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New application of highland barley cyanidin oxygen methyltransferase gene

A technique of application and gene fragment, applied in the field of new application of highland barley cyanidin oxygen methyltransferase gene, achieving the effect of excellent application prospect

Active Publication Date: 2022-07-29
AGRI RES INST TIBET ACADEMY OF AGRI & ANIMAL HUSBANDRY SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Existing technology is still unable to achieve a large amount of synthesis and modification of anthocyanins by genetic engineering and other means

Method used

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  • New application of highland barley cyanidin oxygen methyltransferase gene
  • New application of highland barley cyanidin oxygen methyltransferase gene
  • New application of highland barley cyanidin oxygen methyltransferase gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 2

[0047] Example 2 Construction of Transgenic Tobacco

[0048] In this example, a transgenic tobacco containing the target gene (SEQ ID NO. 1) was constructed. Specifically include the following steps:

[0049] ①Transform the transient expression vector containing the target gene (transient expression vector pEAQ, from John InnesCentre) into Agrobacterium (EHA105);

[0050] ②Pick positive Agrobacterium clones into 500ul LB containing the corresponding antibiotics (kn), and cultivate for 20-24 hours;

[0051] ③ Transfer 200ul to 5ml of LB containing the corresponding antibiotic (kn), shake at 220rpm at 28°C to about OD=2.0.

[0052] ④The cells were collected by centrifugation at 10,000 rpm at room temperature for 2 min. The cells were resuspended with the transformation buffer prepared in advance, and shaken for 3 h. The components and concentrations of the buffer working solution were as follows: 10 mM MES (pH 5.7), 10 mM MgCl2, 100 μUDP-glucose.

[0053] ⑤ Take the prepared ...

Embodiment 1

[0059] The target protein with a molecular weight of 69 kDa and a GST tag prepared by the method in Example 1.

[0060] 1.2 Enzyme activity detection

[0061] in Tris-HCl buffer (100 mM, pH 7.4) containing 200 μM cyanidin 3-O malonyl glucoside as methyl acceptor, 100 μM thioadenosylmethionine as methyl donor and 500 ng The total volume of purified protein was 100 μl for the in vitro methyltransferase assay. After 10 min of incubation, 300 μL of ice-cold methanol was added to stop the reaction. The reaction mixture was then filtered through a 0.2 μm filter (microporous) before being used for LC-MS analysis.

[0062] 2. Results

[0063] After the above-mentioned catalytic reaction, the product was passed through LC-MS / MS, and it was shown that the generated substance was paeoniflorin O-malonyl glucoside ( figure 2 ). It shows that the HOVUSG2104500 protein of the present invention has the ability to catalyze the conversion of cyanidin oxymethylation into paeoniflorin O-mal...

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Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to a novel application of a highland barley cyanidin oxygen methyltransferase gene. The invention provides a cyanidin oxygen methyltransferase gene HOVUSG2104500 from highland barley, a recombinant vector of the gene, a recombinant bacterium of the gene and a new application of cyanidin oxygen methyltransferase. The invention also provides a preparation method of the transgenic plant for producing the paeoniflorin 3-O malonyl glucoside. According to the cyanidin oxygen methyltransferase disclosed by the invention, thioadenosylmethionine can be used as a methyl donor, cyanidin 3-O malonyl glucoside is used as a methyl receptor, and the cyanidin 3-O malonyl glucoside is synthesized, so that the cyanidin oxygen methyltransferase has an excellent application prospect.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a new use of a highland barley bluebonnet oxygen methyltransferase gene. Background technique [0002] The important active ingredient anthocyanin not only has the functions of anti-oxidation, scavenging free radicals, inhibiting inflammation and anti-cancer, but also preventing chronic diseases. At present, anthocyanins mostly exist in the form of glycosylation modification in plants, but there are few reports on the genes responsible for the modification of anthocyanins. [0003] The Qinghai-Tibet Plateau is the domestication area of ​​highland barley, and the highland barley germplasm resources are extremely rich. Colored highland barley is a kind of precious highland barley germplasm resources, mainly including black barley, purple barley, blue barley and so on. Colored highland barley is mainly enriched with a large amount of anthocyanins, and the i...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12P19/60C12N9/10C12N15/70C12N15/84A01H5/00A01H6/82
CPCC12N9/1007C12P19/60C12N15/70C12N15/8205Y02A50/30
Inventor 羊海珍许从萍王玉林顿珠加布危文波甘雅文高利云于明寨德青卓嘎原红军徐齐君卓嘎巴桑玉珍曾兴权
Owner AGRI RES INST TIBET ACADEMY OF AGRI & ANIMAL HUSBANDRY SCI
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