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Application of ssl2084 gene in synthesis of medium and long chain fatty acids

A technology of medium and long chain fatty acids and fatty acids, applied in the field of genetic engineering

Active Publication Date: 2020-04-03
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

The role of ACP and medium and long chain fatty acid synthesis in Synechocystis PCC 6803 has not been reported so far

Method used

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  • Application of ssl2084 gene in synthesis of medium and long chain fatty acids
  • Application of ssl2084 gene in synthesis of medium and long chain fatty acids
  • Application of ssl2084 gene in synthesis of medium and long chain fatty acids

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0088] The ssl2084 gene cDNA fragment, the Pcpc560 promoter fragment, the homologous recombination upstream fragment slr1285U cDNA fragment and the homologous recombination downstream fragment slr1285D cDNA fragment were isolated and cloned to construct the overexpression vector of the Synechocystis sp. PCC6803 ssl2084 gene.

[0089] Cloning of the Pcpc560 promoter fragment of Synechocystis sp. PCC 6803: According to the Synechocystis sp. PCC 6803 (accession number: BA000022, AP012205) registered in GenBank, Pcpc560 was used as the promoter, and the primers were designed:

[0090]Pcpc560-Sal I-F: 5'-AATGTCGACACCTGTAGAGAAGAGTCCCT-3';

[0091] Pcpc560-R: 5'-TGAATTAATCTCCTACTTGAC-3'.

[0092] The amplification system is as follows:

[0093] High Fidelity (HiFi) PCR SuperMix II 10 μL, Pcpc560-SalI-F 1 μL, Pcpc560-R 1 μL, Synechocystis PCC 6803 genomic DNA 1 μL, ddH 2 O 7 μL, the total reaction system is 20 μL.

[0094] The amplification procedure is as follows:

[0095] Pre-...

Embodiment 2

[0133] Construction of Synechocystis sp. PCC6803ssl2084 gene overexpression vector:

[0134] The homologous recombination upstream arm slr1285U gene fragment obtained in Example 1 was digested with KpnI to recover the slr1285U gene fragment; the plasmid pBluescript SK T1T2 was digested with the same method to recover vector fragment 1. The recovered slr1285U gene fragment and vector fragment 1 were ligated to transform Escherichia coli DH5α. By screening positive clones, the early vector was obtained and named pBluescript SK T1T2-slr1285U.

[0135] The connection reaction system is:

[0136] T 4 DNA ligase 1 μL, carrier fragment 3 μL, target fragment 3 μL, 10×T 4 DNA ligation Buffer 2μL, ddH 2 O 11 μL, the total reaction system is 20 μL. Ligation overnight at 16°C.

[0137] The homologous recombination downstream arm slr1285D gene fragment prepared in Example 1 was digested with SacI to recover the slr1285D gene fragment; the same method was used to digest the plasmid pB...

Embodiment 3

[0149] PCR detection of ssl2084 gene overexpression algae strain

[0150] Using transgenic Synechocystis PCC 6803 and wild-type Synechocystis PCC6803 containing the expression vector p5S1285UDssl2084 as materials, the total DNA was extracted for PCR detection and analysis. The specific method is as follows:

[0151] Neutral phenol reagent (purchased from Invitrogen) was used to extract DNA from the mutant strain overexpressing the ssl2084 gene and the wild-type Synechocystis sp. PCC 6803 by shaking glass beads. The specific operation steps are as follows: take 50mL OD 730 =1.8 cyanobacteria, 4 DEG C, 5000rpm centrifuge 10min to collect algal cells, add 0.4mL neutral phenol to connect 0.4mL BG-11 liquid medium, then add an appropriate amount of glass beads (purchased from sigma company) with a diameter of about 0.17mm to There is 0.5 mL of suspension above the glass bead interface. Vibrate with a vortex shaker at the maximum speed for 1min, centrifuge at 11900rpm at 4°C for ...

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Abstract

The invention relates to application of ssl2084 gene in synthesis of medium and long chain fatty acids. The ssl2084 is a protein capable of promoting synthesis of the medium and long chain fatty acidsin synechocystis, and has an amino acid sequence shown as SEQ ID NO. 2. The invention further relates to application of the ssl2084 gene in synthesis of medium and long chain saturated fatty acids ofthe synechocystis. For the first time, the invention discloses an important role of the ssl2084 gene of the synechocystis sp. PCC 6803 in synthesis of the medium and long chain saturated fatty acidsat low temperature. By increasing expression of the ssl2084 gene in the synechocystis sp. PCC 6803 by utilizing an overexpression method, contents of C12:0, C16:0 and C18:0 in mutant strains are significantly increased; and moreover, the contents of the C12:0, the C16:0 and the C18:0 in the mutant strains are further increased after low-temperature treatment, so that the recognition that it is easy to synthesize unsaturated fatty acids at low temperature in the field is changed.

Description

technical field [0001] The invention relates to the application of the ssl2084 gene in synthesizing long-chain fatty acids, in particular to the application of the ssl2084 gene in Synechocystis PCC6803 to synthesize long-chain saturated fatty acids in Synechocystis sp., belonging to the technical field of genetic engineering. Background technique [0002] Fatty acid is one of the basic components of organisms and has important physiological functions. They are not only the main components of cell membrane lipids, but also important energy substances, and the precursors of some signal molecules, which can be distributed on the surface of the body together with other substances to prevent mechanical damage and heat dissipation. In addition, it is also closely related to cell recognition, species specificity and tissue immunity. [0003] Polyunsaturated fatty acids (PUFAs) refer to straight-chain fatty acids with more than two double bonds and 16-22 carbon atoms. It is one of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/405C12N15/31C12N1/13C12N15/66C12P7/64C12R1/89
CPCC07K14/405C12N15/66C12P7/6463
Inventor 陈高钟怀荣路晓媛戴美学张燕李国卫游银伟边斐钟珂罗泽宇
Owner BIOTECH RES CENT SHANDONG ACADEMY OF AGRI SCI
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