Screening And Functional Verification Of Differentially Expressed Genes During The Development Of Chicken Pectoral Muscles Tissue

Chicken occupies an important position in the structure of Chinese meat consumption,meat trait has been the research hotspot in the field of chicken genetic breeding.Local chicken species have unique genetic resources in China.How to analyze the molecular regulation mechanism of muscle growth from the oveall level of functional transcriptome and regulatory transcriptome will become an important breakthrough in chicken genetic improvement.To analyse the transcript levels of muscle tissue at different growth and development stages helps to understand the molecular regulation mechanisms of this tissue involved in the growth and development related genes and regulatory factors.This research treated chicken as the research material to systematically screen and analyze the mRNAs and lncRNAs of breast muscle tissues by RNA-seq technique.We screened the lncRNAs and mRNAs related to muscle growth and constructed the molecular regulatory networks,and preliminary analyzed the relationship between lncRNAs and mRNAs on the aspect of regulating muscle growth and development.In addition,among the differentially expressed genes,GPR133 and SH3BP5 were selected to perform functional verification at the cellular level.Firstly,two transcriptional isoforms of GPR133 gene associated with muscle growth and development were obtained by cloning techniques.Then,the study verified the function of two GPR133 transcriptional isoforms and SH3BP5 gene at the cellular level.The main results are as follows:(1)A total of 817.74 Gb raw data were obtained from 49 sequencing libraries,approximately 706.32 Gb of clean data was retained after quality control.Finally,74%～88.8% of the high quality reads were aligned to the chicken genome.A total of 17905 coding protein mRNAs and 28032 lncRNAs were identified after the Clean data comparison and assembly,including 9594 genic lncRNAs and 15946 intergenic lncRNAs.Compared with lncRNAs,the mRNAs transcripts exhibit longer length,more exons and higher expression levels.In terms of chromosome distribution,most of the mRNAs and lncRNAs are distributed on the large chicken chromosome with a distinct tendency.According to the hierarchical clustering and the principal component analysis of mRNAs,and the correlation analysis of mRNAs and lncRNAs,the correlation between the biological replicates of each sample was high.(2)1020,294,165 and 299 genes were found to highly expressed in the midst of embryonic development,late embryonic development,vigorous development and adult respectively.Along with the growth and development process,most of the differentially expressed mRNAs and lncRNAs show a general downward trend;263 and 131 specifically expressed mRNAs and lncRNAs were screened in skeletal muscle at day 300.By further screening,we obtained 82 mRNAs and 34 lncRNAs which were strongly associated with skeletal muscle growth and development.By functional prediction of the skeletal muscle-specific expression or strongly correlated with growth and development mRNAs,we found that most of those genes encoding proteins are significantly enriched in transcriptional regulation or biological processes and signaling pathways associated with skeletal muscle growth and development and metabolism,suggesting that these genes may be involved in muscle development and metabolic processes.(3)Five co-expressed network maps and some core mRNAs and lncRNAs were obtained by constructing co-expression regulatory network of skeletal muscle-specific mRNAs and lncRNAs,some of these lncRNAs have a direct interaction with one or more mRNAs.For example,lncRNAs TU124209 directly contacts with MYOG,and TU60015 have a direct interaction with SRBD1 and MYOC(myocilin).(4)In the present study,two chicken GPR133 isoforms were identified by a combination of reverse transcription PCR(RT-PCR)and rapid amplification of c DNA 5′-ends(5′ RACE).Thereinto,GPR133-va and GPR133-vb were respectively 3385 and 3289 bp in size.Sequence analysis shows that GPR133-va retains all 26 exons,whereas,GPR133-vb is resulting from the AS(Alternative splicing)modules of exon 4 skipping.The predicted protein structures of both isoforms contain three conserved domains,including Lam G and GPS at the N-terminus,and the seven-transmembrane domain 7TM at the C-terminal.Two chicken GPR133 isoforms were detected in chicken 31 organ tissues,the result showed that two chicken GPR133 isoforms were primarily expressed in most organ tissues.(5)Through inhibition of two chicken GPR133 isoforms in chicken satellite cells,we found that the number of satellite cell proliferation is reduced,and the expression level of proliferative and differentiated marker genes was significantly reduced.(6)We found that overexpressing SH3BP5 gene in chicken satellite cells leads to the decrease of satellite cells.And overexpressing SH3BP5 gene reduces the expression of the cell proliferation marker gene CCNB2 and increases the expression of P21 gene.In addition,the overexpression of SH3BP5 gene inhibited the expression of myogenic differentiated marker genes.The results showed that SH3BP5 gene could inhibit the proliferation and differentiation of chicken satellite cells.This study preliminary analyzed the molecular differences of chicken muscles at different stages of growth and development from the transcriptome level,and constructed the molecular regulatory networks for lncRNAs and mRNAs.Moreover,we preliminary verified the function of differentially expressed genes including the different transcriptional isoforms of GPR133 and SH3BP5.This study enriches the transcriptional genetic information in the embryonic and post-embryonic stages of poultry,and provides genetic resources and molecular theoretical basis for the innate selection of local chicken breeds in China.