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Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS

A synergistic, genetic technology, applied in the field of plant genetic engineering

Active Publication Date: 2014-07-30
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

The content of folic acid in soybean is high, whether there is a synergistic effect of soybean GTPCHI and ADCS on the synthesis of folic acid has not been reported yet

Method used

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  • Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS
  • Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS
  • Method for increasing plant folate content by using synergistic effect of transferred soybean genes of GTPCHI and ADCS

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 2. PCR amplification to obtain the GmGTPCHI gene fragment and its sequence analysis

[0035] 100 mg of soybean leaves were taken, and total RNA was extracted by Trizol method. After being treated with DNase I, the purity and concentration of RNA were detected, and 1 μg was used to obtain cDNA with the reverse transcription kit First strand cDNA systhesis (purchased from Fermentas). Primers were designed using the EST sequence of soybean GTPCHI gene as a template, and soybean cDNA was used as a template for amplification. A band of about 700 bp was obtained, which was sequenced as the fragment sequence of soybean GTPCHI, and the sequence was assembled and spliced ​​with the 5'-end EST and 3'-end EST sequence in Vector NTI. Use GTPCHI-3FW and GTPCHI-7RV as primers to carry out PCR amplification respectively (see Table 1 for primer sequences), and obtain products of about 1400bp. These fragments are recovered with an agarose gel recovery kit (TIANGEN) and connected to p- ...

Embodiment 2

[0045] 2. PCR amplification to obtain the GmADCS gene fragment and its sequence analysis

[0046] 100 mg of soybean leaves were taken, and total RNA was extracted by Trizol method. After being treated with DNase I, the purity and concentration of RNA were detected, and 1 μg was used to obtain cDNA with the reverse transcription kit First strand cDNA systhesis (purchased from Fermentas). Primers were designed using the EST sequence of soybean ADCS gene as a template, and soybean cDNA was used as a template to amplify. A band of about 1300bp was obtained. After the fragment was recovered, it was connected to the p-EASY-T1 vector, and 8 positive clones were picked for sequencing and compared with 3'EST. It was found that the sequencing results of 6 clones were consistent and matched with 3'EST. The size is 1352bp. The obtained 1352bp sequence was compared with the published soybean genome sequence, and it was found that it was highly consistent with a sequence of soybean chromos...

Embodiment 3

[0056] The seeds were cultured in the greenhouse for 20 days, and the leaves were taken to extract DNA. Design GmGTPCHI and GmADCS gene-specific primers (Table 4), and use DNA as a template for PCR amplification. The PCR reaction system is as follows:

[0057]

[0058] PCR reaction conditions: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 30s, annealing at 55°C for 30s, Taq amplification at 72°C for 30s, 35 cycles, PCR products were detected by 1% agarose electrophoresis, and a fragment of about 400bp was obtained as the target band . figure 1 Shown are partial electropherograms of PCR amplification, in which 4, 5, 6, 7, 8, 10, and 11 are plants transformed into the GmGTPCHI gene, and 1, 2, 4, 5, 6, 7, 8, 9, and 11 12 and 12 are plants transformed into GmADCS gene, therefore, 4, 5, 6, 7, 8 and 11 are plants transformed into two target genes at the same time.

[0059]The RNA Trizol method was further used to extract the total RNA of the above-mentioned ...

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Abstract

The invention relates to the field of plant genetic engineering, in particular to a method for increasing plant folic acid content by utilizing the synergistic effect of transgenic soybean GTPCHI and ADCS genes. According to the present invention, the gene sequence of the soybean folic acid synthesis key enzyme GTPCHI is shown in SEQ ID No.1, and the sequence of the ADCS gene is shown in SEQ ID No.2. The synergistic effect of transgenic soybean GTPCHI and ADCS genes can significantly increase the content of folic acid in transgenic plants, therefore, the present invention can be used as a method for producing high-content folic acid transgenic plants, and humans can directly obtain natural folic acid by eating such plants. It solves the lack of folic acid intake and overcomes the adverse side effects caused by taking folic acid tablets for a long time.

Description

technical field [0001] The invention relates to the field of plant genetic engineering, in particular to a method for increasing plant folic acid content by utilizing the synergistic effect of transgenic soybean GTPCHI and ADCS genes. 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: Figure 11 shown. [0003] 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 to form tetrahydrofolate (THF), 5 -Methyl THF, 5-formyl THF, 5,10-methylene THF and various folic acid...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84
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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