Remission method for studying heavy metal cadmium poisoning of corn

Abstract

The invention belongs to the technical field of plant heavy metal pollution remediation, and discloses a relieving method for studying the poison of corn by heavy metal cadmium (Cd), which comprises the following steps: step 1, setting an experimental environment, step 2, treating seeds and accelerating germination, step 3, preparing a nutrient solution, step 4, carrying out water culture on seedlings, step 5, setting a control group, step 6, carrying out water culture on the seedlings, and step 7, carrying out water culture on the seedlings. Photosynthetic gas exchange parameters, growth indexes and resistance-related metabolite indexes are measured; and 6, obtaining an experiment conclusion. The research shows that silicon (Si) and melatonin (MT) have a relatively strong synergistic effect on relieving Cd stress, and the combined application of Si and MT is an effective strategy for relieving Cd toxicity of corn in a Cd stress environment; research results show that under Cd stress, combined application of Si and MT significantly promotes growth of corn and reduces absorption and accumulation of Cd by corn, and the effect of combined application of Si and MT is greater than that of single application, which has important practical significance in improving the tolerance of plants to Cd stress.

Description

Technical Field

[0001] The invention belongs to the technical field of plant heavy metal pollution remediation, and specifically relates to a method for studying the alleviation of corn poisoning by heavy metal cadmium. Background Art

[0002] Corn is China's second largest crop, and my country's corn production accounts for 19% of the world's total corn production. Therefore, ensuring corn quality is crucial for food safety and human health. However, corn production is threatened by heavy metal contamination in the soil, especially cadmium (Cd). Cd is one of the most dangerous environmental pollutants and has strong biotoxicity to the human body, which endangers human health. Cd seriously affects plant growth, causing leaf yellowing, reduced chlorophyll and photosynthesis, etc., and also has substantial adverse effects on crop quality and yield. Most importantly, Cd can easily accumulate in edible tissues through absorption and transportation by plant roots. Eating crops contaminated with Cd can cause serious health problems and even endanger life.

[0003] In recent years, the application of beneficial elements and signaling molecules is an economical and effective strategy to alleviate heavy metal stress in plants, but most studies focus on alleviating Cd-induced growth and photosynthesis inhibition by upregulating the antioxidant defense system. Few studies focus on alleviating Cd toxicity from the expression of maize genes. Therefore, this application is aimed at studying the mitigation of maize toxicity by heavy metal cadmium in the direction of focusing on Cd uptake and the expression of Cd-related genes. Summary of the invention

[0004] The purpose of the present invention is to provide a method for alleviating the poisoning of corn by heavy metal cadmium, so as to solve the problems raised in the above background technology.

[0005] In order to achieve the above object, the present invention provides the following technical solution: a method for studying the mitigation of heavy metal cadmium poisoning in corn, comprising the following method: Step 1: Experimental environment setting: The experiment was carried out in a tissue culture room equipped with an automatic temperature and humidity control system; the temperature was set at 26 °C, the humidity was set at 60%, and the light intensity was 400 μmol m -2 s -1 , photoperiod was 14 / 10 h (day / night); Step 2: Seed treatment and germination: Select seeds with full and uniform grains, disinfect them with 1% sodium hypochlorite for 10 minutes, and then rinse the seeds repeatedly with distilled water several times to remove residual disinfectant; Sow the seeds evenly on the soaked filter paper, ensure sufficient water supply, cover with plastic wrap to maintain temperature and humidity, and place the sealed seeds in a 25°C incubator for germination in the dark for 5 days; Step 3: Prepare the nutrient solution: Use a container with a size of 40 cm × 30 cm × 15 cm, with each pot containing 5 L of nutrient solution; adjust the pH of the nutrient solution to 5.6-5.8; The ingredients of the nutrient solution are: NH4NO3, KH2PO4, MgSO4, KCl, CaCl2, Fe-EDTA, MnCl2, ZnSO4, CuSO4, (NH4)6Mo7O 24 , H3BO4; Step 4: Seedling hydroponics: After 5 days, select corn seedlings with consistent mesocotyl and root lengths, and transfer the selected seedlings to 1 / 4 Hoagland nutrient solution for hydroponics; Step 5, set up control groups: (1) blank control group; (2) Cd addition group; (3) exogenous Si application group; (4) exogenous melatonin application group; (5) exogenous Si and melatonin application group; (6) exogenous Si and Cd application group; (7) exogenous melatonin and Cd application group; (8) exogenous Si, melatonin and Cd application group; Step six, measuring photosynthetic gas exchange parameters, growth indexes and resistance-related metabolite indexes; Step six: draw experimental conclusions.

[0006] Preferably, in the determination of photosynthetic gas exchange parameters in step 6, the net photosynthetic rate (Pn, μmol m -2 · s -1 ) and transpiration rate (Tr, mmol H2O m -2 · s -1 ), the measurement time is two hours.

[0007] Preferably, the method for determining the growth index in step six is: S1, root length and plant height of maize seedlings were measured three weeks after the plants were planted; S2, the maize roots were immersed in 5 mM EDTA-Na2 solution for 15 min to remove Cd attached to the roots, then rinsed with deionized water 5 times and the surface water was dried with absorbent paper; S3, separate the above-ground and underground parts and weigh the fresh weight on a scale; S4, fresh weight was then placed in an oven at 105 °C for 1 h, and then dried at 80 °C to constant weight, and the dry weight was measured.

[0008] Preferably, the determination of resistance-related metabolite indicators in step six is ​​specifically divided into: chlorophyll content determination, hydrogen peroxide content determination, malondialdehyde content determination, cell membrane damage degree determination, anthocyanin content determination and cadmium distribution analysis under different treatment conditions.

[0009] Preferably, the step of determining the chlorophyll content is: A1, take 0.2 g of fresh plant sample frozen at -80 °C, extract chlorophyll with 80% acetone on ice in dark conditions, grind into a homogenate, and extract in the dark until the plant leaves turn completely white; A2, the extract was centrifuged at 5000 g, 4 °C, for 10 min; A3, after centrifugation, the supernatant was measured spectrophotometrically at 470 nm, 646 nm and 663 nm using a spectrophotometer.

[0010] Preferably, the step of determining the hydrogen peroxide content is: B1, grind 0.05 g of the frozen sample into a homogenate and transfer it to a 1.5 mL centrifuge tube, add 500 μL of 0.1% trichloroacetic acid precooled at 4 °C, and shake well; B2, then centrifuged at 13 000 g for 15 min at 4 °C, and the supernatant was transferred to a new tube; B3, add 400 μL PBS buffer to the supernatant, shake well, react for 10 minutes, measure the absorbance at 390 nm, and calculate the amount of H2O2 using the standard curve.

[0011] Preferably, the malondialdehyde content can indicate the degree of plant membrane lipid peroxidation, and the steps of determining the malondialdehyde content are: C1, weigh about 0.2 g of fresh sample frozen at -80 °C, add 2 mL of 10% TCA and a small amount of quartz sand, and grind to a homogenous slurry; C2, then add 8 mL TCA and grind, centrifuge the homogenate at 400 r / min for 10 min, and take the supernatant as the sample extract.

[0012] C3, aspirate 2 mL of the supernatant after centrifugation, add 2 mL of 0.6% TBA solution, mix well, and react in a boiling water bath for 15 min; C4, cool rapidly and then centrifuge, take the supernatant and measure the absorbance of the supernatant at 532 nm, 600 nm and 450 nm.

[0013] Preferably, the cell membrane damage degree determination is to measure the damage degree of corn leaf cell membrane by electrical conductivity (EC); staining can indicate the content of H2O2 in plant tissue. The cell membrane damage degree determination steps are: D1, washing the fresh leaf surface with deionized water to remove dust and electrolytes; D2, transfer it to a sealed tube filled with 20 mL of deionized water and incubate it at 40 °C for 30 minutes at 100 rpm; use a conductivity meter to measure the initial conductivity of the solution and record it as R1; then boil the sample in a boiling water bath for 10 minutes, cool it to room temperature, and record the EC value of the solution as R2; The histochemical method for detecting H2O2 and superoxide anion content is based on the fact that DAB staining can indicate the H2O2 content in plant tissues; the specific steps are: E1, prepare 100 mL of 1 mg / mL DAB staining solution and adjust the pH to 3.8 with 0.2 mol / L HCl; E2, in each treatment, the leaves of corn at the same leaf position were punched and then dyed; the punched leaves were completely immersed in DAB dye solution, and the leaves were vacuumed because they were sulfur-water-based; E3, placed on a shaker in the dark for 6 hours to allow full contact with the staining solution; E4, decolorization was performed with 95% ethanol in a boiling water bath; after the water bath, the leaves were rinsed with distilled water and the staining of the leaves was observed; E5, dissolve 0.1 g NBT in 50 mmol / L phosphate buffer to make the volume 50 mL; E6, for each treatment, the leaves of corn at the same leaf position were punched and then stained; after vacuuming, the leaves were stained in the dark for 3 hours; then, ethanol decolorization was performed, and the leaves were rinsed with distilled water to observe the staining of the leaves.

[0014] Preferably, the method for measuring anthocyanin content is as follows: 0.2 g of fresh leaf sample is placed in a 2 mL centrifuge tube, 1 mL of extract is added, and the ratio of the extract is methanol: hydrochloric acid: water = 25:5:70; extract in the dark for 4 hours, centrifuge at 15000 g for 10 min; measure the absorbance of the supernatant at 525 nm (A525) and 657 nm (A657) with a spectrophotometer, and use the extract as a blank control; The steps of cadmium distribution analysis under the different treatment conditions are as follows: F1, corn seedlings under different treatment conditions. After 2 weeks of treatment, the roots of the plants were washed with 5 mmol / L EDTA-Na2 for several times, each time for 15 minutes, and then washed three times with deionized water; F2, the aboveground and underground parts of corn were dried at 110 °C and then dried to constant weight at 80 °C; F3, grind the dried plant sample into powder, place it in a digestion tube, digest it with a mixture of HNO3 and HClO4, then add 5 mL of HNO3 and leave it overnight; F4, heat the sample in a digestion furnace at 100 °C until it turns yellow, cool the digestion solution and add 2 mL HClO4, and heat at 180 °C until it becomes colorless and transparent; F5 diluted the digested liquid to an equal volume of 25 mL; used atomic absorption spectrometry to analyze the content of cadmium in the aboveground and underground parts of corn plants and the distribution of cadmium in corn tissues.

[0015] Preferably, the mitigation method is applied to corn planting, and exogenous Si and MT are used to alleviate the toxicity of heavy metal cadmium to corn. The above research process proves that the combined application of Si and MT alleviates Cd stress, and corn shows higher biomass and plant height; the application of Si, MT or both can increase the chlorophyll content of corn under Cd stress; the application of Si and MT alleviates the oxidative damage caused by Cd stress in corn by increasing the activity of antioxidant enzymes; the combined application of Si and MT reduces the absorption of Cd in roots and leaves; the application of Si and MT treatments both significantly increased the expression level of the ZmHMA3 gene.

[0016] The beneficial effects of the present invention are as follows: The present study proves that silicon (Si) or melatonin (MT) plays a very important role in alleviating abiotic stress in plants. In this study, maize seedlings were treated with Cd, Si and MT to explore the effects of Cd, Si and MT on maize growth, photosynthesis, antioxidant substances, Cd content and absorption, and Cd-related transporter gene expression. The results showed that Cd stress led to a decrease in maize biomass, oxidative damage and Cd accumulation compared with the control plants without Cd treatment. Compared with the Cd treatment alone, the combination of Si and MT promoted the growth of maize plants under Cd stress, improved the photosynthetic capacity and antioxidant capacity, and reduced the Cd content and Cd absorption in maize roots and leaves. QRT-PCR analysis further found that under Cd stress, the expression of ZmHMA2 and ZmHMA3 genes in maize leaves treated with Si and MT was downregulated by 66.5% and upregulated by 394.1%, respectively, compared with the Cd treatment alone, which helped to reduce the transfer of Cd to the upper tissues of maize and enhance the sequestration of Cd in the vacuole.

[0017] This study showed that Si and MT have a strong synergistic effect in alleviating Cd stress. Under Cd stress environment, the combined application of Si and MT is an effective strategy to alleviate Cd toxicity in corn. The results showed that under Cd stress, the combined application of Si and MT significantly promoted the growth of corn, reduced the absorption and accumulation of Cd in corn, and the effect of combined application of the two was greater than that of single application, which has important practical significance for improving plant tolerance to Cd stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The present invention is a simplified schematic diagram of the research method process; Figure 2 The figure is a graph showing the effects of silicon and melatonin on the phenotype (A), dry weight (B) and plant height (C) of maize under cadmium stress; Figure 3 The figure is the effect of silicon and melatonin on photosynthesis (A) and chlorophyll content (B) of corn seedlings under cadmium stress; Figure 4 This is a graph showing the effects of silicon and melatonin on hydrogen peroxide (A), malondialdehyde (B), electrical conductivity (C) and anthocyanin (D) content in corn seedlings under cadmium stress; Figure 5 This is a graph showing the effects of silicon and melatonin on the contents of SOD (A) and POD (B) in corn seedlings under cadmium stress; Figure 6 The figure is the effect of silicon and melatonin on the cadmium content in the root (A) and leaf (B) and the absorption in the root (C) and leaf (D) of corn seedlings under cadmium stress; Figure 7 This is a diagram showing the effects of silicon and melatonin on ZmHMA2 and ZmHMA2 gene expression in corn seedlings under cadmium stress. DETAILED DESCRIPTION

[0019] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] like Figures 1 to 7 As shown, the embodiment of the present invention provides a method for studying the mitigation of heavy metal cadmium poisoning in corn, comprising the following method: Step 1: Experimental environment setting: The experiment was carried out in a tissue culture room equipped with an automatic temperature and humidity control system; the temperature was set at 26 °C, the humidity was set at 60%, and the light intensity was 400 μmol m -2 s -1 , photoperiod was 14 / 10 h (day / night); Step 2: Seed treatment and germination: Select seeds with full and uniform grains, disinfect them with 1% sodium hypochlorite for 10 minutes, and then rinse the seeds repeatedly with distilled water several times to remove residual disinfectant; Sow the seeds evenly on the soaked filter paper, ensure sufficient water supply, cover with plastic wrap to maintain temperature and humidity, and place the sealed seeds in a 25°C incubator for germination in the dark for 5 days; Step 3: Prepare the nutrient solution: select a container with a size of length × width × height = 40 × 30 × 15, with each pot containing 5L of nutrient solution; adjust the pH of the nutrient solution to 5.6-5.8; The ingredients of the nutrient solution are: NH4NO3, KH2PO4, MgSO4, KCl, CaCl2 (note that MgSO4 is mentioned repeatedly in the ingredients, which may need to be checked and adjusted), Fe-EDTA, MnCl2, ZnSO4, CuSO4, (NH4)6Mo7O 24 , H3BO4; Step 4: Seedling hydroponics: After 5 days, select corn seedlings with consistent mesocotyl and root lengths, and transfer the selected seedlings to 1 / 4 Hoagland nutrient solution for hydroponics; Step 5. Set up control groups: (1) blank control group (CK); (2) Cd group (Cd); (3) exogenous Si (0.5 mM) group (determined by pre-experimental screening at different concentrations); (4) exogenous melatonin (100 μM) group (determined by pre-experimental screening at different concentrations); (5) exogenous Si and melatonin group (Si+Mel); (6) exogenous Si and Cd group (Si+Cd); (7) exogenous melatonin and Cd group (Mel+Cd); (8) exogenous Si, melatonin and Cd group (Si+Mel+Cd).

[0021] Step six, measuring photosynthetic gas exchange parameters, growth indexes and resistance-related metabolite indexes; Step six: draw experimental conclusions.

[0022] In step 6, the net photosynthetic rate (Pn, μmol m -2 · s -1 ) and transpiration rate (Tr, mmol H2O m -2 · s -1 ), the measurement time is 2 hours.

[0023] Among them, the determination method of the growth index in step six is: S1, root length and plant height of maize seedlings were measured three weeks after the plants were planted; S2, the maize roots were immersed in 5 mM EDTA-Na2 solution for 15 min to remove the cadmium attached to the roots, then rinsed with deionized water for 5 times and the surface water was dried with absorbent paper; S3, separate the above-ground and underground parts and weigh the fresh weight on a scale; S4, fresh weight was then placed in an oven at 105 °C for 1 h, and then dried at 80 °C to constant weight, and the dry weight was measured.

[0024] Among them, the determination of resistance-related metabolite indicators in step six is ​​specifically divided into: chlorophyll content determination, hydrogen peroxide content determination, malondialdehyde content determination, cell membrane damage degree determination, anthocyanin content determination and cadmium distribution analysis under different treatment conditions.

[0025] The steps for determining the chlorophyll content are as follows: A1, take 0.2 g of fresh plant sample frozen at -80 °C, extract chlorophyll with 80% acetone on ice in dark conditions, grind into a homogenate, and extract in the dark until the plant leaves turn completely white; A2, the extract was centrifuged at 5000 g, 4 °C, for 10 min; A3, after centrifugation, the supernatant was measured spectrophotometrically at 470 nm, 646 nm and 663 nm using a spectrophotometer.

[0026] Wherein, the steps of determining the content of hydrogen peroxide are: B1, grind 0.05 g of the frozen sample into a homogenate and transfer it to a 1.5 mL centrifuge tube, add 500 μL of 0.1% trichloroacetic acid precooled at 4 °C, and shake well; B2, then centrifuged at 13 000 g for 15 min at 4 °C, and the supernatant was transferred to a new tube; B3, add 400 μL PBS buffer to the supernatant, shake well, react for 10 minutes, measure the absorbance at 390 nm, and calculate the amount of H2O2 using the standard curve.

[0027] Among them, the malondialdehyde content can indicate the degree of plant membrane lipid peroxidation. The steps for determining the malondialdehyde content are: C1, weigh about 0.2 g of fresh sample frozen at -80 °C, add 2 mL of 10% TCA and a small amount of quartz sand, and grind to a homogenous slurry; C2, then add 8 mL TCA and grind, centrifuge the homogenate at 400 r / min for 10 min, and take the supernatant as the sample extract.

[0028] C3, take 2 mL of the supernatant from the centrifugation (control: 2 mL of distilled water), add 2 mL of 0.6% TBA solution, mix well, and react in a boiling water bath for 15 min; C4, cool rapidly and then centrifuge, take the supernatant and measure the absorbance of the supernatant at 532 nm, 600 nm and 450 nm.

[0029] Among them, the degree of cell membrane damage is measured by electrical conductivity (EC) to measure the degree of damage to the cell membrane of corn leaves; staining can indicate the content of H2O2 in plant tissues. The steps for determining the degree of cell membrane damage are as follows: D1, washing the fresh leaf surface with deionized water to remove dust and electrolytes; D2, transfer it to a sealed tube filled with 20 mL of deionized water and incubate it at 40 °C for 30 minutes at 100 rpm; use a conductivity meter to measure the initial conductivity (EC) of the solution and record it as R1; then digest the sample in a boiling water bath for 10 minutes, cool it to room temperature, and record the EC value of the solution as R2; The histochemical method for detecting H2O2 and superoxide anion content is based on the fact that DAB staining can indicate the H2O2 content in plant tissues; the specific steps are: E1, prepare 100 mL of 1 mg / mL DAB staining solution and adjust the pH to 3.8 with 0.2 mol / L HCl; E2, in each treatment, the leaves of corn at the same leaf position were punched and then dyed; the punched leaves were completely immersed in DAB dye solution, and the leaves were vacuumed because they were sulfur-water-based; E3, placed on a shaker in the dark for 6 hours to allow full contact with the staining solution; E4, decolorize with 95% ethanol in a boiling water bath; after the water bath, rinse the leaves with distilled water and observe the staining of the leaves; NBT staining can indicate O in plant tissues. 2- Content; E5, dissolve 0.1 g NBT in 50 mmol / L phosphate buffer (pH 7.5) to make the volume reach 50 mL; E6, for each treatment, the leaves of corn at the same leaf position were punched and then stained; after vacuuming, the leaves were stained in the dark for 3 hours; then, ethanol decolorization was performed, and the leaves were rinsed with distilled water to observe the staining of the leaves.

[0030] The method for anthocyanin content is as follows: 0.2 g of fresh leaf sample is placed in a 2 mL centrifuge tube, 1 mL of extract is added, and the extract composition ratio is methanol: hydrochloric acid: water (v:v:v) = 25:5:70; extract in the dark for 4 hours, centrifuge at 15000 g for 10 min; measure the absorbance of the supernatant at 525 nm (A525) and 657 nm (A657) with a spectrophotometer, and use the extract as a blank control; The steps for cadmium distribution analysis under different treatment conditions are as follows: F1, corn seedlings under different treatment conditions. After 2 weeks of treatment, the roots of the plants were washed with 5 mmol / L EDTA-Na2 for several times, each time for 15 minutes, and then washed three times with deionized water; F2, the aboveground and underground parts of corn were dried at 110 °C and then dried to constant weight at 80 °C; F3, grind the dried plant sample into powder, place it in a digestion tube, digest it with a mixture of HNO3 and HClO4, then add 5 mL of HNO3 and leave it overnight; F4, heat the sample in a digestion furnace at 100 °C until it turns yellow, cool the digestion solution and add 2 mL HClO4, and heat at 180 °C until it becomes colorless and transparent; F5 diluted the digested liquid to an equal volume of 25 mL; used atomic absorption spectrometry to analyze the content of cadmium in the aboveground and underground parts of corn plants and the distribution of cadmium in corn tissues.

[0031] Among them, this mitigation method is applied to corn planting, using exogenous Si and MT to alleviate the toxicity of heavy metal cadmium to corn. The above research process proves that the combined application of Si and MT alleviates Cd stress, and corn shows higher biomass and plant height; the application of Si, MT or both can increase the chlorophyll content of corn under Cd stress; the application of Si and MT alleviates the oxidative damage caused by Cd stress in corn by increasing the activity of antioxidant enzymes; the combined application of Si and MT reduces the absorption of Cd in roots and leaves; the application of Si and MT treatments significantly increased the expression level of the ZmHMA3 gene.

[0032] Effects of Si and MT on phenotype and growth of maize plants under Cd stress: Cd stress seriously affects the growth of maize, and leaves turn yellow. Under Cd-free conditions, compared with the control (CK), Si and MT alone or in combination had no significant effect on maize phenotype, biomass, and plant height. Compared with CK, Cd treatment significantly reduced maize root dry weight, leaf dry weight content, and plant height by 28.9%, 30.7%, and 37.2%, respectively. Under Cd stress, compared with Si or MT alone, the combined application of Si and MT alleviated Cd stress, and maize showed higher biomass and plant height; Effects of Si and MT on photosynthetic rate and chlorophyll content of maize seedlings under Cd stress: Under the condition of no Cd treatment, the application of Si, MT alone or the combined application of the two had no significant effect on the photosynthetic rate and chlorophyll content of maize. Cd treatment significantly reduced the photosynthetic rate and chlorophyll content of maize, decreasing by 44% and 50.6%, respectively. However, Si or MT treatment alleviated the decrease in photosynthetic rate and chlorophyll content induced by Cd stress, and the combined application of Si and MT resulted in a higher photosynthetic rate of maize under Cd stress than that of Si or MT alone. The photosynthetic rates of Si + Cd, MT + Cd and Si + MT + Cd treatments were 32.9%, 22.5% and 59.5% higher than those of Cd treatment, respectively. Exogenous application of Si, MT or the simultaneous application of the two can increase the chlorophyll content of maize under Cd stress; Effects of Si and MT on oxidative products and anthocyanin contents in maize seedlings under Cd stress: Si or MT treatment had no significant effect on H2O2, MDA, conductivity and anthocyanin content. Cd treatment significantly increased H2O2, MDA, conductivity and anthocyanin content. The anthocyanin content was the highest under Cd stress, which was consistent with the content of H2O2, MDA and conductivity, indicating that anthocyanin and its oxidation products may be a stress response to Cd stress. Si or MT alone or in combination could alleviate the increase of H2O2, MDA, conductivity and anthocyanin content caused by Cd stress. In addition, Cd stress significantly reduced the activities of SOD and POD enzymes in maize leaves, which were 28.8% and 54.6% lower than those in CK, respectively. The application of Si or MT or their combination could increase the activities of SOD and POD in maize leaves under Cd stress. The activities of SOD and POD in Si + MT + Cd treatment were higher than those in Cd treatment, indicating that exogenous application of Si and MT can alleviate the oxidative damage caused by Cd stress in maize by increasing the activities of antioxidant enzymes.

[0033] Effects of Si and MT on Cd content and uptake in maize seedlings under Cd stress: Cd treatment resulted in the highest Cd content in maize roots and leaves, and the Cd content in roots of Si + MT + Cd treatment was 54.6% lower than that of Cd treatment. Exogenous application of Si or MT significantly reduced the Cd content in maize leaves. Compared with Cd treatment, the combined treatment of Si and MT significantly reduced the Cd content in maize leaves (75%), followed by Si + Cd (64.1%) and MT + Cd (51.6%). The absorption of Cd by roots and leaves in each treatment was similar to the results of Cd content in roots and leaves. The application of Si, MT or combined application affected the absorption of Cd by roots and leaves. Among them, the Cd absorption amount of Si + MT + Cd treatment was the lowest. Compared with the application of Cd alone, the combined application of Si and MT reduced the absorption of Cd in roots and leaves by 67.5% and 80.3%, respectively.

[0034] Effects of Si and MT on the expression of ZmHMA2 and ZmHMA3 genes in maize under Cd stress: Cd treatment significantly increased the expression of ZmHMA2 gene in maize leaves and significantly decreased the expression of ZmHMA3 gene. Si and MT or the combination of the two treatments decreased the expression of ZmHMA2 gene, among which the combination of Si and MT significantly downregulated the expression level of ZmHMA2 gene (66.5%). However, Si and MT treatments significantly increased the expression level of ZmHMA3 gene, which was 394.1% higher than that of Cd treatment.

[0035] It should be noted that, in this article, relational terms such as first and second, etc. are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the terms "include", "comprise" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or device.

[0036] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for alleviating the poisoning of corn by heavy metal cadmium, characterized in that: The following methods are included: Step 1: Experimental environment setting: The experiment was carried out in a tissue culture room equipped with an automatic temperature and humidity control system; the temperature was set at 26 °C, the humidity was set at 60%, and the light intensity was 400 μmol m -2 s -1 , photoperiod was 14 / 10 h; Step 2: Seed treatment and germination: Select seeds with full and uniform grains, disinfect them with 1% sodium hypochlorite for 10 minutes, and then rinse the seeds repeatedly with distilled water several times to remove residual disinfectant; Sow the seeds evenly on the soaked filter paper, ensure sufficient water supply, cover with plastic wrap to maintain temperature and humidity, and place the sealed seeds in a 25℃ incubator for germination in the dark for 5 days; Step 3: Prepare the nutrient solution: Use a container with a size of 40 cm × 30 cm × 15 cm, with each pot containing 5 L of nutrient solution; adjust the pH of the nutrient solution to 5.6-5.8; The ingredients of the nutrient solution are: NH4NO3, KH2PO4, MgSO4, KCl, CaCl2, Fe-EDTA, MnCl2, ZnSO4, CuSO4, (NH4)6Mo7O 24 , H3BO4; Step 4: Seedling hydroponics: After 5 days, select corn seedlings with consistent mesocotyl and root lengths, and transfer the selected seedlings to 1 / 4 Hoagland nutrient solution for hydroponics; Step 5, set up control groups: (1) blank control group; (2) Cd addition group; (3) exogenous Si application group; (4) exogenous melatonin application group; (5) exogenous Si and melatonin application group; (6) exogenous Si and Cd application group; (7) exogenous melatonin and Cd application group; (8) exogenous Si, melatonin and Cd application group; Step six, measuring photosynthetic gas exchange parameters, growth indexes and resistance-related metabolite indexes; Step six: draw experimental conclusions.

2. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 1, characterized in that: In the determination of photosynthetic gas exchange parameters in step 6, the net photosynthetic rate and transpiration rate of corn leaves are determined using a Li-6800 portable photosynthetic analysis system for two hours.

3. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 1, characterized in that: The method for determining the growth index in step 6 is: S1, root length and plant height of maize seedlings were measured three weeks after the plants were planted; S2, the maize roots were immersed in 5 mM EDTA-Na2 solution for 15 min to remove Cd attached to the roots, then rinsed with deionized water five times and the surface water was dried with absorbent paper; S3, separate the above-ground and underground parts and weigh the fresh weight on a scale; S4, fresh weight, placed in an oven at 105℃ for 1 hour, then dried at 80℃ to constant weight, and the dry weight was measured.

4. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 1, characterized in that: The determination of the resistance-related metabolite indicators in step six is ​​specifically divided into: chlorophyll content determination, hydrogen peroxide content determination, malondialdehyde content determination, cell membrane damage degree determination, anthocyanin content determination and cadmium distribution analysis under different treatment conditions.

5. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 4, characterized in that: The steps of determining the chlorophyll content are: A1, take 0.2 g of fresh plant sample frozen at -80℃, extract chlorophyll with 80% acetone on ice in dark conditions, grind into homogenate, and extract in the dark until the leaves of the plant turn completely white; A2, the extract was centrifuged at 5000 g, 4 °C, for 10 min; A3, after centrifugation, the supernatant was measured spectrophotometrically at 470 nm, 646 nm and 663 nm using a spectrophotometer.

6. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 4, characterized in that: The steps of determining the content of hydrogen peroxide are: B1, grind 0.05 g of the frozen sample into a homogenate and transfer it to a 1.5 mL centrifuge tube, add 500 μL of 0.1% trichloroacetic acid precooled at 4°C, and shake well; B2, then centrifuged at 13 000 g for 15 min at 4 °C, and the supernatant was transferred to a new tube; B3, add 400 μL PBS buffer to the supernatant, shake well, react for 10 minutes, measure the absorbance at 390 nm, and calculate the amount of H2O2 using the standard curve.

7. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 4, characterized in that: The malondialdehyde content can indicate the degree of plant membrane lipid peroxidation, and the steps of determining the malondialdehyde content are: C1, weigh 0.2 g of fresh sample frozen at -80 °C, add 2 mL of 10% TCA and a small amount of quartz sand, and grind to homogenous slurry; C2, add 8 mL TCA and grind, centrifuge the homogenate at 400 r / min for 10 min, and take the supernatant as the sample extract; C3, aspirate 2 mL of the supernatant from the centrifugation, add 2 mL of 0.6% TBA solution, mix well, and react in a boiling water bath for 15 min; C4, cool rapidly and then centrifuge, take the supernatant and measure the absorbance of the supernatant at 532 nm, 600 nm and 450 nm.

8. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 4, characterized in that: The cell membrane damage degree determination is to measure the damage degree of corn leaf cell membrane by electrical conductivity (EC); staining can indicate the content of H2O2 in plant tissue. The cell membrane damage degree determination steps are as follows: D1, washing the fresh leaf surface with deionized water to remove dust and electrolytes; D2, transfer it to a sealed tube filled with 20 mL of deionized water and incubate it at 40 °C for 30 minutes at 100 rpm; use a conductivity meter to measure the initial conductivity of the solution and record it as R1; then boil the sample in a boiling water bath for 10 minutes, cool it to room temperature, and record the EC value of the solution as R2; The histochemical method for detecting H2O2 and superoxide anion content is based on the fact that DAB staining can indicate the H2O2 content in plant tissues; the specific steps are: E1, prepare 100 mL of 1 mg / mL DAB staining solution and adjust the pH to 3.8 with 0.2 mol / L HCl; E2, in each treatment, holes were punched in the same leaf position of corn leaves and then dyed; the punched leaves were completely immersed in DAB dye solution, and the leaves were vacuumed because of their sulfur content; E3, placed on a shaker in the dark for 6 hours to allow full contact with the staining solution; E4, decolorization was performed with 95% ethanol in a boiling water bath; after the water bath, the leaves were rinsed with distilled water and the staining of the leaves was observed; E5, dissolve 0.1 g NBT in 50 mmol / L phosphate buffer to make the volume 50 mL; E6, for each treatment, the leaves of corn at the same leaf position were punched and then stained; after vacuuming, the leaves were stained in the dark for 3 hours; then, ethanol decolorization was performed, and the leaves were rinsed with distilled water to observe the staining of the leaves.

9. The method for alleviating the toxicity of heavy metal cadmium to corn according to claim 4, characterized in that: The method for determining the anthocyanin content is as follows: 0.2 g of fresh leaf sample is placed in a 2 mL centrifuge tube, 1 mL of extract is added, and the extract has a composition ratio of methanol: hydrochloric acid: water = 25:5:70; the extract is performed in the dark for 4 hours, and centrifuged at 15,000 g for 10 minutes; the absorbance of the supernatant at 525 nm (A525) and 657 nm (A657) is measured by a spectrophotometer, and the extract is used as a blank control; The steps of cadmium distribution analysis under the different treatment conditions are as follows: F1, corn seedlings under different treatment conditions. After 2 weeks of treatment, the roots of the plants were washed with 5 mmol / L EDTA-Na2 for several times, each time for 15 minutes, and then washed three times with deionized water; F2, the aboveground and underground parts of corn were dried at 110 °C and then dried to constant weight at 80 °C; F3, grind the dried plant sample into powder, place it in a digestion tube, digest it with a mixture of HNO3 and HClO4, then add 5mL of HNO3 and leave it overnight; F4, heat the sample at 100 °C in a digestion furnace until it turns yellow, cool the digestion solution and add 2 mL HClO4, and heat at 180 °C until it becomes colorless and transparent; F5, dilute the digested liquid to an equal volume of 25 mL; use atomic absorption spectrometry to analyze the content of cadmium in the aboveground and underground parts of corn plants, and analyze the distribution of cadmium in corn tissues.

10. Application of a method for studying the mitigation of heavy metal cadmium poisoning in corn according to claims 1-9: the mitigation method is applied to corn planting, and exogenous Si and MT are used to alleviate heavy metal cadmium poisoning in corn. The above research process proves that the combined application of Si and MT alleviates Cd stress, and corn shows higher biomass and plant height; the application of Si, MT or both can increase the chlorophyll content of corn under Cd stress; the application of Si and MT alleviates the oxidative damage caused by Cd stress in corn by increasing the activity of antioxidant enzymes; the combined application of Si and MT reduces the absorption of Cd in roots and leaves; the application of Si and MT treatments both significantly increased the expression level of the ZmHMA3 gene.

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