Screening And Function Analysis Of MyoD I Tissue-specific Promoter In Guanling Cattle

The gene of MyoD I?MyoGenic differentiation I?,which is the key positive controlling gene in the growth skeletal muscle and a member of myogenic regulatory factors family,was cloned in 1987,firstly.The gene of MyoD I from cattle is located in the 15th chromosome and the total length of MyoD I is 2648 bp,which has three exons.MyoD I acts as a essential switch in the process of formation of muscle-specific transcriptional regulation and can foster the activation of transcription by combining with the promoter region of myogenin,creatine kinase CK,myosin,desmin and other genes.The promoter is an essential regulatory element and plays a key role in the high level expression of exogenous gene.However,the main issue facing the application of promoter is how to screen the exogenous gene with high efficiency and stable expression and strict time-space specificity to overcome the defect of insufficient specific promoter and lower regulatory ability.Conventional breeding technology has marked great accomplishments in heterosis utilization and cultivation of the excellent species.However,technological difficulties,especially the long cycle of selection and the slow genetic progress,have always existed in breeding new varieties.Transgenic breeding breaks the limitation among different biological species and can directly express exogenous gene in animals,cultivating a new variety in a short time.Transgenic breeding can take advantage of all the possible genetic variation and can greatly enhance the speed and the range of genetic improvement,meanwhile it also can create some new-type products based on the needs of people.Given this,the experiment regards MyoD I gene of Guanling cattle as the research object to study tissue-specific for MyoD I gene as well as activity and function of MyoD I gene promoter by real-time quantitative PCR?qRT-PCR?,vector construction,cell transfection,transgenic mice model construction and others.Main results for this study are as follows:1?The study of differential expression in different tissues and period of MyoD I gene in Guanling cattle.This study detected the expression of MyoD I in longest back muscle,leg muscles,heart,liver,fat and small intestine tissues of Guanling cattle by qRT-PCR.Meanwhile,the expressing rules of longest back muscle in foetal phase,18months and 30 months were also analyzed.The result showed that the MyoD I expressed at high level in longest back muscle and in muscle tissues.And after the birth of cattle,the MyoD I expression in the longest back muscle increased with growing.It is inferred that the high expression of MyoD I may be due to the enrichment of muscle in longest back muscle,resulting in the high the gene expression.2?The aim of this study is to analyze the activity of 4 MyoD I promoters with different lengths in Guanling cattle and determine preliminarily the strong activity gene promoter.The PCR procedures of amplifying 4 MyoD I promoters with different lengths in Guanling cattle were conducted with the DNA template extracted from Guanling cattle blood and the phosphorylated 5'end of the primers.And then the product accurately replaced the CMV region of pEGFP-N3 vector and got eukaryotic expression vector pEGFP-N3-MyoD I.The recombinant plasmid was transiently transfected into mouse C2C12 cells by liposome method.The promoter activity of MyoD I promoter was verified by Luminous intensity of mouse C2C12 cells.Eukaryotic expression vector pEGFP-N3-MyoD I was successfully constructed,and four the activities of the four promoters were all observed.The activity of P₃ promoter is the highest,so P₃ promoter is regarded as the strong activity gene promoter.3?In order to study expression of MyoD I gene promoter in mice,the transgenic mouse embryos glowing green fluorescence was gained by pronuclei microinjection method.It can provide technical support for the building of transgenic mice technology platform.