Effects Of Drosophila TSMR1 On Promoter Activity Of BMP Signal Members

Cardiovascular disease has become one of the most common malignant diseases.The research shows that the vertebrate heart development is a complex process regulated by gene specific,accurate expression of numerous transcription factors involved in the regulation,so to find a cure for the disease by studying the molecular mechanism of heart development has become urgent to scientists worldwide problem solving.In recent years,BMP and its Drosophila homolog Dpp have been shown to play an important role in the regulation of a number of cardiac transcription factors such as Tinman and cardiac differentiation.Lack of Dpp in Drosophila embryos caused lack of heart tissue and back muscle precursor cells,and failed to form the heart mesodermsuggesting that BMP signal pathway plays an important role in the process of the development of heart and body wall muscle.This paper aims to investigate whether the Drosophila TSMR1 gene has an effect on the transcriptional activity of the promoter of the BMP signal members by using the two luciferase reporter system in the molecular level.BMP signaling pathway members include gene Dpp,type I receptor gene Tkv,and the specific receptor gene Mad in the Smad family of BMP signaling pathway,Med and suppressor gene Dad.Themain content of the research is using molecular cloning method to construct luciferase reporter vector,detecting the gene TSMR1 regulation activity to BMP signal members' promoters and determing which section of these promoters having roles in regulation.The main results are as follows:1.Firstly,we cloned 3000 bp promoter region upstream of the start codon ATG of Dpp,Tkv,Med,Dad and Mad genes to the luciferase reporter gene plasmid vector p Gl3-basic by one-step cloning connecting method,and successfully constructed the recombinant report plasmids including p Gl3-Dpp-luc,p Gl3-Tkv-luc,p Gl3-Med-luc,p Gl3-Dad-luc and p Gl3-Mad-luc.At the same time,the recombinant expression plasmid TSMR1 with myc tag p CMV-myc-TSMR1 was constructed.2.Through the analysis of double luciferase reporter gene activity,it was found that the Drosophila TSMR1 gene can significantly enhance the transcription activity of Dpp,Med,Tkv and Dad genes,while inhibit the transcriptional activity of Mad gene.3.In order to analyse which section of these BMP members' promoters having roles,each 3 genes 5 'promoter region of Dpp,Dad,Med was divided into 6 sections and was amplified by PCR,and different truncated length detaching promoter luciferase reporter gene plasmid was successfully constructed by one step of cloning method.4.Dual luciferase reporter system detection analysis showed that,TSMR1 controlinging core promoter region of Dpp is located in-493bp-33 bp,Med core promoter is located in-396 bp +105bp,Dad core promoter region is located in-1299bp-762 bp,suggesting TSMR1 gene may be mainly combined to these core promoter regions to activate the transcriptional activity of Dpp,Dad,and Med.