Zinc finger protein transcription factor gene RkMSN4 and application thereof

A technology of transcription factors and zinc finger proteins, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve problems such as the lack of polyunsaturated fatty acids

Active Publication Date: 2017-10-20
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The zinc finger protein MSN4 is a type of transcription factor. Many studies have shown that under different environmental stress conditions, it can promote the transcription of stress adaptation-related genes in host cells, thereby promoting the host's adaptability to environmental stresses such as high salt and high osmotic pressure. However, currently There is no research report on the promotion of polyunsaturated fatty acid synthesis by zinc finger protein MSN4 under low temperature conditions

Method used

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  • Zinc finger protein transcription factor gene RkMSN4 and application thereof
  • Zinc finger protein transcription factor gene RkMSN4 and application thereof
  • Zinc finger protein transcription factor gene RkMSN4 and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0015] Embodiment 1: Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) YM25235 zinc finger protein transcription factor RKMSN4 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: RkMSN4-F: 5'-AAT GGATCC ATGGCGCCTGCTCCCCGT-3' (SEQ ID NO: 3)

[0018] Primer 2: RkMSN4-R: 5'-ATC GATATC TCACGGCATCGCCCACACCT-3' (SEQ ID NO: 4)

[0019] ( GGATCC for Bam H I restriction site, GATATC for Eco RV restriction site)

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

[0021]

[0022] PCR amplification conditions were: 95°C pre-denaturat...

Embodiment 2

[0023] Example 2: Construction of recombinant expression plasmid pRH2034RkMSN4

[0024] The cDNA measured in Example 1 is used as a template to carry out PCR amplification, and the reaction primer combination, reaction components and amplification conditions are as follows:

[0025] Primer 1: RkMSN4-F: 5'-AAT GGATCC ATGGCGCCTGCTCCCCGT-3' (SEQ ID NO: 3)

[0026] Primer 2: RkMSN4-R: 5'-ATC GATATC TCACGGCATCGCCCACACCT-3' (SEQ ID NO: 4)

[0027] ( GGATCC for Bam H I restriction site, GATATC for Eco RV restriction site)

[0028] The PCR amplification system is as follows (50 μL):

[0029]

[0030] PCR amplification conditions were: 95°C pre-denaturation for 5 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 64°C for 30 s, extension at 72°C for 3 min, and finally 10 min at 72°C. The gene fragment obtained by the above PCR and the expression vector pRH2034 were used Bam H I and Eco Perform double digestion with RV endonuclease, recover the digeste...

Embodiment 3

[0031] Example 3: RKMSN4 Genes and Polyunsaturated Fatty Acid Synthesis Experiments in Low Temperature Environment

[0032] 1. Agrobacterium-mediated transformation of Rhodosporidium YM25235

[0033] The recombinant plasmid pRH2034RkMSN4 was transformed into Rhodosporidium YM25235 strain by Agrobacterium-mediated transformation method, and the transformants were selected with YPD medium containing Hygromycin B (HygromycinB) at a final concentration of 150 μg / mL; Genomic DNA was extracted to verify the transformants by PCR .

[0034] 2, RKMSN4 Analysis of Genes and Changes of Fatty Acid Content in Rhodosporidium Yeast

[0035] After the transgenic strain YM25235 / pRH2034RkMSN4 and the control strain YM25235 / pRH2304 were first cultured at 30°C for 24 h, one was cultured at 30°C for 24 h, and the other was immediately transferred to 15°C for cold treatment for 24 h; The total fatty acids in bacterial cells are methylated. The sample was carried out to gas chromatographic ana...

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Abstract

The invention discloses a zinc finger protein transcription factor gene RkMSN4. The nucleotide sequence of the gene is represented by SEQ ID NO:1, and an amino acid sequence coded by the gene is represented by SEQ ID NO:2. The gene is separated from Rhodosporidium kratochvilovae YM25235 and can regulate the transcription factor of a delta<12 / 15>-fatty acid dehydrogenase gene RKD12, used for catalyzing the conversion of oleic acid into linoleic acid (LA) and alpha-linolenic acid (ALA), in the Rhodosporidium kratochvilovae. The gene provides a reference for the study of the low temperature environment resistance mechanism of the hodosporidium kratochvilovae, improves the content of polyunsaturated fatty acids comprising the LA and the ALA through reconstructing microbes in a gene engineering way, has a good application prospect and a good economic benefit in the industrial production of PUFAs, and lays a foundation for the large-scale commercial production of the PUFAs.

Description

technical field [0001] The invention belongs to the fields of biotechnology and genetic engineering, and relates to a zinc finger protein transcription factor gene RKMSN4 and its recombinant expression vector, specifically related to from Rhodosporidium yeast ( Rhodosporidium kratochvilovae ) Zinc finger protein transcription factor gene cloned in YM25235 RKMSN4 And the gene is connected to the carrier and transferred into yeast, and its ability to regulate the production of polyunsaturated fatty acid by yeast is studied, so as to provide reference for the large-scale production of polyunsaturated fatty acid. Background technique [0002] Polyunsaturated fatty acids (PUFAs) refer to fatty acids containing two or more C = C and a carbon chain length of 18-22 carbon atoms. According to the first C = C appears on the 3rd or 6th carbon atom from the methyl atom, it is usually divided into n-3 and n-6 ​​series. Polyunsaturated fatty acids have a variety of important physiolo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/31C07K14/39C12N15/81C12P7/64
CPCC12N15/815C12P7/6427C07K14/39
Inventor 张琦何强魏云林林连兵季秀玲
Owner KUNMING UNIV OF SCI & TECH
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