Study Of The Fuction Of Lats2 Gene In Hippo Signal Pathway In Skeletal Muscle Growth And Development In Hu Sheep

According to Lats2 gene sequence of the Human being and mouse published in GenBank, we designed primers on the conserved domains and used RT-PCR technique to amplify the CDS sequence of Lats2 gene in muscle of Hu sheep. The bio informatics of CDS sequence of Hu sheep Lats2 gene was analyzed to find its overall similarity with other animals and forecast its protein structure and physicochemical property. Vector construction methods and cell transfection techniques were used to construct pEGFP-C1-Lats2 eukaryotic expression vector and transfect skeleton satellite cells, in order to explore the change of the skeleton satellite cells after the over-expression of lats2 gene. RT-PCR method was used to analyze Lats2 gene relative expression between different growth stages, genders and skeletal muscles The purpose is to reveal Lats2 regulatory mechanism of Hu sheep muscle growth process. The results were followed as these:1. The sequence of CDS of the Hu sheep Lats2 gene was 3048bp including ATG start codon and TGA stop codon. The cloned Hu sheep Lats2 gene CDS coding sequence had an overall similarity with a comparable region of wild boar(82.2%), Human being(78%), house mouse(75.3%), brown Rat(74.9%), xenopus(58.9%), zebrafish(36.1%), the amino acid sequence of Lats2 protein of the Hu sheep was 80%、75.6%、72.9%、73.3%、56.3%、61.0% accordingly. The sequence of CDS of the Hu sheep Lats2 gene encoded 1123 amino acids, unstable and water-soluble protein and its relative molecular weight was 126332.1 Da with no potential splice site of signal peptide. The Lats2 protein had 2 strong transmembrane helical structure from the inside-out,6 strong transmembrane helical structure from the out-inside contained 45 phosphorylation sites,4 glycosylation sites, one S-TKc domain and one S-TK-X domain. The subcellular localization of Lats2 protein was in the mucleus.2. pEGFP-C1-Lats2 eukaryotic expression vector was constructed successfully. The eukaryotic expression vector of Lats 2 and Lats 1 gene was transfected into skeleton satellite cells and expressed in the cell successfully. After the over-expression of Lats-2 gene, the relative expression of Lats1、MSTN、MyoG gene incresed. THus, the relative expression of YAP1、 MyHC Ⅰ、MyHC ⅡA、MyHCⅡX gene decresed and there was no significant difference (P> 0.05). After the over-expression of Lats-1 gene, the relative expression of Lats2 gene incresed and the relative expression of YAP1、MSYN、MyoG、MyHCⅠ、MyHC ⅡA and MyHC ⅡX gene decresed, but there was no significant difference (P>0.05)3. In different muscle tissue expression analysis, Lats2 gene had the highest relative expression in the digitorum longus extensor muscle; In different growth stages expression analysis, Lats2 gene had the lowest relative expression in 2-day-old,and lower in 2-month-old and 6-month-old; In different genders expression analysis,the relative expression of rams were higher than ewes in 2-day-old. The results of the correlation in Lats2, YAP1、MSTN、MyoG、 MyHC Ⅰ、MyHC ⅡA and MyHC ⅡXexpression showed that Lats2 expression had a relevance with the muscle fiber development.