Soybean folate synthesis key enzymes GTPCHI and their genes and use

A key enzyme gene, key enzyme technology, applied in soybean folic acid synthesis key enzyme GTPCHI and its gene and application fields

Active Publication Date: 2013-06-05
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

According to the report of the United States Department of Agriculture (2001), compared with cereals, fruits, vegetables, and dairy products, soybean folic acid content is the highest, which shows that there may be GTPCHI gene in soybean, and it may have higher enzyme activity, but So far, there is no report on the cloning of GTPCHI gene in soybean

Method used

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  • Soybean folate synthesis key enzymes GTPCHI and their genes and use
  • Soybean folate synthesis key enzymes GTPCHI and their genes and use
  • Soybean folate synthesis key enzymes GTPCHI and their genes and use

Examples

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Effect test

Embodiment 1

[0160] Example 1, Cloning and sequence analysis of Gm3gGTPCHI and Gm8gGTPCHI genes

[0161] 1. Obtain the EST sequence of the soybean GTPCHI gene

[0162] Since the GTPCHI genes from different species should have a certain degree of homology, or there are conserved structural domains. To this end, the CDS sequence of the GTPCHI gene of Arabidopsis was compared with the EST sequence of soybean on NCBI, and a total of 14 EST sequences were obtained. Four of them have a higher identity with the 5' end of the CDS sequence of Arabidopsis GTPCHI (AtGTPCHI), and they are named 5'EST-1, 5'EST-2, 5'EST-3, and 5'EST-4, respectively. By comparing them, it was found that the 5'EST-2 / 3 / 4 sequences were basically the same, and there were only differences in the length of the sequences. The other 10 EST sequences have a high consistency with the 3' end of the CDS sequence of AtGTPCHI, and they are named 3'EST-1~10 respectively. After comparing and analyzing these 10 ESTs, it is found that ...

Embodiment 2

[0174] Example 2, the expression pattern of Gm3gGTPCHI and Gm8gGTPCHI genes in soybean

[0175] The transcription levels of two genes Gm3gGTPCHI and Gm8gGTPCHI in different soybean tissues and organs were analyzed by RT-PCR. Soybean roots, stems, and leaves grown for 8 weeks were taken, and total RNA was extracted by the Trizol method, reverse-transcribed into cDNA as a template, and soybean actin was used as an internal standard to design specific primers GmActin11F and GmActin11R (see Table 2 for primer sequences). According to the CDS sequences of the two genes, gene-specific primers Gm3gGTPCHI-FSP and Gm3gGTPCHI-RSP and Gm8gGTPCHI-FSP and Gm8gGTPCHI-RSP were designed respectively (see Table 2 for the primer sequences). The results showed that the three genes could be detected in roots, stems and leaves. To transcription, among them, Gm3gGTPCHI expressed the highest in roots, followed by stems and leaves; Gm8gGTPCHI expressed the highest in stems, followed by roots, leaves ...

Embodiment 3

[0178] Example 3, identification of folic acid content in Arabidopsis transgenic Gm3gGTPCHI and Gm8gGTPCHI genes

[0179] The CDS sequences of soybean Gm3gGTPCHI and Gm8gGTPCH were respectively constructed on the plant overexpression vector pH2GW7, driven by the cauliflower mosaic virus (CaMV) 35S promoter, and the recombinant vectors pH2G W7-Gm3gGTPCHI and pH2G W7-Gm8gGTPCHI were obtained.

[0180] These two vectors were transformed into Agrobacterium LBA4404 by electroporation, and the correct clones identified by restriction enzyme digestion were selected, and the wild-type Arabidopsis was infected by the Agrobacterium-mediated flower dipping method, and the harvested seeds were recorded as the T0 generation. The seeds of the T0 generation were first screened on the resistance medium of 1 / 2MS+hygromycin B (25mg / L), and 13 positive green seedlings (referred to as the T1 generation) were obtained from the two infection events, and moved into the greenhouse for 20 days. Days, ...

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Abstract

The invention relates to the field of plant gene engineering, and specially, relates to soybean folate synthesis key enzymes GTPCHI and their genes and use. Amino acid sequences of the two soybean folate synthesis key enzymes GTPCHI are respectively shown in the formula SEQ ID No.1 and the formula SEQ ID No.3. Through the expression of the genes of the soybean folate synthesis key enzymes GTPCHI,folate content of a transgenic plant is obviously improved. Therefore, the soybean folate synthesis key enzymes GTPCHI lay the foundation for plant folate content improvement, enable people to directly get natural folate for daily needs through diet, and are very meaningful to relieve folate intake lack of people.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to GTPCHI, a key enzyme for folic acid synthesis in soybean, and its gene and application. Background technique [0002] Folic acid (folates) is the general term for tetrahydrofolate (THF) and its derivatives. It is a class of water-soluble B vitamins, also known as vitamin B9, vitamin B11, and vitamin M. Its chemical structure is as follows: [0003] [0004] Among them, THF consists of three parts: a pteridine ring (2-amino-4-hydroxy-6-methylpteridine), pABA p-aminobenzoic acid, and poly-glutamic acid (poly-Glu) (1-8 A Gluλ link), also known as pteroylglutamic acid. One-carbon units of different oxidation states, such as methyl, methylene, methylene, formyl, attached to N5 of the pteridine ring, N10 of pABA or bridged both to form tetrahydrofolate (THF4), 5 -Methyl THF4, 5-formyl THF4, 5,10-methylene THF4 various folic acid derivatives. THF and its derivatives, as th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00
Inventor 张春义梁秋菊范云六
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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