CN108593566A - The method for assessing corn variety drought tolerance based on nanogold SPR optical characteristics - Google Patents

Abstract

The invention discloses it is a kind of based on nanogold SPR optical characteristics assess corn variety drought tolerance method, including the Stress treatment of corn seedling, the preparation of maize leaf Aqueous extracts, the preparation of nanogold system, the detection of antioxidant activity, Drought Resistance in Maize assessment and etc..Drought tolerance of the present invention by Application of micron in corn variety is assessed, and applications to nanostructures field has been expanded；Compared with traditional Drought Resistance in Maize appraisal procedure detection defence enzyme activity, field experiment; this method utilizes the drought tolerance of corn total antioxidant activity assessment corn under stress conditions; have the characteristics that simple and easy to do, accurate quick, and method implementation process is free of toxicant, it is environmentally protective.

Description

Method for evaluating drought tolerance of maize varieties based on nano-gold SPR optical properties

technical field

The invention relates to a method for evaluating drought tolerance of maize varieties.

Background technique

Corn is the main food crop that is related to the national economy and people's livelihood in my country, and it is also a crop that is more sensitive to moisture. Drought is the main factor restricting the production and development of corn in my country. With the warming of the climate and the intensification of drought accompanied by the greenhouse effect, about 40% of the corn planting land in my country is affected by the drought every year and the yield is reduced by 20%-30%. Therefore, drought-tolerant maize varieties are becoming more and more important in agricultural production. At present, in addition to field experiments, the methods for evaluating the drought tolerance of maize varieties mainly focus on the detection of morphological indicators and protective enzyme activities, such as the detection of relative water content, superoxide dismutase SOD activity, peroxidase POD activity, etc. Although the results are reliable, the method is complicated and time-consuming. Therefore, it is of great significance to establish an accurate, fast and convenient method for evaluating drought tolerance of maize varieties.

When maize suffers from drought stress, a large number of active oxygen free radicals will be produced in the cells, causing the metabolic imbalance of active oxygen free radicals. The accumulated free radicals will attack biological macromolecules, cause DNA damage, affect the stability and synthesis of proteins, and lead to abnormal metabolism. Plants cause irreversible damage. Existing studies have shown that after drought-tolerant maize varieties are subjected to drought stress, their ability to regulate free radical metabolism is significantly higher than that of weak drought-tolerant varieties, and the degree of stress damage suffered is relatively light. It can be seen that the drought tolerance of maize is positively correlated with its ability to regulate free radical metabolism under stress conditions, and the total antioxidant activity of plants can reflect its ability to regulate free radical metabolism. Therefore, the total antioxidant activity of maize after drought stress can be detected. Activity assessment of drought tolerance in different maize cultivars.

Metal nanomaterials refer to metal materials that have at least one dimension in nanometer size (0.1-100 nm) in three-dimensional space or are composed of them as basic units. When light acts on metal nanomaterials, due to the collective oscillation of conduction band electrons, a strong surface plasmon resonance (SPR) absorption peak will appear in a specific wavelength range. The position and strength of the absorption peak depend on the size of the nanoparticle, Parameters such as shape, aggregation state, and composition. Based on this characteristic of metal nanomaterials, the antioxidant active substances in plant extracts have a reducing effect and can mediate the synthesis of nano-gold, and the total antioxidant activity of plants is detected according to the changes in SPR optical properties before and after the formation of nano-gold. At present, there have been a large number of related studies on the use of nanomaterials to detect the total antioxidant activity of plants, including tea, vegetables, fruit juices, and traditional Chinese medicines. No studies have been reported so far to evaluate it.

Contents of the invention

The purpose of the present invention is to provide a simple, accurate and fast method for evaluating the drought tolerance of corn varieties based on the SPR optical properties of nano-gold.

Technical solution of the present invention is:

A method for evaluating the drought tolerance of corn varieties based on nano-gold surface plasmon resonance optical properties is characterized in that it comprises the following steps:

(1) Stress treatment of corn seedlings: Select different varieties of corn seeds to carry out water cultivation in the turnover box, and perform drought stress treatment when the seedlings grow to the third leaf fully unfolded. The drought stress is simulated by PEG, and the Replace the nutrient solution with 18% PEG6000 to completely soak the root system of the plant for 4 hours;

(2) Preparation of water extract of corn leaves: dry the corn leaves collected after drought stress treatment in a constant temperature incubator at 55°C for 2 h, crush the dried corn leaves with a tissue crusher, and then use a Sieve the powder with a 40-mesh sieve of 4.5mm, and place the collected powder in a desiccator for later use; accurately weigh 0.05g of the above powder material into a 10mL centrifuge tube, add 8mL of distilled water, heat in a constant temperature water bath at 90°C for 15 minutes, and then cool naturally to room temperature. Add filter paper to filter in the funnel, centrifuge at 5000rpm/min for 10 min, take the supernatant to obtain 6.25g/L leaf water extract;

(3) Preparation of nano-gold system: Prepare 3.5×10 ^-4 M AuCl ₄ solution of chloroauric acid, 3.7×10 ^-3 M hexadecyltriethylammonium bromide solution, 2×10 ^-4 M sodium citrate solution and pH=8 phosphate buffer solution, in which hexadecyltriethylammonium bromide was incubated in a constant temperature water bath at 45°C for 15 minutes before use; take a 10mL test tube and add 3mL of Phosphate buffer, 100 μL of chloroauric acid, 600 μL of CTAB and 300 μL of sodium citrate solution, mixed thoroughly to obtain the nano-gold sol preparation system (volume 4 mL);

(4) Detection of antioxidant activity: Add 1 mL of water extract of corn leaves to the prepared nano-gold system, mix well, place in a constant temperature water bath at 35°C for 10 minutes, and test its UV-visible absorption after natural cooling to room temperature Spectrum; the antioxidant active substances in corn leaves have a reducing effect, and can reduce Au ⁴⁺ to Au ⁰ to form gold nanoparticles. Gold nanoparticles have obvious characteristic absorption peaks in the ultraviolet-visible spectrum. The antioxidant activity is positively correlated, so the total antioxidant activity of different varieties of corn leaf water extracts can be evaluated according to the spectral changes before and after adding the nano-gold system;

(5) Maize drought tolerance evaluation: The free radical metabolism regulation ability of maize under drought stress can represent the ability of this variety of maize to cope with stress damage, that is, the total antioxidant activity of maize after stress can reflect its drought tolerance. , so the total antioxidant activity of maize can be detected according to the change of the SPR optical properties of the formed gold nanoparticles, so as to evaluate the drought tolerance of maize.

Corn seedlings after stress treatment are used to prepare the corn leaf extract, and the part above the base of the first leaf of the seedlings is collected as material.

The advantages of the present invention are:

(1) Applying nanomaterials to the drought tolerance assessment of maize varieties expands the application field of nanomaterials.

(2) Compared with the traditional corn drought tolerance evaluation method of detecting protective enzyme activity and field experiments, this method uses the total antioxidant activity of corn under stress conditions to evaluate the drought tolerance of corn, which is simple, accurate and fast , and the implementation process of the method does not contain toxic substances, and is environmentally friendly.

Description of drawings

The present invention will be further described below in conjunction with drawings and embodiments.

Figure 1 is a schematic diagram of the entire process of corn seedling cultivation, drought stress treatment, and material collection.

Fig. 2 is the infrared spectrogram of Zhengdan 958 corn leaf water extract (6.25g/L) after drought stress treatment.

Figure 3 is the UV-Vis spectrum of water extracts (0.5g/L) of different varieties of corn leaves; among them: 1. Yuyu 22; 2. Zhengdan 958; 3. Denghai 662; 4. Jundan 20.

Figure 4 is the UV-Vis spectrum of Zhengdan 958 water extract (6.25g/L) before (water extract) and after (mixed solution) added to the nano-gold preparation system after drought stress treatment.

Figure 5 is a schematic diagram of TEM characterization of gold nanoparticles obtained by reacting Zhengdan 958 corn leaf water extract (6.25g/L) with chloroauric acid system after drought stress treatment.

Figure 6 is the UV-Vis spectrum of the nano-gold solution obtained by the reaction of Zhengdan 958 corn leaf extracts with different concentrations and the chloroauric acid system after drought stress treatment; where: e → a concentration of the aqueous extracts is 4.25 g/L in turn , 5.25 g/L, 6.25 g/L, 7.25 g/L, 8.25 g/L.

Fig. 7 is a graph showing the linear relationship between the addition concentration of Zhengdan 958 corn leaf water extract treated with drought stress and the UV-Vis spectral absorbance value of nano gold formed by the reaction system.

Fig. 8 is after the drought stress treatment, the UV-Vis spectrogram of different kinds of corn aqueous extracts after adding the chloroauric acid system; Wherein: 1, Zhengdan 958; 2, Jundan 20; 3, Denghai 662; 4, Yu Jade 22.

Fig. 9 is a photo of different varieties of corn when drought stress is continuously treated for 15 days; wherein: 1. Zhengdan 958; 2. Jundan 20; 3. Denghai 662; 4. Yuyu 22.

Detailed ways

The present invention includes but is not limited to the above embodiments, and any equivalent replacement or partial improvement made under the spirit and principles of the present invention will be considered within the protection scope of the present invention.

Example 1 Preparation of corn leaf water extract

Stress treatment of corn seedlings: Select different varieties of corn seeds and carry out water cultivation in the turnover box. When the seedlings grow to the third leaf fully unfolded, the drought stress treatment is carried out. The drought stress is simulated by PEG, and the nutrient solution in the turnover box is Replace with 18% PEG6000 to completely soak the root system of the plant for 4 hours;

Collect the part above the leaf base of the first leaf of corn seedlings treated with drought stress (see Figure 1), dry and crush with a tissue crusher, and sieve through a 40-mesh corn leaf powder (see Figure 1), weigh 0.05 Add 8mL of distilled water to a 10mL centrifuge tube, heat in a constant temperature water bath at 90°C for 15min, cool down to room temperature naturally, put filter paper in the funnel to filter and centrifuge at 5000rpm for 10min, the supernatant is corn with a concentration of 6.25g/L leaf extract. Pour the water extract into a 10ml small beaker and treat it in a freeze dryer for 4 days to remove moisture, take the dried sample solid, add a small amount of KBr in a mortar and grind it into powder, then use a tablet machine to tablet, and carry out infrared spectrum analysis ( See Figure 2); after diluting the concentration of the extract to 0.5g/L, detect the UV-visible UV-Vis spectrum of the corn leaf water extract itself (see Figure 3). The analysis of infrared spectrum and UV-Vis spectrum shows that the water extract of corn leaves contains polyphenols with antioxidant activity, and there is no characteristic absorption peak of nano-gold in the range of 400-800nm in the UV-Vis spectrum.

Example 2 Preparation of nano-gold sol preparation system

Prepare 3.5×10 ^-4 M chloroauric acid AuCl ₄ solution, 3.7×10 ^-3 M cetyltriethylammonium bromide CTAB solution, 2×10 ^-4 M sodium citrate solution and pH = 8 phosphate buffer, in which the CTAB solution needs to be incubated in a constant temperature water bath at 45°C for 15 minutes before the experiment. Take a 10mL test tube, add 3mL phosphate buffer solution with pH=8, 100μL chloroauric acid solution, 600μL CTAB and 300μL sodium citrate solution in sequence, and mix thoroughly to obtain the nano-gold sol preparation system solution (volume 4mL).

Example 3 Evaluation of Drought Tolerance of Maize Varieties

The first step is to detect the total antioxidant activity. Take two 10mL test tubes, first add the nano-gold preparation system (4mL) prepared in Example 2 respectively, then add 1mL of the corn leaf water extract prepared in Example 1 to one, and add 1mL of distilled water to the other as a blank , reacted in a constant temperature water bath at 35°C for 10 minutes, and after natural cooling to room temperature, the absorbance was detected by an ultraviolet-visible spectrophotometer. The greater the absorbance value, the stronger the total antioxidant activity of the variety of corn (see Figure 4). The second step is to evaluate the drought tolerance of maize. The ability to regulate free radical metabolism of maize after drought stress is positively correlated with its drought tolerance, that is, the higher the total antioxidant activity of maize varieties after drought stress, the stronger the drought tolerance. According to the test results of the total antioxidant activity, the Drought tolerance is assessed. After the corn water extract was added to the nano-gold system for mixed reaction, the color of the system became light pink, and the UV-Vis spectrum showed an absorption peak at around 650nm. The system was characterized by TEM transmission electron microscopy, which indicated the formation of nano-gold particles (see Figure 5).

Example 4 Detection of Total Antioxidant Activity of Corn Leaf Water Extracts at Different Concentrations

Get 5 10mL centrifuge tubes, add 0.034g, 0.042g, 0.05g, 0.058g, 0.066g corn leaf powder respectively and mark, prepare leaf water extract according to Example 1, the obtained concentration is 4.25 g/L, 5.25 g/L, respectively. g/L, 6.25 g/L, 7.25 g/L, 8.25 g/L, and then detect the total antioxidant activity by the nano-gold detection system, the method is the same as the first step in Example 3. The total antioxidant activity was evaluated according to the position and absorbance value of the UV-Vis spectrum absorption peak after the aqueous extracts of corn leaves with different concentrations were added to the nano-gold preparation system (see Figure 6). In the experiment, it was found that with the increase of the concentration of the corn leaf extract, the UV-visible absorption peak of the gold nanoparticles formed by the system increased, so as to establish the correlation between the concentration of the corn leaf water extract and the absorbance value, and found that the concentration and absorbance had a good relationship. Correlation, and a positive correlation, indicating that the total antioxidant activity of the sample increases with the concentration of the extract (see Figure 7).

Example 5 The Drought Tolerance Strength Evaluation of Different Varieties of Maize

The experiment selected four corn varieties Zhengdan 958, Jundan 20, Denghai 662 and Yuyu 22 suitable for planting in Henan Province. The research showed that Zhengdan 958 was drought-tolerant, Jundan 20 and Denghai 662 were moderately drought-tolerant, and Yuyu 22 Drought sensitive. Take 4 10mL test tubes, mark different corn varieties respectively, add the above-mentioned 4mL nano-gold detection system solution, add 1mL corresponding to the drought stress treatment, and obtain the water extract of corn leaves obtained by Example 1 according to the label, mix well and store at 35°C React in a constant temperature water bath for 10 minutes, let it stand naturally to room temperature, and detect the absorbance by a UV-visible spectrophotometer. From the ultraviolet-visible spectrum of the reaction between the water extracts of different varieties of corn leaves and the nano-gold preparation system (see Figure 8), it can be seen that the absorption peaks of different varieties of corn are different, indicating that the total antioxidant activity of corn varieties is different under drought stress. Arranged in descending order are Zhengdan 958, Jundan 20, Denghai 662, and Yuyu 22. According to the higher the total antioxidant activity under drought stress, the higher the drought tolerance of the variety, the drought tolerance of different maize varieties can be evaluated. The evaluation was consistent with the results of other maize drought tolerance tests and the actual drought tolerance performance of these maize varieties under continuous drought stress (see Figure 9).

Claims (2)

1. a kind of method based on the nanometer gold surface plasmon resonance optical property evaluation corn variety drought resistance, it is characterized in that: comprise the following steps: (1) Stress treatment of corn seedlings: Select different varieties of corn seeds to carry out water cultivation in the turnover box, and perform drought stress treatment when the seedlings grow to the third leaf fully unfolded. The drought stress is simulated by PEG, and the Replace the nutrient solution with 18% PEG6000 to completely soak the root system of the plant for 4 hours; (2) Preparation of water extract of corn leaves: dry the corn leaves collected after drought stress treatment in a constant temperature incubator at 55°C for 2 h, crush the dried corn leaves with a tissue crusher, and then use a Sieve the powder with a 40-mesh sieve of 4.5mm, and place the collected powder in a desiccator for later use; accurately weigh 0.05g of the above powder material into a 10mL centrifuge tube, add 8mL of distilled water, heat in a constant temperature water bath at 90°C for 15 minutes, and then cool naturally to room temperature. Add filter paper to filter in the funnel, centrifuge at 5000rpm/min for 10 min, take the supernatant to obtain 6.25g/L leaf water extract; (3) Preparation of nano-gold system: Prepare 3.5×10 ^-4 M AuCl ₄ solution of chloroauric acid, 3.7×10 ^-3 M hexadecyltriethylammonium bromide solution, 2×10 ^-4 M sodium citrate solution and pH=8 phosphate buffer solution, in which hexadecyltriethylammonium bromide was incubated in a constant temperature water bath at 45°C for 15 minutes before use; take a 10mL test tube and add 3mL of Phosphate buffer, 100 μL of chloroauric acid, 600 μL of CTAB and 300 μL of sodium citrate solution, mixed thoroughly to obtain the nano-gold sol preparation system; (4) Detection of antioxidant activity: Add 1 mL of water extract of corn leaves to the prepared nano-gold system, mix well, place in a constant temperature water bath at 35°C for 10 minutes, and test its UV-visible absorption after natural cooling to room temperature Spectrum; the antioxidant active substances in corn leaves have a reducing effect, and can reduce Au ⁴⁺ to Au ⁰ to form gold nanoparticles. Gold nanoparticles have obvious characteristic absorption peaks in the ultraviolet-visible spectrum. The antioxidant activity is positively correlated, so the total antioxidant activity of different varieties of corn leaf water extracts can be evaluated according to the spectral changes before and after adding the nano-gold system; (5) Maize drought tolerance evaluation: The free radical metabolism regulation ability of maize under drought stress can represent the ability of this variety of maize to cope with stress damage, that is, the total antioxidant activity of maize after stress can reflect its drought tolerance. , so the total antioxidant activity of maize can be detected according to the change of the SPR optical properties of the formed gold nanoparticles, so as to evaluate the drought tolerance of maize. 2. the method for evaluating the drought tolerance of corn varieties based on nano-gold surface plasmon resonance optical properties according to claim 1 is characterized in that: when preparing the corn leaf extract, adopt the corn seedlings after the stress treatment, and collect the seedling plants first The part above the leaf base is the material.