| According to Latsl gene sequence of the human being and cattle published in GenBank,we designed primers on the conserved domains and used RT-PCR technique to amplify the CDS sequence of Latsl gene in muscle of hu sheep.The CDS sequence of hu sheep Latsl gene was analyzed by using the DNAMAN software and online tool.Using the PCR-SSCP to detect the single nucleotide polymorphisms (SNP) of Latsl gene and study the relationship between the gene and the muscle growth traits of hu sheep;RTFQ-PCR method was used to analyze Latsl relative expression between different growth stages, genders and skeletal muscles (longissimus dorsi muscle,soleus muscle,gastrocnemius muscle,extensor digitorum longus);RT-PCR and vector construction methods were used to construct pEGFP-Cl-Latsl eukaryotic expression vector. The purpose is to reveal Latsl regulatory mechanism of hu sheep muscle growth process.The results were followed as these:1.The sequence of CDS of the hu sheep Latsl gene was3372bp encoded1123amino acids including ATG start codon and TGA stop codon.The Latsl gene of hu sheep encoded unstable and water-soluble protein and its relative molecular weight was126332.1Da.The cloned hu sheep Latsl gene CDS coding sequence had an overall similarity with a comparable region of cattle(98.58%), human being (92.27%), house mouse (86.68%),norway rat (86.63%), dog (70.91%), xenopus (69.39%), zebrafish(58.79%), the amino acid sequence of Latsl protein of the hu sheep was over83%with cattle, human being, house mouse, norway rat, dog, xenopus, zebrafish.The Latsl protein had a strong transmembrane helical structure from the inside-out and from the outside-in,contained87phosphorylation sites,,11glycosylation sites,one UBA domain,one S-TKc domain and one S-TK-X domain.The subcellular localization of Latsl protein was in the mucleus.The secondary structure of Latsl protein was mainly composed of random coil.Forecast Latsl had a key role in replication and transcription process.2.We designed eight pairs of primers in eight exons of Latsl gene to analysis its polymorphisms and its relationship with muscle growth traits in hu sheep.we found the exon4of Latsl gene had one point mutation (Câ†'T) at73bp,and there were two genotypes AA andAB. The average weight of birth weight, weaning weight and6-month weight with genotype AA was higher than that with genotyoe AB in the exon4of Latsl gene, but the difference was not significant.3.In different muscle tissue expression analysis,Latsl gene had the highest relative expression in the gastrocnemius muscle,and lowest in longissimus dorsi muscle; In different growth stages (2-day-old,2-month-old and6-month-old) expression analysis,Latsl gene had the highest relative expression in2-day-old,and lower in2-month-old and6-month-old; In different genders expression analysis,the relative expression of rams were higher than ewes in2-day-old.There were no significant differences between rams and ewes in different muscles in2-month-old.The relative expression of ewes were higher than rams in6-month-old. The results of the correlation in Latsl,YAP1,MyHC â… ,MyHC II A,MyHC â…¡X,MSTN,MyoG expression showed that Latsl expression had a relevance with the muscle fiber development, it maybe a candidate gene for muscle growth regulation.4.1t was confirmed that the hu sheep Latsl CDS was correctly inserted into eukaryotic expression vector by PCR,restriction digestion and sequencing. This study settled a experimental basis for exploring Latsl regulation mechanism in the process of myoblasts proliferation and differentiaton. |
PDF Full Text Request