Transcriptome Analysis Of Humerus And Preliminary Study Of The Candidate Genes Associated With Bone Metabolism In Laying Hens

At present,the housing system of laying hens in China is dominated by large-scale,standardized and intensive cages.The laying hen reared in traditional cages has weakened bone quality because of the environmental and behavioral welfare and limitation of movement.The medullary bone contributes 35-40%of the calcium for eggshell formation for laying hen producing 1 egg per day.Meanwhile,the age-related remodeling ability of medullary bone is decreasing.Once the amount of calcium supplied by the medullary bone is insufficient,the cortical bone is absorbed and decomposed to release calcium,resulting in a reduction of bone mass,an increase of bone fragility,and weakness of bone quality,and then causing bone problems such as fractures.Therefore,the bone metabolism balance of medullary bone plays an important role in the bone health and performance of laying hens.This study was conducted to improve the bone quality,prolong the duration,and maintain the bone metabolism balance of laying hens.The transcriptome analysis was used to screen genes associated with bone formation of laying hens,and gene cloning and bioinformatics analysis of related genes were conducted,which can provide a theoretical basis for its regulation mechanism.In this study,Taihang chicken at 41 weeks of age,with similar body weight and the humerus with and without medullary bones,were selected for transcriptome sequencing,and high expression and differentially expressed genes related to bone metabolism were screened.The Hyline Grey laying hens at 75-79 weeks of age were selected for exploring the relationship of expression level,genotype of two genes?the bone metabolism related gene BSP and differentially expressed gene OCN?and the bone breaking strength and egg quality,to reveal the regulatory mechanism of production in laying hens.The Illumina HiSeq high-throughput sequencing platform and bioinformatics were used for transcriptome of Taihang chicken humerus tissues.Results showed that a total of24,849,249,25,339,839 and 23,747,540 pairs of high-quality clean reads were obtained for three replicates of the humerus with medullary bone,respectively.26,914,956,22,251,587and 24,509,445 pairs of high-quality clean reads for three replicates of the humerus without medullary bone,respectively.The ratio of clean reads mapping on the reference genome was greater than 60%,and the number of clean reads aligned to multiple locations in the reference genome was less than 0.6%.The result of gene expression analysis showed that the expression level of the top 10 genes in each sample was above 10³ orders of magnitude,and the high level was up to 10⁴ orders of magnitude.Among them,BSP_FPKM?Y1?=8520.11,and the BSP expression of the other samples was also above 3.0×10³,indicating that BSP was highly expressed in humerus.There were 281 differentially expressed genes,including 236 up-regulated and 45 down-regulated,in the humerus without medullary bone compared with the humerus with medullary bone Among them,OCN_Log2FC=4.05,indicating that OCN was a differentially expressed gene in humerus with and without medullary bone.The coding region of OCN gene was cloned and analyzed to investigate the biological characteristics.The promoter of OCN gene was amplified to identify variation and the promoter activity.The expression level of the gene was measured by RT-PCR technique.The bone breaking strength and eggshell quality of the Hyline grey laying hens were detected.Results showed that there were two types of OCN protein,TI and TII.The deletion of GVAGAPP at amino acid position 55-61 was for TII type.The amino acid numbers of TI and TII proteins were 97 and 90,respectively.Both were hydrophilic unstable proteins and had better fluidity,and mainly located outside the cell.Signal peptide cleavage site located at 20-21 amino acids,and no transmembrane region was predicted.The secondary structural elements of protein included alpha helix,extended strand and random coil.What's more,it was detected that the promoter of OCN gene in Hyline grey laying hens consisted three haplotypes,I,II and V.In addition,haplotype II was the main haplotype with high transcriptional activity,resulting in elevated level of OCN gene expression.Compared with haplotype I,the eggshell strength was significantly increased?P<0.01?,and the eggshell weight was significantly reduced?P<0.05?.The coding region of BSP gene was cloned and analyzed to investigate the biological characteristics.The promoter of BSP gene was amplified to identify variation and the promoter activity.The expression level of the gene was measured by RT-PCR technique.The bone breaking strength and eggshell quality of the Hyline grey laying hens were detected.Results showed that synonymous mutations,153 G>A,294 G>A,510 G>A,513G>A and 651 A>G,and missense mutations,611-612 TG>CA?204 V>A?,644 T>C?215V>A?,529-540 CAG deletion and GAAGAGGAA insertion?178 2E/3E insertion?,were presented in BSP gene in Hyline grey laying hens,which were corresponded with five types?PI,PII,PIII,PIV and PV?of BSP proteins.The amino acid numbers of PI,PII,PIII,PIV and PV were 276,278,279,278 and 279,res pectively.All of them were acidic hydrophilic proteins,unstable,poor in fluidity,and mainly located outside the cell.Signal peptide cleavage site was located at 16-17 amino acids,and no transmembrane region was predicted.The secondary structural elements of protein included alpha helix,extended strand,beta turn and random coil.In this study,the 1,399 bp?-1257/+142?candidate promoter region of BSP gene in Hyline grey laying hens was successfully cloned.Six haplotypes?Hap1-Hap6?in the BSP gene promoter?-1257/+142?was found,among which Hap1-Hap4 correspond to CC genotype,and Hap5 and Hap6 correspond to TT genotype.The-202/+142 region was identified as the core promoter region of chicken BSP gene.The core promoter region activity of CC genotype was significantly higher than TT genotype?P<0.01?,which resulted in elevating the level of BSP gene expression.Compared with the TT genotype,the relative strength of humerus,femur and tibia and the eggshell strength of the CC genotype were significantly reduced?P<0.05?.The eggshell weight was significantly increased as well?P<0.01?.Pearson correlation analysis showed that the correlation coefficient of relative strength was 0.819?P=0.001?between tibia and humerus,while it was 0.505?P=0.033?between tibia and femur.In conclusion,in terms with the relative strength of tibia of Hyline grey laying hens,a positive correlation was presented in the relative strength of humerus and femur.In summary,the promoter of OCN gene in Hyline grey laying hens included three haplotypes?I,II and V?.Haplotype II was the primary haplotype,and there was significant difference between haplotype I and haplotype II in the transcriptional activity,gene expression levels and eggshell quality of the corresponding individuals.BSP gene promoter?-1257/+142?in Hyline grey laying hens included six haplotypes?Hap1-Hap6?,and the core promoter region was-202/+142.Individuals with different genotypes at the644 T>C locus had significant differences in promoter activity,gene expression levels,bone breaking strength,and eggshell strength.The relative strength of the tibia of Hyline grey laying hens was positively correlated with the relative strength of the humerus and femur.