Isolation, Location Of Pig Muscle Segment Homeobox Gene MSX1 And The Identification Of Its Effective SiRNA

Pork is the major source of animal protein in human diet, and the research onporcine growth and meat quality traits has been the emphasis of animal breeding andgenetics researchers. The application of modern molecular biology, molecular geneticsand quantitative genetics and other techniques to research on molecular mechanisms ofpig skeletal muscle growth and development, leads to better understanding of animalmuscle growth and development pattterns, thereby promotes animal genetic improvementof meat production traits.MSX1 (muscle segment homeobox homolog 1), which is a nuclear transcriptioninhibitor, binds to the TATA box and/or other factors to form a nuclear transcriptioncomplex, is one of the main regulatory genes invoved in the organ development. Study inmodel organisms such as mouse showed that MSX1 coordinated with Myogenicregulatory factors MyoD,Pax3 and so on, regulates differentiation of skeletal muscle cell.In this study, MSX1 is selected as a candidate gene for skeletal muscle growth anddevelopment reearch. And the study in cDNA cloning, chromosome assignment,spatio-temporal expression profile anslysis, subcellular location and identification ofeffective siRNA combining bioinformatics methods and molecular biology techniques,lays a foundation for further research of the gene on porcine skeletal muscle cellproliferation and differentiation. The results obtained are as follows:1). The human MSX1 cDNA was used to search for homologous pig expressed sequencetags(ESTs) in the NCBI 'EST-other' database, then assembled them into EST contig.Primers were designed based on the contig information and to clone the complete MSX1gene CDS sequence and 3'-untranslated region (3'-UTR) containing AATAAA sequence.2). Radiation hybrid (RH) and somatic cell hybrid (SCHP) were used for chromosomeregion and precise location of porcine MSX1 gene. The results showed that MSX1 waslocated to SSC8 (1/2 p21)-p23, closely linked to Microsatellite marker S0353(LOD=10.22), then linked to Microsatellite marker SW2410 and S0098.3). MSX1 gene tissue distribution was detected by the semi-quantitative RT-PCR in sevendifferent tissues from a Tongcheng pig heart, liver, spleen, lung, kidney, skeletal muscleand adipose. The results showed that this gene expressed extensively in the detectedtissues, and highest in spleen, then in heart and skeletal muscle. 4). MSX1 gene temporal epression was detected in Tongcheng fetus skeletal muscle at 33days, 65 days, 90 days gestation, 2 and 28 days and 33 days, 65 days, 90 days gestation ofLandrace pig, the results showed that the gene expression was decreased gradually in theembryonic stage, following increased in two days after birth and then decreased.5). The pig MSX1 and GFP fusion protein expression vector was constructed, andtransfected into IB cells. The fusion protein distribution was detected using the lasercofocal scanning microscropy, the result showed that fusion protein were mainlyexpressed in nucleus.6). ShRNA expression vectors and Stealth siRNA of chemical synthesis were used toscreen effective siRNA sequences which can target porcine MSX1 gene, two effectivesequence were obtained.