| Muscovy black feather duck is an excellent meat duck breed with good meat quality and high protein content.However,due to the influence of wild nature,brooding often occurs resulting in low egg production performance.The egg production during the entire breeding period is about 155.Muscovy ducks have strong brooding due to their natural wild nature,and strong brooding will lead to brooding behaviors in Muscovy ducks,which will cause follicle atresia and gradually reduce the duck’s egg production performance,which has a serious impact.The development of Muscovy duck breeding industry.Therefore,improving the egg production performance of black Muscovy duck is a key point to promote the development of Muscovy duck breeding industry.In this study,transcriptome sequencing was performed on the ovaries of low-yielding ducks in black Muscovy ducks before laying,egg-laying peaks,and high-yielding periods,and aimed to explore the relevant genes that affect the egg-laying performance of black Muscovy ducks.The differentially expressed genes were screened out by analyzing the results of transcriptome sequencing,and the tissue-specific expression and expression profiles of different grades of the selected genes were studied to further determine the key genes that affect ovarian development.Finally,according to tissue expression and follicular expression.As a result,one of the genes was selected for subsequent experimental studies of cloning and functional analysis.The main research content and results of this article:1.Screening of Differential Genes in Ovary Transcriptome of Muscovy DucksBy analyzing the ovarian sequencing results of the three egg-laying periods of the black Muscovy duck,combined with the GO enrichment of genes and the KEGG pathway to analyze egg-laying-related pathways,the data of the three periods can be divided into two groups for comparison,which are the pre-production and the KEGG pathway.A total of3,020 DEGs were found during the high-yield period,of which 1,019 were up-regulated genes and 2001 were down-regulated genes;the other group was a comparison between the high-yield group and the low-yield group during the high-yield period.A total of 176 DEGs were found,of which 51 were up-regulated genes.125 down-regulated genes.2.Tissue-specific expression of genes related to ovarian development and the expression patterns of different grades of folliclesFBXO5,ZP4,and TGFβ3 with higher differential expression were selected from a large number of DEGs to conduct specific expression identification of black Muscovy duck tissue and study the expression profile of different grades of ovarian follicles.The expression levels of these three genes in 8 tissues including ovary,heart,liver,spleen,lung,kidney,stomach,and breast muscle were detected by RT-q PCR technology.The results showed that these three genes were expressed in various tissues,and they were also expressed in ovaries.The relative expression level of FBXO5 was significantly higher than the other 6 tissues,thus successfully verifying that the selected gene is a gene that affects ovarian development;RT-q PCR technology was used to detect the expression level of the gene in different grades of follicles: FBXO5 gene is in grade The expression in anterior follicles is the highest in the period of rhubarb follicles larger than 8 mm in size,which is much higher than the other three periods of pre-grade follicles.The expression levels of the other three periods are not significantly different,but with Follicles grow larger and the expression level tends to increase;ZP4 gene expression is the highest in the period of small white follicles,and the expression level decreases as the follicles grow larger,and the gene is almost not expressed in the period of rhubarb follicles;TGFβ3 gene It is expressed in the four stages.As the follicle develops,the expression of this gene gradually decreases,and the expression amount between each stage is significantly different.Based on the quantitative results and literature reading,the functions of the selected three genes can be preliminarily inferred,which also provides basic knowledge for subsequent research.3.Cloning and bioinformatics analysis of FBXO5FBXO5 was selected from the three genes,and the full-length sequence of FBXO5 gene was obtained according to transcriptome sequencing.The full-length CDS region of FBXO5 was successfully cloned and a simple bioinformatics analysis was performed on the gene.The CDS region of FBXO5 was obtained by cloning.The total length is 1278 bp,and 421 amino acids are encoded in total.Through the analysis of the affinity and hydrophobicity of the protein,it is understood that the protein of the gene is a hydrophilic protein,and the gene cannot guide the newly synthesized protein to the secretion pathway;the secondary structure with the highest proportion is obtained when predicting the secondary structure of the gene protein It is a random coil,and the secondary structure with the lowest proportion is β-turn,and a three-dimensional structure diagram of the protein has been successfully established.4.Functional identification of FBXO5 gene in Muscovy duckBased on the successful construction of the FBXO5 cloning vector,an overexpression vector was successfully constructed,and the ovarian granulosa cells of Muscovy ducks were successfully isolated and cultured,and the function of FBXO5 was successfully verified at the level of primary granulosa cells.The relative expression of apoptosis and proliferation-related marker genes after FBXO5 gene overexpression was detected by q PCR.It can be seen from the results that after the overexpression of FBXO5 gene,the expression levels of the pro-apoptotic marker genes BAX and MYC selected in the treatment group were significantly lower than those of the control group.The anti-apoptotic marker gene BCL-2was significantly lower than that of the control group.It can be said that the expression level has increased to a certain extent,which can indicate that FBXO5 gene does not promote cell apoptosis;the expression of proliferation marker genes PCNA and CDK2 in the treatment group is significantly higher than that in the control group,which can indicate that FBXO5 can promote cells proliferation.WB technology also successfully verified that the constructed FBXO5 expression vector can be successfully translated into protein at the cellular level.In summary,this experiment successfully screened out DEGs related to ovarian development based on the results of sequencing the ovarian transcriptome of Muscovy ducks,and initially explored the effect of FBXO5 gene on the proliferation of follicular granulosa cells.The research on egg production performance has more important significance. |
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