Research On Expression Correlation Analysis Between Sheep YAP1and Other Genes Related With Muscle Growth And The Construction Of YAP1Eukaryotic Expression Vector

In this experiment, Hu sheep was researched as the object, gene cloning method was used to clone full-length cDNA sequence of sheep YAP1and biological analysis software was used to analyze the YAP1sequence homology between sheep and other species, physicochemical properties, secondary and tertiary structure; RT-PCR、vector construction and site-directed mutagenesis methods were used to construct pEGFP-C1-YAP1and pEGFP-C1-YAP1S42A eukaryotic expression vector; RTFQ-PCR method was used to analyze YAP1relative expression between different genders, growth stages and skeletal muscles (longissimus dorsi muscle, soleus muscle, gastrocnemius muscle, extensor digitorum longus). The purpose is to reveal YAP1regulatory mechanism of sheep muscle growth process in embryonic period and postnatal period. The results were followed as these:1. The full-length of YAP1gene was1712bp, including1212bp coding sequence encoding403amino acids. The homology analysis found that the nucleotide sequence and amino acid sequence of sheep YAP1gene share the highest78.77%and92.51%identity with the Cricetulus YAP1while there was no large difference between Human and Chimpanzee. The amino acid sequence analysis revealed that the YAP1gene of sheep encoded water-soluble protein and its relative molecular weight was44079.0Da, isoelectric point was4.91, was a kind of hydrophilic non-transmembrane protein. Subcellular localization of YAP1was in the mucleus, and it did not belong to the secreted protein. The YAP1protein contained33phosphorylation sites,7glycosylation sites and2WW domain. The secondary structure of YAP1was mainly composed of random coil. The tertiary structure of domain area of YAP1protein showed a forniciform helix structure. Forecast YAP1had a key role in regulatory functions process.2. YAP1expression had significant differences in different skeletal muscles, which expressed greatest in gastrocnemius muscle, and lowest in longissimus dorsi muscle; the comparison in2-day-old,2-month-old and6-month-old, YAP1expressed significant much greater with the growth stages. YAP1expressed no significant difference in ram and ewe. The results of the correlation in YAP1,MyHC Ⅰ, ⅡA, ⅡX,MSTN,MyoG expression showed that YAP1was significantly negative related with MyHC Ⅰ and significantly positive related with MyHC Ⅱ X,MSTN,MyoG. It revealed that YAP1expression had a relevance with the muscle fiber development after birth, and YAP1may involved in MyHCs transformed, furthermore,it maybe a candidate gene for muscle growth regulation.3. It was confirmed that the sheep YAP1CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine by PCR, restriction digestion and sequencing. This study settled a experimental basis for exploring YAP1regulation mechanism in the process of myoblasts proliferation and differentiaton.