CN116875489A - A preparation method and application of a salt-dissolving and growth-promoting compound microbial inoculant - Google Patents

Abstract

The invention discloses a preparation method and application of a salt-dissolving growth-promoting compound microbial agent, comprising the following steps: e.coli BL-EF and E.cholerae Rs-2 are respectively activated and cultured, and when the bacterial strain of the E.coli BL-EF is grown to the end of the logarithmic phase, IPTG is added for low-temperature induction. Respectively diluting the obtained escherichia coli BL-EF fermentation liquor and escherichia coli Rs-2 fermentation liquor which are used for efficiently expressing ACC deaminase by using physiological saline to prepare escherichia coli BL-EF fermentation liquor and escherichia coli Rs-2 fermentation liquor which are used for efficiently expressing ACC deaminase, and then mixing the escherichia coli BL-EF fermentation liquor and the escherichia coli Rs-2 fermentation liquor which are used for efficiently expressing ACC deaminase according to a volume ratio of 3:1, mixing to obtain the salt-dissolving and growth-promoting compound microbial agent. The preparation method and the application of the salt-dissolving and growth-promoting compound microbial agent can improve the stress resistance of crops, promote the increase of the biomass content of the crops and the growth of stems and leaves, strengthen the accumulation of the biomass of the crops, and improve the economic value of the crops on the whole level.

Description

A preparation method and application of a salt-dissolving and growth-promoting compound microbial inoculant

Technical field

The present invention relates to the technical field of agricultural microorganism production, and in particular to a preparation method and application of a salt-solubilizing and growth-promoting compound microbial inoculant.

Background technique

Soil salinization is one of the most important factors limiting crop growth and yield worldwide, which greatly hinders the sustainable development of modern agricultural economy. It is estimated that by 2050, half of the world's farmland will be salinized. Secondary salinization of soil is also intensifying. Nearly 33% of irrigated farmland worldwide is undergoing secondary salinization due to unreasonable disposal measures, and it continues to intensify. In arid and semi-arid regions of the world, low rainfall, high evaporation, irrigation with low saline water, and inefficient water management also exacerbate soil salinization problems.

Ethylene, as a gaseous plant response hormone, is closely related to plant fruit ripening, seed germination and root formation. It is one of the five endogenous hormones of plants. However, when plants face biotic stresses, such as diseases, insect pests, and pathogenic bacteria; or abiotic stresses, drought, floods, salinity, etc., ethylene will be maintained at a high level. Excessive ethylene will cause plant leaves to wither and cells to undergo apoptosis. , and even cause plant death. Therefore, reducing the accumulation level of ethylene in plants under stress has become the key to improving plant growth, development and stress resistance. ACC deaminase-producing bacteria play an important role in mitigating the harm caused by excessive ethylene. 1-Aminocyclopropane-1-carboxylic acid deaminase, also known as ACC deaminase (ACCDeaminase), EC: 4.1.99.4, is encoded by the ACC deaminase gene (acdS). Microorganisms that produce ACC deaminase can catalyze the cleavage of ACC, the precursor of ethylene, to generate α-ketobutyric acid and ammonia. They can not only obtain some of the C and N source materials they need, but also reduce the stress of plants due to stress to a certain extent. The large amount of ethylene produced enhances plants' ability to adapt to the environment.

At present, the synthesis of ACC deaminase basically relies on the production of growth-promoting bacteria in the plant rhizosphere, and there are few plants that can produce ACC deaminase on their own. ACC deaminase has been found in many microorganisms, such as Enterobacter cloacae, Achromobacter, Rhizobium, etc. Fungi can also secrete and express ACC deaminase, but its expression frequency in fungi is lower than that in bacteria. The activity of ACC deaminase produced by plants and plant rhizosphere growth-promoting bacteria is generally low. Therefore, in order to alleviate the stress level faced by plants and enhance the stress resistance of plants, large amounts of chemical fertilizers are applied to the fields, but the effect is minimal and aggravates the problems of soil salinization and compaction.

Using genetic engineering methods to synthesize recombinant engineering bacteria that efficiently express ACC deaminase can effectively alleviate the high ethylene levels produced by plants in response to salt stress.

Contents of the invention

The purpose of the present invention is to provide a preparation method and application of a salt-resolving and growth-promoting compound microbial inoculant, which can improve the stress resistance of crops, promote the increase in crop biomass content and the growth of stems and leaves, enhance the accumulation of crop biomass, and enhance the overall biomass accumulation of crops. Improve the economic value of crops to a certain extent.

In order to achieve the above object, the present invention provides a preparation method for salt-reducing growth-promoting compound microbial inoculant, which includes the following steps:

S1. Activate E. coli BL-EF by streaking on the LB solid plate, pick a single colony for culture, place the single colony in the liquid LB medium, activate and culture it at 37°C for 12 hours, and then inoculate it into new colonies at a ratio of 1%. In LB liquid medium, when the Escherichia coli BL-EF culture cells grow to the end of the logarithmic phase, IPTG is added for induction to obtain an Escherichia coli BL-EF fermentation broth that highly expresses ACC deaminase;

S2. Activate Enterobacter halleri Rs-2 by streaking on the LB solid plate, pick a single colony for culture, place the single colony in the liquid LB medium, activate and culture it at 37°C for 12 hours, and then inoculate it at a ratio of 1%. In the new LB liquid culture medium, culture it at 37°C for 48 hours to obtain the Enterobacter hosei Rs-2 fermentation broth;

S3. Use physiological saline to dilute the E. coli BL-EF fermentation broth and Enterobacter hosei Rs-2 fermentation broth that are obtained in step S1 and step S2 to efficiently express ACC deaminase. BL-EF bacterial liquid and Enterobacter hordii Rs-2 bacterial liquid, and then mix the E. coli BL-EF bacterial liquid that highly expresses ACC deaminase and Enterobacter hordii Rs-2 bacterial liquid in volume proportions to obtain salt solution Growth-promoting compound microbial agent.

Preferably, the content of Escherichia coli in the composite microbial inoculant is 15 to 20 billion CFU/mL, the content of Enterobacter hosei is 5 to 7 billion CFU/mL, and the ACC deaminase activity is 5.42 to 5.78 U/mg.

Preferably, the Escherichia coli BL-EF is constructed using Escherichia coli BL21 (DE3) as the host strain and pET-28a (+) as the expression vector, and BamHI and Hind III enzyme cutting sites are used to double-cleave the ACC deaminase gene. And the vector pET-28a(+) was ligated, and the ligated product was transferred into E. coli BL21(DE3) for stable expression.

Preferably, the components of the liquid LB culture medium are: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, sterilized at 121°C for 20 minutes, and adjusted to 7.0.

Preferably, in step S1, IPTG is added for induction, the induction time is 24 hours, the induction temperature is 12°C, and the IPTG concentration is 0.4mM.

Preferably, in step S3, the volume ratio of the E. coli BL-EF bacterial liquid that highly expresses ACC deaminase and the Enterobacter hosei Rs-2 bacterial liquid is 3:1, and the pH of the composite microbial agent is neutral.

Preferably, in step S3, the ratio of physiological saline to the fermentation broth of Escherichia coli BL-EF that highly expresses ACC deaminase is 1:50, and the ratio of physiological saline to the fermentation broth of Enterobacter hosei Rs-2 is 1:50.

The invention also provides the application of a salt-resolving and growth-promoting compound microbial agent in improving the stress resistance of crops.

Therefore, the present invention adopts the preparation method and application of the above-mentioned salt-solubilizing and growth-promoting compound microbial inoculant, and its technical effects are as follows:

(1) A composite microbial agent containing high ACC deaminase activity is provided, which is obtained by compounding the fermentation broth of two specific strains of Escherichia coli BL-EF and Enterobacter hodori Rs-2, in which Escherichia coli BL-EF During the fermentation process, IPTG needs to be added within a specific time to induce the bacteria to produce ACC deaminase.

(2) The highly active ACC deaminase produced during the fermentation process can effectively alleviate the ethylene levels in crops under environmental stress, and can promote the increase in crop biomass content and the growth of stems and leaves.

(3) During the growth and development process of Enterobacter hosei Rs-2, it also produces indoleacetic acid, siderophilin, phosphatase and other substances that can promote plant growth and development. It has a strong phosphorus-dissolving and growth-promoting function. Improving the stress resistance of crops can also reduce the use of chemical fertilizers, promote crop growth and development, enhance the accumulation of crop biomass, and increase the economic value of crops on an overall level.

The technical solution of the present invention will be further described in detail below through the accompanying drawings and examples.

Description of the drawings

Figure 1 is a gel electrophoresis diagram of the PCR product;

Figure 2 is a verification diagram of double enzyme digestion of transformant plasmid;

Figure 3 shows the effect of the addition of BL-EF on nutrient elements in tomato rhizosphere soil;

Figure 4 shows the content of nutrients in the soil after treatment with CK and the optimal ratio of compound inoculants.

Detailed ways

The technical solution of the present invention will be further described below through the drawings and examples.

Unless otherwise defined, technical terms or scientific terms used in the present invention shall have the usual meaning understood by a person with ordinary skill in the field to which the present invention belongs.

It is obvious to those skilled in the art that the present invention is not limited to the details of the above-described exemplary embodiments, and that the present invention can be implemented in other specific forms without departing from the gist or essential characteristics of the present invention. Therefore, the embodiments should be regarded as illustrative and non-restrictive from any point of view, and the scope of the present invention is defined by the appended claims rather than the above description, and it is therefore intended that all claims falling within the claims All changes within the meaning and scope of equivalent elements are encompassed by the present invention, and any reference signs in a claim should not be construed as limiting the claim involved.

In addition, it should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity, and those skilled in the art should take the specification as a whole. , the technical solutions in each embodiment can also be appropriately combined to form other implementations that can be understood by those skilled in the art. These other embodiments are also covered by the scope of the present invention.

It should also be understood that the specific embodiments described above are only used to explain the present invention, and the protection scope of the present invention is not limited thereto. Any person familiar with the technical field can use the technology disclosed in the present invention within the scope of the present invention. Any equivalent substitution or change of the scheme and its inventive concept shall be covered by the protection scope of the present invention/invention.

Techniques, methods and devices known to those of ordinary skill in the relevant art may not be discussed in detail, but where appropriate, such techniques, methods and devices should be considered a part of the specification.

The disclosure contents of the prior art documents cited in the specification of the present invention are incorporated by reference into the present invention in their entirety, and are therefore part of the disclosure content of the present invention.

Embodiment 1

Preparation of E. coli BL-EF

1. Design primers EF-F and EF-R. The upstream and downstream primers introduce restriction endonuclease cleavage sites BamHI and HindIII respectively.

PCR primer EF-F: GTCGCGGATCCATGAACCTGAATCGTTTTAAACG;

EF-R: GTCGCGGATCCTCAGCCGTTGCGAAACAAGAAGC;

The amplification template is the ACC deaminase gene, and the reaction conditions for its amplification are as shown in the table:

Table 1 PCR reaction system

Reagents volume 10×PCR buffer 5μL dNTP 4μL DNA template 1μL upstream primer 2μL Downstream primer 2μL DNA polymerase 0.3μL Double distilled water Upto50μL

The PCR product was subjected to 0.8% agarose gel electrophoresis to obtain a 1017 bp band (ACC deaminase gene). The electrophoresis pattern is shown in Figure 1, where 1-4: EF011160 PCR product; M: DNA Ladder.

2. Use the gel recovery kit to perform gel recovery and purification of the target PCR product.

3. The plasmid pET-28a(+) and the recovered ACC deaminase gene were digested with BamHI and Hind III and then ligated to construct a recombinant plasmid.

4. Transform the recombinant plasmid into competent E. coli BL21 (DE3) cells, heat shock at 42°C for 60 seconds, spread on a plate containing 30 μg/mL kanamycin, and culture at 37°C for 12 hours. Positive transformants were selected, the transformant plasmid was extracted, and double enzyme digestion was performed to verify that the constructed strain was the target recombinant strain BL-EF. As shown in Figure 2, 1-4: pET28a-EF011160; M: DNALadder. In Figure 2, after the recombinant plasmid was digested with BamHI and Hind III, the obtained band size was about 5163bp (plasmid pET-28a(+ ) and 1017bp (ACC deaminase gene) after enzyme digestion, indicating that the recombinant strain containing the ACC deaminase gene was successfully obtained.

ACC deaminase activity assay

Take out the glycerin-preserved E. coli BL-EF and Enterobacter hodori Rs-2 from the -80°C refrigerator, activate them by streaking on LB solid plates, pick single colonies for culture, and then culture the single colonies in liquid LB The base was activated at 37℃ for 12h. Re-inoculate into new LB liquid medium at a ratio of 1%. When E. coli BL-EF is cultured to the end of the logarithmic phase, 0.4mM IPTG is added for induction. The induction time is 24h and the induction temperature is 12°C.

The components of liquid LB medium are: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, sterilized at 121°C for 20 minutes, and adjusted to pH 7.0.

The components of LB solid medium are: peptone 10g/L, yeast extract 5g/L, sodium chloride 10g/L, agar 20g/L, sterilized at 121°C for 20 minutes, and adjusted to pH 7.0.

Draw the OD ₅₉₅ value of the bovine serum albumin standard sample and the α-ketobutyric acid standard curve to calculate the ratio of enzyme activity to protein content per unit time (U·mg ^-1 ), which is the ACC deaminase activity. The pure culture of each strain was collected by centrifugation at 5000 rpm for 10 min at 4°C for 24 h, washed and centrifuged twice with ADF culture medium, and the bacteria were resuspended in ADF culture medium and cultured at 28°C and 200 rpm for 24 h to induce the production of ACC deaminase. Collect the cells by centrifugation at 4°C, wash twice with 0.1mol/LTris-HCl buffer (pH 7.6), resuspend in 600μL 0.1mol/LTris-HCl buffer (pH 8.5), add 30μL toluene and quickly Vortex for 30 s to disrupt cells. Take 400 μL of cell extract containing toluene, add 50 μL of 0.5 mol/LACC and mix well. Then add 50 μL of 2mmol/LFeSO ₂ and 1.5mL of 0.1mol/LTris-HCl, mix, fully ventilate, seal, and place in a 30°C water bath for 30 minutes. Then add 0.5mL 4mol/L HCl to terminate the reaction. Take 1 mL of the reaction solution, add 0.2 mL of 0.1% 2,4-dinitrophenylhydrazine and 2 mmol/L HCl solution, mix well, 30°C for 15 min, finally add 1 mL of 2 mol/L NaOH, mix well, and measure the absorbance at 540 nm. Each group was run in parallel and double-distilled water was used instead of the enzyme extract as a blank control, repeated three times. The enzyme activities of Enterobacter horsi Rs-2 and Escherichia coli BL-EF were 0.80U/mg and 5.60U/mg respectively. The ACC deaminase activity in E. coli BL-EF was higher than that in Enterobacter hordii Rs-2, which was 7 times that of Enterobacter hordii Rs-2.

Escherichia coli BL-EF salt solubilization and growth promotion test

The fermentation broth of E. coli BL-EF that highly expresses ACC deaminase is diluted with physiological saline at a ratio of 1:50 and can be directly used for crop irrigation. The experiment set up 4 groups of controls, namely:

T1: Escherichia coli BL21(DE3), irrigated with double distilled water

T2: E. coli BL-EF, irrigated with double distilled water

T3: E. coli BL21(DE3), irrigated with saline (100mM NaCl solution)

T4: E. coli BL-EF, irrigated with saline (100mM NaCl solution)

The irrigation method is: use seedling trays to grow seedlings, transplant the tomato seedlings when they grow to about 10cm, start watering the bacterial solution every other day, water 50mL of diluted bacterial solution in each pot, water the bacterial solution every 5 days, and regularly use water or the same amount of saline (100mM NaCl) to water potted plants twice a day. Relevant indicators were measured after about 30 days of growth.

Each group of experiments was conducted in 5 parallel experiments, and the growth status of tomatoes was observed in the light greenhouse. During this period, each investigation group poured an equal amount of water into the experimental pots to keep the soil moist. When the tomatoes in the experimental group grow to 10cm, treatment begins. Tomato plant height, root length, fresh weight, and dry weight were investigated, and the results are shown in Table 2.

Table 2 Tomato morphological index of each experimental group

test group Plant height(cm) Root length(cm) Fresh weight (g) Dry weight(g) T1 18.27±2.37bc 11.9±1.64b 0.89±0.23bc 0.09±0.02d T2 22.57±3.05a 16.93±1.56a 1.39±0.17a 0.13±0.03cd T3 14.6±0.92bc 10.01±1.45b 0.71±0.11c 0.06±0.01bc T4 20.02±2.24ab 15.65±2.16a 1.19±0.19ab 0.09±0.01bc

From the data in the table above, E. coli BL-EF can alleviate the salt stress faced by tomato plants, and can increase the plant height, root length, fresh weight and dry weight of tomato plants with or without salt stress. It has the function of detoxifying salt and promoting growth.

The contents of nutrient elements in the rhizosphere soil of tomato seedlings in the four groups of experiments were detected. The test results are shown in Figure 3. Adding E. coli BL-EF can promote the content of alkaline hydrolyzed nitrogen, available phosphorus and available potassium in the soil under salt stress, but the effect is not significant. Escherichia coli BL-EF significantly promoted the contents of water-soluble calcium and water-soluble magnesium in tomato rhizosphere soil under salt stress. Under no stress, inoculation of E. coli BL-EF has no significant effect on the contents of alkaline hydrolyzable nitrogen, available phosphorus, and available potassium, but has a significant effect on the accumulation of water-soluble calcium and water-soluble magnesium in the rhizosphere soil. Therefore, E. coli BL-EF can promote the content of nutrients in the soil and significantly increase the accumulation of water-soluble calcium and water-soluble magnesium under salt stress for plant utilization.

Preparation of compound microbial inoculant and salt-dissolving growth promotion test

The compound microbial inoculant is prepared from the fermentation broth of Escherichia coli that efficiently expresses ACC deaminase and the fermentation broth of Enterobacter hodori at a volume ratio of 3:1. The pH of the compound microbial inoculant is neutral.

The content of Escherichia coli in the compound microbial inoculant is 15 to 20 billion CFU/mL, the content of Enterobacter hordii is 5 to 7 billion CFU/mL, and the ACC deaminase activity is 5.42 to 5.78 U/mg.

The E. coli that efficiently expresses ACC deaminase is E. coli BL-EF. E. coli BL-EF is constructed using E. coli BL21 (DE3) as the host strain and pET-28a (+) as the expression vector, using BamH I and Hind III enzymes. The ACC deaminase gene and the vector pET-28a(+) were digested with double enzymes at the cutting site and ligated, and the ligated product was transferred into E. coli BL21(DE3) for stable expression;

Enterobacter hordii is Enterobacter hordii Rs-2.

Take out the glycerin-preserved E. coli BL-EF and Enterobacter hodori Rs-2 from the -80°C refrigerator, activate them by streaking on LB solid plates, pick single colonies for culture, and then culture the single colonies in liquid LB After the medium was activated at 37°C for 12 hours, it was inoculated into new LB medium at a ratio of 1%. When E. coli BL-EF was cultured to the end of the logarithmic phase, 0.4mM IPTG was added for induction. After continuing to culture at 12°C for 24 hours, the fermentation broth was obtained.

Take out the glycerin-preserved E. coli BL-EF and Enterobacter hodori Rs-2 from the -80°C refrigerator, activate them by streaking on LB solid plates, pick single colonies for culture, and then culture the single colonies in liquid LB After the base was activated at 37°C for 12 hours, it was inoculated into new LB medium at a ratio of 1%. The culture environment was maintained at 37°C and the culture time was 48 hours to obtain a fermentation broth.

Use physiological saline to dilute the two fermentation liquids at a ratio of 1:50, which can be directly used for crop irrigation. The bacterial liquids of Escherichia coli BL-EF and Enterobacter hosei Rs-2 are compounded in the ratios of 1:1, 1:2, 1:3, 2:1, and 3:1 to obtain different proportions of bacterial liquids.

The pot trial included 8 different treatments:

T1 (CK, salt stress)

T2 (salt stress + E. coli BL-EF)

T3 (salt stress + Enterobacter hordii Rs-2)

T4 (salt stress + Escherichia coli BL-EF: Enterobacter hordii Rs-2 = 1:1)

T5 (salt stress + Escherichia coli BL-EF: Enterobacter hordii Rs-2 = 1:2)

T6 (salt stress + Escherichia coli BL-EF: Enterobacter hordii Rs-2 = 1:3)

T7 (salt stress + Escherichia coli BL-EF: Enterobacter hordii Rs-2 = 2:1)

T8 (salt stress + E. coli BL-EF: Enterobacter hordii Rs-2 = 3:1).

The selected salt tolerance concentration of tomatoes is 100mM and the lethal concentration is 250mM, so 100mM NaCl was selected for the salt tolerance experiment on tomatoes. The treatment method for salt stress is: use seedling trays to grow seedlings, transplant the tomato seedlings when they grow to about 10cm, start watering the bacterial solution every other day, water 50mL of bacterial solution in each pot, water the bacterial solution once every 5 days, regularly use water or Water the potted plants with the same amount of salt water (100mM NaCl) twice a day. Relevant indicators were measured after about 30 days of growth.

Each group of experiments was conducted in 5 parallel experiments, and the growth status of tomatoes was observed in the light greenhouse. During this period, each investigation group poured an equal amount of water into the experimental pots to keep the soil moist. When the tomatoes in the experimental group grow to 10cm, treatment begins. Tomato plant height, root length, fresh weight, and dry weight were investigated. The results are shown in Table 3 below.

Table 3 Tomato morphological index of each experimental group

test group Plant height(cm) Root length(cm) Fresh weight (g) Dry weight(g) T1 11.97±1.75d 7.57±1.58c 0.72±0.14c 0.05±0.01d T2 17.85±1.32ab 8.65±1.98bc 1.01±0.16b 0.09±0.02cd T3 14.98±0.30bcd 8.46±2.52c 0.86±0.10bc 0.09±0.02bc T4 15.74±1.35abc 8.5±1.27c 0.97±0.11b 0.10±0.02bc T5 13.57±1.71cd 13.83±1.51ab 0.87±0.15bc 0.12±0.03ab T6 13.89±1.93cd 9.13±1.63bc 0.78±0.17bc 0.09±0.03bc T7 14.63±1.28bcd 16.77±6.83ab 0.94±0.14bc 0.11±0.02bc T8 18.34±2.16a 18.56±2.92a 1.30±0.17a 0.15±0.02a

It can be seen from the data in the table above that test group 8 has the strongest salt-resolving growth-promoting effect and the strongest stress resistance compared to the other 7 groups. Through significance analysis, the tomato growth of group 8 was the best, and it was significantly better than that of group 1, and had better anti-stress growth-promoting effect than single bacteria. Therefore, it was determined that both bacteria had anti-stress and growth-promoting effects, and the optimal ratio of the two bacteria was E. coli BL-EF: Enterobacter hodori Rs-2 as 3:1.

The content of nutrients in the soil treated with CK and the optimal proportion of compound inoculants was detected. The test results are shown in Figure 4.

Compared with the CK treatment, the compound bacterial agent significantly increased the contents of alkaline hydrolyzable nitrogen, available phosphorus, available potassium, water-soluble calcium and water-soluble magnesium in the soil, playing an important role in maintaining plant homeostasis and alleviating ion poisoning faced by plants. Effectively improve the utilization of nutrients in the soil by crops.

Therefore, the present invention adopts the above-mentioned preparation method and application of a salt-dissolving and growth-promoting compound microbial inoculant, and provides a compound microbial inoculant containing high ACC deaminase enzyme activity, which can pass through Escherichia coli BL-EF and Enterobacter hordii Rs. -2 The fermentation broth of two specific strains is obtained by compounding. During the fermentation process of E. coli BL-EF, IPTG needs to be added within a specific time to induce the bacteria to produce ACC deaminase; the highly active ACC deamination produced during the fermentation process The enzyme can effectively cleave the ethylene levels in crops under environmental stress, and can promote the increase in crop biomass content and the growth of stems and leaves; Enterobacter hosei Rs-2 also produces indole acetic acid and siderophilin during its growth and development. , phosphorus-dissolving enzymes and other substances that can promote plant growth and development have strong phosphorus-dissolving and growth-promoting functions. While improving crop stress resistance, they can also reduce the use of chemical fertilizers, promote crop growth and development, and enhance crop biomass accumulation. , to improve the economic value of crops on an overall level.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that: The technical solution of the present invention may be modified or equivalently substituted, but these modifications or equivalent substitutions cannot cause the modified technical solution to depart from the spirit and scope of the technical solution of the present invention.

Claims (8)

1. A method for preparing a salt-solubilizing and growth-promoting compound microbial inoculant, which is characterized by comprising the following steps: S1. Activate E. coli BL-EF by streaking on the LB solid plate, pick a single colony for culture, place the single colony in the liquid LB medium, activate and culture it at 37°C for 12 hours, and then inoculate it into new colonies at a ratio of 1%. In LB liquid medium, when the Escherichia coli BL-EF culture cells grow to the end of the logarithmic phase, IPTG is added for induction to obtain an Escherichia coli BL-EF fermentation broth that highly expresses ACC deaminase; S2. Activate Enterobacter halleri Rs-2 by streaking on the LB solid plate, pick a single colony for culture, place the single colony in the liquid LB medium, activate and culture it at 37°C for 12 hours, and then inoculate it at a ratio of 1%. In the new LB liquid culture medium, culture it at 37°C for 48 hours to obtain the Enterobacter hosei Rs-2 fermentation broth; S3. Use physiological saline to dilute the E. coli BL-EF fermentation broth and Enterobacter hosei Rs-2 fermentation broth that are obtained in step S1 and step S2 to efficiently express ACC deaminase. BL-EF bacterial liquid and Enterobacter hordii Rs-2 bacterial liquid, and then mix the E. coli BL-EF bacterial liquid that highly expresses ACC deaminase and Enterobacter hordii Rs-2 bacterial liquid in volume proportions to obtain salt solution Growth-promoting compound microbial agent. 2. A method for preparing a salt-dissolving and growth-promoting compound microbial inoculant according to claim 1, characterized in that the content of Escherichia coli in the compound microbial inoculant is 15 to 20 billion CFU/mL, and the content of Enterobacter hordii is 5 to 7 billion CFU/mL, ACC deaminase activity is 5.42 to 5.78 U/mg. 3. A kind of salt-dissolving and growth-promoting compound microbial inoculant preparation method according to claim 1, characterized in that the Escherichia coli BL-EF uses Escherichia coli BL21 (DE3) as the host bacteria and pET-28a (+) For the construction of expression vector, the ACC deaminase gene and vector pET-28a(+) were digested with BamHI and HindIII enzyme sites and ligated, and the ligated product was transferred into E. coli BL21(DE3) for stable expression. 4. A method for preparing a salt-solubilizing and growth-promoting compound microbial agent according to claim 1, characterized in that the liquid LB culture medium composition is: peptone 10g/L, yeast extract powder 5g/L, sodium chloride 10g/L. L, sterilized at 121°C for 20 minutes, pH 7.0. 5. A method for preparing a salt-dissolving and growth-promoting compound microbial agent according to claim 1, characterized in that, in step S1, IPTG is added for induction, the induction time is 24h, the induction temperature is 12°C, and the IPTG concentration is 0.4mM. . 6. A method for preparing a salt-solubilizing and growth-promoting compound microbial agent according to claim 1, characterized in that, in step S3, the E. coli BL-EF bacterial liquid that efficiently expresses ACC deaminase and Enterobacter hodori Rs The volume ratio of -2 bacterial liquid is 3:1, and the pH of the compound microbial agent is neutral. 7. A method for preparing a salt-dissolving and growth-promoting compound microbial agent according to claim 1, characterized in that in step S3, the ratio of physiological saline to the E. coli BL-EF fermentation broth that efficiently expresses ACC deaminase is 1:50, the ratio of physiological saline to Enterobacter horsi Rs-2 fermentation broth is 1:50. 8. The application of a salt-resolving and growth-promoting compound microbial agent in improving the stress resistance of crops.