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Tartary buckwheat sourced emodin glycosyltransferase as well as coding gene and application thereof

A technology of glycosyltransferase and emodin, applied in the direction of transferase, application, genetic engineering, etc., can solve the problems of unclear and blank metabolic mechanism

Pending Publication Date: 2022-03-01
INST OF CROP SCI CHINESE ACAD OF AGRI SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the metabolic mechanism of each anthraquinone compound such as emodin in tartary buckwheat is still unclear, especially the glycosylation reaction of anthraquinone such as emodin in buckwheat plants is still almost blank
In addition, so far there is no report on the specific 8-OH glucosylation reaction of anthraquinones such as emodin in buckwheat and its functional application

Method used

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  • Tartary buckwheat sourced emodin glycosyltransferase as well as coding gene and application thereof
  • Tartary buckwheat sourced emodin glycosyltransferase as well as coding gene and application thereof
  • Tartary buckwheat sourced emodin glycosyltransferase as well as coding gene and application thereof

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Experimental program
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Effect test

Embodiment 1

[0042] Example 1 Cloning of FtUGT74L2 CDS

[0043] The present embodiment adopts the method of gene cloning to clone and obtain the glycosyltransferase FtUGT74L2 gene from tartary buckwheat (Pinku No. 1) sterile seedling and carry out gene sequence information analysis, specifically as follows:

[0044] Select two-week-old seedlings of Pinku No. 1, take 50-100 mg of the seedlings, add liquid nitrogen to fully grind, and use the Trizol method to extract total RNA. Using this RNA as a template, use III 1st Strand cDNA Synthesis Kit (+gDNAwiper) kit (Nanjing Novizan Biotechnology Co., Ltd.) was used for reverse transcription to obtain the cDNA of the seedlings.

[0045] Design specific primers according to the ORF of FtUGT74L2:

[0046] FtUGT74L2-F: 5'-ATGAAGGTAGTTCCCGGG-3',

[0047] FtUGT74L2-R: 5'-AGCCCTTAACTCCTTCACAA-3';

[0048] PCR amplification was carried out using Pinku 1 cDNA as a template to obtain the CDS sequence of the target gene:

[0049] The PCR program was ...

Embodiment 2

[0051] Example 2 Expression of FtUGT74L2 gene in tartary buckwheat hairy root and detection of expression level

[0052] The FtUGT74L2 gene is operably constructed in the expression control sequence to obtain the plant expression vector pCAMBIA 1302-FtUGT74L2 containing the FtUGT74L2 gene. Specifically, the construction method of the plant expression vector pCAMBIA 1302-FtUGT74L2 includes:

[0053] Homologous recombination primers were designed, and the full-length sequence of FtUGT74L2 was amplified by PCR using the FtUGT74L2-T vector as a template and OE-FtUGT74L2-F / R as primers.

[0054] Upstream primer: 1302-FtUGT74L2-NcoI-F:

[0055] 5'-ccatggATGAAGGTAGTTCCCGGG-3'

[0056] Downstream primer: 1302-FtUGT74L2-BglII-R:

[0057] 5'-agatctAGCCCTTAACTCCTTCACAA-3'.

[0058] After digestion, recovery and ligation transformation, the full-length sequence of FtUGT74L2 was inserted forward into the downstream of the CaMV35S promoter of the pCAMBIA-1302 vector, and the overexpressi...

Embodiment 3

[0063] Amino acid sequence analysis and comparison of embodiment 3 emodin glycosyltransferase

[0064] Blast the amino acid sequence of emodin glycosyltransferase FtUGT74L2 gene in NCBI database to obtain the GTs protein sequence of other species, use Bioxm software to translate the amino acid sequence, and predict its protein size and isoelectric point information.

[0065]The length of the CDS of the gene of emodin glycosyltransferase FtUGT74L2 is 897bp (SEQ ID No.1), encoding 298 amino acids (SEQ ID No.2) with a protein molecular weight of 33.3KDa, and the isoelectric point (pI) of the encoded protein is 5.18 . Using NCBI's blast to analyze the conserved amino acid domain of FtUGT74L2 online, it was found that it has a glycosyltransferase (PSPG) domain, and the encoded protein belongs to the UGT-type glycosyltransferase ( image 3 a). The UGT protein sequences of other species were obtained by Blast comparison in the NCBI database, and the clustering tree was constructed ...

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Abstract

The invention discloses fagopyrum tataricum sourced emodin glycosyltransferase as well as a coding gene and application thereof. According to the invention, a novel glycosyl transferase gene in a tartary buckwheat anthraquinone synthetic route is cloned from tartary buckwheat; a genetic engineering means is further utilized, a buckwheat explant is subjected to genetic transformation through the glycosyl transferase coding gene sequence, and an overexpressed transgenic buckwheat hairy root is obtained; the recombinant emodin glycosyl transferase protein is obtained through prokaryotic expression, and in-vitro enzyme activity detection and catalytic verification show that the glycosyl transferase can efficiently convert anthraquinone compound emodin into corresponding glycoside. By applying the glucosyltransferase provided by the invention, a large amount of emodin corresponding glucoside can be synthesized through an in-vitro bioengineering method, a new method is provided for commercially producing and utilizing emodin and glucoside compounds thereof, and the method is reliable in effect, low in cost, efficient in production process, green, safe and free of environmental pollution.

Description

technical field [0001] The present invention relates to glycosyltransferase and its encoding gene, especially relates to the emodin glycosyltransferase and its encoding gene isolated from tartary buckwheat, the present invention further relates to their 8-OH position of anthraquinone substance The application in glycoside catalysis belongs to the field of emodin glycosyltransferase and its coding gene and application. Background technique [0002] Tartary buckwheat (Fagopyrum tataricum) belongs to the buckwheat crop of the dicotyledonous Polygonaceae (Polygonaceae), and tartary buckwheat is rich in secondary metabolites, such as flavonoids (rutin, quercetin and isoquercetin, etc.), inositol and Anthraquinones. Among them, anthraquinone compounds have antitumor, antibacterial, antioxidative, diuretic and hemostatic functions. Anthraquinones in natural medicines mainly exist in the free form and in the form of binding to glycosides, and often exist in the form of glycosides ...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/82C12P19/56A01H5/00A01H6/00
CPCC12N9/1051C12N15/8243C12P19/56
Inventor 张凯旋周美亮赵辉范昱丁梦琦胡永平
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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