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: 2013-01-30
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 eff...

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

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] 1. Obtain the EST sequence of the soybean GTPCHI gene

[0033] 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 to obtain the EST sequence of the GTPCHI gene of soybean.

[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 seq...

Embodiment 2

[0043] 1. Obtain the EST sequence of soybean ADCS gene

[0044] The CDS sequence of ADCS gene of Arabidopsis was compared with the EST sequence of soybean on NCBI to obtain the EST sequence of soybean GTPCHI gene.

[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 consis...

Embodiment 3

[0054] The CDS sequences of soybean GmGTPCHI and GmADCS were respectively constructed into plant overexpression vectors, both of which were driven by cauliflower mosaic virus (CaMV) 35S promoter. Among them, the CDS sequence of soybean GmGTPCHI was recombined into pH2GW7 through the Gateway system, and the CDS sequence of soybean folic acid synthesis key enzyme gene GmADCS was constructed between the restriction sites Nco I and Pml I of the plant overexpression vector pCAMBIA1302 to obtain the recombinant expression vector pCAMBIA1302- GmADCS. Wild-type Arabidopsis was infected by Agrobacterium-mediated floral staining.

[0055] Transfer the acquired T to GmGTPCHI 0 Generation-positive Arabidopsis plants G1-G4 were used as male parents, and T0-generation positive Arabidopsis plants A1-5 obtained into GmADCS were used as female parents, and the following hybrid combinations were carried out: G1xA1, G1xA2, G2xA3, G2xA4, G3xA2, G4xA5, and seed pods after hybridization were harv...

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Abstract

The invention relates to the field of plant gene engineering, and specifically relates to a method for increasing plant folate content by using a synergistic effect of transferred soybean genes of GTPCHI and ADCS. The gene sequence of GTPCHI synthetic key enzyme of the soybean folate is shown as SEQ ID No. 1; and the gene sequence of ADCS is shown as SEQ ID No. 2. The synergistic effect of the transferred soybean genes of GTPCHI and ADCS can obviously increase the folate content in transgenic plants. Therefore, the method can be used as a method for producing the transgenic plants with high folate content. People can obtain natural folates directly by eating the plants; phenomenon that folate intake is lack can be solved; and adverse side effects brought by long-term taking folate tablets can be overcome.

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 12 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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IPC IPC(8): C12N15/84A01H1/02A01H5/00
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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