study,based on the characteristics that mating Tetrahymena loses the ability to ingest particles and the newly produced sexual progeny does not recover this ability within several hours,we added the Fe3 O4 magnetic nanoparticles to the mating population of T.thermophila at the late stage of sexual reproduction and only the unmated parent cells would become magnetic because they still had the ability to ingest the magnetic particles,which made possible an efficient isolation of sexual progeny under the action of magnetic field.By optimizing the time point at which cell population was passed through the separation column,the diameter of the column and the quality of iron/quartz powder in the column,we could make the purity and yield of the sexual progeny both more than 98%.The established method can also be used for the separation and purification of the sexual progeny of other Tetrahymena or ciliate species.
引文
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Blackburn E H,Gall J G.1978.A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena.Journal of Molecular Biology,120(1):33-53.
Brownell J E,Zhou J,Ranalli T,et al.1996.Tetrahymena histone acetyltransferase A:a homolog to yeast gcn5p linking histone acetylation to gene activation.Cell,84(6):843-851.
Bruns P J,M?ller K M,Leick V.1980.Magnetic purification of mating Tetrahymena.Carlsberg Research Communications,45(1):29-33.
Chen X J,Feng W S,Yu Y H.2014.Comparisons among six strains of Tetrahymena by microcalorimetry.Journal of Thermal Analysis and Calorimetry,115(3):2151-2158.
Dickinson J R,Graves M G,Swoboda B E P.1976.Cyclic AMPmetabolism in the cell cycle of Tetrahymena pyriformis.FEBSLetters,65(2):152-154.
Gorovsky M A.1973.Macro-and micronuclei of Tetrahymena pyriformis:a model system for studying the structure and function of eukaryotic nuclei.The Journal of Protozoology,20(1):19-25.
Greider C W,Blackburn E H.1985.Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.Cell,43(2):405-413.
Hamilton E P,Orias E.2000.Genetic crosses:setting up crosses,testing progeny,and isolating phenotypic assortants.Methods Cell Biology,62(62):219-228.
Kruger K,Grabowski P J,Zaug A J,et al.1982.Self-splicing RNA:autoexcision and autocyclization of the ribosomal RNAintervening sequence of Tetrahymena.Cell,31(1):147-157.
Orias E,Singh D,Meyer E.2017.Genetics and epigenetics of mating type determination in Paramecium and Tetrahymena.Annual Review of Microbiology,71(1):133-156.
Rasmussen L.1976.Nutrient uptake in Tetrahymena pyriformis.Carlsberg Research Communications,41(3):143-167.
Blackburn E H,Gall J G.1978.A tandemly repeated sequence at the termini of the extrachromosomal ribosomal RNA genes in Tetrahymena.Journal of Molecular Biology,120(1):33-53.
Brownell J E,Zhou J,Ranalli T,et al.1996.Tetrahymena histone acetyltransferase A:a homolog to yeast gcn5p linking histone acetylation to gene activation.Cell,84(6):843-851.
Bruns P J,M?ller K M,Leick V.1980.Magnetic purification of mating Tetrahymena.Carlsberg Research Communications,45(1):29-33.
Chen X J,Feng W S,Yu Y H.2014.Comparisons among six strains of Tetrahymena by microcalorimetry.Journal of Thermal Analysis and Calorimetry,115(3):2151-2158.
Dickinson J R,Graves M G,Swoboda B E P.1976.Cyclic AMPmetabolism in the cell cycle of Tetrahymena pyriformis.FEBSLetters,65(2):152-154.
Gorovsky M A.1973.Macro-and micronuclei of Tetrahymena pyriformis:a model system for studying the structure and function of eukaryotic nuclei.The Journal of Protozoology,20(1):19-25.
Greider C W,Blackburn E H.1985.Identification of a specific telomere terminal transferase activity in Tetrahymena extracts.Cell,43(2):405-413.
Hamilton E P,Orias E.2000.Genetic crosses:setting up crosses,testing progeny,and isolating phenotypic assortants.Methods Cell Biology,62(62):219-228.
Kruger K,Grabowski P J,Zaug A J,et al.1982.Self-splicing RNA:autoexcision and autocyclization of the ribosomal RNAintervening sequence of Tetrahymena.Cell,31(1):147-157.
Orias E,Singh D,Meyer E.2017.Genetics and epigenetics of mating type determination in Paramecium and Tetrahymena.Annual Review of Microbiology,71(1):133-156.
Rasmussen L.1976.Nutrient uptake in Tetrahymena pyriformis.Carlsberg Research Communications,41(3):143-167.