Laying Curve Fitting Of Black-feather Muscovy Duck And Selection,Cloning And Sequence Analysis Of Laying-related Genes

Muscovy duck is a typical lean duck species.It has more than 300 years of breeding history in China,and it is currently raised most in Fujian province.It has many advantages,such as fast growth,strong resistance,tender meat and so on,but the average annual egg production is only 150.Its broodiness and low fertility which seriously affects economic benefits.Therefore,this study first explored the laying rule by fitting the laying curve of the black-feather muscovy duck,and then screened the differentially expressed genes for black-feather muscovy duck by transcriptome sequencing technology,cloned the laying-related genes,and revealed the molecular mechanism of laying in different periods which provided basic data for the reproductive performance of black-feathered muscovy duck.1.Laying curve fitting of black-feather muscovy duckIn this experiment,1,245 black-feather muscovy ducks aged 26 to 48 weeks were used as the research objects.The Wood,Mc Millan and Yang-Ning models and the Logistic,Gompertz and Von Bertalanffy models were used to fit the laying rate and cumulative egg production curve,respectively.The results show that the Yang-Ning model is the best in the laying rate curve,and R2 is 0.938;in the cumulative egg production curve,the Von Bertalanffy model R2 reaches 0.999,which is the best effect.2.Sequencing and analysis of ovarian transcriptome of black-feather muscovy duck at different laying periodsThe ovaries of three female ducks were collected at the early,peak and late laying stages of the black-feathered muscovy duck,and the differentially expressed genes were screened by pairwise comparison of transcriptome sequencing for GO and KEGG enrichment analysis.It can be known from the results that a total of 63.6G data was obtained in this sequencing,with an average Q30 of 94.31%,and the sequencing sample quality was better.A total of 1,683 DEGs were obtained in the three comparison groups.The up-and down-regulated genes of BL-vs-BE,BL-vs-BP,and BP-vs-BE were 41 and 60,835 and 255,and 260 and 730,respectively.There were 32,20 and 424 in each group,and 11 DEGs were expressed in three groups.Through GO enrichment analysis,it was found that the biological processes related to developmental reproduction include reproduction,developmental process,and reproductive process,involving 237 related genes.Through KEGG enrichment analysis,we mainly focus on signaling pathways such as ovarian steroid production and steroid hormone biosynthesis.13 DEGs were selected and detected by real-time quantitative PCR,and the results were consistent with the transcriptome results.3.Cloning and sequence analysis of laying-related genes of black-feather muscovy duckBased on the transcriptome sequencing results,a gene(BL-vs-BE: HOXA10;BP-vs-BE: Htr A3;BL-vs-BP: St AR)was selected for each group for cloning and sequence analysis.Prokaryotic expression of Htr A3 gene and observation of its recombinant protein.The coding regions of three genes were successfully cloned with a total length of 1,095 bp,1,401 bp and 852 bp.We obtained the amino acid sequence,understood its physicochemical properties,and the secondary and tertiary structure of the protein,and constructed a phylogenetic tree.HOXA10 has a conserved Homeobox domain,which is involved in the transcriptional regulation of key developmental processes and binds DNA through a helix-turn-helix(HTH)structure.Htr A3 has three conserved domains,namely IGFBP,KAZAL and deg P＿htr A＿DO,which regulate different physiological activities.Both Homeobox and KAZAL domains are involved in the TGF-β metabolic pathway.St AR has a START domain and participates in lipid transport.The Htr A3 prokaryotic expression vector was successfully constructed and the recombinant protein was obtained.In summary,the Yang-Ning and Von Bertalanffy models can establish a standard laying curve for black-feather muscovy duck,and provide a basis for improving laying performance.The transcriptome technique was used to identify the differentially expressed genes of black-feather muscovy duck at different periods,and cloned three laying-related genes,which provided a reference for the black-feather muscovy duck’s laying process.