Dynamic Changes And Function Of N6-methyladenosine Modification In MRNA During Preimplantational Development Of Pig Embryos

N6-methyladenosine(m6A)as a most common RNA methylation modification involved in many developmental events such as cell differentiation and gamete formation.But there is no report on the expression and function of it during preimplantational development of pig embryos.In this study,we try to reveal the dynamic expression of m6 A during early embryonic development.It provides a basis for enriching the understanding of the biological functions about it.The specific test and results are as follows:(1)m6A immunofluorescence staining was performed on each stage embryo.It was found that m6 A modification level presents a dynamic trend.It rise up at first and then go down,among them,the m6 A modification level was highest in the blastocyst stage.(2)Sequence analysis of m6 A modified enzyme encoding genes.pMETTL3,pMETTL14,and pWTAP were found on chromosomes 7,8,and 6,which were 18.8 kb,34.31 kb,and 26.78 kb;pFTO and pALKBH5 were located on chromosomes 6 and 8,and were 410.34 kb ?22.79 kb.These genes have high homology with human and rat in nucleotide and amino acid sequence.(3)q-PCR detected the expression of the m6 A modification enzyme encoding gene in each stage of embryos and found that METTL3 and FTO expression was the highest in 1-cell,and the expression levels of METTL14,WTAP and ALKBH5 were the highest in 2-cell.(4)Using RNA interference and microinjection techniques to inject pMETTL3 siRNA into MII oocytes and the results showed that after siRNA interference,the embryo rate of day 5,Day 6 and Day 7 in the interference group was significantly decreased(21.17±5.10 vs 9.12±2.28,56.80±5.61 vs 26.38±4.72%,59.90±5.32% vs 34.47±6.15 %).(5)Cycloleucine was added to the embryonic in vitro culture solution PZM3,and the blastocyst rate in the experimental group was significantly reduced(P<0.05).In summary,this study revealed the m6 A modification pattern during the early embryonic development of pigs for the first time,demonstrating that m6 A modification is required for post-transcriptional development during early embryonic development in pigs.It could provide a theoretical basis for further mechanical research.