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Identification and analysis of gene cloning and enzymatic function of terpenoid metabolic pathways of MEP DXS gene

Author: YangYanPing
Tutor: GaoWenYun
School: Northwestern University
Course: Biochemistry and Molecular Biology
Keywords: MEP pathway DXS Arabidopsis thaliana Pseudomonas Aeruginosa Triosephosphate isomerization
CLC: Q78
Type: Master's thesis
Year: 2012
Downloads: 152
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Abstract


Terpenoid is a class of secondary metabolites which is widespread in nature with a variety of structural variation. They play not only important biological roles in all forms of life, but has been widely used in industry, medicines and healthcare products. There are two biosynthetic pathways for all natural terpenoids. one is the classical mevalonate (MVA) pathway, and the other is the recently established methylerythritol phosphate (MEP) pathway.1-Deoxy-D-xylulose5-phosphate synthase (DXS) is one of the key enzymes in the MEP biosynthetic pathway, also a rate-limiting enzyme of the pathway. It is generally accepted that only D-glyceraldehyde3-phosphate (D-GAP) can be utilized as natural substrate by DXS to synthesize DXP in the presence of pyruvate and thiamine pyrophosphate (TPP).Studies of this research group have shown that DXS from Escherichia coli and Rhodobacter capsulatus can also take dihydroxyacetone phosphate (DHAP), the other triosephosphate and the isomer of D-GAP as its substrate to biosynthesize DXP. Furthermore, the bacteria DXS possesses triosephosphate isomerization activity. In this thesis, we explored whether DXS from different sources, e.g. from higher plants also possesses the same triosephosphate isomerization activity.We first prepared Pseudomonas aeruginosa DXS (pao-DXS) by using E. coli BL21(DE3)-paodxs cells and then used affinity chromatography and gel to purify the product. The function of DXS was subsequently tested. As a result, we obtained DXS with good purity and high activity on the synthesis of DXP. Meanwhile, The enzyme also showed triosephosphate isomerization activity.On the basis of the above work, we tried to prepare DXS from Arabidopsis thaliana. RNA was reverse cDNA extracted from seedlings of A. thaliana. The cDNA as a template, using RT-PCR method, primers were designed based on A. thaliana dxs gene coding sequence, The A. thaliana segmented dxs gene, divided into qdxs, and hdxs were cloned, then the cloning vector by double digestion, using the ligase, the natural connection of the target fragment in the cloning vector, successfully obtained A. thaliana encoding DXS gene dxs, we constructed the prokaryotic cloning vector of the pMD(?)18-T-Atdxs and prokaryotic .expression vector pET-15b-Atdxs.Explores the A.thaliana prokaryotic expression vectoL PET-15b-Atdxs was transformed into the prokaryotic expression strain BL21(DE3) to establish the optimization of expression conditions.And the recombinant enzyme At-DXS functional studies are in progress.

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