Subcellular Localization Of Chicken Bromodomain-Containing Protein Families (BRDs) And Their Expression Characteristics In Different Chicken Tissue Development

Bromodomain was first found by amino acid sequences comparative analysis of Drosophila Brahma protein.In addition to regulating gene transcription,bromine-containing domain proteins(BRDs)are also associated with growth development,and disease occurrence.BRDs mainly contain seven members,including BRD1,BRD2,BRD3,BRD4,BRD7,BRD8,and BRD9.Although BRDs have been widely studied in human and mice,the subcellular localization of chicken BRDs and their expression in various tissues of chicken embryos at different developmental stages and chicken ovarian tissues are still unclear.In this study,chicken BRDs genes were first cloned,sequenced and analyzed.In addition,the subcellular localization of chicken BRDs,and the expression characteristics of BRDs genes in different tissues during the development of chicken embryos and in the hen reproductive system at different egg-laying peak periods were also investigated.The main findings are as follows:1.The CDS region of chicken BRDs genes was successfully amplified and the recombinant eukaryotic expression vectors of chicken BRDs genes were also correctly constructed.The results of bioinformatics analysis showed that the CDS regions of chicken BRD1,BRD2,BRD3,BRD4,BRD7,BRD8 and BRD9 genes were 3561 bp,2340bp,2169 bp,4137bp,1956 bp,2811bp and 1953 bp in length,respectively,which encoded 1186,779,722,1378,651,936 and 650 amino acids,respectively.Chicken BRDs were rich in secondary structures,which were mainly composed of irregular coils and α-helices.The homology of BRDs among chickens and human and mammals was 76.1%-91.4%,of which the BD domains of BRDs were the most conserved(84.5%-100.0%).Fluorescence observation results revealed that chicken BRDs were all localized in the nucleus.2.Eight tissues including eyeball,brain,heart,liver,lung,muscle stomach,breast muscle and leg muscle of Guizhou yellow chickens were collected at embryonic period 14 days(E14 d),and hatched period 1 day(1 d),7 days(7 d),14 days(14 d),Total RNA was extracted from the above tissues and cDNA was synthesized by reverse transcription method.The expression characteristics of BRDs genes in different tissues during the development of chicken embryo were examined by the quantitative real-time PCR(q RT-PCR).The results showed that BRDs genes were expressed in all the collected tissues at E14 d,1d,7 d and 14 d.The expression of BRDs genes in the eyeballs was stable or slightly increased from E14 d to 1d,increased significantly from 1d to 7d,and decreased again from 7d to 14 d.BRDs genes show different trends in other tissues.The above results indicated that chicken BRDs genes were expressed in different tissues at different stages of chicken embryo growth and development,but showed different expression levels and expression patterns,which layed the foundation for the subsequent study of the mechanism of BRDs genes regulating the development of chicken related tissues.3.Six tissues including ovary,fimbriae of uterine tube,albumin secreting part of uterine tube,isthmus of uterine tube,uterus part of uterine tube and vaginal part of uterine tubewere were collected from Guizhou yellow chicken and Changshun green shell laying hens(about 200 days of age)during peak laying period,respectively.Total RNA was extracted from each tissue and cDNA was synthesized by reverse transcription.The expression levels of chicken BRDs genes in each tissue of the reproductive system during the peak laying period were detected by q RT-PCR,with chicken GAPDH gene as the reference gene.The results showed that the chicken BRDs genes were expressed in the selected reproductive system tissues of the two chicken breeds.Of which BRD1,BRD2,BRD3,BRD4,BRD7 and BRD8 genes had the highest expression levels in the ovary of Guizhou yellow chickens,while BRD9 gene expression level was highest in the isthmus of uterine tube of Guizhou yellow chickens.In addition,BRD1,BRD3,BRD7,BRD8 and BRD9 genes had the highest expression levels in the ovary of Changshun green shell laying hens,while BRD2 gene and BRD4 gene expression levels were highest in the uterus part of uterine tube and the vaginal part of uterine tube,respectively.However,the expression of BRDs genes except for BRD3 gene in thr ovary of Guizhou yellow chickens was much higher than that in Changshun green shell laying hens.These results layed the foundation for further studying the association of chicken BRDs genes with chicken egg production.