Exploring The Composition And Protein Interaction Of SOLO-Containing Cohesion Complex In Meiosis Of Drosophila Melanogaster

Diptera cyclorrhpha is one of the four pests and widely distributes in the world.Flies in residential areas can carry hundreds of bacteria and a variety of viruses through mechanical contact.While other kinds of flies,like Ceratitis capitata and Liriomyza sativae,belong to the worldwide quarantine pests because of the wide range of hosts,and easy to spread with the transportation of agricultural products,which brings great threat to people’s health and agricultural production.Meiosis is a special mode of division adopted by sexual reproduction individuals in eukaryotes.During meiosis,the cohesion between sister chromatids is necessary to maintain normal levels of homologous recombination and accurate chromosome separation of chromosomes.Two of them,ORD and SOLO,play an important role in maintaining the stability of the synaptonemal complex for the female Drosophila and guiding the correct pairing and separation of the homologous chromosomes in meiosis I and sister chromatids in meiosis II for the female and male.The absence of ORD and SOLO will lead to the phenomenon of chromosome nondisjunction（NDJ）.Because of the high homology and conservation of the meiotic cohesin in cycloschizoa insects,the meiosis cohesin in Drosophila melanogaster can be studied to provide a theoretical basis for the biological control of fly pests and other diptera insects.1.The total RNA of w¹¹¹⁸strain was extracted and the Strand c DNA was synthesized by reverse transcription.The ord fragment was amplified by PCR,and the p ET-sumo-ORD recombinant plasmid was constructed by homologous recombination.The His-ORD fusion protein was then induced by IPTG and identified by SDS-PAGE and Western blot.The results showed that the size of His-ORD fusion protein is about69 KDa,mainly in the form of inclusion body.Phylogenetic tree analysis suggested that the gene is closely related to the ord in Drosophila mauritiana,Drosophila sechellia and Drosophila simulans.Bioinformatics analysis showed that the predicted isoelectric point（PI）of ORD protein is 5.69,the average hydrophobicity（GRAVY）is-0.309,which is a hydrophilic protein,and its secondary structure is mainly alpha-helix and random coil.The protein interaction network analysis indicated that ORD interactes with INCENP,and colocalizes with CONA and CG13541.2.By using Gateway cloning technology,Entry cloning and LR expression vector cloning,the Venus::ORD transgenic flies were obtained.Then the chromosome localization experiments of transgenic strains was performed.In order to verify whether inserted fragments were correctly expressed in Drosophila,we chose transgenic strains integrated on chromosome 3 to do the rescue experiment.The results showed that the nucleotide sequence of ORD plasmid is 99.6%homologous with the reference sequence NP＿001286760.Alignment of amino acid sequences of proteins revealed differences in amino acids at 444 position（D-E）.Because both of them were acidic,the subsequent experiments would not be affected.Chromosome location experiment showed that there are four stains integrated on chromosome 3 and one integrated on chromosome.The results of rescue experiment showed that the frequency of NDJ was 0.17%in the wild type and 41.85%in the ORD mutant.However,the frequency of NDJ was 0 after inserting Venus::ORD fragment into the ORD mutant,which proved that the ORD mutant was fully rescued by the exogenous Venus::ORD and integrated fragments were correctly expressed in Drosophila melanogaster.3.Through genetic hybridization and immunofluorescence experiments,the fluorescence signals of Venus::ORD in SOLO mutants during mitosis and meiosis were observed,and the interaction between SOLO and ORD was judged by observing whether they could be colocalized during mitosis and meiosis.The results showed that ORD in SOLO mutant are correctly localized and not significantly affected during mitosis and meiosis,and the localization of SOLO and ORD in mitotic and meiosis cell nucleus were not consistent,indicating that SOLO and ORD may not exist in the same complex,and SOLO does not participate in the regulation of ORD.4.The Drosophila ovaries were dissected,and the total protein was extracted from ovaries.Through immunocoprecipitation and mass spectrometry,the proteins that would interact with SOLO had been screened.The results showed that after CO-IP experiment,Western blot verified that there was specific FH::SOLO protein band in the experiment group and no band in the control group,which proved that CO-IP experiment was successful,so samples were sent for mass spectrometry analysis.Through mass spectrometry analysis,31 potential proteins interacting with SOLO have been screened out.Among them,SOLO has been proved to interact with SMC1 and SMC3 in previous study,firmly indicating that the potential interaction proteins of SOLO obtained in this experiment is highly reliable.Through subsequent bioinformatics analysis,it is found that SOLO may play significant roles in the three protein complexes.