The Isolation And Identification Of Differentially Expressed Genes In Pekin Duck Breast Muscle And Their Association With Muscle Breast Development

Pekin duck is a world-famous meat breed. The Z-type lean Pekin duck, one line of Pekinduck, is a ideal model for the study of skeletal muscle development due to its characteristicsof high feed conversion efficiency and high lean meat. However, the study about skeletaldevelopment of Z-type lean Pekin duck is still rare currently. The formation and reparation ofskeletal muscles is much more than the sum of its individual regulators. Therefore, it is thebasis of exploring the mechanism of skeletal muscle development that picking out thedifferentially expressed genes of various developmental stages in Pekin duck breast musclesbased on the analysis of large-scale data and clearing the relationship between thedifferentially expressed genes and muscle development.The differentially expressed genes in Pekin duck breast muscles were isolated andidentified using suppression subtractive hybridization (SSH) and RNA-seq technology. Then,some classic regulators of skeletal muscle development, such as MSTN, IGF1and FOXO3,were picked out and their association with breast muscle traits of Pekin duck were performed.In addition, MUSTN1gene, a gene found later and may be a regulator of skeletaldevelopment, was picked out to study its molecular characteristics, to detect itsspatiotemporal expression patterns in different tissues and developmental stages and toexplore its association with muscle development. pcDNA3.1（+）-MUSTN1, the Eukaryoticexpression veyctor of MUSTN1gene, was built and was vivo transfected into the breastmuscle of Pekin duck to find out the role of MUSTN1gene in Pekin duck breast muscle. Theresults of this paper were listed as follow.1, The isolation and identification of the differentially expressed genes indifferentdevelopmental stages of Pekin duck breast muscle using suppression subtractivehybridization (SSH)The SSH libraries of breast muscle of Pekin duck at2and6weeks of age were built usingSSH technology. Totally,257differentially expressed genes, including165genes were theidentified genes in other animals and92unkown genes, was found in these two libraries. Anumber of differentially expressed genes were expressed higher in the breast muscle at6 weeks of age than that at2weeks of age and they were involved in energy metabolism andprotein synthesis. The expression levels of23differentially expressed genes in the breastmuscles of2,4,6, and8weeks of age were detected using qRT-PCR technology. Theexpression patterns of these23genes in the breast muscles of2,4,6, and8weeks of age canbe divided into3groups: those whose expression levels descended continuously, those whoseexpression levels increased firstly and then descended, and those whose expression levelsascended continuously.2, The isolation and identification of the differentially expressed genes in the breastmuscle of Pekin duck using RNA-seqAfter RNA-seq sequencing of the breast muscles and skin fats at2,4, and6weeks of ageand the quality control of initial data, the retained high quality reads of the RNA-seq datafrom the breast muscles and skin fat at2,4, and6weeks of age of Pekin duck ranged from29million to45million after sequencing and quality control of sequences and total high qualityreads (combine the6tissues) reached to218million. The bioinformatics analysis showed thatthe obtained high quality reads were distributed along with the reference genes of duck, thehigh quality reads covered89.4%of duck annotated genes and the RPKM values of thesehigh quality reads were consistent with the expression levels of genes determined byqRT-PCR. The differentially expressed genes found between tissues or developmental stagesranged from few hundred to few thousand. However, there were more the differentiallyexpressed genes between tissues than that between developmental stages. The GO and KEGGanalysis showed that differentially expressed genes were significantly enriched in differentGO terms and a number of differentially expressed genes, such as IGF1R, IGF1, MSTN,TGFβ3, TGFβ1, TGIF1, FOXO6and FOXO3were significantly enriched in those pathwaysinvolved in the regulation of skeletal development or fat deposition. For example, there were214differentially expressed genes were enriched in MAPK pathway.3, Characterization of myostatin gene (MSTN) of Pekin duck and the association of itspolymorphism with breast muscle traitsThe expression levels of MSTN gene in the breast muscle of Pekin duck at1day,2,4,6,8week of age increased from1day to4week of age. However, the expression level of thisgene at6week of age was significantly lower than that at4and6week of age using qRT-PCRtechnology. After molecular cloning and bioinformatics analysis, it was found that thefull-long CDS of Pekin duck MSTN gene contained1128bp and the protein encoded by thisgene shared high similarity with other invertebrates. MSTN gene encoded two alter splicingisoforms: the first one encoded an AA sequence with375AA, another one only contained315AA. In addition,3SNP mutation sites were found in MSTN gene CDS. The breast breadth of ducks with BB genotype was significantly higher than the ducks with AA genotype at T129CSNP site. At the T952C SNP site, ducks with the AB genotype owned significantly longerlength of fossilia ossis mastoid than those with the BB genotype.4, The expression profiles of IGF1and FOXO3and developmental differences of breastand leg muscle in Pekin duck during postnatal stages.The paraffin of breast and leg muscle at2day,2,4, and6weeks of age showed that thegrowth rate of breast muscle is lagged behind that of leg muscle. For breast muscle, the fastestgrowth stage was4~6week of age, but the fastest growth stage was2~4week of age for legmuscle. The results of using qRT-PCR detection showed that the highest expression levels ofIGF1in breast muscle and leg muscle were appeared at6weeks of age and2week of agerespectively. Conversely, the minimum values of FOXO3expression levels were appeared at6weeks of age and2week of age respectively.5, Characterization of MUSTN1gene and its relationship with skeletal muscledevelopment at postnatal stages in Pekin ducksAfter the clone of cDNA and DNA, sequence assembly and analysis of MUSTN1gene, itwas found that the full-long CDS of Pekin duck MUSTN1gene encoded a protein with78AA. MUSTN1gene entire DNA sequence was composed by3exons and2introns and anumber of transcription factor-binding sites were found in its promoter region. The expressionmaximum points of MUSTN1in breast and leg muscle were at5and3week of age. Aftervivo transfecting pcDNA3.1（+）-MUSTN1, the Eukaryotic expression vector of MUSTN1gene, into the breast muscle of Pekin duck, it can be expressed effectively in the breast muscleof Pekin duck and lead to significantly change in the expression levels of other regulators ofskeletal muscle hypertrophy.In conclusion, the differentially expressed genes in breast muscle of Pekin duck wereisolated and identified. The gene characteristics of classic regulators of skeletal muscledevelopment and the newly found MUSTN1gene were explored and their association withskeletal muscle development were detected. The results of this paper will provide a basis forexploring the mechanism of skeletal muscle development.