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Protein kinase HvMPK4a related to barley powdery mildew resistance and encoding gene and application of protein kinase HvMPK4a

A protein kinase, powdery mildew resistance technology, applied in application, genetic engineering, plant genetic improvement and other directions, can solve problems such as phytotoxicity, affecting crop yield and quality, and achieve the effect of enhancing resistance, improving and improving germplasm resources

Inactive Publication Date: 2016-07-27
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Triazole fungicides not only have a bactericidal effect, but also have a plant growth regulating effect, so phytotoxicity often occurs during use, affecting the yield and quality of crops
When pathogenic bacteria invade, PAMPs from pathogenic bacteria are recognized by plant cell surface receptors PRRs, activate the MAPK cascade reaction, phosphorylate downstream substrate molecules, and initiate PTI (Chisholmetal., 2006; Tena et al., 2011), while MAPK in barley The pathway has not been studied, and there are few reports on the application of MAPK signaling pathway to control barley fungal diseases

Method used

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  • Protein kinase HvMPK4a related to barley powdery mildew resistance and encoding gene and application of protein kinase HvMPK4a
  • Protein kinase HvMPK4a related to barley powdery mildew resistance and encoding gene and application of protein kinase HvMPK4a
  • Protein kinase HvMPK4a related to barley powdery mildew resistance and encoding gene and application of protein kinase HvMPK4a

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] Example 1 Expression profile analysis of HvMPK4a during the infection of barley by Erysipha hordei

[0054] 1. Preparation of plant material.

[0055] After the seeds of barley variety P01 are sown, wait for about 1 week, cut off the flag leaves, and place them on a petri dish containing a medium (1% agar, 100mg / L bemimidazole), and recover for 24 hours. Physiological race K1 or A6 of powdery mildew of barley were inoculated respectively, and leaf materials were collected at different time points after inoculation.

[0056] 2. Extraction of plant total RNA.

[0057] Grind 300 mg of barley leaves quickly in liquid nitrogen to powder, add 1 mL of TRizol, shake well and centrifuge at 12,000 rpm at 4°C for 10 minutes; transfer the supernatant to a new centrifuge tube, add 1 / 5 of the volume of TRizol in chloroform, and mix well at room temperature Let it stand for 3 minutes to wait for its separation, and then centrifuge at 12000g for 15 minutes at 4°C. Transfer the super...

Embodiment 2

[0065] Cloning of embodiment 2 barley HvMPK4a gene and its localization analysis in barley cells

[0066] 1. Cloning of HvMPK4a gene.

[0067] The 7-day-old seedling leaves of barley variety GP were taken, RNA was extracted, and cDNA was obtained by reverse transcription. The reverse transcription system and process are as follows: RNA 2 μg, Oligo-dT 1 μL, add DEPC-H 2From 0 to 12 μL, mix the above solution in a centrifuge tube, denature at 70°C for 10 minutes, then cool on ice for 2 minutes, then add 2 μL of M-MLVBuffer, 1 μL of dNTPmix, 0.5 μL of RNase inhibitor, 1 μL of M-MLV, and add DEPC-H to the centrifuge tube 2 O to 20 μL, incubate at 42°C for 1 hour, and incubate at 70°C for 15 minutes to obtain reverse-transcribed cDNA.

[0068] The primers required for the PCR amplification of the cloned HvMPK4a gene are as follows:

[0069] Forward: 5'-ATGGACACCTCCGGCGGCGGCGGC-3';

[0070] Reverse: 5'-GTAGGGCGGATCAGGGTTAAAT-3'.

[0071] The PCR reaction system is: KODbuffer 5μ...

Embodiment 3

[0083] Example 3 Overexpression of HvMPK4a in Barley Epidermis Cells Resistance to Barley Powdery Mildew

[0084] 1. Carrier construction.

[0085] The full length of the above-mentioned HvMPK4a gene was constructed into the pUBI-Adaptor-NOS vector by enzyme digestion and ligation to generate pUbi-HvMPK4a.

[0086] 2. Transient overexpression of pUbi-HvMPK4a mediated by particle gun.

[0087] Gold powder was prepared as indicated above. Mix the plasmid DNA and the GUS reporter gene expression plasmid pUbi-GUS equimolarly, add deionized water to 5 μL; add the plasmid DNA mixture to 50 μL gold powder, and add 50 μL 2.5 M CaCl dropwise while shaking 2 , and then quickly add 20 μL 0.1M spermidine, and continue shaking for 3 minutes. Let stand for 1 minute to settle, centrifuge for 2 seconds, discard the supernatant, wash with 140 μL 70% ethanol and 140 μL 100% ethanol respectively, then add 12 μL 100% ethanol and shake and mix well.

[0088] 3. Inoculate powdery mildew and cou...

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Abstract

The invention relates to the field of molecular biology and molecular breeding, and in particular relates to protein kinase HvMPK4a related to barley powdery mildew resistance and an encoding gene and an application of the protein kinase HvMPK4a. The protein kinase HvMPK4a related to barley powdery mildew resistance is protein formed by an amino acid sequence shown in SEQ ID No.1. Through analysis on expression profiles of the HvMPK4a, the expression quantity of the HvMPK4a in the infection process of powdery mildew on barley significantly ascends, which indicates that the HvMPK4a participates into the process that the barley prevents infection of the powdery mildew and plays the biological function. Through transient overexpression on the HvMPK4a and inoculation of non-affinity powdery mildew through barley epidermal cells, the resistance of the barley on the powdery mildew is significantly reduced; the HvMPK4a is silenced in the barley and affinitive powdery mildew is inoculated through virus-induced gene silencing, so that the resistance of the barley on the powdery mildew is enhanced; and the resistance of the barley on the powdery mildew is negatively regulated and controlled by the HvMPK4a.

Description

technical field [0001] The invention relates to the field of molecular biology and molecular breeding, in particular to protein kinase HvMPK4a related to barley powdery mildew resistance, its coding gene and application. Background technique [0002] Barley is one of the main food crops widely grown in the world, and its sown area in the world is second only to wheat, rice and corn, ranking fourth. Barley can be used as raw material for food, feed and beer brewing, and has important economic value. Barley powdery mildew (barleypowderymildew) is one of the main fungal diseases of barley, which is caused by Blumeria graminis f.sp.hordei. The pathogenic fungus belongs to the order Ascomycetes Erysiphales, and it mainly lives obligately in the epidermis cells of the host barley leaves. It can invade the above-ground organs of the barley plant, but mainly the leaves and leaf sheaths. When the disease is severe, the barley stems and ears Department is also violated ( 1994, Cri...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/12C12N15/54C12N15/11
Inventor 薛朋娅沈前华张玲韩新运
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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