Expression Pattern Of Mep50 And Its Interaction With Prmt5 In Medaka, Oryzias Latipes

The mep50 gene was firstly cloned in medaka (Oryzias latipes),then studied its expression pattern in medaka.Finally, the functional site between MEP50 and PRMT5 had been researched in the thesis.The length of mep50 gene was 1005 bp, encoding 334 amino acid residues, including six WD40 repeat sequences,the first three WD40 were end with tryptophan-aspartic acid (W-D),which were conservative.Total RNA of different tissues in mature medaka and different embryonic developmental stages were extracted,then using the methods of semi-quantitative PCR and real-time fluorescent quantitative PCR to get the expression pattern of mep50 in medaka.The results showed that the expression quantity of the mep50 which in the spleen and the ovary is high(P<0.05)and significant high (P<0.01) compared with the other tissues,which hinted that methylation plays an important role in the immune response and gender development in medaka.Different embryonic developmental stages data showed the expression pattern of mep50 changed from high to low(after the gastrula stage),until the seedling stage change to high.All of the results hinted mep50 was a kind of maternal genes,which played an important role in oocyte formation and the early embryonic development,maintained the normal growth of embryo until the birth comes,and it expressed sustainedly and stably in varioue tissues.The embryo in situ hybridization confirmed that mep50 was widely expressed in early embryos.After emergence,the mep50 was expressed in eyes,brains,gill,fins(pectoral fins, anal fins,tail fins) with high level,which confirmed that the mep50 was widely expressed in tissues and organs.Finally,the functional sites which interaction MEP50 with PRMT5 were explored in the paper by using the methods of the two-yeast-hybridization,throught cloned different WD40 truncation sequences and mutation sequences of OLMEP50 constructed the two-hybrid recombinant vectors of pGBKT7,then transformed into AH 109 yeast strain with pGADT7-olprmt5 and screened the positive bacterias in the four short board(His-Ade-Trp-Met) by the reported genes(HIS3,ADE2,LACZ,MEL1).The results showed that the WD40 domain was the key region interact OLMEP50 with OLPRMT5 and the maximum active domain of MEP50 and PRMT5 was the third WD40 in OLMEP50.The third WD40 lacking of WD two-hybrid further confirmed that OLMEO50-OLPRMT5 interaction site was the WD site.It had be the key site for MEP50-PRMT5 to develop the maximum activity.This research first revealed the expression pattern of MEP50 and its interactional site with PRMT5 in medaka,and it would provide the valuable references for study the effect of the methylation in development and immune system.