A method and application of regulating unsaturated fatty acid synthesis in Synechocystis

A technology for unsaturated fatty acids and Synechocystis, which is applied in the field of regulating the synthesis of unsaturated fatty acids in Synechocystis, and can solve problems such as the content of unsaturated fatty acids cannot be further increased.

Active Publication Date: 2021-08-10
BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] Although this method can increase the content of unsaturated fatty acids in Synechocystis sp., it cannot further enhance the content of unsaturated fatty acids.

Method used

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  • A method and application of regulating unsaturated fatty acid synthesis in Synechocystis
  • A method and application of regulating unsaturated fatty acid synthesis in Synechocystis
  • A method and application of regulating unsaturated fatty acid synthesis in Synechocystis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] spkD gene cloning and insertion into quick connection vector and spkG gene cloning and insertion into quick connection vector:

[0074] Using the primer pair to carry out electrophoresis after PCR amplification of the Synechocystis sp. PCC6803 genomic DNA, the length of the spkD gene fragment is 1.9kb. The spkD gene sequence is SEQ ID NO.17, which is verified to be correct and recovered by cutting the gel; the primer pair is as follows:

[0075] spkD-F: 5'-ACTTACCCGTTCTGATTGA-3' SEQ ID NO.1

[0076] spkD-R: 5'-TAACCATTGATAA GCAGAT-3'SEQ ID NO.2;

[0077] Synechocystis PCC6803 genomic DNA was subjected to electrophoresis after PCR amplification with a primer pair, and the spkG gene fragment length was 1.9kb. The spkG gene sequence was SEQ ID NO.18, which was verified to be correct, and the gel was cut and recovered; the primer pair was as follows:

[0078] spkG-F: 5'-AGACTTTTCTCTATTGCCTC-3' SEQ ID NO.3

[0079] spkG-R: 5'-GGACCCAAATCCAGAAGAC-3' SEQ ID NO.4.

[0080] T...

Embodiment 2

[0087] Construction of spkD gene knockout (insertion inactivation) and spkG gene knockout (insertion inactivation) plasmids:

[0088] The 007-spkD plasmid was digested with EcoR I to obtain 007-spkD-EcoR I. The restriction site of EcoR I is at 0.9kb of the spkD gene fragment, the first 0.9kb is used as the upstream arm of homologous recombination, and the latter 1.0kb (the length of the spkD gene fragment is 1.9kb) is used as the downstream arm of homologous recombination. The 007-spkG plasmid was digested with BamHI to obtain 007-spkG-BamHI. The restriction site of BamHI is at the first 0.9kb of spkG gene. The first 0.9 kb was used as the upstream arm of homologous recombination, and the last 1.0 kb (the length of the spkG gene fragment was 1.9 kb) was used as the downstream arm of homologous recombination.

[0089] Digest the plasmid pBluescript-Kan (providing a kanamycin resistance fragment) with EcoR I endonuclease, and recover the kanamycin fragment (1.2kb) by electroph...

Embodiment 3

[0092] The acquisition of spkD gene knockout (insertion inactivation) mutant strain and spkG gene knockout (insertion inactivation) mutant strain:

[0093] Take the logarithmic culture period (OD 730 =0.6) (Culture condition: BG-11 liquid medium, 30°C, light condition 40μmol·m -2 ·s -1 ) Synechocystis PCC6803 culture solution 30ml, at room temperature, centrifuge at 4500g for 8min, discard the supernatant; add fresh BG-11 liquid medium to wash once, centrifuge, remove the culture medium, add fresh BG-11 liquid medium to final concentration OD 730 =4.8, and immediately used for transformation, the specific transformation process; the algae liquid collected is divided into 1.5ml EP tubes (400 μl per tube), and each tube adds 5 μg of plasmid (respectively for the plasmid 007-spkD- prepared in Example 2) kan and 007-spkG-kan), and incubated for 6 hours under low light conditions (light conditions: 10 μmol·m -2 ·s -1 , the temperature is 30°C), during which the mixture was sli...

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Abstract

The present invention relates to the application of a serine / threonine kinase gene in the synthesis of unsaturated fatty acids in Synechocystis sp. spkD gene knockout mutants and spkG gene knockout mutants in Synechocystis sp. PCC6803; the invention is disclosed for the first time The spkD and spkG genes in Synechocystis sp. PCC6803 play an important role in changing the synthesis of unsaturated fatty acids in Synechocystis sp. C18:2, C18:3n6, C18:3n3, and C18:4 The content of iso-fatty acid was significantly lower than that of the wild-type Synechocystis; two genes, spkD and spkG, were negatively correlated with the production of polyunsaturated fatty acids in Synechocystis in the early stage of cultivation, and it was necessary to coordinate the expression of spkD and spkG in the next step to improve The content of polyunsaturated fatty acids such as linoleic acid, alpha-linolenic acid and stearidonic acid in cyanobacteria lays the foundation.

Description

technical field [0001] The invention relates to a method for regulating and controlling the synthesis of unsaturated fatty acids in Synechocystis and its application, belonging to the field of biotechnology. Background technique [0002] Cyanobacteria are a class of prokaryotes with photoautotrophic ability. Cyanobacteria cells can grow autotrophically, can survive without relying on external organic matter, can use simple inorganic matter to synthesize organic matter to provide energy for life activities, and proteins produced by exogenous gene expression can be fully folded and assembled, which means that cyanobacteria have almost no Inclusion bodies are formed, and most cyanobacteria and their cell extracts are non-toxic to humans and animals, and are good recipients for transgenic research. Synechocystis sp. PCC6803 (Synechocystis sp. PCC6803), as a single-celled cyanobacteria, has the characteristics of simple structure, simple genetic background, easy culture, conveni...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P7/64C12N1/21C12N15/74C12R1/01
CPCC12P7/6427C12N9/12C12Y207/11C12N15/74
Inventor 陈高钟怀荣曹月蕾范仲学崔晓艳于金慧李燕乐路晓媛
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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