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Transcriptional regulatory factor PfaR for promoting synthesis of shewanella EPA and application of transcriptional regulatory factor PfaR

A technology of transcription regulator, Shewanella, applied in application, bacterial peptide, genetic engineering, etc., to achieve the effect of increasing yield

Pending Publication Date: 2022-07-15
INST OF AQUATIC LIFE ACAD SINICA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, the research on the gene clusters of Shewanella EPA synthesis has been reported. The methods to increase the EPA content are mainly to change the carbon source and nitrogen source, lower the temperature, and exogenously express gene clusters. However, the EPA synthesis transcription regulator PfaR increases the EPA content. The method has not been reported yet, we confirmed for the first time that PfaR plays a positive regulatory role in Shewanella EPA synthesis by gene-directed knockout and overexpression

Method used

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  • Transcriptional regulatory factor PfaR for promoting synthesis of shewanella EPA and application of transcriptional regulatory factor PfaR
  • Transcriptional regulatory factor PfaR for promoting synthesis of shewanella EPA and application of transcriptional regulatory factor PfaR
  • Transcriptional regulatory factor PfaR for promoting synthesis of shewanella EPA and application of transcriptional regulatory factor PfaR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Example 1: Cloning of the Shewanella Transcriptional Regulator PfaR Gene

[0019] 1. Shewanella genome extraction

[0020] The experimental material was the pure cultured Shewanella W3-18-1 (Shewanella sp.W3-18-1) in the laboratory, and the genome was extracted using the OMEGA bacterial DNA extraction kit.

[0021] 2. PfaR gene cloning

[0022] Using genome as template, the upstream and downstream primers of PfaR gene, PfaR-F:GCCATTTTACGAGAGCACGTC and PfaR-R:ACGATGGCAACAGGCATATCT, were designed as primers for PCR reaction. PCR amplification and reaction conditions were carried out in accordance with the instructions. After the reaction, the PCR product was separated by 1% agarose gel electrophoresis to obtain a length of about 1050bp. After purification and recovery, the product was sent to Sangon Bioengineering for sequencing. The result is shown in SEQID NO.1, and the length of the nucleotide sequence is 870bp. Its encoded protein sequence is shown in SEQ ID NO. 2,...

Embodiment 2

[0023] Example 2: Functional verification of the Shewanella PfaR gene

[0024] 1. The effect of PfaR on EPA synthesis-related genes

[0025] 1.1 Knockout of PfaR gene

[0026] 利用基因定点敲除技术敲除希瓦氏菌W3-18-1中的pfaR基因,设计PfaR上下游引物PfaR-5O:accgcatgcgatatcgagctcTCGAGCCCTGTTTGTTGCTT,PfaR-5I:AAAGAGTCTCCACGGGTCGAGAGTCTCATCTCAGGCGTGG,PfaR-3I:CCACGCCTGAGATGAGACTCTCGACCCGTGGAGACTCTTT和PfaR-3O:gtggaattcccgggagagctcCTTTGGCGACAATCAGCGAC (Italic lowercase is homology arm), first use the genome as a template, use PfaR-5O and PfaR-5I, PfaR-3O and PfaR-3I as primers to amplify 5' and 3' fragments, and then use 5' and 3' The 'fragment was used as the template, and PfaR-5O and PfaR-3O were used as primers to carry out Cross sover PCR to obtain the target fragment ΔpfaR lacking PfaR. Single-enzyme digested SacⅠ-treated pDS3.0 (Wan, VerBerkmoes et al. 2004) was recombined with the target fragment ΔpfaR using the ligase-independent single-fragment rapid cloning kit from Nanjing Novizan Biotechnology Company...

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Abstract

The invention discloses a transcriptional regulatory factor PfaR for promoting synthesis of shewanella eicosapentaenoic acid (EPA, C20: 5 omega 3) and application of the transcriptional regulatory factor PfaR, the nucleotide sequence of the transcriptional regulatory factor PfaR is as shown in SEQ ID NO.1, gene knockout and real-time fluorescent quantitative PCR (RT-qPCR) prove that the PfaR serves as a positive regulatory factor to participate in regulation and control of shewanella eicosapentaenoic acid synthesis at low temperature, and the PfaR can be used for promoting synthesis of shewanella eicosapentaenoic acid (EPA, C20: 5 omega 3). Meanwhile, it is proved that the EPA content of shewanella at low temperature can be increased through overexpression of the transcriptional regulatory factor PfaR. Therefore, the transcriptional regulatory factor PfaR promotes the shewanella to synthesize the EPA at low temperature, and plays an important role in related genetic improvement for improving the synthesis of the shewanella EPA.

Description

technical field [0001] The invention belongs to the technical field of molecular microorganisms, and in particular relates to the application of a PfaR transcriptional regulator in regulating the low temperature of Shewanella to promote EPA synthesis. Background technique [0002] Eicosapentaenoic acid (EPA; C20:5ω3) is an ω3 long-chain polyunsaturated fatty acid, which is used in lowering blood pressure and cholesterol, maintaining cardiovascular health, treating rheumatoid arthritis, preventing depression, ensuring Prenatal health and treatment of osteoporosis have better effects. Ordinary consumers get n-3LC-PUFAs by eating seafood and other products, and often eat some deep-sea fish to get enough n-3LC-PUFAs. The human serum level survey found that most people lack these essential fatty acids. [0003] The increasing human demand leads to overfishing, and the sustainable development of fishery resources needs to be ensured. Moreover, environmental pollution has led to s...

Claims

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Application Information

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IPC IPC(8): C12N15/31C07K14/195C12N15/74C12P7/6432
CPCC07K14/195C12N15/74C12P7/6427
Inventor 邱东茹魏贺红余佃贞
Owner INST OF AQUATIC LIFE ACAD SINICA
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