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Method of blocking gene clusters of Saccharopolyspora pogona based on homologous recombination of linear fragments

A homologous recombination technology of Saccharopolyspora spp., applied in the field of gene cluster blocking of S. spp., can solve problems such as retention and difficulty in exogenous plasmid transformation, and achieve the effect of increasing production

Active Publication Date: 2019-08-16
HUNAN NORMAL UNIVERSITY
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  • Description
  • Claims
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Problems solved by technology

[0006] At present, the genome editing work of Saccharopolyspora mentabilis mainly stays at the single gene level, and there are no reports on the blockage of large fragments of gene clusters, and the transformation of exogenous plasmids is also relatively difficult

Method used

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  • Method of blocking gene clusters of Saccharopolyspora pogona based on homologous recombination of linear fragments
  • Method of blocking gene clusters of Saccharopolyspora pogona based on homologous recombination of linear fragments
  • Method of blocking gene clusters of Saccharopolyspora pogona based on homologous recombination of linear fragments

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Embodiment 1

[0041] (1) Specific medium formula and culture conditions

[0042] (1) S. pogona seed activation medium CSM (Complete synthetic medium): TrypticSoyBroth4.5g / L, Glucose 0.9g / L, Yeast extract 0.3g / L, MgSO 4 .7H 2 O 0.22g / L. Inoculate the monoclonal or 2% strain preservation solution into a 150 mL shake flask equipped with 20 mL LCSM, and incubate at 30°C and 280r / min for 48 h;

[0043] (2) S. pogona synthetic fermentation medium (Synthetic fermentation medium, SFM): Glucose20.0 g / L, Yeast extract 4.0 g / L, Tryptone 4.0 g / L, KNO 3 1.0 g / L, K 2 HPO 4 •3H 2 O 0.5g / L, MgSO 4 •7H 2 O 0.5 g / L, FeSO 4 •7H 2 O 0.01 g / L. Inoculate 2% strain activation solution into a 300mL shake flask filled with 50mL SFM, and culture at 30°C and 280r / min for 12 days.

[0044] (3) R5 medium was used for protoplast transformation: sucrose 103 g / L, glucose 10 g / L, casamino acids 0.1 g / L, Yeast extract 5 g / L, K 2 SO 4 0.25 g / L, MgCl 2 •6H 2 O 10.12g / L, TES 5.73 g / L, trace element solution 2 ...

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Abstract

A method of blocking gene clusters of Saccharopolyspora pogona based on homologous recombination of linear fragments comprises the steps of (1) finding, from genome of Saccharopolyspora pogona, ura4 acting as a negative screening marker gene; (2) designing a sequence having the size of 1 kb at each end of the gene ura4 to act as homologous arms to carry out ura4 gene replacement; (3) fusing upstream and downstream homologous arms of the gene ura4 to an apramycin-resistant gene by means of fusion PCR (polymerase chain reaction), bonding with plasmid pOJ260 to obtain recombinant plasmid pOJ260-ura4; (4) transferring a protoplast containing the recombinant plasmid pOJ260-ura4 to Saccharopolyspora pogona, knocking out the gene ura4 to obtain a ura4-deleted strain S. pogona-delta ura4; subjecting the ura4-deleted strain S. pogona-delta ura4 to Saccharopolyspora pogona gene cluster blocking through the homologous recombination of linear fragments. The method herein is suitable for acquiringa positive monoclone with a target gene cluster blocked without constructing a vector or introducing an exogenous recombinase; non-target gene clusters of Saccharopolyspora pogona can be blocked moreefficiently.

Description

technical field [0001] The invention relates to a method for blocking gene clusters of Saccharopolyspora tentaciformis, in particular to a method for blocking gene clusters of Saccharopolyspora tentaciformis based on homologous recombination of linear fragments. Background technique [0002] Butenyl spinosyns are a class of macrolide compounds with insecticidal activity produced by Saccharopolyspora spp., and have stronger insecticidal activity and insecticidal spectrum than spinosyns, such as against The worldwide quarantine pests and important agricultural pests that are difficult to control, the tobacco budworm, have a very good biological control effect, and have become one of the most promising new biopesticides. However, the ability of wild-type Saccharopolyspora spp. to synthesize spinosyns is weak and the fermentation period is long, which restricts the large-scale industrial production and application of spinosyns. Therefore, how to shorten the fermentation cycle a...

Claims

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Application Information

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IPC IPC(8): C12N15/80C12N15/90C12N1/15C12R1/645
CPCC12N15/80C12N15/902C07K14/37Y02A50/30
Inventor 夏立秋穰杰何昊城丁学知孙运军
Owner HUNAN NORMAL UNIVERSITY
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