CN103636416B - MgCl 2promoting the application in plant nitrate nitrogen absorption - Google Patents

Abstract

The invention discloses a new application of MgCl ₂ , that is, the application of MgCl ₂ as a plant nitrate nitrogen absorption promoting agent. During use, adopt the MgCl solution that the amount concentration of substance is 130-180 μ mol/L to carry out _pretreatment 6-8h to plant, then plant is placed in the farmland wastewater containing nitrate nitrogen, observe the growth index of plant; Experimental result shows , Plants treated with MgCl ₂ can enhance the absorption capacity of nitrate nitrogen in farmland wastewater, and enhance the activities of enzymes related to nitrogen metabolism such as plasma membrane H ⁺ -ATPase, nitrate reductase, and glutamine synthetase. MgCl ₂ can promote plant Nitrogen uptake and assimilation.

Description

Application of MgCl2 in Promoting Absorption of Nitrate Nitrogen by Plants

technical field

The invention belongs to the field of promoting the absorption of nitrate nitrogen by plants, and in particular relates to a new application of inorganic chemical reagent _MgCl2 in promoting the absorption of nitrate nitrogen by plants.

Background technique

In recent years, the maturity and popularization of facility cultivation technology has accelerated the development of agriculture in our country. At present, China's greenhouse agricultural production area continues to expand. In 2011, the domestic protected horticultural cultivation area exceeded 2.4 million hm ² , ranking first in the world. However, in the production process, the environmental problems caused by the greenhouse environment are more serious than those in the open air environment. Facility vegetables and flowers have a short cultivation cycle, high multiple cropping index, and single planting structure. In addition, the protected cultivation soil cannot be washed by rainwater all year round. Generally, secondary salinization will occur to varying degrees after 3-5 years of use. The salinity anion in the facility soil is mainly NO ₃ ^- , which is the main obstacle factor for facility vegetable production. The way to desalinize the uncultivable land is to soak the farmland with water, and further make the wastewater containing a large amount of nitrogen enter the rivers and lakes, further causing the eutrophication of the water body. The above not only involves resources and ecological environment issues, but also is closely related to the sustainable development of agriculture. It is an urgent problem to be solved in the current facility vegetable production. Therefore, it is of great significance to invent an efficient, simple, cheap and acceptable plant nitrate nitrogen uptake accelerator.

Canna indica ( Canna indica ) is a bulbous flowering plant in the family Cannaceae. It is a perennial erect herb that likes high temperature and sunlight. It has a long flowering period, strong adaptability and high ornamental value. In recent years, with the development of constructed wetland sewage treatment technology, people's research on it shows that the root system of canna is relatively developed, and the removal effect of TN, TP, and COD is better. It has become a common plant for the treatment of eutrophic water bodies and has wide applications. and promotion prospects.

Magnesium participates in the entire growth process of all living organisms, and is regarded by soil and fertilizer scholars as the fourth essential nutrient element for plants after nitrogen, phosphorus, and potassium. Magnesium is known to be an important component of chlorophyll and the only metal element in chlorophyll. Magnesium is the basic element and the most active part of various enzymes, widely involved in the metabolic process of plants, and is an indispensable part of plants forming starch and protein. The effect of magnesium on plants has become a hot issue in international agronomy and soil fertilizer research. Existing studies have shown that the uptake and assimilation of nitrogen by plants is a very complicated process. Nitrate nitrogen is one of the main forms of inorganic nitrogen absorbed by plants. After being absorbed by plant roots, it needs to undergo a series of actions such as nitrate reductase, nitrite reductase, glutamine synthetase, and glutamic acid synthase to be absorbed by plants. assimilation. Plasma membrane H ⁺ -ATPase is an important functional protein on the plant cell membrane. It is called the "master enzyme" in the process of plant life activities. It can establish a transmembrane proton electromotive force and promote the transmembrane transport of ions and nutrients. Nitrate reductase (NR), which can convert nitrate in the body into nitrite, is the first step in the process of nitrate assimilation and is the rate-limiting enzyme of nitrogen metabolism. Glutamine synthetase (GS) is one of the key enzymes in plant nitrogen metabolism, it catalyzes the condensation of glutamic acid (Glu) and _NH3 to form glutamine (Gln) in the nitrogen assimilation cycle, and participates in the synthesis of nitrogenous compounds in plants. metabolic pathways. At present, the research on the mechanism of nitrogen uptake by plants has made great progress, but there is no direct and effective method to improve the uptake of nitrogen by plants. In actual production, people mainly increase the nitrogen absorption of plants by increasing the use of nitrogen fertilizer, but the effect is very limited and causes the above-mentioned problems.

Contents of the invention

The purpose of the present invention is to provide a new application of _MgCl2 as a plant nitrate nitrogen absorption accelerator, that is, the application of _MgCl2 in the efficient and rapid promotion of plant nitrate nitrogen absorption and assimilation.

In order to realize the above-mentioned purpose of the present invention, technical scheme of the present invention is as follows:

(1) Prepare a MgCl ₂ solution with a concentration of 130-180 μmol/L;

(2) Place canna seedlings with a plant height of 30-40cm in the wetland soil in the above step (1) MgCl ₂ solution, and pretreat the canna roots for 6-8 hours;

(3) The pretreated plants were placed in farmland wastewater with different concentrations of nitrate nitrogen, the absorption rate of nitrate nitrogen by plants was observed, and the activities of key enzymes for nitrate nitrogen absorption and assimilation were measured.

The _MgCl2 accelerator provided by the invention is convenient to use and has low cost; the accelerator significantly increases the absorption and assimilation of nitrate nitrogen by plants. The invention opens up a new way to promote plant nitrogen absorption and assimilation, helps scientific and technical workers to study the molecular mechanism of _MgCl2 stimulating plant growth, and has broad prospects in agricultural production.

Beneficial effects of the present invention: the _MgCl2 accelerator for promoting plant nitrogen assimilation described in the present invention has the characteristics of low investment, simple operation and high efficiency. At normal temperature, _MgCl2 is an ideal accelerator for promoting plant nitrate nitrogen absorption Magnesium chloride pretreatment can increase the activity of nitrogen absorption and assimilation-related enzymes such as H ⁺ -ATPase, nitrate reductase and glutamine synthetase on the canna cell membrane, and improve the efficiency of nitrogen absorption and assimilation; Soil salinization caused by fertilization is of great significance to the sustainable development of agriculture.

Description of drawings

Fig. 1 is the result schematic diagram of canna purification wastewater efficiency before and after adding promotor 150 μ mol/L MgCl of the present invention treatment 8h, wherein _A picture is without MgCl Treatment canna root is to the absorption result of nitrate _; B picture is through _MgCl After treatment Results of nitrate uptake by canna root;

Fig. 2 is a schematic diagram of the effect of adding accelerator MgCl ₂ on canna plasma membrane H ⁺ -ATPase activity in the present invention, wherein A is the result of plasma membrane H ⁺ -ATPase activity in canna roots before and after MgCl ₂ treatment, and CK is without MgCl ₂ Treated canna; B is the result of plasma membrane H ⁺ -ATPase activity in canna leaves before and after MgCl ₂ treatment, and CK is canna without MgCl ₂ treatment;

Fig. 3 is a schematic diagram of the effect of adding accelerator MgCl of the present invention _on the H ₊ ^-pump activity of canna root plasma membrane, wherein _A is the control group without MgCl treatment, represented by CK; B is the experiment treated with MgCl Group;

Fig. 4 is that the present invention adds promotor MgCl ₂ to canna nitrate reductase active result schematic diagram, wherein A picture is MgCl nitrate reductase activity result in canna root before and after treatment, CK is the canna without MgCl ₂ treatment _; B picture is the result of nitrate reductase activity in canna leaves before and after MgCl ₂ treatment, and CK is canna without MgCl ₂ treatment;

Fig. 5 is a schematic diagram of the effect of adding accelerator MgCl of the present invention _on the activity of canna glutamine synthetase, wherein _A is the result of glutamine synthetase activity in canna roots before and after MgCl treatment, and CK is the canna without _MgCl treatment _; Figure B is the result of glutamine synthetase activity in canna leaves before and after treatment with MgCl , and CK is the canna without MgCl treatment _;

Fig. 6 is a schematic diagram of the results of canna purification wastewater efficiency before and after ₆ hours of treatment with the addition of accelerator 130 μmol/L MgCl, wherein _A is the result of absorption of nitrate by canna roots without MgCl treatment _; B is after MgCl treatment Results of nitrate uptake by canna root.

Figure 7 is a schematic diagram of the results of canna purification wastewater efficiency before and after ₇ hours of treatment with the addition of accelerator 180 μmol/L MgCl in the present invention, wherein picture _A is the result of absorption of nitrate by canna root without MgCl treatment _; picture B is after treatment with MgCl Results of nitrate uptake by canna root.

Detailed ways

The present invention will be described in further detail below by accompanying drawing and embodiment, but protection scope of the present invention is not limited to described content. The methods in the examples are carried out according to conventional operations unless otherwise specified, and the reagents used are conventional commercially available reagents or reagents prepared according to conventional methods unless otherwise specified.

Embodiment ₁ : promote the MgCl of canna nitrate nitrogen absorption and assimilation The application of promotor, comprises the steps:

(1) The experimental material is canna seedlings; select canna seedlings with a plant height of 30-40cm in the wetland soil and dig them out, put them in tap water for hydroponics, change the water every two days, and cultivate them for about 30 days until new roots grow up for use this experiment;

(2) Prepare a MgCl ₂ solution with a concentration of 150 μmol/L;

(3) Place (1) middle canna in the above step ( ₂ ) MgCl solution, and pretreat canna roots for 8 h; then transfer the treated plants to a volume of 3 L of farmland wastewater containing different concentrations of nitrate nitrogen At the same time, the canna without magnesium chloride pretreatment was used as a control (CK), and it was also placed in 3L of farmland wastewater containing different nitrate nitrogen concentrations; Influence.

Embodiment 2: adopt the canna after the processing of embodiment 1 to verify MgCl _The impact of accelerator on the absorption rate of nitrate in canna comprises the following steps:

At the three time points of 2h, 12h, and 22h of treatment, 5 mL of farmland wastewater treated with canna in the experimental group and the control group were taken, and 2 drops of aluminum hydroxide suspension were added to allow flocculation. After centrifugation, the supernatant was used for nitrification. Determination of nitrogen concentration, using a quartz cuvette with an optical path length of 10 mm, using fresh deionized water as a reference, and detecting at wavelengths of 220 nm and 275 nm respectively, the corrected value of the absorbance of nitrate nitrogen is OD220 value Subtract 2 times the OD275 value, and check the nitrate nitrogen concentration from the standard curve. Each treatment was repeated three times, and the average value of the measurement results was taken; this method was used to verify the effect of the use of MgCl ₂ activator on the rate of nitrate absorption by canna.

Figure 1 shows that A is the result of nitrate absorption by canna roots without _MgCl2 treatment, and B is the result of nitrate absorption by canna roots after _MgCl2 treatment. The results of the effect of _MgCl2 treatment on the nitrate absorption rate of cannas showed that after cannas were treated with the _MgCl2 treatment agent of the present invention, the nitrate absorption rate of canna seedlings was significantly higher than that of the control group without _MgCl2 treatment.

Embodiment 3: Adopt the canna after the processing of embodiment 1 to verify MgCl _The impact of accelerator on canna plasma membrane H ⁺ -ATPase activity comprises the following steps:

A. After treating wastewater with different concentrations of nitrate nitrogen, take 0.5 g of canna root and leaf tissue under different treatments, freeze them quickly with liquid nitrogen, grind them into powder with liquid nitrogen, and add 1 mL of plasma membrane extract (0.25 mol/ L sorbitol, 1 mmol/L EDTA, 5 mmol/L MgSO ₄ , 10 mmol/L Tris-HCl pH7.4); the homogenate was centrifuged at 9000 g, 4 °C for 20 min to remove the precipitate, and the supernatant was washed at 30000 g, 4 °C Centrifuge for 1h; collect the precipitate and suspend it in 0.5mL of the above-mentioned extract, and the obtained is plasma membrane protein;

B. Determination of plasma membrane protein concentration by Brodford method: add 5 μL of plasma membrane protein to 800 μL of distilled water and mix well, then add 200 μL of commercially available Brodford solution, detect protein concentration at OD595 wavelength, and calculate the corresponding concentration of 500 μg of plasma membrane protein volume;

C. The determination of H ⁺ -ATPase activity was carried out in a 0.5 mL reaction system; the reaction system contained 50 mmol/L BTP/MES, 5 mmol/L MgSO ₄ , 50 mmol/L KCl, 0.02% dodecyl poly Ethylene glycol ether (w/v), 50 mmol/L KNO ₃ , 1 mmol/L (NH ₄ ) ₂ MoO ₄ , 1 mmol/L NaN ₃ , 4 mmol/L ATP-Na ₂ , add 500 μg of plasma membrane Start the reaction after the protein extract;

After the reaction mixture was placed in a water bath at 30°C for 30min, 1 ml of reaction termination solution (2% H ₂ SO ₄ (v/v), 5% SDS (w/v) and 0.7% (NH ₄ ) ₂ MoO ₄ (w After /v)), 50 μL of 10% Vc (w/v) was added immediately and left at room temperature for 20 min, and the absorbance value at a wavelength of 700 nm was measured.

D. Calculate the release content of inorganic phosphorus according to the standard curve of inorganic phosphorus. The activity of 1 unit of plasma membrane H ⁺ -ATPase is defined as: under the reaction conditions of 30 ℃, every mg of protein catalyzes the decomposition of ATP and releases the microscopic amount of inorganic phosphate within 1 minute. number of moles.

Figure 2 shows, A is the change of H ⁺ -ATPase activity in the plasma membrane of canna roots before and after _MgCl2 treatment, wherein the canna without _MgCl2 treatment is the control group, represented by CK, and the canna treated with _MgCl2 is the test group; B The picture shows the changes of plasma membrane H ⁺ -ATPase activity in canna leaves before and after MgCl ₂ treatment. The canna without MgCl ₂ treatment is the control group, denoted by CK, and the canna treated with MgCl ₂ is the test group. The results of the effect of MgCl ₂ treatment on canna plasma membrane H ⁺ -ATPase activity showed that after canna was treated with the MgCl ₂ treatment agent of the present invention, the plasma membrane H ⁺ -ATPase activity in roots and leaves of canna seedlings was significantly higher than that of the control group CK.

Embodiment 4: adopt the canna after the processing of embodiment 1 to verify MgCl _The impact of accelerator on H ⁺ -pump activity in canna root comprises the following steps:

A. After treating wastewater with different concentrations of nitrate nitrogen, take 0.5 g of canna roots under different treatments, freeze them quickly with liquid nitrogen, grind them into powder with liquid nitrogen, and add 1 mL of plasma membrane extract (0.25 mol/L sorbitol , 1 mmol/L EDTA, 5 mmol/L MgSO ₄ , 10 mmol/L Tris-HCl pH7.4); the homogenate was centrifuged at 9000g at 4°C for 20 min to remove the precipitate, and the supernatant was centrifuged at 30000g at 4°C for 1h; Collect the precipitate and suspend it in 0.5ml of the above-mentioned extract, and the obtained is plasma membrane protein;

B. Determination of plasma membrane protein concentration by Brodford method. Add 5 μL of plasma membrane protein extract to 800 μL of distilled water and mix well, then add 200 μL of commercially available Brodford solution, detect the protein concentration at OD595 wavelength, and calculate the volume corresponding to 100 μg of plasma membrane protein;

C. 1.5 ml of the reaction system contains 5 mmol/L BTP/MES (pH 6.0), 12 μmol/L AO, 300 mmol/L KCl, 250 mmol/L sucrose, 0.5 mmol/L EGTA (use BTP to adjust the pH to 6.0 ), 1 mmol/L NaN ₃ , 1 mmol/L Na ₂ MoO ₄ , 50 mmol/L KNO ₃ , 0.05% lauryl polyethylene glycol ether (w/v) and 100 μg plasma membrane protein; The detergent lauryl polyethylene glycol ether was used to invert the membrane in situ, and the reaction mixture was left at room temperature for 20 min, and then 5 mmol/L ATP/BTP (pH 6.0) was added to start the reaction;

D. The pumping of protons from the inside to the outside of the membrane is carried out according to the method of measuring the quenching of the absorbance value of acridine orange (AO) at 492 nm; through this method, the use of MgCl ₂ activator to verify the H ⁺ -pump activity in canna root Influence.

Figure 3 shows that A is the control group that has not been treated with MgCl ₂ , and B is the experimental group that has been treated with MgCl ₂ . The results of the effect of MgCl ₂ treatment on the H ⁺ -pump activity of canna roots showed that after canna was treated with the MgCl ₂ treatment agent of the present invention, the H ⁺ -pump activity of canna roots was significantly higher than that of the control group CK.

Embodiment 5: adopt the canna after the processing of embodiment 1 to verify MgCl _The impact of accelerator on canna nitrate reductase activity comprises the steps:

A. After treating wastewater with different concentrations of nitrate nitrogen, take 0.5 g of canna root and leaf tissues under different treatments, freeze them quickly with liquid nitrogen, grind them into powder with liquid nitrogen, and then add 1.5 mL of extract (25 mmol/L phosphate buffer solution pH7.4, 1 mmol/L EDTA, 10 mmol/L cysteine), grind and homogenate, transfer to 2 mL EP tube, centrifuge at 4000 rpm, 4 ℃ for 15 min, and remove the precipitated supernatant to obtain enzyme extraction liquid;

B. Take 0.2 mL of enzyme extract in a 2 mL EP tube, add 0.5 mL of 0.1 mmol/L KNO ₃ phosphate buffer and 0.3 mL of NADH solution, mix well, and bathe in water at 25°C for 30 min. After the water bath is over, add 0.5 mL of sulfonamide to terminate Reaction, then add 0.5 mL α-naphthylamine, mix well, develop color for 15 min, then use a spectrophotometer to perform colorimetric measurement at 520 nm, record the OD value, and use no NADH (add 0.2 mL water) as a blank control ;

C, calculate the nitrite nitrogen content in the reaction solution according to the standard curve, and represent the nitrate reductase activity with the micrograms of nitrite nitrogen produced per gram of fresh weight per hour;

Figure 4 shows that A is the change of nitrate reductase activity in canna roots before and after MgCl ₂ treatment, and the canna without MgCl ₂ treatment is the control group, which is represented by CK, and the canna treated with MgCl ₂ is the test group; B is MgCl ₂ Changes of nitrate reductase activity in canna leaves before and after treatment. Canna without MgCl ₂ treatment is the control group, denoted by CK, and canna treated with MgCl ₂ is the test group. The results of the influence of _MgCl2 treatment on the activity of canna nitrate reductase showed that after canna was treated with the _MgCl2 treatment agent of the present invention, the activity of nitrate reductase in roots and leaves of canna was significantly higher than that of the control group CK.

Embodiment 6: adopt the canna after the processing of embodiment 1 to verify MgCl _The impact of accelerator on canna glutamine synthetase activity, comprises the steps:

A. After treating wastewater with different concentrations of nitrate nitrogen, take 0.5 g of canna root and leaf tissues under different treatments, freeze them quickly with liquid nitrogen, grind them into powder with liquid nitrogen, add 1.5 mL of extract to grind and homogenate, and transfer them Centrifuge at 4500 rpm/min at 4 °C for 10 min in a centrifuge tube, discard the precipitate, take the supernatant and centrifuge again at 4500 rpm for 10 min, the precipitate is the chloroplast part, take the supernatant and centrifuge at 12000 rpm for 20 min at 4 °C, the precipitate is mitochondria part, the supernatant is the crude enzyme solution;

B. Use the Brodford method to measure the protein concentration of the crude solution, add 5 μL of GS protein crude enzyme solution to 800 μL of distilled water and mix well, then add 200 μL of commercially available Brodford solution, detect the protein concentration at OD595 wavelength, and calculate 0.7 mL Soluble protein content of crude enzyme solution;

C. Add 0.7 mL of crude enzyme extract and 0.7 mL of ATP (40 mmol/L) solution to 1.6 mL of reaction solution B (80 mmol/L hydroxylamine hydrochloride, 80 mmol/L MgSO ₄ , 20 mmol/L sodium glutamate , 20 mmol/L cysteine, 2 mmol/L EGTA, 0.1 mol/L Tris-HCl pH7.4), invert and mix well, let stand at 37 ℃ for 30 min, add 1ml chromogen (0.2 mol/L TCA , 0.37 mol/L FeCl ₃ and 0.6 mol/L HCl mixture), invert to mix, centrifuge at 5000 rpm for 10 min, take the supernatant and read the absorbance at 540nm. Add 0.7mL reaction solution A (80 mmol/L MgSO ₄ , 20 mmol/L sodium glutamate, 20 mmol/L cysteine, 2 mmol/L EGTA, 0.1 mol/L Tris -HCl pH7.4) as a blank control;

D. GS activity (A/mg protein h) = OD540nm/(soluble protein content × 0.5) to calculate the enzyme activity; use this method to verify the effect of MgCl ₂ accelerator on the activity of glutamine synthetase in various tissues of canna.

Fig. 5 shows, and A graph is the change of glutamine synthetase activity in canna root before and after MgCl ₂ treatment, wherein the canna without MgCl ₂ treatment is a control group, expresses with CK, and through MgCl ₂ The canna of treatment is test group; B picture is Changes of glutamine synthetase activity in canna leaves before and after MgCl ₂ treatment. The canna without MgCl ₂ treatment is the control group, denoted by CK, and the canna treated with MgCl ₂ is the test group. The results of the effect of _MgCl2 treatment on the activity of canna glutamine synthetase showed that after canna was treated with the _MgCl2 treatment agent of the present invention, the activity of glutamine synthetase in canna roots and leaves was significantly higher than that of the control group CK.

Embodiment ₇ : promote the MgCl of canna nitrate nitrogen absorption and assimilation The application of accelerator, comprises the steps:

(1) Prepare a MgCl ₂ solution with a concentration of 130 μmol/L;

(2) Select canna seedlings with a plant height of 30-40cm in the wetland soil and dig them out, put them in tap water for cultivation, change the water every two days, and cultivate them for about 30 days until new roots grow up for this experiment;

(3) Canna root in (2) was placed in the above step ( ₁ ) MgCl solution for 6 h pretreatment; then the treated plants were transferred to a volume of 3 L of farmland wastewater containing different concentrations of nitrate nitrogen Simultaneously with the canna that has not been pretreated with magnesium chloride as contrast (CK), also be placed in the farmland waste water that volume is 3L containing different nitrate nitrogen concentrations; Measure MgCl Accelerator absorbs nitrate rate to canna, method is the same as embodiment ₂ .

Figure 6 shows that A is the control group that has not been treated with MgCl ₂ , and B is the experimental group that has been treated with MgCl ₂ . The results of the influence of _MgCl2 treatment on the nitrate absorption rate of cannas showed that after cannas were treated with the _MgCl2 treatment agent of the present invention, the nitrate absorption rate of canna seedlings was higher than that of the control group CK.

Embodiment ₈ : promote the MgCl of canna nitrate nitrogen absorption and assimilation The application of accelerator, comprises the steps:

(1) Prepare a MgCl ₂ solution with a concentration of 180 μmol/L;

(2) Select canna seedlings with a plant height of 30-40cm in the wetland soil and dig them out, put them in tap water for cultivation, change the water every two days, and cultivate them for about 30 days until new roots grow up for this experiment;

(3) Canna root in (2) was placed in the above step (1) MgCl ₂ solution, and canna roots were pretreated for 7 h; then the treated plants were transferred to a volume of 3 L of farmland wastewater containing different concentrations of nitrate nitrogen Simultaneously with the canna that has not been pretreated with magnesium chloride as contrast (CK), also be placed in the farmland waste water that volume is 3L containing different nitrate nitrogen concentrations; Measure MgCl Accelerator absorbs nitrate rate to canna, method is the same as embodiment ₂ .

Figure 7 shows that A is the control group that has not been treated with MgCl ₂ , and B is the experimental group that has been treated with MgCl ₂ . The results of the influence of _MgCl2 treatment on the nitrate absorption rate of cannas showed that after cannas were treated with the _MgCl2 treatment agent of the present invention, the nitrate absorption rate of canna seedlings was higher than that of the control group CK.

Claims (1)

1. The application of _MgCl2 as a plant nitrate nitrogen absorption accelerator is characterized in that: the _MgCl2 solution with a substance concentration of 130-180 μmol/L is used to treat the plants for 6-8 hours, and then the plants are placed in a nitrate-containing state. In farmland wastewater containing nitrogen, the concentration of nitrate nitrogen in the wastewater and the growth indicators of plants are detected.