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Wheat UDP-glucosyltransferase TaUGT6 and application thereof

A technology of glucosyl and transferase, applied in the field of plant genetic engineering, can solve problems such as unreported

Active Publication Date: 2019-06-18
JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Wheat UDP-glucosyltransferase TaUGT6 is a member of wheat glycosyltransferase family 1, the gene sequence has been published with the wheat genome sequence, but the use of this gene, especially in the degradation of DON toxin and the improvement of plant DON tolerance Applications in unreported

Method used

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  • Wheat UDP-glucosyltransferase TaUGT6 and application thereof
  • Wheat UDP-glucosyltransferase TaUGT6 and application thereof
  • Wheat UDP-glucosyltransferase TaUGT6 and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Cloning of embodiment 1 wheat UDP-glucosyltransferase gene TaUGT6

[0029] The spikelet RNA of the wheat variety Sumai No. 3 at the heading stage was extracted using the SV Total RNA Isolation System kit (Promega, product number: Z3100), and then the cDNA was obtained using the reverse transcription kit (TAKARA company, product number: 6210A).

[0030] The cDNA sequence of the TaUGT6 gene was obtained from the public website http: / / plants.ensembl.org / . Design and synthesize primers for amplifying the TaUGT6 gene, the forward primer is 5'-CGGGATCCATGGCTGTCCACGACGAGCC-3' (its nucleotide sequence is shown in SEQ ID NO.3), and the reverse primer is 5'-CGGAATTCGCTGGCCTGGATGTCTTGGC-3' ( Its nucleotide sequence is shown in SEQ ID NO.4). Using the primer pair and high-fidelity Mix (Novazyme, Cat. No.: P511), PCR amplification was performed using cDNA as a template.

[0031] The PCR reaction system is 2× Master Mix 25 μl, cDNA template 2 μl, SEQ ID NO.3 and SEQ ID NO.4 prim...

Embodiment 2

[0033] Example 2 Application of wheat UDP-glucosyltransferase TaUGT6 in the degradation of DON toxin

[0034] 1. Construction of prokaryotic expression vector of TaUGT6

[0035] Digest the T-TaUGT6 plasmid obtained in Example 1 with BamH I (NEB, Cat. No. R3136) and EcoR I (NEB, Cat. No. R3101), and recover the target gene fragment; The prokaryotic expression vector pGEX-4T-1 was used to recover the vector fragment. Connect the target gene fragment into the above-mentioned prokaryotic expression vector, and obtain the prokaryotic expression vector pGEX-TaUGT6 of TaUGT6, and verify the pGEX-TaUGT6 vector with BamH Ⅰ and EcoR I, as shown in figure 2 shown.

[0036] 2. Transform Escherichia coli, induce protein expression and protein purification

[0037] The pGEX-TaUGT6 plasmid was transformed into Escherichia coli BL21(DE3) (Quan Shi Jin Company, catalog number: CD601). Add the BL21 bacterial liquid containing the positive clone to the LB liquid medium containing Amp at a rat...

Embodiment 3

[0045] Example 3 Application of Wheat UDP-glucosyltransferase TaUGT6 in Degrading Toxin Content in DON Contaminated Grain or Whole Wheat Flour

[0046] Wheat UDP-glucosyltransferase TaUGT6 enzyme solution (2ml) preparation: the recombinant protein 20 μ g that embodiment 2 step 2 obtains, the UDP-glucose that final content is 5mM, the 2-mercaptoethanol that final content is 14mM, Tris-HCl (pH =7.0) to make up to 2ml.

[0047] In the above enzyme solution, 2-mercaptoethanol can protect the activity of the enzyme; Tris-HCl can maintain a relatively stable enzymatic reaction environment.

[0048] Wheat UDP-glucosyltransferase TaUGT6 enzyme solution (protein concentration 10 μg / ml) was sprayed on wheat grains contaminated by scab ( Figure 5 A) or whole wheat flour ( Figure 5 C), place it at 30°C for 4 hours, and use the vomitoxin ELISA detection kit (Beijing Huaan Maike Co., Ltd., article number: HEM0896 / HEM0848), and quickly detect the DON content according to the product inst...

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Abstract

The invention discloses wheat UDP-glucosyltransferase TaUGT6 and an application thereof to DON (deoxynivalenol) degradation and improvement of DON tolerance of plants. The nucleotide sequence of the wheat UDP-glucosyltransferase TaUGT6 is represented as SEQ ID NO.1, and the protein sequence encoded by the wheat UDP-glucosyltransferase TaUGT6 is represented as SEQ ID NO.2; an in-vitro enzyme activity experiment proves that the protein can transform DON into D3G; DON content can be remarkably reduced by spraying the protein to highly-polluted wheat grains and whole wheat flour; with the adoptionof an agrobacterium mediated genetic transformation method, the gene is excessively expressed in arabidopsis, endurance capacity of transgenic arabidopsis for DON is improved, and the new applicationof the enzyme to DON degradation and resistance of plants to DON accumulation and fusarium head blight is proved.

Description

technical field [0001] The invention relates to the application of wheat UDP-glucosyltransferase gene, especially the application of the gene in the degradation of DON toxin and the improvement of plant DON tolerance, belonging to the technical field of plant genetic engineering. Background technique [0002] Wheat head blight (Fusarium head blight, FHB) is a major fungal disease caused by Fusarium graminearum as the main pathogen, and it is also one of the main diseases that endanger the development of my country's wheat industry. In recent years, due to climate warming and changes in farming systems, the incidence of wheat scab in my country has continued to expand. At present, the frequent occurrence area has expanded to the southern Huanghuai wheat region, and the occurrence of the disease is also obvious in the northern Huanghuai, southwest, and northwest wheat regions. aggravated. Fusarium scab can lead to a decline in wheat yield, generally causing a 10%-15% yield red...

Claims

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Application Information

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IPC IPC(8): C12N15/54C12N9/10C12N15/70C12N15/82A23L5/20A01H5/00A01H6/20
Inventor 何漪马鸿翔张旭吴磊姜朋刘祥
Owner JIANGSU ACADEMY OF AGRICULTURAL SCIENCES
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