Research About The Spatiotemporal Influences Of Incubation Temperature On Genomic Methylation Of Duck Embryo And Transcriptional Regulation Of Related Genes

Incubation temperatures is one of important external environmental factors for shaping of avian, appropriate change of incubation temperature can affect the skeletal muscle development of birds during both embryonic stages and post-hatch stages. We suspected that genome epigenetic mechanisms may play an important role in mentioned above physiological process, that is, incubation temperature changes may affect the genome methylation levels of duck embryo, thereby affecting gene transcriptions, and ultimately result in changes of animal phenotypes. Therefore, in present study, we intended to amplify genes partial sequences of DNA methyltransferases (DNMTS), methyl-binding proteins (MBPS) gene families in duck and design specific quantitative primers and study the spatiotemporal influences of incubation temperature on genomic methylations of duck embryos. Then, we selected some genes that transcriptional levels were significantly affected by incubation temperature from skeletal muscle development related genes and analyzed the possible effects of promoter methylation status and on gene expression, we discussed the possible mechanisms that incubation temperature affect embryonic development and adult phenotypes from the perspective of epigenetic modification. The main results are as follows:(1) We obtained partial coding sequences of DNMT1, DNMT3A, DNMT3B, MBD3 and MBD5 by cloning. Their length are 507,481,443,387 and 525bp respectively. Analysis the corresponding sequence homology with the chicken in Genebank reached 91%-99%.(2) DNMT1, DNMT3A, DNMT3B, MBD3 and MBD5 genes expressions were detected in leg muscle, breast muscle, heart, liver and spleen in duck embryos. These results show that DNA methyaltion may involve in regulating tissue formation. Genes expressions were high in breast muscle, these results show that DNA methylation may have special significance in myogenesis. Furthermore, DNMTS and MBPS genes expressions were higher in spleen tissue, these results show that DNA methylation levels is relatively high in tissue that cell division is very strong.(3) Incubation temperature had marked effects on genes expressions of DNMT1, DNMT3A and DNMT3B during the early embryonic stages (P<0.05), which may prove that DNA methyaltion levels change more dynamic during the early embryonic stages. MBD3 and MBD5 have similar patterns of gene expression in both control groups and treatment groups during the whole incubation stages, which indicate that incubation temperature has the same effect on transcriptional levels of genes.(4) Incubation temperature in early embryonic stages could significantly increase the expressions of genes related to skeletal muscle development (P<0.05), while along with the extension of the incubation time, temperature manipulation could significantly inhibit the levels of gene expressions in Pax3, Pax7, Sixl, Akirin2, MyoD, MyoG, Mfy5, MCK and TNNC1 (P<0.05). These results indicate that the hatching temperature significantly affect regulated gene expressions is characterized by stages.(5) During the early and middle incubation stages, the methylation levels of TNNC1 gene’promoter CpG islands in treatment group are significantly lower than the control groups (P<0.05). During the early incubation stages, the methylation levels of Akirin2 gene’ promoter CpG islands are significantly lower than control groups (P<0.05). These results indicated that the raising of incubation temperature could inhibit methylate of promoter, while could promote gene expressions.