Method for determining Guanling cattle MyoDI gene core promoter by using dual-luciferase report gene

Abstract

The invention discloses a method for determining a Guanling cattle MyoDI gene core promoter by using a dual-luciferase report gene. The method comprises the following steps: constructing a recombinant vector containing a Guanling cattle MyoDI gene promoter full-length fragment and a subcloning fragment; culturing skeletal muscle growth-related cells and planking; preparing a promoter recon / liposome mixture, and carrying out transfection on the cells obtained in the step (2); carrying out dual-luciferase activity detection on the transfection cells obtained in the step (3) by using the dual-luciferase report gene. The method has the advantages of high sensitivity, fastness in detection speed and low cost and does not need radioactive isotopes and the like; as a used pGL3-Basic report gene does not comprise the promoter and an enhancer, the expression of the luciferase report gene can be used for detecting the starting activity of inserted promoters and comparing the intensity of the promoters, and further a core promoter region is preliminarily determined.

Description

technical field [0001] The invention relates to the field of molecular genetics, in particular to a method of using dual luciferase reporter genes to determine the MyoDI Methods for gene core promoters. Background technique [0002] MyoDI Its full name is myogenic differentiation Ⅰ, and its Chinese name is myogenic determinant. It is an important positive regulatory gene in the process of skeletal muscle growth and development. In the regulation of myogenic muscle-specific gene transcription, MyoDI Acting as a master switch, it can bind to the promoter regions of myogenin, creatine kinase CK, myosin, desmin and other genes to play an important regulatory role and promote their transcriptional activation. MyoDI As the best model to study cell differentiation, the mediated muscle differentiation mechanism has become the focus of many scholars' research and is particularly important. At present, there are many related studies on the expression and regulation of this gene in ...

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Problems solved by technology

At present, there are many related studies on the expression and regulation of this gene in the myocyte formation mechanism of mice, chickens and pigs at home and abroad. There have been some studies on cattle, including genetic variation and function studies, but mainly focused on post-transcriptional and translational levels, MyoDI The molecular mechanism of gene transcription regulation is still unclear

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