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Use of a histidine kinase gene hisk2301

A histidine kinase and gene technology, applied in the field of biotechnology and genetic engineering, can solve the problems of no research reports on polyunsaturated fatty acids

Active Publication Date: 2020-07-10
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, HK plays an important regulatory role in many cell biological processes, but there is no research report on HK promoting the synthesis of polyunsaturated fatty acids

Method used

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  • Use of a histidine kinase gene hisk2301
  • Use of a histidine kinase gene hisk2301
  • Use of a histidine kinase gene hisk2301

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) YM25235 histidine kinase gene Hisk2301 gene cloning

[0016] The total RNA of Rhodosporidium YM25235 was extracted with the OMEGA kit E.Z.N.A Fungal RNA Kit, and the cDNA was synthesized with the reverse transcription kit Takara First Strand cDNA Synthesis Kit. According to the transcriptome sequence of Rhodosporidium YM25235, specific primers (primer 1 and primer 2) were designed for PCR amplification; the primers, components and amplification conditions used in the reaction were as follows:

[0017] Primer 1: Hisk2301-F: 5'- CACACCGC GGATCC ATGCCGCAGACGCC-3' (SEQ ID NO: 3)

[0018] Primer 2: Hisk2301-R: 5’- CTGACCGC GATATC TCACTGCGGGTCGTT-3' (SEQ ID NO: 4)

[0019] ( GGATCC for Bam H Ⅲ Restriction site, GATATC for EcoR V restriction site);

[0020] The PCR amplification system is as follows (50 μL):

[0021]

[0022] PCR amplification conditions: pre-denaturation at 95°C for 5 m...

Embodiment 2

[0023] Example 2: Construction of Hisk2301 overexpression vector pRHHisk2301

[0024] Using the reverse-transcribed YM25235 cDNA as a template, using Hisk2301-F and Hisk2301-R as primers to amplify the Hisk2301 coding sequence, the obtained Hisk2301 fragment was about 3888 bp in size, and the amplified Hisk2301 fragment was subjected to Bam H I. EcoR Ⅴ After cutting with two restriction endonucleases, connect it to the expression vector pRH2304 to obtain the recombinant plasmid pRHHisk2301 (Figure 2). The obtained recombinant plasmid was transformed into Escherichia coli DH5α for amplification, and then verified by colony PCR to extract the recombinant plasmid and use Bam H I. EcoRⅤ Carry out double enzyme digestion verification on pRHHisk2301. The results showed that the recombinant plasmid pRHHisk2301 produced two bands of about 3.8 kb and 10 kb after double digestion (the second lane in Figure 3). The size of the fragments was consistent, preliminarily indicating th...

Embodiment 3

[0025] Example 3: Analysis of the relationship between the Hisk2301 gene and low-temperature synthesis of polyunsaturated fatty acids

[0026] 1. The recombinant plasmid pRH2034-Hisk2301 was transformed into Rhodosporidium YM25235 strain

[0027] The recombinant plasmid pRH2034-Hisk2301 was transformed into the Rhodosporidium YM25235 strain by the lithium acetate transformation method, and the transformants were selected with the YPD medium containing Hygromycin B (HygromycinB) at a final concentration of 150 µg / mL; the genomic DNA was extracted and verified by PCR son.

[0028] 2. Analysis of Hisk2301 gene and fatty acid content of Rhodosporidium yeast

[0029] Two strains of transgenic Rhodosporidium YM25235 transformed into the empty plasmid pRH2304 and the recombinant plasmid pRHHisK2301 were cultured at 30°C for 24 hours, and then the cultures were immediately placed at 15°C for 24 hours for cold treatment. After the cold treatment, the fatty acids of the two transgenic ...

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Abstract

The invention discloses uses of a histidine kinase gene Hisk2301, particularly applications of a histidine kinase gene Hisk2301 in production of polyunsaturated fatty acids at a low temperature. According to the present invention, polyunsaturated fatty acids are produced at a low temperature with the histidine kinase gene Hisk2301, and the contents of the polyunsaturated fatty acids such as LA and ALA are improved by modifying microorganisms through genetic engineering, such that the good application prospects and economic benefits are provided for the industrial production of polyunsaturated fatty acids, and the foundation is established for the large-scale industrial production of polyunsaturated fatty acids.

Description

technical field [0001] The invention belongs to the field of biotechnology and genetic engineering, and relates to a histidine kinase gene His 2301 uses. Background technique [0002] Oleaginous yeast cells are rich in polyunsaturated fatty acids (PUFAs), PUFAs have a wide range of physiological functions in the human body, and changes in their synthesis can lead to a variety of diseases, such as cardiovascular disease, obesity, and non-insulin-dependent diabetes , hypertension, neurological diseases and cancer. PUFAs also have important functions in cell membrane biology. The relative content of PUFAs in cell membrane lipids has an important impact on the physical properties of most biological membranes, such as the fluidity of cell membranes. Stable fluidity is very important for maintaining the normal function of cell membranes. role. The traditional sources of PUFAs have problems in terms of yield, quality, production cost, and safety, which cannot meet the large dema...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N9/12C12N15/81C12P7/64C12R1/645
CPCC12N9/12C12N15/815C12P7/6427C12Y207/13003
Inventor 张琦肖虎魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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