Application of OsAPBP2 protein to promotion of synthesis of plant folic acid

A folic acid and plant technology, applied in the biological field, can solve the problems of insufficient substrates of folic acid synthesis and metabolism, and folic acid metabolism disorder.

Inactive Publication Date: 2017-12-15
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 people can increase the folic acid content of plants by overexpressing key enzymes of the folate synthesis pathway in plants, single expression of a certain enzyme in the folate synthesis pathway often leads to enrichment of the substrates of the folate synthesis branch of the enzyme, while the bottom of the other folate synthesis metabolism branch Insufficient food, causing folic acid metabolism disorder in plants

Method used

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  • Application of OsAPBP2 protein to promotion of synthesis of plant folic acid
  • Application of OsAPBP2 protein to promotion of synthesis of plant folic acid
  • Application of OsAPBP2 protein to promotion of synthesis of plant folic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0070] Embodiment 1, the preparation of turning OsAPBP2 rice

[0071] 1. Construction of OsAPBP2 plant overexpression vector

[0072] (1) Genomic DNA of rice Zhonghua 11 was extracted.

[0073] (2) Design and synthesize the following primers (the underlined sequence is the enzyme recognition site):

[0074] Upstream primer: 5'-GC TCTAGA ACCATGTGCGGCGGAGCAATCATC-3';

[0075] Downstream primer: 5'-AT GTC GAC GTAGGCACCAGCTGCCATGA-3'.

[0076] (3) Using the genomic DNA in step (1) as a template and using the upstream primer and downstream primer in step (2) as primers to perform PCR amplification to obtain the OsAPBP2 gene.

[0077] (4) Use Xba I and Sal I to double digest the PCR product obtained in step (3) to obtain a gene fragment; Xba I and SalI to double digest pCAMBIA1307 to obtain a large vector fragment; connect the gene fragment to a large vector fragment to obtain a recombinant Plasmid, named as pCAMBIA1307-OsAPBP2, pCAMBIA1307-OsAPBP2 structure diagram as shown...

Embodiment 2

[0095] Example 2, detection of folic acid content in trans OsAPBP2 rice

[0096]1. Select the seeds of T3 generation OsAPBP2-transformed rice and wild-type rice Zhonghua 11, grind them into powder, and store them at 4°C for later use.

[0097] 2. Dilute 1 mg / ml folic acid solution with folic acid extraction buffer to standard solutions with concentrations of 100 ng / ml, 50 ng / ml, 20 ng / ml, 10 ng / ml, 5 ng / ml, and 1 ng / ml.

[0098] The folic acid extraction buffer is a solution obtained by mixing vitamin C sodium salt (sigma A7631) with 5mM phosphate buffer, wherein the mass fraction of vitamin C sodium salt in the folic acid extraction buffer is 1%. The formula of 5mM phosphate buffer is as follows: NaH 2 PO 4 (sigma, 17844) 0.419g / L, Na 2 HPO 4 2H 2 O (sigma, 71633) 0.269g / L, pH 6.0-6.5.

[0099] 3. Take 300 μl of folic acid solutions of different concentrations obtained in step 2, cook in boiling water for 10 minutes, cool on ice for 10 minutes, and mix well. Add 100 μl...

Embodiment 3

[0105] Example 3, Detection of expression levels of key folic acid synthesis genes in rice transfected with OsAPBP2

[0106] 1. The leaves of 3-week-old T3 transgenic OsAPBP2 rice and wild-type rice Zhonghua 11 were selected, washed with water, and the excess water was blotted with absorbent paper, then quickly frozen with liquid nitrogen and stored at -80°C.

[0107] 2. Take about 0.1 g of leaves, quickly grind them into powder with liquid nitrogen, add 1 ml of TRIZOL (Invitrogen, product number 15596026) to extract total RNA according to the instructions.

[0108] 3. Use a reverse transcription kit (TaKaRa, catalog number: DRR014A) to reverse transcribe the RNA obtained in step 2 into cDNA.

[0109] 4. Carry out quantitative PCR using the cDNA in step 3 as a template to detect the expression levels of key genes OsGDPCHI, OsDHNA, OsADCS, OsADCL, OsDHFR1, OsDHFR2 and OsFPGS1 in rice folic acid synthesis, and analyze the regulatory effect of OsAPBP2 protein on key genes of foli...

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Abstract

The invention discloses application of OsAPBP2 protein to promotion of synthesis of plant folic acid. The OsAPBP2 protein provided by the invention is protein shown as a) or b) or c) or d): a) protein with an amino acid sequence shown as a sequence 2; b) fused protein obtained by connecting an end N and/or an end C of the protein shown as the sequence 2 with a label; c) protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequence shown as the sequence 2, and has the same function; d) protein which has 75 percent or more than 75 percent of homology with the amino acid sequence shown as the sequence 2 and has the same function as the sequence 2. An experiment proves that the OsAPBP2 protein can be used for identifying and combining with rice folic acid to synthesize a key gene OsADCS promoter to regulate and control the expression of a rice folic acid synthesis key gene and further regulate and control the content of the folic acid in rice, and plays an important role in the synthesis of the plant folic acid.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the application of OsAPBP2 protein in promoting plant folic acid synthesis. Background technique [0002] Folic acid, also called vitamin B9, is an essential B vitamin for animals and humans. As a one-carbon donor, folic acid participates in the synthesis of purine and thymine, which further affects the synthesis of DNA and RNA; as a one-carbon carrier, folic acid is also related to the metabolism of amino acids such as glycine, serine and histidine; in addition, folic acid is also involved in hemoglobin and adrenaline The synthesis of methyl compounds such as , choline and creatine plays an important role in improving megaloblastic anemia and fetal neural tube development. Although folic acid has such an important role in the living body, it cannot be synthesized by humans and needs to be ingested from the daily diet. However, the folic acid content in rice and other m...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8243
Inventor 张执金张海文权瑞党王娟赵辉黄荣峰
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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