Identification Of Differentially Expressed Genes Of Pituitary Tissues Between Tibetan Chicken And Ross Broiler Using RNA-seq Technology

In order to explore the key genes of affecting the early rapid growth of poultry,by sequencing and comparing the RNA expression profiles of pituitary tissues between slowly growing Tibetan chickens and fast-growing Ross broilers,the candidate genes related to growth and development were screened,and the selected candidate genes were further studied.42-day-old female Tibetan chicken and Ross broilers were selected as research objects,and 3 replicate samples were selected for each breed of chicken.The RNA expression profiles of pituitary tissues of Tibetan chicken and Ross broiler were identified by RNA-seq technique and bioinformatics method.Among the differentially expressed genes,GO(Gene Ontology)and KEGG analysis were used to screen candidate genes of affecting early growth and development.Then,the sub-cellular localization of the candidate gene was performed by Western blot,and the tissue expression profile and time-series expression profile of the candidate gene were constructed by real-time fluorescent quantitative PCR.The results show: 1.Sequencing results of pituitary tissue expression profiles of 42-day-old female Tibetan chicken and Ross broilersEach transcript Q30 was above 90%,indicating that the transcript library was good quality.The new transcripts predicted in each transcript are all above 700 bp,with the longest containing 30 exons and the shortest containing only 1 exon.The IR(intron retention)variable shear model was predominant(≈50%)in two breeds of chicken.Among the transcripts,the conversion type(A-G/C-T)SNP locus accounted for 76%,and the transversion type SNP locus(A-C/A-T/C-G/G-T)SNP locus accounted for 24%.2.Results of GO and KEGG analysis of pituitary tissue expression profiles for Tibetan chicken and Ross broilersThe analysis revealed that there were 151 significant differentially expressed genes in the pituitary tissue expression profile,including 77 up-regulated genes and 74 down-regulated genes(Tibetan chicken Vs Ross broilers).Annotating the differentially expressed genes and attributes of gene product(GO annotation),it was found that 22 genes were annotated in the cell components,corresponding to 48 functional subclassifications,20 genes were annotated in the molecular function,corresponding to 48 functional sub-categories,23 genes were annotated in the biological process,corresponding to 242 functional sub-categories.The differentially expressed genes involved in the major metabolic pathways and signal transduction pathways were annotated(KEGG annotation).It was found that 138 of the 151 significant differentially expressed genes were enriched into 147 KEGG pathways,including lipid metabolism,energy metabolism,carbohydrate metabolism,cell growth and death,signaling and other important metabolic pathways and signaling pathways.3.Enrichment results in the development process entry(GO:0032502)A total of 9 significant differentially expressed genes were enriched in the developmental process entry(GO: 0032502),including 5 up-regulated genes(FST,GH,LHFPL5,BPIFB7,NPY)and 4 down-regulated genes(PTHLH,EGR1,IGF2BP1,SHH)(Tibetan chicken Vs broiler).The analysis showed that down-regulation of EGR1,IGF2BP1,and SHH genes are important factor leading to slow growth of Tibetan chickens in the early stage.4.qPCR results on expression profilingEight genes(EGR1,FST,FKBP5,GH,LOC419390,PPARG,ZBTB16,SHH)were randomly selected from the differentially expressed genes,and the expression profiles were verified by real-time quantitative PCR(qPCR).The qPCR results were consistent with the transcription results,indicating that the expression profiles were reliable.5.Subcellular localization results of FST and EGR1 proteinsSubcellular localization of FST and EGR1 proteins by Western blot,the results showed that FST was mainly localized in the cytoplasm in the pituitary,hypothalamic,pectoral and leg muscle tissues of Tibetan chicken and Ross broiler,while EGR1 protein was mainly located in the nucleus.6.Construction results of FST and EGR1 gene expression profilesThe FST and EGR1 were the highest expression in pituitary tissues at the any age of Tibetan chicken and Ross broiler,which was significantly higher than other tissues(hypothalamus,chest muscle and leg muscle tissue)(P<0.01).From 1 to 42 days old,except for Tibetan hens,the expression levels of FST and EGR1 in Ross broiler tissues(pituitary,hypothalamus,chest muscles and leg muscles)were gradually higher than those in Tibetan chickens.Construct a temporal expression profile:The FST temporal expression profile of Tibetan chickens(1~150 days old)showed that FST showed a trend of increasing first and then decreasing in pituitary tissues.In the hypothalamic tissue,FST is the first to decline,then rise,and then decline.FST has a higher expression level in muscle tissue at 1 day of age,but decreases sharply in the pre-developmental stage(1 to 42 days)and then maintains a stable level;The temporal expression of FST in Ross broiler(1~42 days old)was consistent with the expression changes in Tibetan chickens.The EGR1 temporal expression profile of Tibetan chicken(1~150 days old)showed that the expression level of EGR1 in pituitary tissue first decreased,then increased,and then decreased,and the expression level was highest at 70 days.In the hypothalamic tissue,the expression level of EGR1 showed a decrease first,then increased and then remained stable.In muscle tissue,expression level of EGR1 is higher at 1 day old,but it decreases sharply at 1 to 42 days of age and then remains stable;Ross broiler from 1 to 42 days old,the expression of EGR1 in pituitary and hypothalamic tissue showed an upward trend,and EGR1 in muscle tissue showed a downward trend.By sequencing the expression profiles of pituitary tissues of Tibetan chickens and Ross broilers,the nine genes with significant differential expression were found.Among them,five genes(FST,GH,LHFPL5,BPIFB7,NPY)were up-regulated,and four genes(PTHLH,EGR1,IGF2BP1,SHH).)were down-regulated.(Tibetan chicken Vs Ross broiler).Analysis of the nine genes revealed that down regulation of the EGR1,IGF2BP1,and SHH gene is an important factor leading to slow growth in early Tibetan chickens.These phenomena are consistent with the biological characteristics of Tibetan chickens growing under the alpine conditions of the Qinghai-Tibet Plateau,and are also consistent with the fact that Tibetan chickens are less well-bred.The differences in expression of FST and EGR1 gene in pituitary tissues between Tibetan chicken and Ross broiler are consistent with that growth pattern of the rate of Ross broilers growth reached a peak at 42 days of age and the rate of Tibetan chicken growth reached a peak at 70 days of age.This is also consistent with the growth pattern of most domestic chicken.Therefore,FST,EGR1,IGF2BP1,and SHH genes are important candidate genes for the early growth rate of domestic chicken breeds.