Method for improving content of 5-methyltetrahydrofolate by gene knockout

A methyltetrahydrofolate and gene knockout technology, applied in the fields of genetic engineering and molecular biology, can solve folic acid deficiency, changes in DNA methylation level and pattern, accelerate the development of colon cancer and prostate cancer, and precancerous lesions, etc. problem, achieve the effect of increasing folic acid content and alleviating hidden hunger

Active Publication Date: 2020-04-21
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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Problems solved by technology

At present, some countries and regions usually adopt measures such as supplementing folic acid tablets to improve the situation of folic acid deficiency, but excessive intake of artificially synthesized folic acid will lead to changes in the level and pattern of DNA methylation in the human body, and accelerate the development and precancerous development of colon cancer and prostate cancer The occurrenc

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  • Method for improving content of 5-methyltetrahydrofolate by gene knockout
  • Method for improving content of 5-methyltetrahydrofolate by gene knockout
  • Method for improving content of 5-methyltetrahydrofolate by gene knockout

Examples

Experimental program
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Effect test

Embodiment 1

[0027] Example 1 Corn ZmGFT1 Construction of gene knockout vector and acquisition and identification of transgenic plants

[0028] (1) The leaves of maize inbred line C01 were collected, the total RNA of maize was extracted by Trizol method, and the cDNA of maize was obtained with RevertAid First Strand cDNA Synthesis Kit (Thermo). Using maize cDNA as a template, Primer1 (SEQ ID NO.5) / Primer2 (SEQ ID NO.6) as amplification primers, the CDS full-length of the GRMZM2G124863 gene was amplified ( figure 1 A).

[0029] (2) Referring to the results of the sequencing sequence, design two target sites in the genomic region corresponding to the GFT1 exon ( figure 1 T1 and T2 of B, design website: http: / / cas9.cbi.pku.edu.cn / index.jsp), these two targets have common characteristics in the genome, NGG at the 3' end (N is A , T, C, G any base), the sequence is SEQ ID NO.3 and SEQ ID NO.4.

[0030] (3) Referring to the method of gene knockout vector construction (Li CX, Liu CL et al.,...

Embodiment 2

[0040] Example 2 ZmGFT1 Detection of gene knockout maize transcription level

[0041] Will ZmGFT1 The negative transgenic T0 generation plants with gene knockout and no CRISPR / Cas9 recombinant plasmid were selfed to obtain stable genetic ZmGFT1 Gene knockout T1 generation line. Will ZmGFT1 The gene knockout T1 generation plants were selfed, and the grain materials 25 days after pollination were taken, frozen in liquid nitrogen, and ground into powder, and total RNA was extracted using a total RNA extraction kit (purchased from Beijing Yuanpinghao Biotechnology Co., Ltd.). RevertAid First Strand cDNA Synthesis Kit (Thermo) was used to obtain cDNA. Transgenic plants were detected by qRT-PCR detection kit (purchased from Beijing Quanshijin Biotechnology Co., Ltd.) ZmGFT1 gene transcription levels. PCR reaction system: DNA template 1 μl, Primer9 (SEQ ID NO.13) 0.4 μl, Primer10 (SEQ ID NO.14) 0.4 μl, Dye II 0.4 μl, 2X Buffer 10 μl, ddH 2 O 7.8 μl; PCR amplification program: ...

Embodiment 3

[0042] Example 3 ZmGFT1 Determination of Folic Acid Content in Gene Knockout Maize

[0043] Utilize high performance liquid chromatography mass spectrometry to measure described in embodiment 2 ZmGFT1 Folic acid content in leaves and grains of transgenic maize (including monoglutamic acid-tailed 5-methyltetrahydrofolate, diglutamic acid-tailed 5-methyltetrahydrofolate, triple-glutamic acid-tailed 5-methyltetrahydrofolate). Organs tested included fresh leaves 70 days after planting and fresh seeds 25 days after pollination.

[0044] The result is as Figure 6-8 as shown, ZmGFT1 The content of 5-methyltetrahydrofolate, the main form of folic acid, in the leaves and grains of transgenic maize was significantly higher than that in the control (wild type). In fresh leaves planted for 70 days, the gene knockout mutants had about 2.01-2.23 times more monoglutamic tail 5-methyltetrahydrofolate and 2.23 times more diglutamic tail 5-methyltetrahydrofolate than the wild type. Increa...

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Abstract

The invention provides a method for increasing the content of 5-methyltetrahydrofolate through gene knockout. Corn ZmGFT1 is subjected to gene knockout through a CRISPR/Cas9 technology, and it is found that the content of 5-methyltetrahydrofolate in the corn after gene knockout is remarkably increased. According to the invention, the design of the CRISPR/Cas9 target site of corn ZmGFT1 and the function of the gene can significantly influence the folic acid content; the gene provides reference for CRISPR research of the gene in corn and other crops, provides a theoretical basis for folic acid metabolism of corn and other crops, and relieves the invisible hunger problem of human beings in the aspect of folic acid nutrition by increasing the folic acid content of transgenic plants.

Description

technical field [0001] The invention belongs to the technical fields of genetic engineering and molecular biology, and in particular relates to a method for increasing the content of 5-methyltetrahydrofolate through gene knockout. Background technique [0002] Folate is a donor and acceptor of one-carbon units in living organisms, and is involved in the biosynthesis of purine, formylmethionine-tRNA, thymidylate, glycine, serine, and methionine, and the formazan of DNA and proteins. basicization and other functions. It belongs to the water-soluble B vitamins and is a general term for tetrahydrofolate and its derivatives, including tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methylenetetrahydro Derivative forms such as folic acid. Since humans and other animals cannot synthesize folic acid themselves, dietary intake has become the main source of folic acid for humans. Insufficient folic acid intake can cause many serious health problems, which...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/11A01H5/00A01H6/46
CPCC12N9/1014C12N15/8218C12N15/8243C12Y201/02005
Inventor 姜凌张春义连通梁秋菊王维轩
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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