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The Expression, Purification and Primer Structure Analysis of Protein Eliciter PebC1

Author: LiTang
Tutor: LiuZuo
School: Chinese Academy of Agricultural Sciences
Course: Biochemistry and Molecular Biology
Keywords: PebC1 protein NAC protein family Protein crystallography X-ray diffraction Small-angle scattering Homology modeling
CLC: Q78
Type: Master's thesis
Year: 2010
Downloads: 54
Quote: 0
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Abstract


Elicitor protein is able to induce plant resistance proteins from fungi in a class, with the promotion of plant metabolism and growth, and enhance the activity of plant disease resistance and resilience. PebC1 isolated by our laboratory from gray mold (Botrvtis cinerea) protein stimulate sub (Zhang et al, 2010), in order to further clarify the mechanism of PebC1 functionality, gray mold the PebC1 protein and its interaction βNAC The proteins were with prokaryotic and eukaryotic expression systems for the expression, and further of the PebC1 bcβNAC composite protein; the expressed protein was purified on this basis, right PebC1 protein Preliminary structural analysis. The main results are as follows: 1, informatics the PebC1 protein sequence analysis, speculated that the NAC (Nascent polypeptide Associated Complex) protein family αNAC homologous proteins. Genome by querying the database, get Botrytis cinerea the encoding gene egd1 βNAC (bcβNAC) gene sequence for depth structure study PebC1 protein to lay the foundation. Constructed PebC1 and bcβNAC prokaryotic and eukaryotic expression systems, protein expression showed that PebC1 for prokaryotic systems, and bcβNAC protein expression. The pebC1 Genes and egd1 of gene cloned into the prokaryotic expression vector pET-30 His and pET-30 His MBP were prokaryotic expression; while pebC1 Genes and egd1 of genes were cloned into the carrier pFastBac His GST recombinant baculovirus and eukaryotic expression. 3, successfully expressed the composite PebC1 and bcβNAC protein. Prokaryotic expression system pebC1 Genes and egd1 of gene cloning to two polyclonal locus pETDuet-1 dual expression plasmid, the review body protein and complex protein overexpression; eukaryotic expression system , the use of two recombinant viruses with pebC1 the gene and egd1 gene also infected Hi5 cells for eukaryotic expression, but because PebC1 be degraded, can not express the complex protein. 4 to complete the exploration of the PebC1, bcβNAC complex protein purification conditions. Using affinity chromatography, ion exchange chromatography and molecular sieve chromatography, the three-step method for purification, and explore the purification conditions, and to obtain a high purity of the target protein, to lay the groundwork for the subsequent experiments. 5, by small-angle scattering experiments get the lattice structure PebC1 and bcβNAC model. The purified PebC1 bcβNAC protein in small-angle scattering experiments, collected scattering signal PebC1 with MBP tag bcβNAC in the small angle region, the processing of the experimental data obtained lattice model of the protein. Homology modeling structure analysis combined PebC1 the NAC and UBA domain (Ubiquitin Associated domain), that PebC1 was S-shaped symmetrical distribution NAC dimer formation depression β-barrel structure UBA domain hanging at the side of the . cylindrical model of the mbp-bcβNAC a dot can be roughly divided into two parts, Part 2 was a diameter of 50?; Part 1 is a thickness of 30? flat, consistent with the spatial structure with the MBP protein, thereby suggesting that MBP is located in Part 1. bcβNAC fine structure needed further determined experimentally. 6, the initial crystallization conditions of the filters to obtain a composite body protein. Sitting drop vapor diffusion method, the screening of complex protein crystallization conditions at 20 ℃, and twins Hampton Research Crystal Screen II # 19 and MolecularDimensions MD1-02 34 # conditions for eventually get high quality single crystal X-ray diffraction analysis of three-dimensional structure laid an important foundation.

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