Method for relieving soybean cadmium stress by using 2, 4-epibrassinolide and method for comparing relieving effect of 2, 4-epibrassinolide on soybean cadmium stress

Abstract

The invention relates to the technical field of cadmium pollution control. The invention provides a method for relieving soybean cadmium stress by using 2, 4-epibrassinolide and a method for comparing the relieving effect of 2, 4-epibrassinolide on soybean cadmium stress, and the method comprises the following steps: spraying 2, 4-epibrassinolide with the concentration of 0.1-1.0 [mu] mol / L on soybean leaves, and continuously spraying for 2-5 days. Soybean varieties with large cadmium sensitivity difference are adopted for water culture test, cadmium treatment is performed on seedlings of the two varieties, 2, 4-epibrassinolide pretreatment is performed at the same time, the influence of 2, 4-epibrassinolide pretreatment on growth inhibition and physiological characteristics of cadmium stress of the soybean seedlings is studied, and the cadmium stress resistance of the soybean seedlings is improved. Potential effects of response and resistance mechanisms of plants under cadmium stress in Cd tolerance are discussed, and a theoretical basis is provided for cultivation of cadmium-tolerant and low-cadmium-accumulation soybean varieties.

Description

Technical Field

[0001] The invention relates to the technical field of cadmium pollution prevention and control, and in particular to a method for alleviating soybean cadmium stress by utilizing 2,4-epibrassinolide and a method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress. Background Art

[0002] Cadmium (Cd) is the most common heavy metal pollutant in farmland. The accumulation of Cd has a negative impact on water status, disrupts photosynthesis, and indirectly induces oxidative stress. Cadmium toxicity has been reported in many plants, including peas, kidney beans, cowpeas, and mung beans. It has been reported that jasmonic acid and potassium, melatonin and potassium, nitric oxide and hydrogen sulfide, silicon and nitric oxide, and salicylic acid can regulate plant heavy metal stress. 2,4-Epibrassinolide is a new class of plant hormones, among which 2,4-epibrassinolide (EBR) is the most active class of plant hormones and has been widely used in experimental studies. A large number of studies have shown that EBR plays a variety of roles in regulating plant growth and development, including cell division and proliferation, photomorphogenesis and photosynthesis, and responses to various environmental conditions. Therefore, EBR can induce plant performance under various abiotic stresses such as low temperature, drought, salt, alkali, and heavy metals by increasing photosynthesis capacity and enhancing antioxidant and detoxification capabilities.

[0003] Leguminous cereals are important and staple crops such as wheat, rice and corn, which contribute greatly to human nutrition. Among leguminous plants, soybean (Glycine max (L). Merr.) belongs to the Fabaceae family, Papilionidae subfamily. Different soybean varieties have different tolerance to Cd stress. However, the mechanism of EBR tolerance to Cd remains poorly understood. Summary of the invention

[0004] The purpose of the present invention is to provide a method for alleviating soybean cadmium stress by utilizing 2,4-epibrassinolide and a method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress. Different cadmium accumulation soybean varieties are taken as research objects, and the differences in the effects of EBR on the physiological, biochemical and cytological characteristics of soybean seedlings with different cadmium tolerance are explored.

[0005] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0006] The invention provides a method for alleviating soybean cadmium stress by utilizing 2,4-epibrassinolide, comprising the following steps: spraying soybean leaves with 2,4-epibrassinolide at a concentration of 0.1-1.0 μmol / L for 2-5 consecutive days.

[0007] Preferably, the spraying period is the two-leaf-one-heart period.

[0008] Preferably, the spraying time is 7-9 am and 16-18 pm.

[0009] The present invention also provides a method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress, comprising the following steps:

[0010] (1) A batch of soybean seedlings at the two-leaf and one-heart stage were selected, grouped and hydroponically cultured, and treated with cadmium stress and 2,4-epibrassinolide, respectively. The gas exchange parameters, chlorophyll fluorescence parameters, SDS-PAGE patterns, Rubisco enzyme activity and rbcS gene expression of each group of soybean plants were then detected;

[0011] (2) Another batch of soybean seedlings at the two-leaf and one-heart stage were selected and grouped for hydroponics. They were subjected to cadmium stress and 2,4-epibrassinolide treatments, respectively. The plant height, stem diameter, fresh weight, dry weight, leaf relative water content, leaf enzyme activity, leaf photosynthetic parameters, pigment content, electrical conductivity, soluble sugar content, leaf thickness, upper epidermis thickness, lower epidermis thickness, palisade tissue thickness, spongy tissue thickness, H2O2, and O 2- , the integrity of the cell membrane, and detect whether the cells are viable;

[0012] (3) Organize the data and perform variance analysis.

[0013] Preferably, the method for cultivating soybean seedlings in step (1) and step (2) is: after sowing soybean seeds, cultivate them under the conditions of 22-25° C. and relative humidity of 70%-80%, and the day and night cycle during the cultivation process is 13-15 / 9-11h;

[0014] In the step (1) and the step (2), 1 / 2 Hoagland solution is used in the hydroponic culture.

[0015] Preferably, the specific method of grouping and processing in step (1) is as follows:

[0016] The T1 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0017] The T2 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0018] The T3 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0019] The T4 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0020] The T5 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution;

[0021] The T6 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution;

[0022] The T7 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution;

[0023] The T8 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution.

[0024] Preferably, the detection in step (1) is performed on the 0th, 1st, 2nd, 3rd, 4th, 5th, 6th and 7th day after cadmium stress treatment.

[0025] Preferably, the grouping and treatment method during the cadmium stress treatment in step (2) are as follows:

[0026] ① The soybean seedlings in the control group without cadmium stress were cultivated with only 1 / 2 Hoagland solution;

[0027] ②There are three experiments on cadmium stress treatment, namely:

[0028] Group 1: cultured with 1 / 2 Hoagland nutrient solution containing 10 mg / L CdCl2;

[0029] Group 2: pretreated with 0.1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L CdCl2;

[0030] The third group was pretreated with 0.1 μmol / L 2,4-epibrassinolide only and cultured with 1 / 2 Hoagland solution.

[0031] Preferably, the detection in step (2) is performed on the third day after the cadmium stress treatment.

[0032] The present invention provides a method for alleviating soybean cadmium stress by using 2,4-epibrassinolide and a method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress, comprising the following steps: spraying soybean leaves with 2,4-epibrassinolide at a concentration of 0.1-1.0 μmol / L, and spraying continuously for 2-5 days. The present invention uses soybean varieties with large differences in cadmium sensitivity to conduct hydroponic experiments, treats seedlings of the two varieties with cadmium, and simultaneously performs 2,4-epibrassinolide pretreatment, studies the effects of 2,4-epibrassinolide pretreatment on growth inhibition and physiological characteristics of soybean seedlings under cadmium stress, explores the response of plants under cadmium stress and the potential role of resistance mechanisms in Cd tolerance, and provides a theoretical basis for breeding soybean varieties with cadmium tolerance and low cadmium accumulation. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 The growth of soybean seedlings under EBR and cadmium stress treatments, where ad is the treatment results of Cd concentrations of 0, 5, 10, and 20 mg / L, and ef is the treatment results of groups T1 to T4;

[0034] Figure 2 The changing trends of chlorophyll fluorescence parameters under different concentrations of cadmium stress;

[0035] Figure 3 The changing trend of spectral index under different concentrations of cadmium stress;

[0036] Figure 4 The changing trend of pigment content under different concentrations of cadmium stress;

[0037] Figure 5 Effects of different concentrations of 2,4-epibrassinolide on gas exchange parameters and chlorophyll fluorescence parameters of soybean seedlings under cadmium stress;

[0038] Figure 6 The effects of 0.01, 0.10, 1 μoml / L EBR on Rubisco activity in soybean seedling leaves under cadmium stress;

[0039] Figure 7 The effect of 0.01, 0.10, 1 μoml / L EBR on the expression of rbcS gene in soybean seedling leaves under cadmium stress;

[0040] Figure 8 The effects of 0.01, 0.10, and 1 μmol / L EBR on the protein profile of soybean seedling leaves under cadmium stress;

[0041] Fig. 9 The effect of 0.1μmol / L EBR on soybean morphological characteristics under cadmium stress;

[0042] Fig.10 The effect of 0.1 μmol / L EBR on soybean fresh weight, dry weight, plant height, stem diameter, and leaf length-width ratio under cadmium stress;

[0043] Fig.11 The effect of 0.1μmol / L EBR on soybean pigment indexes such as Chl a, Chlb, Chl a / b, Chl a+b, and Car under cadmium stress;

[0044] Fig.12 The effects of 0.1 μmol / L EBR on peroxidase (POD), malondialdehyde (MDA), catalase (CAT), electrical conductivity (EC) and relative water content (RWC) of soybean plants under cadmium stress;

[0045] Fig.13 Effects of EBR and cadmium stress on photosynthetic parameters of soybean seedlings;

[0046] Fig.14 This is the cross section of soybean leaves after 3 days of cadmium stress with 0.1μmol / LEBR;

[0047] Fig.15 The effect of 0.1μmol / L EBR on soybean tissue chemical staining after 3 days of cadmium stress;

[0048] Fig.16 Soybean P N Correlation analysis with physiological factors. DETAILED DESCRIPTION

[0049] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0050] Example 1 Materials and Methods

[0051] 1. Effects of different concentrations of cadmium stress on soybean plant morphology and physiological indicators

[0052] Soybean seeds were sown in plug trays and cultured in a plant growth chamber at 25 / 22°C, relative humidity (RH) of 70% to 80%, and a day-night cycle of 14 / 10h. When the soybean seedlings grew to the stage of two leaves and one heart, hydroponic experiments were carried out on plants with consistent growth. Four cadmium concentrations were set in this experiment, CK (0mg / L), 5mg / L, 10mg / L, and 20mg / L. The effects of Cd stress on the photosynthetic parameters, pigment content, and spectral index of leaves of cadmium-tolerant soybean seedlings were analyzed at 7, 14, and 21 days.

[0053] The initial fluorescence (Fo), maximum fluorescence yield (Fm) and maximum photochemical quantum yield of PSII (Fv / Fm) were measured using a portable modulated chlorophyll fluorometer (PAM-2500, Walz, Germany) after selecting the plant leaves for each treatment and dark-treating for 30 min. A German CI-710 spectrometer was used to measure and calculate four reflectance spectral indices: photochemical reflectance index (PRI), calculated using the formula (R531-R570) / (R531+R570); carotenoid / chlorophyll ratio index (CCRI), calculated using the formula ((R720-R521) / R521) / ((R783-R705) / R705); structure-insensitive vegetation index (SIPI), calculated using the formula (R800-R445) / (R800+R680); anthocyanin reflectance index 1 (ARI1), calculated using the formula (1 / R550)-(1 / R700).

[0054] 2. Effects of different concentrations of 2,4-epibrassinolide on physiological and biochemical characteristics of soybean under cadmium stress

[0055] EBR (concentrations of 0.01, 0.10, and 1.0 μmol / L) was sprayed on the leaves of soybean seedlings for 3 consecutive days at 7:00-9:00 am and 16:00-18:00 pm. CdCl2·2.5H2O (CAS NO: 7790-78-5, Aladdin) was added to 1 / 2 Hoagland solution with a final concentration of 10 mg / L. Soybean seedlings that were not subjected to cadmium stress (control) were irrigated with only 1 / 2 Hoagland solution.

[0056] There are 8 treatments in this experiment, and the specific treatments are as follows:

[0057] The T1 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0058] The T2 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0059] The T3 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0060] The T4 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium;

[0061] The T5 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution;

[0062] The T6 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution;

[0063] The T7 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution;

[0064] The T8 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution.

[0065] Gas exchange parameters, chlorophyll fluorescence parameters and SDS-PAGE spectra were measured after 0, 1, 2, 3, 4, 5, 6 and 7 days of cadmium treatment. -2 .s -1 , atmospheric CO2 concentration 400μmol.mol -1 , air flow 500μmol.S -1 Gas exchange parameters, including net photosynthetic rate, transpiration rate, stomatal conductance and intercellular CO2 concentration, were measured under the following conditions. SDS-PAGE experiments were performed according to Hu Zhihui (2016) and others. Rubisco enzyme activity was detected using a plant ribulose bisphosphate carboxylase (Rubisco) enzyme-linked immunosorbent assay kit. The expression level of ribulose-1,5-bisphosphate carboxylase small subunit (rbcS) gene was determined according to the method of Wu Chunxing (2024).

[0066] 3. Effects of 0.1 μmol / L EBR on morphological and physiological parameters of soybean plants under cadmium stress

[0067] When the seedlings grow to two leaves and one heart, select seedlings with consistent growth and transplant them into the hydroponic box. The soybean seedlings are subjected to the following stress treatment 7 days after transplanting:

[0068] The order is CK: 1 / 2 Hoagland nutrient solution culture;

[0069] Group 1: cultured with 1 / 2 Hoagland nutrient solution containing 10 mg / L CdCl2;

[0070] Group 2: pretreated with 0.1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L CdCl2;

[0071] The third group was pretreated with 0.1 μmol / L 2,4-epibrassinolide only and cultured with 1 / 2 Hoagland solution.

[0072] EBR pretreatment refers to the treatment of spraying EBR on soybean leaves 3 days before the onset of Cd stress. Soybeans from each treatment group were collected 3 days after Cd stress treatment. The sampling was repeated 3 times for each treatment group. After sampling, they were immediately placed in liquid nitrogen and stored in a -80℃ refrigerator.

[0073] Results and Analysis

[0074] 1. Growth of soybean seedlings under EBR and cadmium stress

[0075] Depend on Figure 1 It can be seen that with the increase of cadmium concentration, the growth of soybean seedlings was inhibited (ad), especially under 10mg / L Cd and 20mg / L cadmium stress, the growth of soybean seedlings was significantly inhibited. Figure 1 (g) (EBR concentration is 0.10 μmol / L). Figure 1 As shown in (eh), compared with the 0, 0.01, and 1 μmol / LEBR treatments, 0.10 μmol / LEBR significantly promoted the growth of soybean plants under cadmium stress.

[0076] 2. Changing trends of chlorophyll fluorescence parameters of soybean seedlings under different concentrations of cadmium stress

[0077] The average values ​​of Y(II) of soybean seedlings at 7 and 14 days after Cd stress were 0.72 and 0.72, which were 2.99% and 3.07% higher than those of the control without Cd stress, with significant differences (P<0.05); the average value at 21 days after stress was 0.67, which was 4.00% lower than that of the control, with extremely significant differences (P<0.01) ( Figure 2 a). After 7d and 14d of stress, the mean Fv / Fm values ​​were 0.81 and 0.81, respectively, which were 0.32% and 0.19% higher than the control, respectively, with no significant difference; after 21d of stress, the mean Fv / Fm value was 0.78, which was 4.06% lower than the control, with a very significant difference (P<0.01) ( Figure 2 b). The average value of Fo at different heavy metal concentrations 7 days after stress was 480, which was 9.00% higher than that of the control, with no significant difference; the average values ​​of Fo at 14 days and 21 days after heavy metal stress were 508.45 and 520.60, respectively, which were 15.71% and 18.48% higher than that of the control, with extremely significant differences (P<0.01) ( Figure 2c). The average Fm values ​​after 7 and 14 days of Cd stress were 2535.25 and 2660.95, respectively, which were 11.28% and 16.79% higher than those of the control, with extremely significant differences (P<0.01); after 21 days of stress, the average Fm value was 2346.10, which was 2.98% higher than that of the control, with no significant difference. Moreover, Fm did not increase significantly at 10 mg / L and 20 mg / L, but increased extremely significantly at 5 mg / L ( Figure 2 d).

[0078] 3. Change trend of spectral index of soybean seedlings under different concentrations of cadmium stress

[0079] The results show that Figure 3 a): PRI increased significantly after 5mg / L and 10mg / L Cd stress. Under 0mg / L and 20mg / L cadmium stress, PRI showed a high-low-high trend. 7d after Cd stress, the average PRI was 0.0467, which increased to 36.95% compared with the control, with a very significant difference (P<0.01); 14d and 21d after Cd stress, the average PRI was 0.0387 and 0.0379, respectively, which increased to 13.49% and 11.14% compared with the control, with no significant difference. The results showed ( Figure 3 b): CCR1 at 7d, 14d, and 21d after Cd stress was significantly higher than that of the control group, which were 79.02%, 37.41%, and 39.86%, respectively (P<0.01). CCR1 at 5mg / L and 20mg / L Cd stress was significantly higher than that of the control group, which were 51.25% and 126.92%, respectively (P<0.01). The results showed that ( Figure 3 c): The average SIPI value of the plants 7 days after Cd stress was 0.7055, which was 1.90% lower than that of the control, with a very significant difference. After 14 days and 21 days of heavy metal stress, the average SIPI values ​​were 0.7199 and 0.7219, respectively, which were 0.97% and 0.38% higher than that of the control, respectively, with no significant difference. The results showed ( Figure 3 d): The average ARI1 values ​​of plants 7d, 14d, and 21d after Cd stress were 0.0091, 0.0097, and 0.0093, respectively, which were not significantly different from the control.

[0080] 4. Changes in pigment content in soybean seedlings under different concentrations of cadmium stress

[0081] When the Cd concentration reached 10 mg / L, the average chlorophyll content of the leaves was 2.87, which was 42.28% lower than the control, and the difference was extremely significant; when the Cd concentration reached 5, the average chlorophyll content of the leaves was 3.60, which was 27.65% lower than the control, and the difference was significant. After 7 and 14 days of Cd stress, there was no significant difference in the chlorophyll content of the leaves compared with the control; after 21 days of Cd stress, the average chlorophyll content of the leaves was 2.18 mg / g, which was 53.24% lower than the control, and the difference was extremely significant ( Figure 4 a). When the Cd concentration reached 10, the average carotenoid content of leaves at different periods reached 0.74, which was 43.88% higher than the control, with a very significant difference; when the Cd concentration reached 5 mg / L and 20 mg / L, the average carotenoid content of leaves at different periods was 0.5 and 0.41, which was 3.23% and 20.64% lower than the control, with no significant difference. The carotenoid content of different Cd concentrations was 0.44 and 0.43 7 and 14 days after stress, which was 33.21% and 34.16% lower than the control, respectively, with a very significant difference. After 21 days of heavy metal stress, the average carotenoid content was 0.65, with no significant difference compared with the control ( Figure 4 b).

[0082] Effects of 5, 0.01, 0.10, and 1 μmol / L EBR on plant morphology, gas exchange parameters, and chlorophyll fluorescence parameters of soybean seedlings under cadmium stress

[0083] Depend on Figure 5 It can be seen that the photosynthetic rates of T1, T2, T3, and T4 began to increase on the second day of cadmium stress and reached the highest value on the fourth day, which were 12.60, 14.31, 16.23, and 14.69 μmol / (m 2 .s), and with the extension of cadmium stress time, the photosynthetic rate gradually decreased to the initial level of about 10μmol / (m2.s). The photosynthetic rates of T6, T7, and T8 gradually increased after cadmium stress, reaching the highest value on the 4th day, which were 16.07, 17.62, and 16.20μmol / (m2.s), respectively. 2 .s), and then gradually decreased. After cadmium stress, the photosynthetic rate of T5 decreased and reached a minimum of 4.76μmol / (m 2 .s), and the photosynthetic rate reached a maximum of 11.94 μmol / (m 2.s). The stomatal conductance of T1, T2, and T3 decreased after the first day of cadmium stress, and began to increase on the second day, while the stomatal conductance of T4 increased after the first day of cadmium stress, and began to decrease after 2 days. In the T5, T6, T7, and T8 treatments where only EBR solution was sprayed, the stomatal conductance of the plants treated with 0.10μmol / L EBR (T7) remained at a high level (not shown in the figure). Compared with the treatment without EBR spraying, spraying EBR can increase the intercellular CO2 concentration of T2, T3, and T4, and the maximum values ​​can reach 340.66, 350.17, and 325.01μmol / (m 2 .s), and can also increase the transpiration rate of T2, T3, and T4. EBR can maintain the Fv / Fm value of plant leaves at a high level. The highest values ​​of Fv / Fm values ​​of T2, T3, and T4 are 0.81, 0.83, and 0.80, respectively. Therefore, under the stress of heavy metal cadmium, EBR can alleviate the reduction of photosynthetic rate. The mitigation effect of different concentrations of EBR is inconsistent. When the concentration of EBR reaches 0.10μmol / L, it can play a significant mitigation effect.

[0084] 6. Effects of 0.01, 0.10, and 1 μmol / L EBR on Rubisco activity in soybean seedling leaves under cadmium stress

[0085] Compared with T1 without EBR, spraying EBR (including 0.01, 0.10, 1 μmol / L) significantly increased the activity of Rubisco carboxylase in soybean leaves after 1 and 5 days of cadmium stress (P<0.05); however, after 3 days of cadmium stress, spraying EBR (including 0.01 μmol / L, 0.10 μmol / L) did not significantly increase the activity of Rubisco carboxylase. The activity of Rubisco carboxylase could only be significantly increased when the concentration of EBR reached 1 μmol / L (P<0.05). In addition, the activity of Rubisco carboxylase in soybean plants (T5, T6, T7, T8) sprayed with EBR alone was higher than that in soybean plants under cadmium stress ( Figure 6 ).

[0086] 7. Effects of 0.01, 0.10, and 1 μmol / L EBR on rbcS gene expression in soybean seedling leaves under cadmium stress

[0087] Compared with T1 without EBR, 0.01 μmol / L EBR solution significantly increased the expression of rbcS gene after 1 day of cadmium stress, while 0.10 and 1 μmol / L EBR solutions did not significantly increase the expression of rbcS gene. After 5 days of cadmium stress, 1 μmol / L EBR solution significantly increased the expression of rbcS gene in soybean leaves ( Figure 7 ).

[0088] 8. Effects of 0.01, 0.10, and 1 μmol / L EBR on SDS-PAGE patterns of soybean seedling leaves under cadmium stress

[0089] After spraying different concentrations of EBR solution on the leaves of soybean seedlings, SDS-PAGE test was performed on the leaves of soybean seedlings on the 0th and 7th days of cadmium stress, and the SDS-PAGE protein expression map was obtained ( Figure 8 ). Compared with the 0th day of cadmium stress, when the spraying concentration was 0.01mol.L -1 When the concentration of EBR solution was 0.10 mol.L -1 and 1mol.L -1 When the protein bands were restored to the original number and gray value (day 0 after cadmium stress) after 7 days of cadmium stress. In addition, the protein profiles of T5, T6, T7, and T8 did not change significantly at 0 and 7 days after stress. Therefore, the 0.10 μmol / L EBR solution can alleviate the expression level of proteins under cadmium stress.

[0090] 9. Effect of spraying EBR on soybean leaf morphology under cadmium stress

[0091] Compared with 0, 0.01, and 1 μmol / L EBR treatments, 0.1 μmol / L EBR promoted the growth of soybean plants under cadmium stress. Cadmium stress inhibited the growth of soybean seedlings. After spraying EBR, the growth state of the seedlings was restored. The growth of the seedlings sprayed with EBR only was promoted ( Fig. 9 ).

[0092] 10. Effect of 0.1 μmol / L EBR on soybean growth under cadmium stress

[0093] Depend on Fig.10It can be seen that under cadmium stress, the fresh weight and dry weight of cadmium-tolerant soybean m6 decreased significantly by 23.00% and 32.00% (P<0.05), and the fresh weight and dry weight of cadmium-sensitive soybean m20 decreased by 15.26% and 16.00%, respectively, and there was no significant difference before and after cadmium stress. The treatment with 0.1μmol / L EBR increased the fresh weight and dry weight of m6 by 7.00% and 16.00%, respectively, and reduced the fresh weight and dry weight of m20 by 12.87% and 19.00%, respectively, and there was no significant difference before and after treatment. Under cadmium stress, the plant height of m6 and m20 decreased significantly, but the plant height did not change significantly before and after spraying EBR. There was no significant difference in stem diameter and leaf length / width ratio between m6 and m20 in T1 (treatment 1, CK), T2 (Cd stress), T3 (Cd+EBR) and T4 (CK+EBR) groups. Therefore, cadmium stress reduced the fresh weight and dry weight of m6 and m20. Short-term application of 0.1 μmol / L EBR could not effectively alleviate the decrease of dry weight and fresh weight of soybean seedlings under cadmium stress, and had no alleviating effect on plant height, stem diameter and leaf length-width ratio.

[0094] 11. Effects of 0.1 μmol / LEBR on photosynthetic pigments in soybean leaves under cadmium stress

[0095] Cadmium stress had no significant effect on Chl a, Chl b, Car, Chl a+b, and Chl a / b in the leaves of m6 and m20 seedlings. After spraying 0.1 μmol / L EBR, the Chl b content in the leaves of cadmium-tolerant soybean m6 seedlings (T3) was significantly reduced by 12.45% compared with the Cd (T2) group, and the Chl a+b content was significantly increased by 17.7%, and other indicators did not change significantly. Compared with the Cd (T2) group, the contents of Chl a and Car (T3) were significantly reduced by 46.26% and 45.42%, respectively, and other indicators did not change significantly. Therefore, the application of EBR can significantly increase the Chl a+b of m6 under cadmium stress, but has no effect on the pigment index of m20 ( Fig.11 ).

[0096] 12. Effects of spraying 0.1 μmol / L EBR on POD, MDA, CAT, EC and RWC of soybean leaves under cadmium stress

[0097] This study measured catalase (CAT) activity, peroxidase (POD) activity, electrical conductivity (EC), malondialdehyde (MDA) content and relative water content (RWC). These physiological indices are often used to indicate oxidative damage in plants. Fig.12As shown in the results, POD, CAT, EC and RWC of leaves of cadmium-tolerant soybean m6 increased significantly under cadmium stress (T2), while MDA content did not change significantly before and after cadmium stress. 0.1μmol / L EBR significantly reduced POD activity, CAT activity, EC and RWC of cadmium-tolerant soybean m6, while MDA content did not change significantly before and after cadmium stress. Under cadmium stress, MDA, CAT, EC and RWC of cadmium-sensitive soybean m20 decreased significantly, and POD increased significantly. 0.1μmol / L EBR significantly reduced POD activity and EC of m20. The above results show that spraying EBR can significantly reduce POD, CAT, EC and RWC of cadmium-tolerant soybean m6, but EBR can only effectively reduce POD activity and EC of cadmium-sensitive soybean m20, and EBR induces a stronger adaptation mechanism of m6.

[0098] 13. Effect of spraying 0.1 μmol / L EBR on photosynthetic parameters of soybean leaves under cadmium stress

[0099] In this study, photosynthetic parameters were measured in the first period (before spraying EBR), the second period (after spraying EBR and before cadmium stress), and the third period (after spraying EBR and after cadmium stress). N , g s , Ci, and E values ​​were the highest in the second stage. In the third stage, P N The value of Cd+EBR(T3) group was 2.53% lower than that of CK group, but the difference was not significant. N The value of P in CK+EBR(T4) group was significantly increased by 285.00% compared with that in Cd(T2) group. N The value was significantly increased by 77.46% compared with the CK group, indicating that spraying EBR can significantly increase the P value of cadmium-tolerant soybean seedlings under cadmium stress. N The P value of the control group can also significantly increase N The P values ​​of cadmium-sensitive soybean seedlings treated with Cd (T2) and Cd+EBR (T3) were significantly higher than those of control (P<0.05). N The values ​​of P in Cd+EBR(T3) group were significantly lower than those in CK group by 52.14% and 59.26%. N The value was significantly lower than that of Cd(T2) group (14.88%). The results showed that spraying EBR could effectively alleviate the cadmium stress-induced cadmium-tolerant soybean P N The value decreased, but it could not effectively alleviate the cadmium-sensitive soybean P N In the third period, the P value of m20 under CK (T1), Cd (T2) and CK+EBR (T4) treatments decreased. N The values ​​were higher than those in the m6 treatment, indicating that the photosynthetic efficiency of the leaves of cadmium-tolerant soybean seedlings was higher than that of the leaves of cadmium-sensitive soybean seedlings under the same treatment.

[0100] 14. Effect of spraying 0.1 μmol / L EBR on the cell structure of soybean leaves under cadmium stress

[0101] The anatomical structure of soybean seedling leaves has typical C3 plant anatomical characteristics, with epidermis and two layers of palisade cells. Fig.14 The results showed that the palisade tissue cells of CK group cadmium-resistant soybean leaves were arranged closely and neatly in a long columnar shape. After cadmium stress, some palisade tissue cells became shortened, arranged unevenly, and had larger intercellular distances. In addition, the cell microstructure images of cadmium-sensitive soybeans showed that compared with the cadmium-resistant soybean group, the palisade tissue cells of each treatment were arranged closely and the palisade tissue thickness increased, which was beneficial to the photosynthesis of the leaves.

[0102] 15. Application of 0.1 μmol / LEBR in soybean leaf histochemical staining under cadmium stress

[0103] When plants respond to stress, they often accumulate reactive oxygen species (ROS). In this study, NBT, DAB, and EB staining were used to detect superoxide content, hydrogen peroxide content, and cell activity in plants. Fig.15 It can be seen that the results of histochemical staining of cadmium-sensitive soybeans showed that some damaged spots appeared on the leaves of the control (CK) after NBT, DAB and EB staining. After cadmium stress (T2), the spots became smaller, and after cadmium + EBR treatment (T3), the spots became larger. After CK + EBR treatment, the damaged spots on the leaves of the control (CK) were reduced, indicating that spraying EBR could not alleviate the damage of cadmium stress to cadmium-sensitive soybean leaves. In contrast, different treatments caused less damage to the leaves of soybean cadmium-tolerant seedlings.

[0104] 16. Simple correlation analysis and multiple linear regression analysis.

[0105] There are many factors that affect photosynthesis. Fig.16 The results showed that stem diameter was significantly positively correlated with Chl b and Chl a+b; Chl a was significantly positively correlated with Chl b, Chl a+b and Car; Chlb was significantly positively correlated with Chl a+b and Car; Chl a / b was significantly negatively correlated with MDA; Chl a+b was significantly positively correlated with Car; g s With Ci, E, P N There is a significant positive correlation.

[0106] Note: Fig.16Height is plant height; Stem diameter is stem diameter; Aspectratio is leaf length-to-width ratio; EC is electrical conductivity; Chl a is chlorophyll a content; Chlb is chlorophyll b content; Chla / b is chlorophyll a / b ratio; Chl a+b is total chlorophyll content; Car is carotenoid content; RWC is relative leaf water content; POD is peroxidase activity; MDA is malondialdehyde content; CAT is catalase activity; gs is stomatal conductance; Ci is intercellular CO2 activity; E is transpiration rate; PN is net photosynthetic rate.

[0107] Multiple linear regression analysis can reflect the soybean P N The correlation between soybean and cadmium-sensitive varieties under different treatments was determined. N According to the Kolmogorov-Smirnov analysis results, when sig.=0.200>0.05, Shapiro-Wilk is suitable for small sample tests, and when sig.=0.171>0.05, that is, P N It obeys normal distribution, so P N Regression analysis was performed on physiological indicators. Physiological indicators such as plant height, stem diameter, leaf length-width ratio, EC, chl a, chlb, chl a / b, chl a+b, car, RWC, POD activity, MDA, CAT activity, E, Ci, gs, etc. were used as X1-X16 to represent the influencing factors, and PN was used as Y for stepwise multiple regression analysis. The best regression equation was: Y=22.417+3.695x16-9.672x3-0.246x11(R 2 =0.995, multiple correlation coefficient R = 0.997, standard error of estimate S = 0.4739, F = 246.854, P < 0.05). The results of the significance test showed that the regression coefficients of X3, X11, and X16 were all less than 0.05, and there were significant differences between the independent variables and the dependent variables. The results of the correlation analysis showed that E was the factor affecting soybean leaf P N The main factors of change, E and P N There is a significant positive correlation. The results of correlation analysis are consistent with the linear regression equation.

[0108] 17. Grey correlation analysis between soybean physiological and biochemical parameters and photosynthetic rate under cadmium stress

[0109] As shown in Table 1, the correlation order between each physiological index and leaf photosynthetic rate is E>g>Ci>stem diameter>length-width ratio>Chla / b>height>RWC>CAT>Car>Chl a>EC>Chl a>MDA>Chl b>POD, among which POD activity has the lowest correlation with photosynthetic rate, with a correlation coefficient of 0.687. Compared with cadmium-tolerant soybean, the correlation between photosynthetic rate and each index of cadmium-sensitive soybean under cadmium stress is higher. Under Cd+EBR treatment (T3), the correlation between photosynthetic rate and each physiological index of cadmium-tolerant soybean is higher than that of cadmium-sensitive soybean.

[0110] Table 1

[0111]

[0112]

[0113] Note: T1, control; T2 (Cd), cadmium stress (10 mg / L); T3 (Cd+EBR), 0.1 μmol / L EBR+10 mg / L cadmium; T4 (CK+EBR), control+0.1 μmol.L -1 EBR; control, spraying an equal amount of water instead of EBR.

[0114] As can be seen from the above embodiments, the present invention provides a method for alleviating soybean cadmium stress using 2,4-epibrassinolide and a method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress, comprising the following steps: spraying soybean leaves with 2,4-epibrassinolide at a concentration of 0.1-1.0 μmol / L, and spraying continuously for 2-5 days. The present invention uses soybean varieties with large differences in cadmium sensitivity to conduct hydroponic experiments, treats seedlings of these two varieties with cadmium, and simultaneously pre-treats them with 2,4-epibrassinolide, studies the effects of 2,4-epibrassinolide pretreatment on growth inhibition and physiological characteristics of soybean seedlings under cadmium stress, explores the response of plants under cadmium stress and the potential role of resistance mechanisms in Cd tolerance, and provides a theoretical basis for breeding soybean varieties with cadmium tolerance and low cadmium accumulation.

[0115] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for alleviating soybean cadmium stress using 2,4-epibrassinolide, characterized in that: The method comprises the following steps: spraying soybean leaves with 2,4-epibrassinolide with a concentration of 0.1-1.0 μmol / L for 2-5 consecutive days.

2. The method according to claim 1, characterized in that The spraying period is the two-leaf and one-heart period.

3. The method according to claim 1, characterized in that The spraying time is 7-9 am and 16-18 pm.

4. A method for comparing the effect of 2,4-epibrassinolide on alleviating soybean cadmium stress, characterized in that: The steps include: (1) A batch of soybean seedlings at the two-leaf and one-heart stage were selected, grouped and hydroponically cultured, and treated with cadmium stress and 2,4-epibrassinolide, respectively. The gas exchange parameters, chlorophyll fluorescence parameters, SDS-PAGE patterns, Rubisco enzyme activity and rbcS gene expression of each group of soybean plants were then detected; (2) Another batch of soybean seedlings at the two-leaf and one-heart stage were selected and grouped for hydroponics. They were subjected to cadmium stress and 2,4-epibrassinolide treatments, respectively. The plant height, stem diameter, fresh weight, dry weight, leaf relative water content, leaf enzyme activity, leaf photosynthetic parameters, pigment content, electrical conductivity, soluble sugar content, leaf thickness, upper epidermis thickness, lower epidermis thickness, palisade tissue thickness, spongy tissue thickness, H2O2, and O 2- , the integrity of the cell membrane, and detect whether the cells are alive; (3) Organize the data and perform variance analysis.

5. The method according to claim 4, characterized in that The method for cultivating soybean seedlings in step (1) and step (2) is as follows: after sowing soybean seeds, culturing them under the conditions of 22-25° C. and relative humidity of 70%-80%, wherein the day and night cycle during the culturing process is 13-15 / 9-11 hours; In the step (1) and the step (2), 1 / 2 Hoagland solution is used in the hydroponic culture.

6. The method according to claim 4, characterized in that The specific method of grouping and processing described in step (1) is as follows: The T1 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium; The T2 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium; The T3 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium; The T4 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L cadmium; The T5 group was sprayed with clean water and cultured with 1 / 2 Hoagland solution; The T6 group was sprayed with 0.01 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution; The T7 group was sprayed with 0.10 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution; The T8 group was sprayed with 1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution.

7. The method according to claim 4, characterized in that The timing of the detection in step (1) is 0, 1, 2, 3, 4, 5, 6 and 7 days after cadmium stress treatment.

8. The method according to claim 4, characterized in that The grouping and treatment methods for the cadmium stress treatment in step (2) are as follows: ① The soybean seedlings in the control group without cadmium stress were cultivated with only 1 / 2 Hoagland solution; ②There are three experiments on cadmium stress treatment, namely: Group 1: cultured with 1 / 2 Hoagland nutrient solution containing 10 mg / L CdCl2; Group 2: pretreated with 0.1 μmol / L 2,4-epibrassinolide and cultured with 1 / 2 Hoagland solution containing 10 mg / L CdCl2; The third group was pretreated with 0.1 μmol / L 2,4-epibrassinolide only and cultured with 1 / 2 Hoagland solution.

9. The method according to claim 4, characterized in that The timing of the detection in step (2) is the third day after the cadmium stress treatment.

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