Application of slr0681 gene in synthesis of synechocystis carotene

A technology of carotene and Synechocystis, applied in the field of genetic engineering

Active Publication Date: 2021-12-21
VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

The relationship between the slr0681 gene and carotenoid sy...

Method used

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  • Application of slr0681 gene in synthesis of synechocystis carotene
  • Application of slr0681 gene in synthesis of synechocystis carotene
  • Application of slr0681 gene in synthesis of synechocystis carotene

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Experimental program
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Effect test

Embodiment 1

[0090] Construction of slr0681 gene knockout (insertion inactivation) plasmid:

[0091](1) Taking the sequences shown in SEQ ID NO.3 and SEQ ID NO.4 in the sequence table as upstream primers respectively, and taking the sequences shown in SEQ ID NO.5 and SEQ ID NO.6 as downstream primers respectively, from Synechocystis sp. PCC 6803 The upstream and downstream fragments of the slr0681 gene were respectively amplified from the genomic DNA;

[0092] (2) The two gene fragments were connected to the T3 cloning vector and then transformed into Escherichia coli DH5α, and the positive clones were screened and sequenced to obtain the slr0681-T3 plasmid;

[0093] (3) the slr0681-T3 plasmid is single-digested with BamH I, and the vector fragment is recovered;

[0094] (4) Use BamH I to digest the pBluescript-Kan vector, and reclaim the kanamycin-resistant fragment;

[0095] (5) transforming into Escherichia coli DH5α after connecting the vector fragment and the kanamycin-resistant fra...

Embodiment 2

[0103] Obtaining of slr0681 gene knockout (insertion inactivation) mutant strain:

[0104] Take the logarithmic culture period (OD 730 =0.6) (Culture condition: BG-11 liquid medium, 30°C, light condition 40μmolphoton m -2 ·s -1 ) wild-type Synechocystis PCC6803 culture solution 4ml, at room temperature, centrifuge at 4,000rmp for 10min, discard the supernatant; add fresh BG-11 liquid medium to wash once, centrifuge, remove the culture solution, and resuspend the algae cell sediment Suspend in 1 mL of BG-11 medium, add 15 μL of the recombinant plasmid (pΔslr0681) prepared in Example 1, and incubate for 6 hours under low light conditions (light conditions: 10 μmol photon m -2 ·s -1 , the temperature is 30°C), during which the mixture was slightly inverted and mixed once. Then, spread the mixed culture of Synechocystis and plasmid on the nitrocellulose filter membrane of BG-11 solid plate, and cultivate it in the light incubator for 24 hours (cultivation condition: 30°C, ligh...

Embodiment 3

[0106] PCR detection of slr0681 gene knockout mutant strain:

[0107] Using wild-type Synechocystis sp. PCC6803 and Δslr0681 as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows: DNA was extracted from wild-type Synechocystis PCC6803 and Δslr0681 using DNA extraction phenol reagent (purchased from Solarbio Company) and glass bead shaking method. The specific operation steps are as follows: take 2mL OD 730 =0.6 cyanobacteria, collect algal cells by centrifugation at 12,000rpm at room temperature for 1min, add 0.4mL 1×TE Buffer and an appropriate amount of glass beads with a diameter of about 0.17mm (purchased from sigma company) until there is 0.5mL of suspension above the glass bead interface . Via a vortex shaker at the maximum speed for 1 min, then add 0.4 mL DNA extraction phenol reagent. Centrifuge at 12,000rpm for 10min, take the supernatant into a new 1.5mL centrifuge tube, add 0.4mL Tris saturated phenol / chlorofo...

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Abstract

The invention relates to application of an slr0681 gene in synthesis of synechocystis carotene. The invention discloses that the slr0681 gene of synechocystis PCC6803 has obvious influence on carotenoid synthesis in a high-salt environment for the first time, an slr0681 gene knockout mutant strain delta slr0681 is constructed by utilizing a homologous recombination method, and a result shows that the growth condition of the delta slr0681 has obvious advantages under a high-salt stress condition, the content of total carotenoid and chlorophyll a is increased, HPLC analysis shows that the content of myxoxanthophyll, zeaxanthine, echinenone and beta-carotene is increased by 1.2 times, 1.1 times, 1.7 times and 1.05 times respectively compared with the content of wild type counterparts, and the expression of psaA, psaB and psaL in PSI and the expression of psbB and psbD genes in PSII are increased to some extent.

Description

technical field [0001] The invention relates to the application of the slr0681 gene in the synthesis of Synechocystis carotenoids, in particular to the application of the slr0681 gene in Synechocystis PCC6803 in the synthesis of Synechocystis carotenoids under high-salt stress conditions, and belongs to the technical field of genetic engineering. Background technique [0002] Cyanobacteria are the earliest photoautotrophic prokaryotes on the earth and one of the most important primary producers. Cyanobacteria cells can grow autotrophically, can survive without relying on external organic matter, and can also use simple inorganic matter to synthesize organic matter to provide energy for life activities. Moreover, most cyanobacteria and their extracts are non-toxic to humans and animals, and are good recipients for transgenic research. Synechocystis sp. PCC 6803 (Synechocystis sp. PCC 6803) is a single-cell cyanobacteria with a natural exogenous DNA transformation system, fas...

Claims

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Application Information

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IPC IPC(8): C12N15/31C12N15/74C12N1/21C12R1/01
CPCC07K14/195C12N15/74
Inventor 陈高耿耘钟怀荣孙秀芹宣宁张婧崔晓艳牛旭东
Owner VEGETABLE RES INST OF SHANDONG ACADEMY OF AGRI SCI
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