Saline-alkali soil improvement method based on microbial regulation and control

Abstract

The invention relates to the technical field of microbial regulation and control, and particularly discloses a saline-alkali soil improvement method based on microbial regulation and control, which comprises the following steps: S1, screening: screening microorganisms with initial tolerance to saline-alkali environment from natural environment; s2, gene editing: carrying out gene modification on the screened microorganisms by utilizing a CRISPR-Cas9 gene editing system; according to the invention, a CRISPR-Cas9 gene editing system is utilized to carry out gene modification on the screened microorganisms, and the viability of the microorganisms in a saline-alkaline environment is remarkably improved by modifying osmotic pressure regulating genes of the microorganisms, so that the microorganisms can survive and play a role in a more severe saline-alkaline environment, and meanwhile, the survival rate of the microorganisms in the saline-alkaline environment is improved. Plant growth hormone related genes generated by microorganisms are introduced, plant growth is promoted, powerful support is provided for yield increase of saline-alkali soil crops, the genetic characteristics of the microorganisms are accurately regulated and controlled in the gene editing step, the function of the microorganisms is enhanced, and more effective microorganism resources are provided for saline-alkali soil improvement.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial regulation, and in particular relates to a saline-alkali land improvement method based on microbial regulation. Background Art

[0002] The improvement of saline-alkali land has always been an important topic in the agricultural field. The key lies in improving soil fertility and reducing salt content to create an environment suitable for crop growth. Traditional improvement methods mostly rely on physical and chemical means, such as irrigation and salt washing, application of gypsum, etc., but these methods are often costly and have limited effects, and may cause damage to soil structure. In recent years, with the development of biotechnology, microbial regulation technology has gradually become a new direction for the improvement of saline-alkali land.

[0003] However, existing microbial regulation technologies mainly rely on the screening and application of microbial agents, but these agents often have problems such as single function and poor adaptability. The emergence of the CRISPR-Cas9 gene editing system has provided new possibilities for the genetic improvement of microorganisms. The system has the advantages of simple operation, high efficiency and high precision. It can achieve site-specific editing of microbial genomes, thereby giving microorganisms new traits and functions. However, the application of CRISPR-Cas9 gene editing technology to the screening and cultivation of saline-alkali land improvement microorganisms still requires solving a series of technical problems, so staff need to improve it. Summary of the invention

[0004] The purpose of the present invention is to provide a saline-alkali land improvement method based on microbial regulation to solve the problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solutions:

[0006] A method for improving saline-alkali land based on microbial regulation comprises the following steps:

[0007] S1. Screening: Screen out microorganisms with preliminary tolerance to saline-alkali environment from the natural environment;

[0008] S2. Gene editing: Genetically modify the selected microorganisms using the CRISPR-Cas9 gene editing system;

[0009] S3, cultivation: cultivating the genetically modified microorganisms to obtain a sufficient number of modified microorganisms;

[0010] S4. Application: Apply the improved microorganisms to saline-alkali soil to improve the saline-alkali soil through the activities of microorganisms;

[0011] S5. Monitoring and evaluation: After applying improved microorganisms, regularly monitor the physical and chemical properties, plant growth and microbial community structure of saline-alkali soil to evaluate the improvement effect and make necessary adjustments.

[0012] Preferably, in step S1, the screening further comprises sequencing the genome of the microorganism and identifying candidate genes related to salt-alkali tolerance, nutrient absorption and plant growth promotion by comparative genomics methods.

[0013] Preferably, in step S2, the genetic modification specifically includes: modifying the osmotic pressure regulating gene of the microorganism to improve its survival ability in a saline-alkali environment; enhancing the expression of the mineral element transport protein gene of the microorganism to improve its absorption capacity of soil nutrient elements; introducing plant growth hormone related genes produced by the microorganism to promote plant growth.

[0014] Preferably, in step S3, the cultivation also includes adaptive domestication of the improved microorganisms to further improve their survival rate and activity in saline-alkali soil.

[0015] Preferably, in step S4, the application amount and application method of the improved microorganisms are adjusted according to the specific salinity level of the saline-alkali land, soil type and plant growth requirements.

[0016] A modified microorganism for improving saline-alkali land, wherein the modified microorganism has enhanced salt-alkali tolerance, nutrient absorption capacity and plant growth promotion effect.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] (1) The CRISPR-Cas9 gene editing system was used to genetically modify the selected microorganisms. By modifying the osmotic pressure regulation gene of the microorganisms, their ability to survive in saline-alkali environments was significantly improved, allowing the microorganisms to survive and function in more severe saline-alkali environments. At the same time, the expression of the mineral element transporter gene of the microorganisms was enhanced, which improved their ability to absorb soil nutrients, helping to improve the fertility of saline-alkali soil. In addition, the introduction of plant growth hormone-related genes produced by microorganisms promoted plant growth and provided strong support for increasing the yield of crops in saline-alkali land. The gene editing step achieved a significant enhancement of the microbial function by precisely regulating the genetic characteristics of the microorganisms, providing a more effective microbial resource for saline-alkali land improvement.

[0019] (2) By screening out microorganisms with preliminary tolerance to saline-alkali environments from the natural environment, effective utilization of microbial resources was achieved. These microorganisms themselves have a certain degree of salt-alkali tolerance, which provides a good foundation for subsequent genetic modification. During the screening process, the genome of the microorganisms was further sequenced, and candidate genes related to salt-alkali tolerance, nutrient absorption and plant growth promotion were identified through comparative genomics methods. This helps to accurately locate the gene sites that need to be modified and points out the direction for subsequent gene editing work. This step not only saves the time and cost of gene editing, but also improves the pertinence and efficiency of genetic modification, laying the foundation for the smooth progress of subsequent steps.

[0020] (3) Cultivating genetically modified microorganisms and obtaining a sufficient number of improved microorganisms is a prerequisite for large-scale application of saline-alkali land improvement. By adaptively domesticating the improved microorganisms, their survival rate and activity in saline-alkali soil are further improved, ensuring the stability and durability of the improved microorganisms in the saline-alkali environment. During the application stage, the application amount and method of the improved microorganisms are adjusted according to the specific salinity, soil type and plant growth requirements of the saline-alkali land, achieving precise policy implementation and improving the improvement effect. Finally, by regularly monitoring the physical and chemical properties of saline-alkali soil, plant growth conditions and microbial community structure, the improvement effect is evaluated and necessary adjustments are made to ensure the continuous optimization and improvement of the improvement method. This step not only helps to grasp the improvement effect in real time, but also provides a scientific basis for subsequent optimization and adjustment, ensuring the continuity and effectiveness of saline-alkali land improvement work. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The figure is a flow chart of the method of the present invention. DETAILED DESCRIPTION

[0022] 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.

[0023] Embodiment 1:

[0024] See also Figure 1 As shown, a method for improving saline-alkali land based on microbial regulation comprises the following steps:

[0025] S1. Screening: Screen out microorganisms with preliminary tolerance to saline-alkali environment from the natural environment;

[0026] S2. Gene editing: Genetically modify the selected microorganisms using the CRISPR-Cas9 gene editing system;

[0027] S3, cultivation: cultivating the genetically modified microorganisms to obtain a sufficient number of modified microorganisms;

[0028] S4. Application: Apply the improved microorganisms to saline-alkali soil to improve the saline-alkali soil through the activities of microorganisms;

[0029] S5. Monitoring and evaluation: After applying improved microorganisms, regularly monitor the physical and chemical properties, plant growth and microbial community structure of saline-alkali soil to evaluate the improvement effect and make necessary adjustments.

[0030] In step S1, the screening also includes genome sequencing of the microorganisms and identifying candidate genes related to salt tolerance, nutrient absorption and plant growth promotion through comparative genomics methods.

[0031] Through genome sequencing and comparative genomics methods, candidate genes related to salt-alkali tolerance, nutrient absorption and plant growth promotion are accurately identified, providing precise targets for subsequent gene editing and screening potential microbial resources for saline-alkali land improvement.

[0032] E is a quantitative indicator of the improvement effect of saline-alkali land, which is used to evaluate the improvement effect after microbial genetic modification.

[0033] G(x) is the salt-alkali tolerance enhancement function of microorganisms after genetic modification, and x represents the degree or number of genetic modification.

[0034] N(y) is the enhancement function of microbial nutrient absorption capacity, and y represents the degree of improvement in the expression of mineral element transporter genes.

[0035] P(z) is the enhancement function of the microbial plant growth-promoting effect, and z represents the expression level of plant growth hormone-related genes.

[0036] T(s) is the salinity function of saline-alkali land, and s represents the specific indicators of saline-alkali land (such as pH value, salt content, etc.).

[0037] M(t) is the influence function of soil type on microbial activity, and t represents the classification index of soil type.

[0038] R(u) is the function of the impact of plant growth requirements on microbial application, and u represents the specific requirements of plant growth.

[0039] D(v) is the application amount function of the modified microorganism, and v represents the application amount or application concentration.

[0040] W(w) is a function of the application method of the improved microorganism, and w represents the specific parameters of the application method.

[0041] Preliminary screening effect evaluation:

[0042]

[0043] Among them, G0 represents the basic value of salinity tolerance of unmodified microorganisms, and T0 represents the initial salinity level of saline-alkali land.

[0044] Evaluation of the effects of genetic modification:

[0045]

[0046] Only the enhancement of salt tolerance and nutrient absorption capacity, as well as the effect of salinity, were considered.

[0047] Comprehensive evaluation of the effects of all factors:

[0048]

[0049] The value range of E is (0, +∞). When E is close to 0, it means that the improvement effect is weak or ineffective; when E is larger, it means that the improvement effect is significant.

[0050] The value range of G(x), N(y), and P(z) is [0,1], which indicates the degree of enhancement of genetic modification, nutrient absorption, and plant growth promotion effects, where 1 indicates the maximum enhancement.

[0051] The value range of T(s), M(t), and R(u) is (0, +∞), which indicates the influence of salinity, soil type, and plant growth requirements on the improvement effect.

[0052] The value ranges of D(v) and W(w) are determined according to actual conditions, but usually D(v) represents the range of application amount or concentration, and W(w) represents the specific parameter range of the application method.

[0053] Although the summation form is not directly used here, the integral can be regarded as the limiting form of continuous summation, and several key factors affecting the improvement effect of saline-alkali land are taken into account. By precisely regulating the genetic characteristics of microorganisms (i.e., G(x), N(y), P(z)), a more efficient evaluation of the improvement effect of saline-alkali land is achieved.

[0054] In step S2, the genetic modification specifically includes: modifying the osmotic pressure regulating gene of the microorganism to improve its survival ability in a saline-alkali environment; enhancing the expression of the mineral element transport protein gene of the microorganism to improve its absorption capacity of soil nutrient elements; introducing the plant growth hormone related gene produced by the microorganism to promote plant growth.

[0055] The CRISPR-Cas9 system is used to genetically modify microorganisms to enhance their salt-alkali tolerance, nutrient absorption capacity and plant growth-promoting effects, achieve customized optimization of microbial functions, and provide efficient and versatile improved microorganisms for saline-alkali land improvement.

[0056] The CRISPR-Cas9 gene editing system is used to genetically modify the selected microorganisms. The specific steps are as follows:

[0057] Design sgRNA: Based on the candidate gene identified in step S1, design a specific sgRNA (single guide RNA) to guide the Cas9 nuclease to the target gene site.

[0058] Construction of gene editing vector: The designed sgRNA sequence and Cas9 nuclease encoding gene are constructed into the gene editing vector to ensure that the vector can be stably expressed in microbial cells.

[0059] Transform microorganisms: Introduce the constructed gene editing vector into the selected microbial cells through appropriate methods (such as electrotransformation, chemical transformation, etc.).

[0060] Screening of successfully gene-edited microorganisms: Screen out successfully gene-edited microorganisms through appropriate screening methods (such as antibiotic resistance screening, PCR testing, etc.).

[0061] Verification of gene editing effects: Perform gene sequencing on the screened gene-edited microorganisms to verify whether the target gene has been edited as expected and evaluate the editing effect.

[0062] During the genetic modification process, the osmotic pressure regulating genes of the microorganisms were specifically modified to improve their survival ability in saline-alkali environments; the expression of the mineral element transporter genes of the microorganisms was enhanced to improve their ability to absorb soil nutrient elements; and the plant growth hormone-related genes produced by the microorganisms were introduced to promote plant growth.

[0063] In step S3, the cultivation also includes adaptive domestication of the improved microorganisms to further improve their survival rate and activity in saline-alkali soil.

[0064] Through adaptive domestication, the survival rate and activity of improved microorganisms in saline-alkali soil can be improved to ensure that they can play the best role in practical applications and provide sufficient number of highly active improved microorganisms for saline-alkali land improvement.

[0065] In step S4, the application amount and application method of the improved microorganisms are adjusted according to the specific salinity of the saline-alkali land, soil type and plant growth requirements.

[0066] According to the specific salinity, soil type and plant growth requirements of saline-alkali land, an intelligent microbial application system is designed to achieve precise application and dynamic regulation of improved microorganisms, realize efficient utilization of improved microorganisms in saline-alkali soil, and improve the accuracy and efficiency of saline-alkali land improvement.

[0067] In step S5, by regularly monitoring the physical and chemical properties of saline-alkali soil, plant growth and microbial community structure, timely evaluate the improvement effect and make necessary adjustments to ensure the continuity and stability of saline-alkali land improvement, and provide a scientific basis and decision-making support for saline-alkali land improvement.

[0068] A modified microorganism for improving saline-alkali land, wherein the modified microorganism has enhanced salt-alkali tolerance, nutrient absorption capacity and plant growth promotion effect.

[0069] The comprehensive effects of the steps of microbial screening, gene editing, cultivation, application, monitoring and evaluation, and the precise regulation of the genetic characteristics of microorganisms by CRISPR-Cas9 gene editing technology.

[0070] The static evaluation formula that does not consider the impact of time changes on the improvement effect is:

[0071]

[0072] The dynamic evaluation formula that takes into account the influence of time factors on the improvement effect but does not include the adjustment of application method and application amount is as follows:

[0073]

[0074] The complete formula taking into account the impact of time changes, adjustments in application method and application rate, and the monitoring and evaluation phase:

[0075]

[0076] IE saline-alkali The value range of is (0,+∞). The larger the value, the better the improvement effect of saline-alkali land. When the value is 0, it means that the improvement is ineffective. As the value increases, the improvement effect gradually increases.

[0077] IE saline-alkali (Improvement Effect of Salinity-Alkali Soil) is the final evaluation indicator.

[0078] It represents the time integral from the start of the implementation of the improvement method (t=0) to the end of the evaluation (t=T), reflecting the dynamic changes of the entire improvement process.

[0079] represents the salt-alkali tolerance GT of n modified microorganisms at time t i (t), nutrient absorption capacity EE i (t) and plant growth promoting effect PA i These functions change with time t, reflecting the dynamic performance of microorganisms during the improvement process.

[0080] AT adapt (t) (Adaptation and Training) is the effect function of adaptive training of improved microorganisms at time t, reflecting the improvement of microbial survival rate and activity during the training process.

[0081] DF apply (t) (Distr ibution and Application Factor) is an adjustment function for the application amount and method of improved microorganisms according to the salinity, soil type and plant growth requirements at time t.

[0082] It represents the time integral of the total number of tests or total microbial input during the improvement process, which is used for normalization.

[0083] EF monitor (t) (Evaluation Factor of Monitoring) is the adjustment factor for the improvement effect based on the monitoring results at the end time T of the evaluation, reflecting the impact of the monitoring and evaluation stage on the final evaluation indicators.

[0084] Embodiment 2:

[0085] See also Figure 1 As shown, a specific microorganism was selected: salt-alkali tolerant Bacillus as the initial screening object. This microorganism itself has strong salt-alkali tolerance and is suitable as a candidate microorganism for saline-alkali land improvement.

[0086] Screening and gene sequencing: The genome of salt-tolerant Bacillus was sequenced, and several key genes closely related to salt tolerance, nutrient absorption and plant growth promotion were identified through comparative genomics methods.

[0087] Gene editing: Using the CRISPR-Cas9 gene editing system, these key genes were edited at specific sites. Specifically, the expression of its osmotic pressure regulation gene was enhanced to improve its survival ability in saline-alkali environments; at the same time, the mineral element transporter gene was optimized to enable it to absorb nutrients in the soil more effectively.

[0088] Cultivation and domestication: The genetically modified salt-alkali tolerant Bacillus is cultivated and adaptively domesticated to make it more adaptable to the saline-alkali soil environment.

[0089] Application and monitoring: The domesticated improved microorganisms were applied to saline-alkali soil, and the physical and chemical properties of the soil, plant growth and microbial community structure were regularly monitored. The results showed that the improved soil fertility was significantly improved and the crop growth was significantly improved.

[0090] Embodiment three:

[0091] See also Figure 1 As shown, a microorganism with potential plant growth-promoting effect, Rhizobium, was selected as the initial screening object and improved in combination with CRISPR-Cas9 gene editing technology.

[0092] Screening and gene editing: Screening and genome sequencing of rhizobia were performed to identify candidate genes related to plant growth promotion. Using the CRISPR-Cas9 system, plant growth hormone-related genes were introduced to enhance their ability to promote plant growth.

[0093] Cultivation and testing: The genetically modified rhizobia were cultivated and tested in a series of tests to verify their improved effects. The results showed that the modified rhizobia can significantly increase the growth rate and yield of crops.

[0094] Field trial: The improved rhizobia were applied to saline-alkali land field trials. Through comparative observation, it was found that the growth of crops in saline-alkali land where the improved microorganisms were applied was significantly better than that of the control group where no application was made.

[0095] First, a variety of microbial samples were collected from the natural environment. Subsequently, the genomes of the screened microorganisms were sequenced using high-throughput sequencing technology. Through comparative genomics methods, candidate genes related to salt-alkali tolerance, nutrient absorption and plant growth promotion were successfully identified.

[0096] In the gene editing stage, the CRISPR-Cas9 gene editing system was used to perform site-specific gene modification on the selected microorganisms. Specifically, the osmotic pressure regulation gene of the microorganism was modified to improve its survival ability in saline-alkali environments; at the same time, the expression of the mineral element transporter gene of the microorganism was enhanced to improve its absorption capacity of soil nutrients; in addition, the plant growth hormone-related genes produced by the microorganism were introduced to promote plant growth.

[0097] After the genetic modification is completed, the modified microorganisms are adaptively domesticated to further improve their survival rate and activity in saline-alkali soil. During the domestication process, the environmental conditions of saline-alkali land are simulated and the salt-alkali concentration is gradually increased so that the microorganisms can gradually adapt and grow stably in a high-salt-alkali environment.

[0098] Next, the improved microorganisms are applied to the saline-alkali soil, and the application amount and method of the improved microorganisms are adjusted according to the specific salinity, soil type and plant growth requirements of the saline-alkali land. In order to ensure the improvement effect, the physical and chemical properties, plant growth and microbial community structure of the saline-alkali soil are regularly monitored to timely evaluate the improvement effect and make necessary adjustments.

[0099] During the test, multiple sets of data were recorded, including the salt content, pH value, organic matter content, plant growth height, biomass and other indicators of saline-alkali soil before and after improvement. At the same time, a comparative analysis was conducted with existing technologies (such as irrigation and salt washing + application of gypsum + microbial fertilizer and other traditional improvement methods). The following are the recorded test data:

[0100]

[0101] By comparing the data in the table, first, in terms of salt content, the salt content of saline-alkali land before improvement was 3.06%, while the salt content of soil after improvement was significantly reduced, even at a low application rate, it was reduced to 2.60%, which is lower than the 2.69% after improvement by the existing technology. This shows that the improvement method can more effectively reduce the salt content of the soil.

[0102] Secondly, in terms of pH value, the pH value of saline-alkali land before improvement was 8.2, which was alkaline. After improvement, the pH value of the soil gradually decreased, which was conducive to the growth of crops. In contrast, although the pH value of the soil after improvement by existing technology also decreased, it was still higher than the improvement effect.

[0103] In terms of organic matter content, the organic matter content of saline-alkali land before improvement was low, only 8.19 g / kg. After improvement by the present invention, the organic matter content increased significantly, up to 12.33 g / kg. This shows that the improvement method can increase soil fertility and provide a better nutritional environment for crop growth.

[0104] Finally, in terms of soybean growth height and biomass, soybeans grown on the improved soil showed a higher growth rate and greater biomass. Even at low application rates, the growth height and biomass of soybeans reached 47.16 cm and 38.46 g / plant, respectively, which were higher than 35.34 cm and 26.61 g / plant before improvement. Compared with the existing technology, the improvement effect is more significant.

[0105] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A method for improving saline-alkali land based on microbial regulation, characterized in that: The following steps are involved: S1. Screening: Screen out microorganisms with preliminary tolerance to saline-alkali environment from the natural environment; S2. Gene editing: Genetically modify the selected microorganisms using the CRISPR-Cas9 gene editing system; S3, cultivation: cultivating the genetically modified microorganisms to obtain a sufficient number of modified microorganisms; S4. Application: Apply the improved microorganisms to saline-alkali soil to improve the saline-alkali soil through the activities of microorganisms; S5. Monitoring and evaluation: After applying improved microorganisms, regularly monitor the physical and chemical properties, plant growth and microbial community structure of saline-alkali soil to evaluate the improvement effect and make necessary adjustments.

2. The method for improving saline-alkali land based on microbial regulation according to claim 1, characterized in that: In step S1, the screening also includes genome sequencing of the microorganisms and identifying candidate genes related to salt tolerance, nutrient absorption and plant growth promotion through comparative genomics methods.

3. The method for improving saline-alkali land based on microbial regulation according to claim 1, characterized in that: In step S2, the genetic modification specifically includes: modifying the osmotic pressure regulating gene of the microorganism to improve its survival ability in a saline-alkali environment; enhancing the expression of the mineral element transport protein gene of the microorganism to improve its absorption capacity of soil nutrient elements; introducing the plant growth hormone related gene produced by the microorganism to promote plant growth.

4. The method for improving saline-alkali land based on microbial regulation according to claim 1, characterized in that: In step S3, the cultivation also includes adaptive domestication of the improved microorganisms to further improve their survival rate and activity in saline-alkali soil.

5. The method for improving saline-alkali land based on microbial regulation according to claim 1, characterized in that: In step S4, the application amount and application method of the improved microorganisms are adjusted according to the specific salinity of the saline-alkali land, soil type and plant growth requirements.

6. A modified microorganism for improving saline-alkali land, applicable to the saline-alkali land improvement method based on microbial regulation according to claims 1 to 6, characterized in that: The modified microorganisms have enhanced salt-alkali tolerance, nutrient absorption capacity and plant growth-promoting effects.

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