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Ophiobolin compound mother nucleus synthetic gene AuOS and application thereof

A kind of pseudochicin and synthetic gene technology, applied in the field of genetic engineering

Active Publication Date: 2015-06-03
WUHAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are only reports of pseudochicin F synthase from Aspergillus clavus NRRL 1 strain

Method used

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  • Ophiobolin compound mother nucleus synthetic gene AuOS and application thereof
  • Ophiobolin compound mother nucleus synthetic gene AuOS and application thereof
  • Ophiobolin compound mother nucleus synthetic gene AuOS and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 AuOS gene cloning

[0031] 1. Extraction of RNA from Aspergillus pylorus strain 094102

[0032] Using the mycelium of Aspergillus pyogenes strain 094102 as the material, the total RNA was extracted with the RNeasy Mini Kit (50) kit (article number 74104) from QIAGEN, Germany, and the specific operation was as follows:

[0033] (1) Estimate the amount of mycelium samples. Typically 50 mg mycelia are required for each microextraction.

[0034] (2) Grind the sample into powder with liquid nitrogen and transfer it to a 1.5 mL plastic centrifuge tube.

[0035] (3) Add 450 μL of Buffer RLT (Lysis buffer) to the centrifuge tube (add 10 μL of β-mercaptoethanol to every 1 mL of Buffer RLT before use), shake on the shaker for 1-3 minutes to mix well (must Shake the solution at the bottom of the tube).

[0036] (4) Centrifuge at 13,000×g for 2 min at 4°C, and transfer the supernatant to another clean 1.5 mL plastic centrifuge tube (the lower organic phase and the m...

Embodiment 2

[0086] Example 2 Expression and purification of AuOS protein

[0087] 1. Contain AuOS Construction of gene expression vector in Escherichia coli

[0088] Build contains AuOS The Escherichia coli expression vector of the gene was transformed into Escherichia coli BL21 (DE3) by the heat shock transformation method for further verification AuOS The function of the gene.

[0089] restriction endonuclease Nde I and Hind Ⅲ From the recombinant plasmid containing the target gene pEASY -Blunt- AuOS Cut out the target fragment, and at the same time use Nde I and Hind Ⅲ Digest the pET28a vector with double enzymes, and after separation by agarose gel electrophoresis, use the OMEGA gel recovery kit to recover the target gene fragment and the large fragment of the pET28a vector respectively. Using T4 DNA ligase to convert AuOS The gene was inserted forward into the pET28a vector, and the ligation product was transformed into Escherichia coli TOP 10 competent cells. A...

Embodiment 3

[0104] Example 3 Functional analysis of AuOS protein

[0105] In order to determine the function of AuOS protein, DMAPP (dimethylallyl pyrophosphate), GPP (geranyl pyrophosphate), FPP (farnesyl pyrophosphate) and GGPP (geranyl Leaf base pyrophosphate) was used as the substrate, and IPP (isopentyl pyrophosphate) was added to design in vitro reactions. Reaction system (50 μL): 220 μM DMAPP (or GPP, FPP, GGPP), 340 μM IPP, 0.1 M Tris-HCl (pH 7.4), 2 mM DTT (dithiothreitol), 5 mM MgCl 2 React with 9.4 μM AuOS at 30 °C for 3 h. After the reaction was completed, it was extracted three times with an equal volume of ethyl acetate, and the organic solvent was blown dry with a nitrogen blower, and 50 μL of ethyl acetate was dissolved for GC-MS (gas chromatography-mass spectrometry) detection. The test results are as follows:

[0106] (1) Using DMAPP and IPP as substrates to carry out in vitro reaction according to the above reaction system, the reaction product was treated by the abo...

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Abstract

The invention discloses an ophiobolin compound mother nucleus synthetic gene AuOS and an application thereof and belongs to the field of gene engineering. The ophiobolin compound mother nucleus synthetic gene AuOS is derived from aspergillus ustus 094102, wherein the gene sequence, the cDNA sequence and the amino acid sequence of coded AuOS protein are respectively shown in SEQ ID NO.1-3. The AuOS protein has the functions of catalyzing the substrate chain length extension and the structure cyclization, and the protein can be reacted with the IPP to generate the ophiobolin compound mother nucleus by taking the DMAPP, GPP, FPP and GGPP as substrates. The invention first discovers the AuOS gene of the aspergillus ustus 094102, wherein the coded gene has the functions of catalyzing the substrate chain length extension and the structure cyclization, the ophiobolin compound mother nucleus can be synthesized by taking the DMAPP as a substrate, and the ophiobolin compound mother nucleus can be used for preparing the ophiobolin compound and researching the biosynthetic pathway.

Description

technical field [0001] The present invention relates to the field of genetic engineering, in particular to a Aspergillus ustus 094102) cloned ophiobolin compound mother nucleus synthesis gene AuOS and its application. Background technique [0002] 1957 Orsenigo from phytopathogenic fungi Bipolaris oryzae The first pseudochicin compound was isolated from M. ORSENIGO. Estrazione e purificazione della cochliobolina, una tossina prodotta da Helmintosporium oryzae . Phytopath. Z. 1957, 29, 189–196), more than 30 species of this type of compound have been discovered so far, and the source strains are all filamentous fungi. [0003] Ophiobolin compounds belong to sesquiterpene compounds, and their structural characteristics are that they all have a three-wheel sequence of C5-C8-C5 ring core structure ( figure 1 ). The so far published pseudochicin compounds with C5-C8-C5 ring core structure mainly include pseudochicin A, C, F, H, M, L (M. ORSENIGO. Estrazione e purificaz...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N1/21C12P1/02C12R1/70
CPCC07K14/38C12N9/1085C12N9/10C12P7/02
Inventor 洪葵孟会影柴沆镇殷如邓子新
Owner WUHAN UNIV
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