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A method for increasing the 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 problems such as folic acid deficiency, DNA methylation level and pattern changes, and cover up vitamin B12 deficiency, etc., to alleviate invisible hunger, Effect of increasing folic acid content

Active Publication Date: 2020-07-14
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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 occurrence of lesions will also mask the lack of vitamin B12
There is also a natural variation in the gene encoding the folic acid metabolism gene methylenetetrahydrofolate reductase in some populations, making it difficult for them to convert ingested folic acid into the active 5-methyltetrahydrofolate
All of these make it difficult to improve the folic acid deficiency in some countries and regions in the short term

Method used

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  • A method for increasing the content of 5-methyltetrahydrofolate by gene knockout
  • A method for increasing the content of 5-methyltetrahydrofolate by gene knockout
  • A method for increasing the 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 corn inbred line C01 were collected, the total RNA of corn was extracted by Trizol method, and the cDNA of corn was obtained with RevertAid FirstStrand cDNA Synthesis Kit (Thermo). Using maize cDNA as a template, Primer1 (SEQ ID NO.5) / Primer2 (SEQ ID NO.6) as amplification primers, amplified the CDS full length of the GRMZM2G124863 gene ( 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., 2017, P...

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. Maize by CRISPR / Cas9 technology ZmGFT1 The gene knockout was implemented, and it was found that the content of 5-methyltetrahydrofolate in maize was significantly increased after the gene knockout. Corn in the present invention ZmGFT1 The design of the CRISPR / Cas9 target site and the discovery that the function of the gene can significantly affect the folic acid content not only provide a reference for the CRISPR research of this gene in maize and other crops, but also provide a theory for the folic acid metabolism of maize and other crops The foundation, by increasing the folic acid content of transgenic plants, alleviates the hidden hunger problem of human beings in folic acid nutrition.

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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Patent Type & Authority Patents(China)
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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