Construction Of Fluorescent Protein Fusion Expression Vectors And Screening Of Abs3-1D Suppressor Mutants

Endomembrance system has a very important significance in eukaryotic cells.Many life activities of cells,such as protein synthesis,substance transport,cell life and apoptosis,all rely on the support and regulation of the endometrium system.Proteins are the basis of cellular life activities,and their synthesis,modification,transportation,and decomposition are all inseparable from the intimal system.It can be said that the life course of proteins is accomplished with the help of the endometrium system or the endometrium system.Therefore,to study the function of a certain protein,an important part is to determine the location of this protein in the endometrial system.As a molecular tag,fluorescent protein occupies a very important position in today's molecular biology experiments.The fluorescent protein,refers to a class of proteins that produce fluorescence.The fluorescent protein is fused with the target protein and expressed in the tissue or cells.Observation by laser confocal microscopy reveals that the protein with the fluorescent protein tag is located within the cell or within a certain area of the tissue,thereby more clearly Track the life course of this protein.In this study,bioinformatics analysis of the newly synthesized fluorescent protein,selection of localization gene construction fluorescent protein fusion expression vector and transient expression of the constructed fluorescent protein fusion expression vector,to provide some new ideas for the study of protein function;Screening of abs3-1D suppressor mutants under dark carbon starvation system to further understand the function of ABS3 protein.The main results and conclusions obtained are as follows:1.By comparing the protein sequence of mScarlet with mRFP and mCherry and the physicochemical parameters of EGFP,YFP,m RFP1,tdTomato,mCherry and mScarlet,the fluorescent tags were selected;12 intimal system localization genes were identified through literature search;2.Twelve intimal system localization genes were connected with pUC18HE-NGFP(Yu et al.2004),pUC18HE-NmCherry,and pUC18-mScarlet NT,respectively.Three groups of36 fluorescent protein fusions were obtained.Expression vectors were transiently transformedinto Arabidopsis mesophyll protoplasts to obtain the transient expression of these fluorescent protein fusion expression vectors;3.The ABS induction of the abs3-1D mutant was used to screen the possible abs3-1D suppressor mutants using the dark carbon starvation inducing system in the induced population and the background was purified.In this experiment,three sets of different fluorescent protein fusion expression vectors located in the intima system were constructed to establish a series of tagging tags for the inner membrane system,which facilitated the localization and function research of the intracellular proteins;through the abs3-1D mutants.The EMS chemical induction and the screening of suppressor mutants were obtained,and possible suppressor mutants were obtained.Materials were obtained for further study of the ABS3 gene.