The Study On Potential SSE Sites Of Cohesion Protein SOLO In Drosophila

Meiosis is a specialized cell division in the sexual reproduction of eukaryotes.It involves one round of chromosome replication and two rounds of cell division.Errors in the meiosis process can lead to nondisjunction of chromosomes（NDJ）,resulting in abnormal sperm or eggs.Cohesion between chromosomes plays an important role in meiosis.Cohesion is provided by a protein complexed-cohesin.Most biological meiotic cohesin are composed of SMC1,SMC3,REC8,and SCC3/SA.Protease SSE is activated during the meiosis anaphase,then cleavage the REC8 subunit to separate chromosomes.In Drosophila,there is no ortholog that can replace the function of REC8.The function of cohesion protein sister on the loose（SOLO）is similar to that of REC8.The C-terminus has a certain homology with the C-terminus of REC8 and the C-terminus of SOLO also binds to SMC1.Moreover,SOLO contains 5 potential SSE cutting sites.In summary,the cohesion protien SOLO may replace REC8 to perform its function.Therefore,this study performed 3 types of site-directed mutations on 5 potential SSE sites in SOLO to investigate whether it can replace REC8 to function in meiosis in drosophila.The three types of mutations at the potential SSE sites of SOLO are M1,M2,and M3,respectively.M1 represents a single amino acid mutation at the 3 sites of SOLO,M2 represents a single amino acid mutation at the 5 sites of SOLO,and the M3represents multiple amino acid mutations at the 5 sites of SOLO.In this study,Site-directed mutagenesis,expression vector construction,embryo microinjection,genetic hybridization,and immunofluorescence are used to study the function of cohesion protien SOLO in meiosis.The experimental results are as follows:1.NDJ statistics of genetic hybridization showed that the M1 mutation type experimental group,positive control group and negative control group had NDJ of0.33%,47.6%and 0.30%,respectively.The M2 mutation type experimental group,the positive control group and the negative control group had NDJ of 0.33%,48.5%and 0.20%,respectively.The M3 mutation type experimental group,the positive control group and the negative control group had NDJ of 0.25%,46.8%and 0.46%,respectively.The above NDJ results showed that the experimental group is much smaller than the positive control group,but is roughly equal to the negative control group,indicating that the M1,M2,and M3 mutation type transgenes played a rescue role in drosophila,and the site-directed mutations on the potential SSE sites of SOLO cannot be causes abnormal chromosome segregation,SOLO may not be the cutting target of SSE and cannot replace REC8.2.It was further verified whether the above three types of mutations were expressed in Drosophila.The male Drosophila testes were dissected to observe the fluorescence of Venus::SOLO^M1,Venus::SOLO^M2,Venus::SOLO^M3.The results showed that both the experimental group and the positive control group could observed green fluorescent bright spots,which proved that the transgenic fruit flies are normally expressed,and also showed that the above NDJ statistical results and conclusions are reliable.In summary,the replacement of Drosophila REC8 may be a complex composed of SOLO and another cohesion protein,and this cohesion protein also has potential SSE sites.Through the study of the mechanism of meiosis cohesion protein of Drosophila,it can provide a theoretical basis for the study of meiosis of Drosophila,and also provide a theoretical reference for the study of meiosis mechanism of other insects.It is of great significance for the research and control of pest sexual reproduction.