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Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance

A technology related to protein and wheat, applied in application, genetic engineering, plant genetic improvement, etc., can solve the problems of easy loss of resistance and high mutation frequency, and achieve the effect of improving disease resistance

Active Publication Date: 2020-09-15
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Both wheat stripe rust and leaf rust pathogens have the characteristics of high mutation frequency, and their physiological races can complete multiple mutations in a short period of time, which makes it easy for single-resistant wheat varieties to lose resistance in a short period of time

Method used

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  • Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance
  • Wheat disease course related protein TaPR1a gene and application thereof in wheat stripe rust and leaf rust resistance

Examples

Experimental program
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Effect test

Embodiment 1

[0021] Embodiment 1, cloning of wheat disease process-related protein TaPR1a gene

[0022] Wheat leaf RNA extraction: RNA extraction was performed using the RNA Extraction Kit (QIAGEN, Hilden, Germany). The second leaf sample of common wheat spring wheat material "JW1" at the seedling stage was rapidly ground into powder in a sterilized mortar with liquid nitrogen, and then used. Prepare the Buffer RLT mixture, add 10 μL β-mercaptoethanol to each milliliter of Buffer RLT, mix it and place it on ice. Take out the ground RNA sample from liquid nitrogen, quickly add 500 μL of Buffer RLT mixture, shake fully, and centrifuge at 10,000 g for 2 min. Use a pipette gun to draw the supernatant, transfer it to a purple spin column, and centrifuge at 10,000 g for 1 min. Transfer the collected liquid into a pink spin column, add pre-cooled anhydrous ethanol (the amount added is 1 / 2 of the collected liquid), invert and mix well, and let stand to precipitate the nucleic acid. Centrifuge f...

Embodiment 2

[0030] Example 2. Construction of TaPR1a gene wheat transgenic vector pLGY-02.

[0031] Construction of the wheat transgenic vector pLGY-02: extract the correctly sequenced TaPR1a-T recombinant plasmid and the plasmid of the pLGY-02 vector, and perform double digestion with restriction endonucleases KpnI+SpeI. The specific digestion system is: plasmid 1.0 μg, KpnI (15U / μL) 1.0 μL, SpeI (10U / μL) 1.0 μL, 10×Buffer 2.0 μL, ddH 2 O to make up to 20 μL. The enzyme digestion mixture was digested in a metal bath at 37°C for 3-5 hours. After electrophoresis detection of the digested products, the target gene fragment and the pLGY-02 vector fragment were recovered from the gel and ligated. Ligation system: target fragment 12 μL, pLGY-02 vector fragment 5 μL, T4DNA ligase 1.0 μL, T4DNA Ligasebuffer 2.0 μL, ddH 2 O to make up to 20 μL. Centrifuge to mix the reagents thoroughly, and connect at 22°C for at least 1 hour or overnight in a refrigerator at 4°C. The ligated product was tra...

Embodiment 3

[0032] Embodiment 3, the preparation of TaPR1a overexpression wheat transgenic plant

[0033] The preparation of Agrobacterium-mediated wheat transgenic materials was completed by Shandong Jinan Bangdi Biological Co., Ltd. The background material for the transformation was common wheat spring wheat material "JW1", and the method of Agrobacterium-mediated transformation of young wheat embryos was used.

[0034] Genomic DNA extraction by SDS method: sample and label; add 1 polishing bead to each tube, pre-cool it with liquid nitrogen, put it in the proofer, grind it with 1100g for 1min, take it out and put it into 600μL Extraction buffer (100mL 0.1M Tris-HClpH= 7.5, 100mL 0.5M EDTA (pH = 8.0, 125mL 10% SDS) were shaken and placed in a water bath at 65°C for 30 minutes; took it out and put it on ice for 15 minutes to cool to room temperature, then added 300 μL of 6MAmmonium Acetate (ammonium acetate), mixed evenly, and placed in 4 Refrigerate at ℃ for 15 minutes, centrifuge at 12...

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Abstract

The invention provides a wheat disease course related protein TaPR1a gene and an application thereof in wheat stripe rust and leaf rust resistance. The invention relates to a genetic sequence, and particularly discloses the wheat pathogenesis related protein TaPR1a gene with a nucleotide sequence as shown in SEQ ID No.1, and a preparation process of a wheat transgenic material for overexpressing the TaPR1a gene. Experiments prove that the TaPR1a gene can significantly improve the resistance level of wheat to wheat stripe rust and wheat leaf rust.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and relates to a wheat disease process-related protein TaPR1a gene and its application in wheat resistance to stripe rust and leaf rust. Background technique [0002] As the main food crop, the quality and output of common wheat seriously affect the food security and social stability of our country. The high and stable yield of wheat is of great significance to the agricultural development of our country. Wheat stripe rust and wheat leaf rust caused by Puccinia striiformis f.sp. tritici and Puccinia triticina respectively are important fungal diseases that seriously affect wheat production in my country. In recent years, due to the increase of planting density and the change of agricultural farming system, the occurrence of wheat stripe rust and leaf rust has become more and more serious, seriously affecting the grain quality and safety of our country. The pathogenic bacteria of w...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C12N15/10C12N15/11A01H5/00A01H6/46
CPCC07K14/415C12N15/8282
Inventor 王逍冬赵姣洁毕伟帅赵淑清苏君庞书勇于秀梅刘大群
Owner HEBEI AGRICULTURAL UNIV.
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