Wheat Process-related Protein tapr1a Gene and Its Application in Wheat Resistance to Stripe Rust and Leaf Rust

A technology for wheat stripe rust and wheat leaves, applied in the fields of 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: 2022-06-24
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 Process-related Protein tapr1a Gene and Its Application in Wheat Resistance to Stripe Rust and Leaf Rust
  • Wheat Process-related Protein tapr1a Gene and Its Application in Wheat Resistance to Stripe Rust and Leaf Rust

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1. Cloning of wheat disease course-related protein TaPR1a gene

[0022] Wheat leaf RNA extraction: RNA extraction was performed using the RNA Extraction Kit (QIAGEN, Hilden, Germany). The second leaf leaf sample of the common wheat spring wheat material "JW1" at the seedling stage was quickly ground into powder with liquid nitrogen in a sterilized mortar, and was ready for use. To prepare the Buffer RLT mixture, add 10 μL of β-mercaptoethanol per ml of Buffer RLT, prepare and use now, and place on ice after mixing. Take out the ground RNA sample from liquid nitrogen, quickly add 500 μL of Buffer RLT mixture, shake well, and centrifuge at 10,000 g for 2 min. Transfer the supernatant to a purple spin column with a pipette and centrifuge at 10,000 g for 1 min. Transfer the collected liquid to the pink spin column, add pre-cooled absolute ethanol (the amount added is 1 / 2 of the collected liquid), invert and mix, and let stand to precipitate the nucleic acid. Imme...

Embodiment 2

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

[0031] Construction of wheat transgenic vector pLGY-02: The TaPR1a-T recombinant plasmid and the plasmid of pLGY-02 vector with correct sequencing were extracted, and double digested with restriction enzymes KpnI+SpeI. The specific digestion system is: plasmid 1.0μg, KpnI (15U / μL) 1.0μL, SpeI (10U / μL) 1.0μL, 10×Buffer 2.0μL, ddH 2 O supplemented to 20 μL. The digestion mixture was digested in a metal bath at 37°C for 3-5h. After the digestion products were detected by electrophoresis, the target gene fragment and the pLGY-02 vector fragment were recovered by gel and ligated. Ligation system: 12 μL of target fragment, 5 μL of pLGY-02 vector fragment, 1.0 μL of T4DNA ligase, 2.0 μL of T4DNA Ligasebuffer, ddH 2 O supplemented to 20 μL. Centrifuge to mix the reagents well, connect at 22°C for at least 1 h or overnight at 4°C. The ligation product was transformed into E. coli, tested by PCR, picke...

Embodiment 3

[0032] Example 3. Preparation of TaPR1a overexpressing wheat transgenic plants

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

[0034] Genomic DNA extraction by SDS method: sample and label; add 1 grinding bead to each tube, pre-cool with liquid nitrogen, balance and put it in the proofing machine, grind at 1100g for 1min, take it out and put it in 600μL Extraction buffer (100mL 0.1M Tris-HCl pH= 7.5, 100mL of 0.5M EDTA pH=8.0, 125mL of 10% SDS) after shaking, put it in a water bath at 65°C for 30min; take it out and put it on ice for 15min to cool to room temperature, add 300μL of 6MAmmonium Acetate (ammonium acetate), mix well, put it in 4 ℃ refrigerator for 15 minutes, centrifuged at 12000g for 15 minutes; ta...

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Abstract

The invention provides a wheat disease process-related protein TaPR1a gene and its application in wheat resistance to stripe rust and leaf rust. The invention relates to a gene sequence, and specifically discloses a wheat disease process-related protein TaPR1a gene, the nucleotide sequence of which is shown in SEQ ID No.1, and a preparation process of a wheat transgenic material overexpressing the TaPR1a gene. The invention verifies through experiments 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 biological genetic engineering, and relates to a wheat disease course-related protein TaPR1a gene and its application in resistance to stripe rust and leaf rust of wheat. Background technique [0002] As the main food crop, the quality and output of common wheat seriously affect my country's food security and social stability. The high and stable yield of wheat is of great significance to my country's agricultural development. 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, which seriously affects the food quality and safety in my country. Both the pathogens of wheat...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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