GSTs activity analysis method suitable for plant holoprotein extracting solution

A technology for activity analysis and extraction, which is applied in the field of biochemistry, can solve the problems of low solubility, low operability, and cover up enzyme-catalyzed reactions, so as to avoid protein denaturation and improve solubility

Inactive Publication Date: 2016-05-25
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In fact, the solubility of CDNB in ​​ethanol is lower, and it is more difficult to dissolve in ethanol / water solution, so the amount of CDNB used in some literatures is actually difficult to achieve, and adding more ethanol will cause protein denaturation; in addition, high CDNB and GSH The background reaction is higher when the content is higher, which will cover the enzyme-catalyzed reaction; there are also some methods that require further manipulation of the whole protein extract to increase the protein content of GSTs
These methods are less practical in experiments

Method used

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  • GSTs activity analysis method suitable for plant holoprotein extracting solution
  • GSTs activity analysis method suitable for plant holoprotein extracting solution
  • GSTs activity analysis method suitable for plant holoprotein extracting solution

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Application of Analytical Method for Comparing GSTs Activity in Tobacco Root and Leaf

[0022] Preparation of reagents: Weigh 0.20255 g of CDNB (molecular weight: 202.55) and dissolve it in 1 ml of acetone to prepare 1 MCDNB solution, and freeze it at -20°C; weigh 0.61466 g of reduced glutathione (GSH, molecular weight: 307.33) and dissolve it in 10ml of the extract was prepared into a 200mM GSH solution, each 1ml portion was frozen at -20°C, and one portion was taken out and thawed on ice before use.

[0023] Preparation of materials: Tobacco leaves and roots grown to the age of 6 were used as materials, and the third and fourth leaves that were fully expanded were collected and used after weighing; the soil at the roots was washed with clean water, dried with filter paper, and weighed until use.

[0024] Preparation of whole protein extract (crude extract): Use phosphate buffer solution (without urea) with a pH of 7.5 to add to tobacco leaves or roots at a ratio of 1...

Embodiment 2

[0039] Application of GSTs Activity Analysis Method in Screening Excellent Plants with High GSTs

[0040] The leaves and roots of Dongnong 248, Dongnong 9702, Dongnong Wannian, and Dongtian 3 corn varieties were used as samples, and the extract was used as a control sample, and GSH and CDNB were used as reaction substrates to determine the GS-CDNB pair. The amount of co-products was used to analyze the activity of GSTs. Specific steps are as follows:

[0041] Reagent preparation reference example 1.

[0042] Preparation of materials: Sow several kinds of corn seeds in water culture seedling trays, and when they reach the age of 3 leaves, collect all the leaves and weigh them for later use; cut off the roots and wash them 3 times in clean water, dry them with filter paper and weigh them .

[0043] The preparation of whole protein crude extract refers to Example 1.

[0044] Refer to Example 1 for enzyme activity detection and activity calculation.

[0045] Experimental resu...

Embodiment 3

[0047] Application of GSTs Activity Analysis Method in Screening Herbicides

[0048]Tobacco was sprayed with different tobacco herbicides to be screened, and the leaves and roots were taken as samples. At the same time, the tobacco leaves and roots without herbicide were used as control samples, and GSH and CDNB were used as reaction substrates to determine the GS-CDNB coupling. The amount of product was used to analyze the activity of GSTs. Specific steps are as follows:

[0049] Reagent preparation reference example 1.

[0050] Material preparation:

[0051] Refer to Example 1 for enzyme activity detection and activity calculation.

[0052] Purchase the commonly used herbicides napropamide and paraquat in tobacco agriculture from the market; when the soil-cultivated tobacco (Nicotianatobacum L.) in the laboratory grows to the age of 6 leaves, spray it on the soil surface of the planted tobacco according to the dosage recommended in the application instructions Or on the ...

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Abstract

The invention discloses a GSTs activity analysis method suitable for a plant holoprotein extracting solution. According to the method, the holoprotein extracting solution of leaves and roots of a plant is used as a sample and a check sample, GSH and CDNB are used as reaction substrates, and the activity of GSTs is analyzed by measuring the quantity of GS-CDNB coupling products. According to the improved method, the solvent of CDNB is changed into acetone from ethyl alcohol, the solubility of CDNB is greatly improved, the use quantity of the solvent is lowered under the same CDNB concentration, and therefore protein degeneration caused by an excessive quantity of the solvent is avoided; meanwhile, the molar ratio of glutathione (GSH) to CDNB is increased through the improved method, and the plant holoprotein extracting solution with low GSTs content can be analyzed more conveniently.

Description

technical field [0001] The invention belongs to the technical field of biochemistry, and in particular relates to a method for analyzing the GSTs activity of plant whole protein extracts. Background technique [0002] Glutathione transferases (GSTs) have a variety of catalytic activities: reduced glutathione (GSH) can be covalently coupled to non-polar compounds containing electrophilic carbon, nitrogen, and sulfur atoms, so that GSTs As a central component, it participates in the metabolic detoxification process of drugs, pesticides and other heterologous substances; GSTs can also catalyze and reduce some metabolites of oxidative stress, such as peroxides, so that GSTs can defend against bacterial infection and abiotic stress play an important role in. Therefore, GSTs activity is used as an important indicator to evaluate the growth status and stress response characteristics of plants in many plant studies. [0003] The analysis method of GSTs activity is mainly based on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48
CPCC12Q1/48
Inventor 周会娜陈霞翟妞刘萍萍陈千思郑庆霞张慧徐国云林福呈
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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