Cloning,expression Localization And Function Of Scp3 And Dmc1 Genes In Silurus Meridionalis

Synaptonemal complex 3（scp3）and DNA meiotic recombinase 1（dmc1）are two key factors in meiosis.In order to study the expression and function of scp3 and dmc1 during meiosis in Silurus meridionalis,30dah（days after hatching,dah）,55dah,and 90dah Silurus meridionalis fry were used as experimental subjects in this experiment.In this experiment,PCR technology was used to clone CDS sequences of scp3 and dmc1 in Silurus meridionalis and the structural and physicochemical properties of DMC1 and SCP3 proteins were analyzed and predicted by bioinformatics.The expression of scp3 and dmc1 in 9 different tissues and 3 different ages were studied by RT-PCR and Real-PCR.The localization of DMC1 and SCP3 in ovary was determined by immunofluorescence technique and GST pull down technique was used to verify the interaction between the two proteins.The results are as follows:1.The Coding Sequence（CDS）of Silurus meridionalis scp3（NCBI accession number:MW075231）and dmc1（NCBI accession number:MF770581）are 723 bp and 1029 bp,encoding 240 and 342 amino acids,respectively.The molecular mass of DMC1 is 37.96k D.The theoretical isoelectric-point is 5.65 and the formula is C₁₆₇₃H₂₆₇₄N₄₅₈O₅₁₅S₁₆.There is no signal peptide and transmembrane structure.The secondary structure includes 50.58%forαhelix,5.26%forβsheet,29.24%for random coil and the extension chain is 14.91%.The molecular mass of SCP3 is27.88k D.The theoretical isoelectric point is 8.92,and the formula is C₁₂₀₇H₁₉₆₂N₃₄₆O₃₈₄S₁₃.There is no signal peptide and transmembrane structure.The secondary structure includes 74.58%forαhelix,0.42%forβsheet,0.42%for random coil,and the extended chain is 0.83%.The result of homology comparison showed that the homology of DMC1 in Silurus meridionalis,Ictalurus Punetaus and Pelteobagrus fulvidraco is as high as 97%,and the homology with Oncorhynchus mykiss,Oncorhynchus mykiss is as high as 92%.The homology of SCP3 with P.fulvidraco and I.punctatus was 89.17%and 87.92%,respectively,and the homology with O.mykiss and D.rerio is as high as 78.42%and 73.44%.In phylogenetic analysis,SCP3 and DMC1 of Silurus meridionalis clong with other fishes,and then clong with tetrapod.2.In the study of female Silurus meridionalis,scp3 and dmc1 were only expressed in the ovary.They were not expressed in brain（B）,intestine（I）,heart（H）,gills（G）,liver（L）,kidney（K）,muscle（Mu）,spleen（S）and other tissues,and the expression of the two genes was the highest at 55dah.SCP3 had a strong positive signal in the nucleus and cytoplasm of Silurus meridionalis oocytes,and DMC1 had a strong positive signal in the nucleus of oocytes.3.In the GST pull-down experiment,GST antibody was used to detect the control group（CK,GST+glutathione-agarose resin+HIS-DMC1）,test group（Exp,GST+glutathione-agarose resin+HIS-DMC1）,GST protein,GST-SCP3recombinant protein,HIS-DMC1 recombinant protein.The results showed that the target band with molecular weight of 50 k D was detected in the experimental group and GST-SCP3 recombinant protein.HIS antibody was used to detect the control group（CK）,test group（Exp）,GST protein,GST-SCP3 recombinant protein,HIS-DMC1 recombinant protein.The results showed that the target band with a molecular weight of 42 k D was detected in experimental group and HIS-DMC1recombinant protein.These results indicated that GST antibody successfully enriched the bait protein GST-SCP3,and bait protein GST-SCP3 pulled down the prey protein HIS-DMC1,indicating that GST-SCP3 and HIS-DMC1 had an interaction in vitro.