Regulation Of Porcine Muscle And Fat Growth And Differentiation By LMNA Gene

Pork is always the most important consumption of meat products in China,the lean-type pig is unable to meet the requirements of the pork flavor as the improvement of people’s living standard, so researchers transform the focus of scientific research into improving pork quality and taste in recent years.Finding out the genes related to the development of muscle or fat through the study of major genes,QTL,SNP,MAS on the excellent traits of pigs.Researchs show LMNA gene affect the development and formation of muscle and fat.LMNA,also called Lamin A/C,is a nuclear lamin,binds to the inner nuclear membrane,participates the formation of the nuclear envelope,maintains the morphology of nucleus,regulates the import and export of the nuclear pore channels,influences the activations and functions of related transcription factors and cell signaling pathways.LMNA gene mutations cause genetic diseases(Laminopathies) associated with growth,regeneration and generation of muscle and fat in human.LMNA mutant and knockout mice cause similar muscle or fat developmental disorders.So we take LMNA gene as our resreach target,construct the pc DNA3.1-s LMNA overexpression vector,synthetise the interferential si RNA of LMNA.In order to study the effects of LMNA gene on muscle and fat development and production,C2C12 and 3T3-L1 were applied to the experiments of overexpression,interference and differentiation of LMNA gene.The main research achievements are as follows.1.We utilize porcine c DNA as a template,the LMNA gene sequences was inserted into p MD18-T vector,using p MD18-T-s LMNA as a template,Cutting the target fragment and pc DNA3.1 vector with the restriction enzymes Nhe I and Kpn I to construct the recombinant vector of pc DNA3.1-s LMNA.Verifications of pc DNA3.1-s LMNA were carryed on by sequencing,PCR and restriction enzyme digestion.The pc DNA3.1-s LMNA was transfected into C2C12 and 3T3-L1 respectively and stable expression in cells.2.The constructed pc DNA3.1-s LMNA and synthetised si RNA were transfected into C2C12 cell, moreover C2C12 was differentiated from 0 day to 6 days after transfections,cells were collected to extract RNA and protein for quantitative PCR and Western Blot experiments.The results showed that LMNA overexpression caused muscle related genes down-regulated,LMNA interference caused muscle related genes up-regulated,Interfering the expression of LMNA inhibited C2C12 cell differentiation.3.The constructed pc DNA3.1-s LMNA and synthetised si RNA were transfected into 3T3-L1 cell,3T3-L1 was differentiated from 0 day to 6 days after transfections,cells were collected to extract RNA and protein for quantitative PCR and Western Blot experiments.The results showed that LMNA overexpression caused fat related genes down-regulated,LMNA interference caused fat related genes up-regulated,Interfering the expression of LMNA inhibited 3T3-L1 cell differentiation.4.Overexpression of LMNA in C2C12 and 3T3-L1 differentiation experiments,myogenesis and lipogenesis related genes up-regulated differently in the second day of differentiation,then stable or down-regulated in following days.These implicate LMNA may promote myoblast and preadipocyte differentiation at the early stage.