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Cultivating method of disease-resistant TaM2K transgenic wheat, and related biological materials of cultivating method

A technology of transgenic plants and disease resistance, applied in genetic engineering, plant genetic improvement, botany equipment and methods, etc., can solve problems such as lack and slow progress

Active Publication Date: 2020-03-27
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, progress in conventional breeding methods for wheat breeding for sheath blight resistance has been slow due to the lack of readily available wheat germplasm resources for sheath blight resistance

Method used

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  • Cultivating method of disease-resistant TaM2K transgenic wheat, and related biological materials of cultivating method
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  • Cultivating method of disease-resistant TaM2K transgenic wheat, and related biological materials of cultivating method

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Experimental program
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preparation example Construction

[0073] The preparation method of bacterial wheat kernels: soak the wheat kernels for 5-6 hours, boil them for 20 minutes, fill up the triangular flask, inoculate the mycelia block of the pathogenic bacteria of wheat sheath blight into the triangular flask after sterilization, and cultivate at a constant temperature of 25°C until The mycelia are densely covered with grains.

[0074] The preparation method of bacterial toothpick: put the toothpick segment upright and fill it with a small beaker, pour liquid MS medium into it, inoculate the mycelia block of wheat sheath blight pathogenic bacteria into the small beaker after sterilization, and cultivate it at a constant temperature of 25°C until the mycelia Densely strewn with toothpicks.

Embodiment 1

[0075] Embodiment 1, sheath blight resistant wheat kinase TaM2K and cloning of its coding gene

[0076] 1. Cloning of TaM2K gene

[0077] The inventors of the present invention isolated and cloned a wheat disease resistance-related protein from sheath blight resistant wheat CI12633, as shown in sequence 1 of the sequence listing, and named it TaM2K protein, which belongs to wheat kinase. The gene encoding TaMK protein is named TaM2K gene, and its cDNA is shown in sequence 2 of the sequence listing. In sequence 2 of the sequence listing, nucleotides 39-1145 are open reading frames.

[0078] Specific cloning steps: extract the total RNA from the stem of wheat CI12633 inoculated with the pathogenic bacteria of wheat sheath blight, and reverse-transcribe the RNA to synthesize the first-strand cDNA according to the procedure of the first-strand cDNA synthesis kit of Tiangen Biochemical Company, as The template for gene cloning was amplified with primers TaM2K-OF: 5'-GGAGCCAGCCTCG...

Embodiment 2

[0093] Embodiment 2, acquisition and disease resistance identification of transgenic TaM2K wheat resistant to sheath blight

[0094] 1. Construction of recombinant expression vector

[0095] 1. Extract the total RNA from the stem of wheat CI 12633 and reverse transcribe to obtain cDNA.

[0096] 2. Using the cDNA obtained in step 1 as a template, using a primer pair composed of TaM2K-O-F1 and TaM2K-O-R1, carry out PCR amplification under the action of the high-fidelity amplification enzyme PrimeSTAR, and recover the PCR amplification product.

[0097] TaM2K-O-F1: 5'-cG GGATCC ATGCGTCCGGGCGGGCCG-3';

[0098] TaM2K-O-R1: 5'-c GAGCTC gtggtggtggtggtggtgGCACGGCGGGGCGGCGAGGG-3'.

[0099] PCR reaction program: 94°C pre-denaturation for 5 minutes; 35 cycles of 98°C for 10s, 60.8°C for 30s, 68°C for 1.5min; 68°C for 10min.

[0100] 3. Take the PCR amplification product obtained in step 2, perform double digestion with restriction endonucleases BamHI and SacI, and recover the dige...

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Abstract

The invention discloses a cultivating method of disease-resistant TaM2K transgenic wheat, and related biological materials of the cultivating method. The invention first protects an important kinase resistant to sheath blight from wheat, and the kinase is named as TaM2K protein, and is a protein composed of an amino acid sequence shown in a sequence 1 or sequence 3 in a sequence listing. A nucleicacid molecule encoding the TaM2K protein also belongs to the protection scope of the invention. The invention also protects a method for cultivating transgenic plants. The method comprises the following steps: introducing a TaM2K gene into a target plant to obtain a transgenic plant with higher disease resistance than the target plant. The method for cultivating transgenic plants with improved disease resistance has important theoretical and practical significance, and can play an important role in genetic improvement of plants.

Description

technical field [0001] The invention relates to a method for cultivating disease-resistant transgenic TaM2K gene wheat and related biological materials. Background technique [0002] More than 40% of the world's population uses wheat as a staple food crop, so wheat plays a pivotal role in ensuring food security. With the increase of planting density, changes in fertilizer and water conditions and farming systems, wheat sheath blight has developed into the largest soil-borne disease in my country's wheat production, and has become an important limiting factor for high and stable wheat yields in my country. [0003] Wheat sheath blight, also known as wheat sharp eyespot. Wheat sheath blight in my country is mainly caused by the saprotrophic fungus Rhizoctonia cerealis. Sheath blight can generally reduce wheat production by 10% to 30%, and severe plots can reduce wheat production by more than 50%. According to the report of the National Agricultural Technology Promotion Stat...

Claims

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Application Information

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IPC IPC(8): C12N9/12C12N15/54C12N15/82A01H5/00A01H6/46
CPCC12N9/12C12N15/8282
Inventor 张增艳王开
Owner INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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