Arabidopsis Thaliana EFR And Drosophila Melanogaster Arabidopsis SCOT Recombinant Protein Exptession,Puriacation And Crystal Research

The thesis will be introduced from two parts. The first part for arabidopsis EFR recombinant protein expression, purification and crystal growth, the second part of drosophila melanogaster protein SCOT recombinant expression, purification and preliminary crystallographic study and determination of enzyme activity.Pathogenic microorganisms and plant under long-term natural environment in common in the process of evolution gradually evolved some characteristic of the innate immune system, the innate immune system consists of a series of complex recognition mechanism, can let the plant through recognition and perception of exogenous pathogenic molecular molecules to start their own defense response against the invaders. Pathogenic microorganisms and plant long-term in nature were the result of evolution, plants develop different levels of the immune system, the use of pathogen associated molecular patterns mediated immune response (PTI, Pattern-triggered immunity), and the effect of the child to participate in the immune response (ETI, Effector-triggered immunity) against different kinds of pathogenic microorganisms infection. Plants use receptor kinases as EFR and FLS2, distinguish between exogenous bacteria pathogen at the same time start the plant innate immune response, however this immune response is often bacterial inhibition effect protein such as protein, enable exogenous pathogenic microorganisms to multiply and cause all sorts of pathological changes in plant body. Plants against exogenous pathogenic microorganisms, in the long-term process of natural evolution produced resistance genes to identify bacteria protein surface effect and relative resistance.Arabidopsis thaliana by a rich blend of Leucine repeated sequences of the receptor protein kinase (LRR-RLK, Leucine rich repeat receptor-like kinase) FLS2(Flagellin-sensing2) to identify the cell surface of Flagellin pattern recognition receptors (PRR). LRR-RLK are major in plant cell membranes by intracellular serine/threonine protein kinase region, transmembrane region and LRR membrane function of a single transmembrane proteins. Arabidopsis thaliana EFR protein is Tu elongation factor (EF-Tu) receptors and bacterial flagellin, plants depend on the receptor kinase (EFR and FLS2) to pathogen invasion and the immune response. Protein EFR and FLS2is one of the research in arabidopsis plants only known two PRRs. The stretch factor found in large Numbers in bacteria Tu (EF-Tu, Elongation factor Tu) protein can be arabidopsis cruciferae and other members of the identification of PAMP. EF-Tu amino acid sequence contains a deeply conservative elf18sequence, the sequence of N end at the front of the acetylation of18amino acids. Elf18sequence length and EF-Tu complete accord mediated by the immune response.Receptor protein kinase in arabidopsis biological EFR (LRR-RLK,EF-Tu receptor), which is responsible for identifying EF-Tu. It belongs to LRRXII family with FLS2. Experimental studies found in efr mutant cancer Agrobacterium to root (Agrobacterium tumefaciens) and has a weak pathogenicity Pst DC3000sex is stronger, so prove that class protein kinase efr to resist exogenous bacteria play a very important role.Currently in plant NB-LRR protein identification of pathogenic microorganisms effect of activation of downstream immune response pathways are still not clear. So we are, construct the arabidopsis EFR (675-1031)-His-TEV-pET28a/EFR (681-1019)-His-TEV-pET28a/EFR (695-1002)-His-TEV-expression plasmid pET28a reorganization。Test expression analysis showed that in addition to EFR (695-1002)-His-TEV-pET28a protein is not expressed, the other two subclones were able to express proteins. By Ni-NTA affinity chromatography purified and found EFR (675-1031)-His-TEV-pET28a/EFR (681-1019)-His-TEV-pET28a expressed target protein soluble. EFR (675-1031)-His-TEV-pET28a and EFR (681-1019)-His-TEV-pET28a expressed proteins by Ni-NTA the purpose of the affinity chromatography and superdex200gel filtration chromatography, get high purity stable uniform protein for protein crystal growth. By sitting drop weather diffusion method, USES the Hampton Research Crystal Screen Ⅰ, Crystal Screen, Salt Ⅱ RxTM1, Salt RxTM2, PEG RxTM1, PEG RxTM2, Emerald Biosystems Wizard, the Wizard Ⅰ and Ⅱ Index, the Index Ⅱ protein Crystal growth Ⅰ kit for protein Crystal filter, after a preliminary screening of EFR (681-1019)-His-TEV-pET28a crystals found in two Crystal growth conditions, repetition, optimization and sds-page detection found two crystals are salty.Amber acyl coenzyme A transferase (SCOT) produced acetyl acetate and amber acyl coenzyme A catalyzed by succinic acid and acetyl acetylcoa. Acetyl acetyl-coa sulfur under the action of enzymes in mitochondria produce into two molecules of acetyl coenzyme A, enters the citric acid cycle.Succinyl CoA transferase (SCOT) on the catalytic succinyl CoA group transferred to acetyl acetate. Acetyl acetate is one of the compounds of ketone bodies. Ketone body produced by the liver, and through the bloodstream to extrahepatic tissues, such as heart and brain, to be used as a kind of energy metabolism of organisms. In the extrahepatic tissue mitochondria, SCOT catalytic reaction to generate acetyl CoA, acetyl acetyl CoA by sulfur solution enzyme into acetyl CoA. Acetyl CoA into the tricarboxylic acid cycle provides energy for metabolism of organisms. Organism SCOT missing cause ketoacidosis.Protein SCOT after combining with the CoA form an intermediate products to the catalytic reaction transition state. By William Jencks to the pig heart protein SCOT of experimental research, found that succinyl CoA on the CoA of ligand and protein SCOT on the305loci on the glutamic acid residue, form an anhydride intermediates. However, the related CoA transferase in drosophila melanogaster research rarely. Extracted so we build the CoA in the drosophila transferase prokaryotic expression plasmid SCOT-His-TEV-pET28a, recombinant plasmid protein induced by IPTG expresses the purpose, target protein through the Ni-NTA superdex200affinity chromatography and gel filtration chromatography, such as proteins purification methods gained more than95%purity and homogeneity better target protein. Through sat drops of atmosphere diffusion method, using the Hampton Research Crystal Screen Ⅰ, Crystal Screen, Salt Ⅱ RxTM1, Salt RxTM2, PEG RxTM1, PEG RxTM2, Emerald Biosystems the Wizard Ⅰ and Ⅱ Index Ⅰ and Index Ⅱ kit for protein Crystal growth, Crystal filter has get and parse out the space protein Crystal structure. By spectrophotometric method at UV310nm measured drosophila melanogaster SCOT under the activity of protease.