| China is the largest country in duck meat production,consumption and export.In 2021,4.10 billion commercial ducks were marketed in China,with a total output value of 101.74 billion yuan.Duck meat,as a high-quality protein source at a reasonable price,is popular among consumers because of its advantages of high protein and low fat.Although the high quality local duck varieties in China have excellent flavor,their growth rate is far less than that of foreign varieties.In order to improve the growth rate of local duck breeds in China,it is very important to study the related genes of duck muscle development.In this study,the breast muscle tissues of 6-week-old ducks with high and low breast muscle percentage from F2 resource population were sequenced by transcriptome sequencing technology,and the differentially expressed genes were analyzed and the functional comments of the differentially expressed genes were made,and the candidate functional genes that may affect muscle growth and development were screened out.The expression trends of 8 randomly selected differential genes were verified by fluorescence quantitative technique.The function of the key gene MYOZ2 on muscle growth was further verified,and the influence of MYOZ2 on muscle development was detected by overexpression and knockout methods at the cellular level.The main results are as follows:1.The transcriptome sequencing results of breast muscle tissue of ducks with high and low breast muscle ratio were analyzed.A total of 16433 positive genes were obtained,and 173 differentially expressed genes were screened according to the standard of difference multiple greater than 2 and P<0.05.There were 65 up-regulated genes and 108 down-regulated genes in the high pectoral muscle ratio group.Perform GO enrichment analysis on the screened differentially expressed genes.The results show that the pathways are closely related to lipid binding in biological processes,and the pathways in molecular functions are clustered in the process of muscle contraction and growth factor stimulation.The differentially expressed genes involved in these GO entries were EDN2,HTR1D,MYL4,SMAD7,TMPRSS6,CER1,FABP3 and CRABP1.KEGG pathway analysis found that the top 20 pathways enriched in these differentially expressed genes were mainly cytokine-cytokine receptor interaction,PPAR signaling pathway,citric acid cycle（TCA cycle）,RIG-Ⅰ-like receptor signaling pathway and Myocardial contraction and other pathways related to fat metabolism and muscle growth,the genes enriched in these pathways are CCL3、L1R2、Cxcr5、FABP3、DHX58、Ca12.Eight differentially expressed genes（EDN2,MB,SMAD7,FABP6,FABP3,CYC,HTR1D,and MYL4）were randomly selected,and the expression of the differentially expressed genes in duck pectoral muscle tissue with high and low pectoral muscle rates was verified by real-time quantitative PCR（qRT-PCR）.The results of qRT-PCR were consistent with those of RNA-seq.The results showed that the differentially expressed genes identified by the RNAseq method were reliable.2.Preliminary functional verification of MYOZ2 gene,the results of dual-luciferase reporter system showed that the core region of MYOZ2 promoter is located at-578bp～0bp.The expression of this gene in duck pectoralis with low pectoral muscle rate was significantly higher than that in high pectoral muscle rate group（P<0.05）,and its expression trend was opposite to that of the myogenic differentiation marker genes ACTB and MyoG;After interference My blasts differentiate and fuse into thicker myotubes.In this study,173 differentially expressed genes that may affect muscle development were screened out and the main pathways involved in differentially expressed genes were identified.Preliminary validation of MYOZ2 showed that MYOZ2 inhibited proliferation and differentiation of myoblasts and negatively regulated muscle development. |
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