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Application of osvtc1-3 protein in promoting vitamin C synthesis in plants

A vitamin and plant technology, applied in the fields of application, plant products, plant gene improvement, etc., can solve the problems of Vc synthesis pathway and regulatory sites are unclear

Inactive Publication Date: 2016-08-17
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

[0005] At present, although the Vc synthesis pathway is relatively clear in the model plant Arabidopsis, the Vc synthesis pathway and regulatory sites in food crops such as rice are still unclear

Method used

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  • Application of osvtc1-3 protein in promoting vitamin C synthesis in plants
  • Application of osvtc1-3 protein in promoting vitamin C synthesis in plants
  • Application of osvtc1-3 protein in promoting vitamin C synthesis in plants

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Embodiment 1, the expression of OsVTC1-3 protein in Escherichia coli

[0048] 1. Construction of OsVTC1-3 prokaryotic expression vector

[0049] (1) Genomic DNA of rice Zhonghua 17 was extracted.

[0050] (2) Design and synthesize the following primers:

[0051] Upstream primer: 5'-GG CATATG ACCATGAAGGCGCTCA-3'

[0052] Downstream primer: 5'-AT GTC GAC CATGACAATCTCGGGCTT-3'

[0053] (The underlined sequence is the enzyme recognition site)

[0054] (3) Perform PCR amplification using the genomic DNA in step (1) as a template, and the upstream primer and downstream primer in step (2) as primers, and the PCR product is the OsVTC1-3 gene. The sequence of the gene is as shown in SEQ ID No. 1, the sequence of OsVTC1-3 protein is shown in SEQ ID No.2.

[0055] (4) NdeI and SalI double digestion of the PCR product obtained in step (3) to obtain the gene fragment; NdeI and SalI double digestion of pET-30a(+) to obtain a large vector fragment; link the gene fragment to t...

Embodiment 2

[0086] Embodiment 2, OsVTC1-3 gene overexpression vector transforms Arabidopsis

[0087] 1. Construction of overexpression vector

[0088] (1) Genomic DNA of rice Zhonghua 17 was extracted.

[0089] (2) Design and synthesize the following primers:

[0090] Upstream primer: 5'-GC TCTAGA ACCATGAAGGCGCTCATT-3'

[0091] Downstream primer: 5'-AT GTC GAC CATGACAATCTCGGGCTT-3'

[0092] (The underlined sequence is the enzyme recognition site)

[0093] (3) Perform PCR amplification using the genomic DNA in step (1) as a template and the upstream primer and downstream primer in step (2) as primers to obtain the OsVTC1-3 gene.

[0094] (4) Digest the PCR product obtained in step (3) with Xba I and Sal I to obtain a gene fragment; XbaI and Sal I double enzyme digest pCAMBIA1307 to obtain a large vector fragment; connect the gene fragment to the large vector fragment to obtain a recombinant Plasmid, named it pCAMBIA1307-OsVTC1-3, the plasmid was sent for sequencing and the result ...

Embodiment 3

[0110] Embodiment 3, OsVTC1-3 gene interference carrier transforms rice

[0111] 1. Construction of interference carrier

[0112] (1) Genomic DNA of rice Zhonghua 17 was extracted.

[0113] (2) Design and synthesize the following primers:

[0114] Upstream primer: 5'-AA CTCGAG GAGGCGAGGCGAAGGATT-3';

[0115] Downstream primer: 5'-GG AGATCT CAACCATATAGCCTGATGACA-3'.

[0116] (The underlined sequence is the enzyme recognition site)

[0117] (3) Perform PCR amplification using the genomic DNA in step (1) as a template, and the upstream primer and downstream primer in step (2) as primers to obtain a PCR amplification product. The nucleotide sequence of the DNA molecule is as shown in SEQ ID No. As shown in .3, this fragment does not contain the cDNA fragment of the OsVTC1-3 gene, and is located in the 3'UTR region of the OsVTC1-3 gene.

[0118] (4) The PCR product obtained in the step (3) of double digestion with XhoⅠ and BglⅡ was obtained to obtain the 3'UTR fragment of ...

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Abstract

The invention discloses an application of an OsVTC1-3 protein in the promotion of synthesis of plant vitamin C and an interference vector. The interference vector is obtained through the steps: inserting a DNA (Deoxyribonucleic Acid) fragment as shown in SEQ ID No.3 between loci XhoI and BglII of a starting vector pUCCRNAi to obtain an intermediate vector 1; inserting a reverse complementary sequence of the DNA fragment as shown in SEQ ID No.3 between loci SalI and BamHI of the intermediate vector 1 to obtain an intermediate vector 2; digesting the intermediate vector 2 by using PstI to obtain a DNA fragment; digesting pCAMBIA 2300 by using the PstI to obtain a klenow fragment of the vector; and connecting the DNA fragment and the klenow fragment of the vector. According to the application, the important role of the OsVTC1-3 protein to the synthesis of plant Vc is proved.

Description

technical field [0001] The invention relates to the application of OsVTC1-3 protein in promoting plant vitamin C synthesis. Background technique [0002] Vitamin C (Vitamin C, Vc) is an important antioxidant and coenzyme in animals and plants, but humans cannot synthesize Vc and need to be ingested from daily diet. Plants are an important source of Vc required by the human body, but the Vc content in plants, especially food crops, is very low, so increasing the Vc content in plants through biotechnology plays an important role in improving the nutritional value of crops. [0003] In animals, the synthesis pathway of Vc is relatively clear. Its synthesis process is from D-glucose through UDP-glucose, UDP-D-glucuronic acid, D-glucuronic acid, L-gulonic acid, L-gulose-1,4-lactone, and finally in ancient Vc is synthesized under the action of lauconolactone oxidase. Humans and some animals cannot synthesize Vc due to changes in the gene encoding gulonolactone oxidase. Unlike ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/84A01H5/00C12N15/54C12N9/12
Inventor 张执金秦华邓载安张传玉王亚云王娟张海文权瑞党黄荣峰
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI