Study On Estrogen Treated Chicken Liver Transcriptome And Estrogen Regulation Molecular Mechanism On The Candidate Genes

The laying performance is one of the most important economic performances for laying hens.Most of the deposited protein and lipids in the eggs are not synthesized directly from the ovaries but livers.Almost all of the lipids are synthesized in chicken livers and transported to the egg yolk.Liver is the most important metabolic organs for chicken.The development of poultry follicles is strongly dependent on estrogen induction,and chicken liver is also an important target organ of estrogen.Estrogen plays a variety of physiological functions as an important steroid hormone,the study of estrogen target gene and regulatory network has become a research focus.In this study,the next generation of high-throughput technique was used to explore the estrogen-treated chicken liver,we obtained a lot of differently expressed genes and constructed the estrogen regulation network in chicken liver.Thyroid hormone response protein SPOT14(THRSP)and melanocortin receptor accessory protein gene(MRAP)were validated both in vivo and in vitro.The molecular mechanism of THRSP regulated by estrogen was studied by Ch IP-q PCR technique.Study 1: Transcriptome of estrogen treated chicken liverIn this study,six 10 weeks old Lushi chickens were randomly divided into two groups,three chickens in each group.One group was injected with 2 mg / kg body weight of 17?-estradiol,the control group was injected with estradiol solvent by the same way.After 12 hours of treatment,the liver was collected and RNA was extracted to construct transcriptome libraries.The libraries were sequenced using the Illumina Hi Seq 2500 platform.After filtering the original sequencing data,the clean reads were mapped to the reference genome,and the results were obtained by statistical analysis.We obtained a large number of estrogen target genes,and these genes were enriched to GO and KEGG database.The main results are as follows:1.The serum TG in the estrogen-treated group was significantly higher than that of the control group.The lipid droplets and apo VLDL-II,APOB m RNA expression in the liver of estrogen treated were also increased.2.Six chicken liver cDNA libraries were successfully constructed,and a total of 883427404 Reads were obtained by sequencing.Through the data filtering,mapping to reference genome and the correlation analysis between the samples showed that the overall quality of RNA-Seq was high-quality,which reached the requirement of further analysis.3.A total of 15556 genes were obtained by transcriptome data and a total of 11964 potential new genes were found by bioinformatics analysis.4.A total of 962 different genes were selected by intergroup analysis,compared with the control group,545 up-regulated and 417 down-regulated gene were obtained.Among the new genes,there were 869 different genes,of which 382 were up-regulated and 486 were down-regulated.5.Through the GO,KEGG enrichment analysis of the differential genes,59 significant GO terms and 14 significant KEGG pathways were enriched.Significantly enriched GO and KEGG were significantly associated with fat metabolism,of which 17 fat-related GO terms and 7 KEGG pathways were associated with lipids.6.Ten genes were randomly selected and verified by q RT-PCR.The results were consistent with transcriptome data,indicating that the transcriptome were reliable.Study 2: Molecular mechanism of THRSP expression regulation by estrogenTHRSP is one of the most important lipogenic activator genes selected in study 1.In order to study the tissue distribution of THRSP,eleven different tissues from six 10 weeks and 30 weeks old Lu Shi chickens were collected.In order to study the expression pattern of THRSP,the liver and abdominal fat tissues from forty 10,15,20,25,30 and 35 weeks old chickens were collected.In order to study the effect of estrogen on THRSP expression,48 chickens were divided into 8 groups(6 in each group).The four groups were injected with 17?-estradiol by 0,0.5,1and2 mg / kg body weight,The liver and abdominal tissues were collected after 12 hours and the other four groups were treated by the same way but collected the tissues after 24 hours.In order to further verify the estrogen effect on THRSP expression in vitro,chicken primary hepatocyte were cultured and treated with 0,1,50 and 100 n M 17?-estradiol.In order to verify whether the estrogen receptor binds to the THRSP promoter,the estrogen-treated liver tissue is used for validation of Ch IP-q PCR.The main results are as follows:1.The results of spatio-temporal expression showed that THRSP was highiy abundance in chicken liver and abdominal fat.The expression of THRSP in different weeks old chicken livers increased with the maturation of chicken,and reached the highest expression level at the peak of laying(30 weeks).2.The expression of THRSP in chicken liver and abdominal fat were significantly up-regulated by estrogen both in vivo and in vitro.3.Estrogen antagonists ICI can effectively inhibit the Induced expression of THRSP in chicken primary hepatocytes.4.Structure analysis of the THRSP gene indicated that there is a conscious estrogen response element(ERE)located in the-2434 –-2422 range of the gene promotor region.Further studies by Ch IP-q PCR proved that the ER? interacts with the putative ERE site.Study 3: Association of Estradiol on Expression of Melanocortin Receptors and Their Accessory Proteins in the Liver of ChickenThe melanocortin receptor accessory proteins(MRAP and MRAP2)are small single-pass transmembrane proteins that regulate the biological functions of the melanocortin receptor(MCR)family.MCRs comprise five receptors(MC1R–MC5R)with diverse physiological roles in mammals.Five MCR members and two MRAPs were also predicted in the chicken(Gallus gallus)genome.However,little is known about their expression,regulation and biological functions.Only MC5 R and MRAP were detected in our RNA-seq in chicken liver,its much difference with mammals,So we studied the seven genes systematically.The material and method were same with the study 2,the mina results are as follows:1.We cloned the MRAP and MRAP2 coding region,sequencing analysis indicated that the coding sequences of MRAP and MRAP2 were entirely identical to the predicted coding sequences of the two genes in Gen Bank.The chicken MRAP gene comprised of three exons and encoded a 120-amino acid MRAP protein and MRAP2 gene encoded a 206-amino acid MRAP2? protein.2.Bioinformatics analyses results revealed that there was no signal peptide but possible transmembrane domain in the MRAP and MRAP2 protein sequence.Amino acid sequence Multiple alignment analysis among species manifested that chicken MRAP shared low identifies with other species(<50%),but the N-terminus and transmembrane region were relatively conserved.What's more,amino acid sequence alignment analysis between chicken MRAP and MRAP2 indicated that MRAP was only 37% identical to MRAP2.3.The molecular evolution and the relationships between the homologs were investigated using phylogenetic methods.The evolution analysis result showed that the phylogeny tree was divided into two phylogenetic groups by MRAP and MRAP2.The avian MRAP and MRAP2 clusters separate from mammalian and reptile MRAP and MRAP2.4.Tissue distribution results showed that MRAP2 can be detected in the adrenal gland and brain tissues,and MC2 R only can be detected in the adrenal gland.Only MRAP and MC5 R can be detected in the liver tissue of chicken.Other genes were extensively expressed in multiple tissues.5.The relative expression levels of MRAP m RNA and protein in 30-week-old chickens were significantly higher than those in 10-and 20-week-old.The relative expression levels of PPAR? m RNA were significantly lower,while MC5 R m RNA was not changed.6.Estrogen treatment can significantly up-regulated the m RNA and protein level of THRSP in chicken liver and down-regulated the m RNA level of PPAR?but not change the expression of MC5 R.7.Estrogen treatment can significantly up-regulated the m RNA level of THRSP in chicken primary hepatocytes and down-regulated the m RNA level of PPAR?but not change the expression of MC5 R.