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Application of maize zmgft1 gene in increasing folic acid content in plants

A corn and gene technology, applied in the fields of genetic engineering and molecular biology, can solve the problems of folic acid deficiency and difficult realization, and achieve the effect of increasing folic acid content and alleviating invisible hunger

Active Publication Date: 2020-01-14
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although effective measures such as food fortification and vitamin supplementation have been implemented in some countries and regions to improve the situation of folic acid deficiency, it is still difficult for underdeveloped countries and regions to achieve

Method used

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  • Application of maize zmgft1 gene in increasing folic acid content in plants
  • Application of maize zmgft1 gene in increasing folic acid content in plants
  • Application of maize zmgft1 gene in increasing folic acid content in plants

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Cloning and Transformation of Example 1 Maize ZmGFT1 Gene

[0038] The leaves of maize inbred line Qi319 were collected, the total RNA of maize was extracted by trizol method, and the cDNA of maize was obtained with RevertAid FirstStrand cDNA Synthesis Kit (Thermo). Using maize cDNA as a template, 4863CDS-BglII-F and 4863CDS-BstEII-R as amplification primers, the full-length CDS of the GRMZM2G124863 gene was amplified ( figure 1 A). The plasmid containing the full-length CDS sequence of GRMZM2G124863 was correctly sequenced and digested with BglII and BstEII, and the fragment was ligated into ubiquitin-pCAMBIA3301 after digestion. The full-length CDS of the Qi319GRMZM2G124863 gene (ZmGFT1 gene) was amplified using the cDNA of the maize inbred line Qi319 (S2069 genotype is G) as a template and ZM-FAB4863-CDS-F and ZM-FAB4863-CDS-R as amplification primers ( figure 1 B). The correct sequenced GRMZM2G124863 full-length CDS was recombined into the overexpression vector ...

Embodiment 2

[0043] Example 2 Expression pattern of ZmGFT1 gene in maize

[0044] Maize inbred line Qi319 was collected from tissues and organs at different development stages, such as roots, stems, shoot tips, young leaves, filaments, and embryos and endosperms of different days of pollination. Total RNA was extracted, and the expression of ZmGFT1 gene in maize was detected by microarray technology. The results showed that the expression of ZmGFT1 gene could be detected in the selected tissues and organs, and the expression level of ZmGFT1 gene was the highest in endosperm 20 days after pollination. The weakest expression of the gene ( figure 2 ).

Embodiment 3

[0045] Example 3 Identification of ZmGFT1 Transgenic Maize and Transgenic Arabidopsis Plants and Determination of Folate Content

[0046] The complete CDS sequence of ZmGFT1 was amplified from maize cDNA, and cloned into overexpression vectors pCAMBIA3301 and pH2GW7 by enzyme digestion and homologous recombination. The constructed ZmGFT1-pCAMBIA3301 was transformed into maize callus through Agrobacterium-mediated genetic transformation to obtain transgenic maize plants. The obtained transgenic plants were identified by PCR ( FIG. 3 ), and the leaf materials of the identified positive plants were taken for folic acid determination. The constructed 73GFT1-pH2GW7 and 319GFT1-pH2GW7 were used to infect col wild-type Arabidopsis thaliana by flower dipping method, and the obtained T0 generation seeds were screened on hygromycin resistance plates. The screened transgenic positive plants were transferred to the culture soil, and when the material was available, the leaves were taken ...

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Abstract

The invention provides application of maize gene ZmGFT1 in increasing folic acid content in plants. 5-Formyltetrahydrofolate content in 513 selfing line materials of a maize natural variant group is measured, the influence of group structure and genetic relationship of the natural variant group is estimated in connection with genotype data mined from RNA-Seq data, gene GFT1 having significant influence on the 5-formyltetrahydrofolate content and located on maize 5th chromosome is located by means of genome-wide association study, and the gene GFT1 encodes glutamate formiminotransferase 1. The invention also provides an SNP marker associated with maize folic acid content and its application. The maize gene ZmGFT1 and discovery of the key SNP site therein having significant influence on the 5-formyltetrahydrofolate content not only provide theoretical basis for studying folic acid metabolism in maize and other crops, but also enable the hidden hunger problem of human in terms of folic acid nutrition to be solved through the increase in the folic acid content of transgenic plants.

Description

technical field [0001] The invention relates to the fields of genetic engineering and molecular biology, in particular to the application of the corn ZmGFT1 gene in increasing the folic acid content of plants. Background technique [0002] Folic acid belongs to the water-soluble B vitamins and is a general term for tetrahydrofolate and its derivatives. There are different forms of folic acid derivatives due to different oxidation states, such as tetrahydrofolate, 5-methyltetrahydrofolate, 5-formyltetrahydrofolate, 5,10-methylenetetrahydrofolate, etc. Folate plays an important role as a donor or acceptor in one-carbon transfer reactions in most species. Folate is involved in the biosynthesis of purine, formylmethionine-tRNA, thymidylate, glycine, serine and methionine, and folic acid is also involved in the synthesis of pantothenate, methylation reaction and histidine metabolism. In plants, folic acid is also related to the biosynthesis of lignin, alkaloids, betalains and c...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/54C12N15/82A01H5/00A01H6/46C12Q1/6895C12N15/11
CPCC12N9/1014C12N15/8243C12Q1/6895C12Q2600/156C12Y201/02005
Inventor 张春义郭文柱梁秋菊范云六
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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