Application of sll0528 gene in improvement of synechocystis PCC6803 oxidative stress tolerance

A PCC6803, oxidative stress technology, applied in the field of industrial microorganisms, can solve problems such as low tolerance, and achieve the effect of wide application prospects

Active Publication Date: 2020-05-19
SOUTH CHINA UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] In order to overcome the disadvantages and deficiencies of the low oxidative stress tolerance of Synechocystis PCC6803 in the prior art, the object of the present invention is to provide an application of the sll0528 gene in improving the oxidative stress tolerance of Synechocystis PCC6803

Method used

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  • Application of sll0528 gene in improvement of synechocystis PCC6803 oxidative stress tolerance
  • Application of sll0528 gene in improvement of synechocystis PCC6803 oxidative stress tolerance
  • Application of sll0528 gene in improvement of synechocystis PCC6803 oxidative stress tolerance

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Embodiment 1

[0045] The acquisition of the sll0528 gene overexpression strain Osll0528, including the construction of the homologous recombination double-crossover plasmid P3031, the homologous exchange between the homologous recombination double-crossover plasmid and the wild-type genome of Synechocystis sp. PCC6803, and the screening and verification of Osll0528:

[0046] (1) Construction of homologous recombination double exchange plasmid P3031

[0047] Using the wild-type Synechocystis sp. PCC6803 genome as a template, primers were designed, with SEQ ID NO: 3 and SEQ ID NO: 4 in the sequence table as the upstream and downstream primers of the target fragment slr2030, and SEQ ID NO: 7 and SEQ ID NO: 8 are The upstream and downstream primers of psbA2 promoter, SEQ ID NO: 9 and SEQ ID NO: 10 are the upstream and downstream primers of sll0528, and SEQ ID NO: 11 and SEQ ID NO: 12 are the upstream and downstream primers of slr2031, amplified by PCR The target genes slr2030, psbA2 promoter, s...

Embodiment 2

[0067] The gene knockout strain Δsll0528 is disclosed in the patent "201811591708.3, the application of the sll0528 gene in improving the ethanol tolerance of Synechocystis sp. PCC6803".

[0068] Phenotype analysis of Synechocystis sp. PCC6803 wild-type WT, knockout strain Δsll0528, and gene overexpression strain Osll0528 under menadione oxidative stress:

[0069]The wild-type WT grown to the logarithmic phase, the knockout strain Δsll0528, and the overexpression strain Osll0528 were used as seed solutions, and were inoculated with 0 μM, 2.5 μM, 5 μM, 7.5 μM, 10 μM, 12.5 μM, 15 μM, and 17.5 μM menadione In the 50mL BG11 medium, the starting OD of each bottle of algae 730 = 0.1. Measure OD on day 3 730 , with algae in 0μM menadione as the control, algae OD under each stress 730 OD 730 For comparison, the OD 730 (%Control) represents the survival rate of WT, Δs110528, and Os110528 under the oxidative stress of menadione at various concentrations, and the survival rate curve...

Embodiment 3

[0077] Phenotype analysis of Synechocystis sp. PCC6803 wild-type WT, gene knockout strain Δsll0528, and gene overexpression strain Osll0528 under hydrogen peroxide oxidation stress:

[0078] The wild-type WT grown to the logarithmic phase, the knockout strain Δsll0528, and the overexpression strain Osll0528 were used as seed solutions and inoculated into 0mM, 1mM, 2mM, 3mM, 4mM, 5mM, 6mM H 2 o 2 In 30mL of BG11 medium, the starting OD of each bottle of algae 730 = 0.24. Measure 12h OD 730 , at 0mM H 2 o 2 The middle algae was used as the control, and the OD of the algae under each stress 730 OD 730 For comparison, the OD 730 (%Control) represents the survival rate of WT, Δs110528 and Os110528 under the oxidative stress of each concentration of hydrogen peroxide, and the survival rate curve is drawn.

[0079] The wild-type WT grown to the logarithmic phase, the knockout strain Δsll0528, and the overexpression strain Osll0528 were used as the seed solution, and were inoc...

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Abstract

The invention discloses application of a sll0528 gene in improvement of synechocystis PCC6803 oxidative stress tolerance and belongs to the field of industrial microorganisms. A sll0528 gene in synechocystis PCC6803 is overexpressed by a homologous recombination method to obtain a synechocystis PCC6803 strain Osll0528 with remarkably improved oxidative stress tolerance. The growing status of the Osll0528 is remarkably superior to that of a wild strain in a BG11 culture medium which is added with menadione having different concentrations (2.5[mu]M-15[mu]M) or hydrogen peroxide having differentconcentrations (1mM-3mM). The oxidative stress tolerance strain has important theoretical and practical meaning and wide application prospect in constructing genetically engineered bacteria with oxidative stress tolerance and genetically engineered strain proximal disks tolerating various environmental stresses.

Description

technical field [0001] The invention belongs to the field of industrial microorganisms, and in particular relates to the application of a sll0528 gene in improving the oxidative stress tolerance of Synechocystis sp. PCC6803. Background technique [0002] Reactive oxygen species (Reactive Oxygen Species, ROS) include singlet oxygen ( 1 o 2 ), superoxide anion (O 2 · - ), hydrogen peroxide (H 2 o 2 ) and hydroxyl radicals (OH·), they are all strong oxidants in cells, which will inhibit the synthesis of proteins and may lead to the cleavage of nucleic acids and the peroxidation of unsaturated fatty acids. [0003] Synechocystis PCC6803 has a simple genetic background, and its genetic information is fully published. It is regarded as a model organism for research on new energy development and new raw material production. Studies have proved that Synechocystis is a new generation of biofuels such as ethanol and alkanes, as well as free fatty acids, industrial Premium produc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/65C12N15/74C12R1/01
CPCC07K14/195C12N15/65C12N15/74
Inventor 陈谷刘秤利许白雪林诗琪周健
Owner SOUTH CHINA UNIV OF TECH
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