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Method for creating wheat seedling drought premature senility physiological phenotype by using cell autophagy inhibitor

An autophagy inhibitor, wheat technology, applied in the field of botany, can solve the problems of long time, cumbersome operation steps, high experimental cost, and achieve the effect of easy operation and observation, and low experimental cost.

Inactive Publication Date: 2021-01-05
CROP RES INST SHANDONG ACAD OF AGRI SCI
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AI Technical Summary

Problems solved by technology

Although these methods are accurate and mature, the experimental operation steps are relatively cumbersome, the cost of the experiment is high, the period of obtaining the desired plant phenotype is relatively long, and the success rate is relatively low
For example, virus-mediated gene silencing technology requires 2 weeks from the construction of viral vectors to infection and observation of phenotypic characteristics, while gene interference or gene editing requires the construction of interference vectors or gene editing vectors, and then genetic transformation to obtain positive results Plant seedlings need at least 3 months, but not every experiment can be successful, the success rate is low and time-consuming

Method used

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  • Method for creating wheat seedling drought premature senility physiological phenotype by using cell autophagy inhibitor
  • Method for creating wheat seedling drought premature senility physiological phenotype by using cell autophagy inhibitor

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Effect test

Embodiment 1

[0023] A method for creating the physiological phenotype of drought premature aging of wheat seedlings by using an autophagy inhibitor, the specific operation steps are as follows:

[0024] 1. Select wheat seeds with plump grains, the same size, and harvested in the same year, and place them in a constant temperature light incubator for 10 days; Set the culture temperature at 25°C, 16 hours of sunshine and 8 hours of darkness;

[0025] 2. 20 wheat seedlings with consistent growth are divided into treatment group and control group, each group of 10;

[0026] 3. The treatment group was placed in 20g / mL PEG-8000 solution, and 2mM, 5mM and 8mM 3-methyladenine were added as three treatment groups, while the control group was only placed in 20g / mL PEG-8000 solution. 8000 solution, the incubation time is 72 hours;

[0027] 4. After the experimental treatment, observe the differences in wheat plant phenotype between the treatment group and the control group.

Embodiment 2

[0029] A method for creating the physiological phenotype of drought and premature aging of wheat seedlings by using an autophagy inhibitor, and a method for determining the phenotypic difference, the specific detection method is:

[0030] 1. According to the conventional method, extract the total protein of the wheat leaves of the treatment group and the control group in Example 1 respectively.

[0031] 2. SDS-PAGE gel electrophoresis of the total protein of wheat leaves, the concentration of the used separating gel is 12%, the voltage is 100 volts, and the electrophoresis time is 1.5 hours.

[0032] 3. Perform Western blot detection on the protein in the above gel according to the existing method, use the polyclonal antibody of wheat ATG8 to identify the target protein in the wheat leaf in the gel, and detect the amount of ATG8-PE formed. The weaker the band, it means The more obvious the inhibitory effect is, the more obvious the drought effect is, and the physiological phen...

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Abstract

The invention belongs to the field of botany, and relates to a method for creating a wheat seedling drought premature senility physiological phenotype by using a cell autophagy inhibitor. The method inhibits the activity of phosphoinositide 3 phosphokinase (PI3K) necessary for formation of an autophagy vesicle membrane structure by using the cell autophagy inhibitor, and the time for creating thewheat seedling drought premature senility phenotype only needs 3 days. Through the method provided by the invention, the autophagy function of wheat cells under drought stress can be simply and quickly verified, and an important methodological basis is also provided for carrying out autophagy research on other plants under drought stress.

Description

technical field [0001] The invention belongs to the field of botany, and relates to a method for creating the physiological phenotype of drought premature aging of wheat seedlings by using a cell autophagy inhibitor. Background technique [0002] The purpose of creating the wheat drought premature senescence phenotype is to provide screening criteria for further exploring the molecular mechanism of autophagy regulating seedling premature senescence and mining excellent anti-aging genes. Numerous studies have shown that drought can induce autophagy in plants. When autophagy occurs, a large number of autophagic vacuoles that remove harmful substances will appear in the cells, and the expression of autophagy-related protein ATG8-PE will increase significantly, thereby improving the drought resistance of plants. At present, the method of blocking autophagy of plant cells under drought stress is mainly through the method of interfering or knocking out genes (ATG8 or ATG18) relat...

Claims

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Application Information

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IPC IPC(8): A01G22/20A01G7/06G01N33/68
CPCA01G7/06A01G22/20G01N33/68G01N2333/415
Inventor 李永波楚秀生崔德周樊庆琦隋新霞黄琛
Owner CROP RES INST SHANDONG ACAD OF AGRI SCI
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