A strain of halobacillus DH111 tolerant and its application in alleviating cadmium toxicity in plants

Abstract

The invention discloses a strain of tolerant bacillus DH111 and its application in alleviating plant cadmium toxicity, belonging to the technical field of heavy metal contaminated soil remediation. The invention discloses a strain of tolerant bacillus DH111, and its deposit number is CGMCC No.26845. The tolerant bacillus DH111 disclosed by the invention has a significant promoting effect on corn seedlings' tolerance to cadmium toxicity, photosynthesis and antioxidant enzyme activity to varying degrees under the heavy metal cadmium stress environment; the tolerant bacillus DH111 can be applied to production, has the effect of promoting plant growth under cadmium stress, and is of great significance for repairing cadmium-contaminated soil and protecting the ecological environment.

Description

Technical Field

[0001] The invention relates to the technical field of heavy metal contaminated soil remediation, and more particularly to a halobacillus tolerant DH111 and application thereof in alleviating cadmium toxicity in plants. Background Art

[0002] Bioremediation technology is a method that uses the metabolic activities of organisms to reduce the content of heavy metals in contaminated soil. It uses the ability of plants, microorganisms or other organisms to absorb, transform or stabilize heavy metals to reduce the concentration of heavy metals in the soil, thereby reducing potential harm to the environment and ecosystem. Functional microorganisms can form a symbiotic relationship with plant roots, enhance the tolerance and absorption capacity of plants to heavy metals, and promote the stabilization and transformation of heavy metals.

[0003] Cadmium is part of the geochemical cycle and exists in the earth's crust, soil, rocks and ores. It can be released into the environment through geological processes, volcanic eruptions, weathering and erosion. The presence of cadmium is related to the interaction and cycle of other elements and plays a certain role in maintaining geochemical balance. It can enter the food chain through plant absorption and transport, thereby affecting the metabolism and biochemical processes in organisms. The presence and transformation of cadmium in organisms are closely related to the growth, development and adaptation of organisms to the environment.

[0004] Cadmium is highly toxic. Excessive accumulation in the soil will have a negative impact on soil microorganisms and biodiversity and cause direct damage. Cadmium can not only inhibit the microbial activity and enzyme activity in the soil, but also destroy the metabolic function of soil microorganisms, thereby reducing the biological activity of the soil. This will lead to the destruction of the soil ecosystem and reduce the fertility and health of the soil. Secondly, cadmium is significantly toxic to plants in terms of absorption and accumulation. Plants absorb cadmium from the soil through their roots and enter the plant body. Cadmium can affect the growth and development of plants, causing plant toxic symptoms such as yellowing leaves and restricted growth. Cadmium has a serious inhibitory effect on plant growth and crop yields, and is easily enriched through the food chain and enters the human body, posing a potential threat to human health. In addition, cadmium also has an adverse effect on the chemical properties and nutrient cycle of the soil. Cadmium can combine with organic and inorganic substances in the soil to form insoluble cadmium compounds, reducing the fertility and water retention capacity of the soil.

[0005] However, cadmium-resistant bacteria have strong tolerance to high concentrations of cadmium and can survive and play a symbiotic role in soils with severe cadmium poisoning. Cadmium-resistant bacteria can improve crop tolerance to cadmium through mycorrhizal symbiosis and promote plant absorption and transport of cadmium, reduce cadmium accumulation in agricultural products, ensure food safety, and achieve sustainable agricultural development. However, in current research, there are few bacteria that can promote plant growth under cadmium poisoning.

[0006] Therefore, providing a strain of halobacillus DH111 and its application in alleviating cadmium toxicity in plants is an urgent problem to be solved by those skilled in the art. Summary of the invention

[0007] In view of this, the present invention provides a salt-tolerant Bacillus DH111 and its application in alleviating plant cadmium toxicity, which has important theoretical and practical significance and important scientific value for achieving sustainable development of agriculture and environment.

[0008] In order to achieve the above object, the present invention adopts the following technical solution:

[0009] A strain of Bacillus halotolerans DH111, with a deposit number of CGMCC No. 26845, has been deposited in the General Microbiology Center of the China Culture Collection Administration, referred to as CGMCC, located at the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is March 17, 2023, and the classification name is Bacillus halotolerans.

[0010] Furthermore, the salt-tolerant Bacillus DH111 is used in cadmium stress resistance.

[0011] Furthermore, the salt-tolerant Bacillus DH111 is used to reduce the cadmium concentration in the environment.

[0012] Furthermore, the salt-tolerant Bacillus DH111 is used to alleviate cadmium toxicity in corn under cadmium stress environment.

[0013] Furthermore, the salt-tolerant Bacillus DH111 is used to promote the increase of corn biomass in a cadmium stress environment.

[0014] Furthermore, the application of the salt-tolerant Bacillus DH111 in promoting the increase of corn biomass.

[0015] Furthermore, the salt-tolerant Bacillus DH111 is used to promote corn photosynthesis in a cadmium stress environment.

[0016] Furthermore, the application of the salt-tolerant Bacillus DH111 in promoting corn photosynthesis.

[0017] Furthermore, the salt-tolerant Bacillus DH111 is used to promote the antioxidant enzyme activity of corn under cadmium stress environment.

[0018] It can be seen from the above technical scheme that, compared with the prior art, the present invention discloses a strain of Bacillus halogenus DH111 and its application in alleviating plant cadmium toxicity. Bacillus halogenus DH111 can tolerate high concentration cadmium stress and can survive in a low concentration cadmium environment (0.3 mM CdCl 2 ) significantly reduced the cadmium content; the tolerant salt Bacillus DH111 significantly promoted the growth of corn seedlings under cadmium stress; the tolerant salt Bacillus DH111 promoted the photosynthesis and antioxidant enzyme activity of corn seedlings to varying degrees under cadmium stress; the tolerant salt Bacillus DH111 can be used in production, has the effect of alleviating plant cadmium toxicity and promoting plant growth, and is of great significance for the remediation of heavy metal contaminated soil and ecological environment protection. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the provided drawings without paying creative work.

[0020] Figure 1 The accompanying drawing shows the colony morphology of the DH111 strain of the present invention;

[0021] Figure 2 The attached figure shows the different concentrations of CdCl 2 The biomass OD of Bacillus halodurans DH111 600 The impact of

[0022] Figure 3 The attached figure shows the different concentrations of CdCl 2 Effect of Halobacillus DH111 on Cd 2+ The influence of content;

[0023] Figure 4 The attached figure shows the different concentrations of CdCl 2 Effect of Halobacillus DH111 on Cd in maize leaves 2+ The influence of content;

[0024] Figure 5 The attached figure shows the different concentrations of CdCl 2 Effects of Bacillus halogenide DH111 on maize biomass;

[0025] Wherein, A: fresh weight of corn; B: dry weight of corn;

[0026] Figure 6 The attached figure shows the different concentrations of CdCl2 Effects of Bacillus halobacterium DH111 on photosynthesis of maize;

[0027] Among them, A: net photosynthetic rate; B: stomatal conductance; C: transpiration rate; D: intercellular CO 2 concentration;

[0028] Figure 7 The attached figure shows the different concentrations of CdCl 2 Effects of Bacillus halobacterium DH111 on antioxidant enzyme activities of maize;

[0029] Among them, A: SOD activity; B: POD activity; C: CAT activity; D: MDA content;

[0030] Figure 3-Figure 7 In the figure, + indicates treatment with strain DH111; - indicates no treatment with strain. DETAILED DESCRIPTION

[0031] 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.

[0032] Example 1 Isolation and identification of halobacillus tolerant DH111 strain

[0033] 1) The DH111 strain was isolated from the soil near the Dingbian Salt Lake in Shaanxi Province by streak culture method. The specific steps are as follows:

[0034] (1) The components of each culture medium are as follows:

[0035] The composition of the inorganic phosphate solid medium is as follows: the composition of the reference NBRIP medium was slightly modified, and the medium contained (per liter) 10g glucose, 5g Ca 3 (PO 4 ) 2 , 0.03 g FeSO 4 7H 2 O, 0.5 g (NH 4 ) 2 SO 4 , 0.3 g MgSO 4 7H 2 O, 0.3gKCl, 0.3gNaCl, 0.03g MnSO 4 and 18 g agar, add distilled water to 1000 ml, and adjust the pH of the agar medium to 7.0-8.0.

[0036] LB solid medium: tryptone 10 g / L, yeast extract powder 10 g / L, sodium chloride 5 g / L, agar 18 g / L, high-pressure steam sterilization at 121°C for 30 min.

[0037] LB liquid culture medium: 10 g / L tryptone, 10 g / L yeast extract powder, 5 g / L sodium chloride, sterilized by high-pressure steam at 121°C for 30 min.

[0038] LB solid selection medium: tryptone 10 g / L, yeast extract powder 10 g / L, sodium chloride 5 g / L, agar 18 g / L, 0.75 mM cadmium chloride, high pressure steam sterilization at 121°C for 30 min.

[0039] LB liquid selection medium: 10 g / L tryptone, 10 g / L yeast extract powder, 5 g / L sodium chloride, (0 mM, 0.3 mM, 0.6 mM, 0.9 mM, 1.2 mM and 1.5 mM) different concentrations of cadmium chloride, 121 ° C, 30 min high pressure steam sterilization.

[0040] (2) Isolation and purification: First, 10 g of saline-alkali soil was weighed and dissolved in 90 ml of sterile water. After shaking culture at 150 rpm for 30 min, the mixture was diluted to 10 -5 . Inoculate 0.2 ml of the dilution solution on an inorganic phosphorus solid culture medium plate, incubate inverted in a 28°C constant temperature incubator for 7 days, select colonies that can produce clear transparent halos and continue to re-streak on the inorganic phosphorus solid culture medium plate 2-3 times to further purify the strain until a single colony grows on the agar plate. Secondly, inoculate the phosphate-solubilizing bacteria isolated by screening on LB solid selective medium, repeat the operation twice, and observe and compare the growth of the strains after 7 days of incubation; in order to determine the Cd 2+ (CdCl 2 ) to the resistance level of bacteria, and 0.75 mM CdCl 2 The inhibitory concentration test was performed and the strains that could grow normally on LB solid selective medium were identified as strains that could tolerate cadmium toxicity and stored on slant at 4°C.

[0041] 2) Pyrosequencing was used to classify and identify the DH111 strain. The specific steps are as follows:

[0042] (1) Preparation of bacterial genomic total DNA: The genomic DNA of the DH111 strain was extracted using a bacterial genomic DNA extraction kit (Tiangen) and used as a template for the PCR reaction.

[0043] (2) PCR amplification of genes:

[0044] The PCR amplification reaction was performed with reference to the reaction system of the TransFast Taq DNA Polymerase Kit instruction manual using bacterial full-length universal primers 27F and 1492R.

[0045] The primer sequences of universal primers 27F and 1492R are as follows:

[0046] 27F: 5'-AGRGTTTTGATYNTGGCTCAG-3'; SEQ ID NO.1;

[0047] 1492R: 5'-TASGGHTACCTTGTTASGACTT-3'; SEQ ID NO. 2.

[0048] The reaction conditions were as follows: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 40 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, for a total of 30 cycles; and extension at 72°C for 10 min.

[0049] (3) PCR product purification, sequencing and analysis

[0050] The PCR amplification product was submitted to the Xi'an Branch of Beijing Qingke Biotechnology Co., Ltd. for sequencing, and the 16S rRNA gene sequence was determined as shown in SEQ ID NO. 3. The sequence returned by sequencing was logged into the website of the National Center for Biotechnology Information (https: / / www.ncbi.nlm.nih.gov / ), and BLAST comparison and identification were performed in the database. The results showed that the homology of the 16S rRNA gene sequence of the DH111 strain and Bacillus halotolerans was 99.58%, so the isolated strain was identified as Bacillus halotolerans.

[0051]

[0052] 3) The morphological identification characteristics of the tolerant Bacillus DH111 strain are as follows:

[0053] The DH111 strain belongs to the Firmicutes, Bacillaceae, Bacillus, and is Gram-positive. It forms white colonies on inorganic phosphorus solid culture media with a convex center and irregular, smooth edges, such as Figure 1 As shown. Glucose, maltose, xylose, sucrose, lactose and galactose can be used as the sole carbon source; ammonium sulfate, diammonium phosphate, sodium nitrate and potassium nitrate can be used as the sole nitrogen source.

[0054] Example 2 Physiological and biochemical characteristics of the tolerant Bacillus DH111 strain

[0055] (1) Starch hydrolysis test

[0056] Preparation of beef extract peptone agar medium containing 0.2% soluble starch: 3g / L beef extract, 5g / L sodium chloride, 10g / L peptone, 2g / L soluble starch, 18g / L agar powder, adjust pH=7-8, sterilize at 121℃ for 20min, pour into a plate in a clean bench for use. Lugo's iodine solution: 1g iodine, 2g potassium iodide, first dissolve potassium iodide with a small amount of deionized water, then add iodine to completely dissolve, and dilute to 300mL with water. Inoculate the strain into the plate culture medium, invert and culture in a 30℃ constant temperature incubator for 3-5 days. After the colony is formed, add Lugo's iodine solution and evenly spread the plate. Observe after a while. If the culture medium is dark blue and a colorless transparent circle appears around the colony, it indicates that the strain can secrete amylase to hydrolyze starch, which is a positive reaction; if there is no transparent circle around the colony, it indicates that the strain cannot hydrolyze starch, which is a negative reaction.

[0057] (2) VP test

[0058] Preparation of culture medium: 5g / L peptone, 5g / L glucose, 5g / L NaCl, adjust pH=7.0-7.2, dispense 10mL into each test tube, sterilize at 121℃ for 20min. Reagents: 40% KOH, 6% α-naphthol anhydrous ethanol solution. In the clean bench, inoculate the strain into the above culture medium, shake and culture at 30℃ constant temperature shaker for 3 days, take 5mL of culture solution, add 300μL 40% KOH solution to the test tube, add 300μL 6% α-naphthol solution, shake vigorously to mix, and then put it in 37℃ constant temperature incubator for 15-30min. If the culture solution turns red, it is a VP positive reaction. Some bacteria need to be placed for 4h before turning red. If it still does not turn red after 4h, it can be judged as a negative reaction.

[0059] (3) MR test

[0060] Culture medium preparation: same as VP test.

[0061] MR reagent: Weigh 0.04 g of methyl red, dissolve it in 60 mL of 95% ethanol solution, and then add 40 mL of ultrapure water to make up to 100 mL.

[0062] In an ultra-clean workbench, inoculate the strain into the above-mentioned culture medium, and culture it in a constant temperature shaker at 30°C for 3 days. Then, use a pipette to draw 5 mL of culture medium into a sterile centrifuge tube, and add 100 μL of MR reagent (color change range pH = 4.2-6.3, color changes from red to yellow). If red appears, it is a positive MR reaction; if it turns yellow, it is a negative reaction.

[0063] (4) Gelatin hydrolysis test

[0064] Preparation of culture medium: 0.5 g peptone, 12 g gelatin, 100 mL deionized water, pH = 7.2-7.4. Dispense 10 mL into each test tube and sterilize at 121°C for 20 min.

[0065] In the clean bench, use a long toothpick to pick up the strain and pierce it into the middle of the gelatin at a depth of about 2 / 3. Culture it at a constant temperature of 20°C for 7 days. Observe the test results every day. If the gelatin itself is liquefied due to the high culture temperature, it should not be shaken. Let it stand in a 4°C refrigerator for 2 hours to solidify. Observe whether the gelatin is liquefied by the bacteria. If it is liquefied, it is a positive reaction for gelatin hydrolysis, otherwise it is a negative reaction.

[0066] (5) Tween 80 hydrolysis test

[0067] Preparation of culture medium: peptone 10g / L, sodium chloride 5g / L, calcium chloride 0.2g / L, Tween 80 10mL / L, agar powder 18g / L, pH = 7-8, sterilize at 121℃ for 20min, invert the plate for use. In the clean bench, inoculate the strain on the culture medium plate, incubate at 30℃ for 3-5 days, and observe. If there is a white halo around the colony, it is a positive reaction, otherwise it is a negative reaction.

[0068] (6) Catalase activity

[0069] Add 600 μL of 3% hydrogen peroxide solution to a 1.5 mL centrifuge tube, use a pipette to draw 400 μL of LB bacterial solution and add it, and observe the generation of bubbles within 30 seconds. The generation of bubbles is a positive reaction for catalase, and the absence of bubbles is a negative reaction for catalase.

[0070] Preparation of LB culture medium: peptone 10g / L, sodium chloride 5g / L, yeast extract powder 10g / L, adjust pH to 7-8, dispense 50mL in 100mL Erlenmeyer flasks, sterilize at 121℃ for 30min. Inoculate DH111 strain into LB liquid medium, shake and culture at 30℃, 180rpm for 3 days, and dilute the final LB culture medium to OD 600 =0.6. All the following experiments that require inoculation of bacterial solution have the same preparation steps.

[0071] (7) H production 2 S capability determination

[0072] Culture medium preparation: peptone 20g / L, NaCl 5g / L, ammonium ferric citrate 0.5g / L, Na 2 S 2 O 3 ·5H 2 O0.5g / L, agar 10g / L, adjust pH=7.2, melt peptone and agar, cool to 60℃, add other ingredients, use a pipette to draw 10mL into test tubes, sterilize at 112.6℃ for 30min, and set aside upright to solidify for use.

[0073] Use a sterilized long toothpick to dip into the LB solution and puncture the H 2 The S test culture medium is placed in a constant temperature incubator at 28°C and cultured for 3 days. If a black precipitation line appears, it indicates that sulfide is produced, which is a positive reaction. Otherwise, it is a negative reaction.

[0074] (8) Determination of the ability to hydrolyze citrate

[0075] Medium preparation: NH 4 H 2 PO 4 1g / L, K 2 HPO 4 1g / L, NaCl 5g / L, MgSO 4 7H 2 O0.2g / L, sodium citrate 2g / L, agar powder 18g / L. Heat and dissolve the above ingredients, adjust the pH to 6.8, then add 1% bromothymol blue ethanol solution indicator, shake well, and it will turn yellow-green. Use a pipette to draw 10mL into a test tube and sterilize at 121℃ for 20min to make a slant culture medium.

[0076] Use a pipette to draw 100 μL of LB bacterial solution and inoculate it into the citrate slant culture medium. Place it in a 28°C incubator and culture it for 3 days. Observe whether the bacteria grow and the color change of the culture medium: yellow or blue indicates a positive reaction; green indicates a negative reaction.

[0077] Starch hydrolysis test, VP test, MR test, gelatin hydrolysis test, Tween 80 hydrolysis test, catalase activity, H production 2 The results of the S ability test and the citrate hydrolysis ability test are shown in Table 1.

[0078] Table 1 Physiological and biochemical characteristics of DH111 strain

[0079]

[0080] Note: +, positive reaction; -, negative reaction.

[0081] Example 3 Determination of cadmium resistance and cadmium removal ability of halobacillus DH111

[0082] (1) Set up liquid culture medium CdCl at pH = 7-8 2 The added concentrations were 0mM, 0.3mM, 0.6mM, 0.9mM, 1.2mM and 1.5mM. 2+ 0.5 mL of DH111 strain culture (LB bacterial solution) was inoculated into LB liquid medium and cultured in a 30°C constant temperature shaker at 180 rpm for 5 days. The horizontal axis represents different culture conditions, and the vertical axis represents the strain propagation index OD 600 Value, see the result Figure 2 .

[0083] according to Figure 2 It can be seen that with the increase of CdCl 2 As the concentration increased, the tolerance of DH111 strain to cadmium stress gradually decreased, that is, the strain propagation capacity OD 600 The value was 0.77 (0 mM CdCl 2 ) has been reduced to 0.02 (1.2mM CdCl 2 ), indicating that the DH111 strain can adapt to a certain concentration of cadmium stress and can survive under low concentrations (0.3 mM CdCl 2 ) It still has good propagation ability under cadmium stress.

[0084] (2) Set up liquid culture medium CdCl at pH = 7-8 2 The added concentrations were 0mM, 0.3mM, 0.6mM, 0.9mM, 1.2mM and 1.5mM. 2+ 0.5 mL of DH111 strain culture (LB bacterial solution) was inoculated into LB liquid medium and cultured in a 30°C constant temperature shaker at 180 rpm for 5 days. At the same time, a blank control was set with no strain added. The horizontal axis was different culture conditions, and the vertical axis was the culture medium Cd. 2+ Content, results see Figure 3 The results showed that different concentrations of CdCl 2 There were some differences in the ability of strains to remove cadmium under different treatments. The ability to remove cadmium under 0.3 mM treatment was the most significant. 2+ The content was significantly reduced by 5.24 mg / L.

[0085] In summary, the DH111 strain can tolerate high cadmium concentrations (1.2 mM CdCl 2 ) and in the presence of cadmium (0.3 mM CdCl 2 ) has good cadmium removal ability.

[0086] Example 4 Effect of strain DH111 on Cd in corn leaves under cadmium stress 2+ Effect of content and biomass

[0087] The river sand was washed with running water to remove the residual mineral nutrients on the surface, and sterilized at 121℃ for 1h to remove the residual microorganisms on the surface. 1.7kg of the treated river sand was weighed and placed in a clean flower pot. The "Zhengdan 958" corn seeds were surface sterilized with 75% anhydrous ethanol for 10min, and then rinsed with sterile water 4-5 times to remove the residual alcohol. Two plants were planted in each flower pot, and 10mL of strain DH111 culture (OD 600 =0.6) three times, each time with an interval of 5 days. In the first month of the growth period, the seedlings were managed with water and Hogland nutrient solution as usual. One month later, cadmium chloride was added, and CdCl 2 Four heavy metal stress conditions with concentrations of 0mM, 4mM, 8mM and 12mM were used for stress treatment. Each flower pot was irrigated with 100mL, and each treatment was repeated four times, for a total of 16 pots of plants. One month after the stress treatment, the plants were harvested, and the cadmium content and biomass of the plant leaves were measured to reflect the response of strain DH111 to cadmium stress. At the same time, a treatment without strain was set as a blank control, and four CdCl 2 The concentration was consistent with the inoculated strain, and each treatment had four biological replicates, totaling 16 pots of plants. 2+ Content see Figure 4 , maize biomass Figure 5 .

[0088] Preparation of Hoagland nutrient solution: 945 mg / L calcium nitrate tetrahydrate, 607 mg / L potassium nitrate, 136 mg / L potassium dihydrogen phosphate, 493 mg / L magnesium sulfate heptahydrate, 2.5 mL / L iron salt solution and 5 mL / L trace element solution.

[0089] Iron salt solution: 2.78 g of ferrous sulfate heptahydrate and 3.73 g of disodium ethylenediaminetetraacetate were dissolved in sterile deionized water respectively, the two were mixed and the final volume was 500 mL, and the pH was adjusted to 5.5.

[0090] Trace element solution: potassium iodide 0.83 mg / l, boric acid 6.2 mg / L, manganese sulfate 22.3 mg / L, zinc sulfate 8.6 mg / L, sodium molybdate 0.25 mg / L, copper sulfate 0.025 mg / L and cobalt chloride 0.025 mg / L.

[0091] according to Figure 4 It can be seen that strain DH111 can significantly reduce the Cd content in corn leaves under medium and high concentrations of cadmium (8mM and 12mM). 2+ The content of cadmium indicates that the inoculated strain DH111 can significantly inhibit the absorption of cadmium by corn, thereby alleviating the stress of cadmium on corn. Figure 5 A and Figure 5 B shows that strain DH111 can significantly increase the fresh biomass of corn under medium and high concentration treatments (8mM and 12mM), and 2 The dry weight biomass of corn was significantly increased in the concentration environment.

[0092] In summary, strain DH111 can not only significantly alleviate the stress and toxic effects of cadmium on maize under medium and high concentrations of cadmium (8mM and 12mM) but also can significantly reduce the toxic effects of cadmium on maize under high concentrations of cadmium (12mM CdCl 2 ) stress significantly promoted the growth and development of corn.

[0093] Example 5 Response of strain DH111 to corn photosynthesis and antioxidant enzyme activity under cadmium stress

[0094] (1) The corn plants in Example 4 were selected, and 3 unfolded leaves were selected from each plant. Each leaf was counted 10 times, and the response of corn photosynthesis (net photosynthetic rate, transpiration rate, stomatal conductance and intercellular carbon dioxide concentration) to cadmium stress was measured using a Li-6400 portable photosynthetic meter (LI-COR Inc., Lincoln, NE, USA). The results are shown in Figure 6 .

[0095] according to Figure 6 It can be seen that strain DH111 can alleviate the adverse effects of cadmium stress on corn photosynthesis. Under cadmium stress treatment, the inoculated strain DH111 can significantly increase the photosynthetic rate of corn; after the inoculation of strain DH111, the net photosynthetic rate under various concentrations of cadmium stress treatment can be significantly promoted. In addition, compared with corn without strain treatment, under low and medium concentrations of cadmium (4mM, 8mM CdCl 2 ) treatment, strain DH111 could significantly promote stomatal conductance; under high concentration of cadmium (12 mM CdCl 2 Under treatment, strain DH111 could significantly increase the transpiration rate; under medium and high concentrations of cadmium (8mM, 12mM CdCl 2) treatment, strain DH111 could significantly reduce the intercellular CO 2 concentration.

[0096] (2) Select corn plants in Example 4 above, take 3g of fresh leaves from each pot and preserve them. Four biological replicates were performed for each treatment, for a total of 32 samples. Extract crude enzyme solution and measure SOD, POD, CAT activities and MDA content. The results are shown in Figure 7 .

[0097] Extraction method of crude enzyme solution: weigh 3g of fresh corn leaves, add about 15mL of crude enzyme extract, grind thoroughly in an ice bath, centrifuge at 10000rpm for 20min, and take the supernatant and refrigerate for later use.

[0098] Preparation of crude enzyme extract: 50mmol·L -1 Prepared with PBS at pH = 7.8, containing 0.1 mmol·L -1 EDTA, 0.3% Triton X-100 and 4% polyvinylpolypyrrolidone (PVPP) (Rohn) by mass concentration, wherein Triton X-100 (Rohn) and PBS are mixed in advance and placed in a 37°C to 40°C water bath for 2 to 3 hours to fully dissolve and mix for use.

[0099] 50mmol·L -1 Preparation of PBS at pH = 7.8:

[0100] Solution A: Prepare 500mL 0.2M NaH 2 PO 4 ·H 2 O (Shanghai test), weigh 13.8 g and dilute to 500 mL;

[0101] Solution B: Prepare 1L 0.2M Na 2 HPO 4 12H 2 0 (Shanghai test), weigh 71.6g / L and dilute to 1L;

[0102] 50mmol·L -1 PBS (pH 7.8): 85 mL of solution A + 915 mL of solution B = 1000 mL, dilute 4X to obtain 4L of 50 mmol / L -1 PBS.

[0103] 1) SOD determination method

[0104] Take 10mL centrifuge tubes and add 3mL of reaction mixture (in 54mL 14.5mmol·L -1 DL-methionine (McLean), add 2 mL of 3 μmol·L -1EDTA, 2mL 2.25mmol·L -1 NBT (McLean) and 2 mL 60 μmol·L -1 Riboflavin solution (McLean)) and crude enzyme solution 0.02 mL, mixed and placed on a transparent centrifuge tube rack, illuminated in a light incubator (8000 Lux), and immediately after 10 minutes of illumination, away from light, and quickly measured A 560 The value was obtained by taking the illumination tube without enzyme solution as control and calculating the percentage of reaction inhibition. One enzyme unit was defined as the unit that could inhibit 50% of the photochemical reaction of NBT.

[0105] Total SOD activity = (A ck -A E )V / (0.5×A ck ×W×V t )

[0106] In the formula

[0107] A ck : Absorbance of control centrifuge tube

[0108] A E : Absorbance of sample tube

[0109] V: total volume of extract (mL)

[0110] V t :Sample volume used in the measurement (mL)

[0111] W: Sample amount (g)

[0112] 2) POD determination method

[0113] The guaiacol method was used. 3 mL of the reaction mixture and 0.05 mL of PBS phosphate buffer were added to a 10 mL centrifuge tube as a zero control; 3 mL of the reaction mixture and 0.05 mL of the crude enzyme solution were added to another 10 mL centrifuge tube, and the mixture was quickly shaken and the A 470 The value was counted once at 0, 30s and 60s respectively, and the enzyme activity unit was expressed as the absorbance change value per gram of fresh weight per minute, and the unit was ΔA470 / min·g·FW. (Reaction mixture preparation: 0.05mL 50mmol·L -1 PBS (pH = 7.8), 0.95 mL 0.2% guaiacol (McLean) and 2 mL 0.2% hydrogen peroxide were mixed evenly)

[0114] POD activity (U) = (ΔA470 × V) / (0.001 × Δt × Vs × W)

[0115] In the formula

[0116] ΔA470: Absorbance change of the reaction mixture (A470)

[0117] Δt: Enzymatic reaction time (min)

[0118] V: Total volume of sample extract (mL)

[0119] Vs: Volume of sample extract taken during measurement (mL)

[0120] W: sample mass (g).

[0121] 3) CAT determination method

[0122] Take 2.9 mL of the reaction mixture and add 0.1 mL of the crude enzyme solution. Quickly invert 2-3 times to mix thoroughly. Immediately measure A 240 The value is counted once at 0, 30s and 60s. It is expressed as the absorbance change per gram of fresh weight per minute (ΔA240 / min·mg·FW). (Reaction mixture preparation: 1mL 0.2% hydrogen peroxide (diluted with PBS with pH = 7.8), add 1.9mL distilled water to dilute)

[0123] The calculation formula is: u / mg=E 240 ×3 / 0.0436×E W

[0124] In the formula

[0125] E 240 : The decrease in absorbance per minute at 240 nm

[0126] E W : Fresh weight of sample in each 0.1mL crude enzyme extract (mg)

[0127] 0.0436: Absorbance of 1 μmol hydrogen peroxide at 240 nm.

[0128] 3: Total volume of the reaction mixture.

[0129] 4) MDA determination method

[0130] 3 mL of crude enzyme solution and 3 mL of 0.6% thiobarbituric acid (McLean) were taken, mixed and capped in a 10 mL centrifuge tube, reacted in a boiling water bath for 15 min, quickly cooled in an ice bath and centrifuged at 4000 rpm for 10 min, and the supernatant was taken to determine A 532 , A 450 value.

[0131] The calculation method is: MDA (μmol -1 )=6.45A 532 -0.56A 450

[0132] MDA (nmol·g -1 )=MDA(μmol -1 )×V / W×1000

[0133] In the formula

[0134] V: Amount of crude enzyme extract used

[0135] W: fresh weight of sample (g).

[0136] according to Figure 7 It can be seen that strain DH111 can reduce the adverse effects of corn on the response to cadmium stress, which leads to increased reactive oxygen content. Compared with corn without strain inoculation, in the absence of CdCl 2 Under stress conditions, strain DH111 could significantly promote the enhancement of SOD activity in maize; at 4 mM CdCl 2 Under stress conditions, strain DH111 could significantly promote the enhancement of POD activity in maize and reduce the MDA content in maize. 2 Under stress conditions, strain DH111 could significantly promote the activities of SOD and POD in maize; under 12 mM CdCl 2 Under stress conditions, strain DH111 can significantly promote the enhancement of SOD and CAT activities in corn. It can be seen that under different concentrations of cadmium stress, the antioxidant enzyme activity of corn showed significant differences, and overall it can alleviate the stress and toxic effects of cadmium on corn.

[0137] In summary, the tolerant Bacillus DH111 strain can alleviate the adverse effects of cadmium stress on corn photosynthesis, promote the enhancement of corn antioxidant enzyme activity, and reduce the damage of reactive oxygen species produced by cadmium stress to corn.

[0138] The above description of the disclosed embodiments enables one skilled in the art to implement or use the present invention. Various modifications to these embodiments will be apparent to one skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of Bacillus halotolerans DH111, characterized in that: Its deposit number is CGMCC No.26845.

2. Use of the halobacillus tolerant DH111 according to claim 1 in reducing cadmium concentration in the environment.

3. Use of the salt-tolerant Bacillus DH111 described in claim 1 in alleviating cadmium toxicity in corn under cadmium stress environment.

4. Use of the halobacillus tolerant DH111 according to claim 1 in promoting the increase of corn biomass under cadmium stress environment.

5. Use of the halobacillus tolerant DH111 according to claim 1 in promoting the increase of corn biomass.

6. Use of the halobacillus tolerant DH111 according to claim 1 in promoting corn photosynthesis under cadmium stress environment.

7. Use of the halobacillus tolerant DH111 according to claim 1 in promoting corn photosynthesis.

8. Use of the halobacillus tolerant DH111 according to claim 1 in promoting the antioxidant enzyme activity of corn under cadmium stress environment.

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