A combination module and its application in improving stress resistance of nitrogen-fixing microorganisms

Abstract

The application provides a combination module and application thereof in improving stress resistance of nitrogen-fixing microorganisms. The combination module comprises, in sequence from upstream to downstream, a constitutive promoter element Pc, a RNA polymerase sigma factor from Pseudomonas AlgU and a molecular chaperone protein from Deinococcus DosH . The application also provides application of the combination module in improving stress resistance of nitrogen-fixing microorganisms. The application obtains the combination module Pc −algU−dosH −algU−dosH , introduces the combination module into an expression vector to construct a recombinant expression vector, and transfers the recombinant vector into nitrogen-fixing bacteria to obtain a nitrogen-fixing recombinant engineering strain. Compared with a single stress resistance module, the combination module can significantly improve the stress resistance of a chassis nitrogen-fixing microorganism under high-temperature and drought stress conditions, and significantly improve the nitrogenase activity and survival ability of the chassis microorganism.

Description

Technical Field

[0001] This invention relates to the field of synthetic biology, specifically to a combinatorial module, a recombinant expression vector, a transformant, and a method for improving the heat and drought resistance of crops. Background Technology

[0002] The complex environment of the plant rhizosphere is a major limiting factor affecting the widespread application of nitrogen-fixing microorganisms in agriculture. Studies have shown that adverse conditions such as high temperature, salinity, and drought limit the field survival ability and nitrogen-fixing activity of nitrogen-fixing microorganisms. Therefore, constructing stress-resistance gene modules in the chassis of nitrogen-fixing microorganisms has important agricultural application value for improving the strains' tolerance to high temperature, salinity, and drought.

[0003] While directed evolution, genetic engineering, and bioprocessing techniques can enhance the stress resistance of microorganisms—for example, CN104059937A discloses a novel use for a protein derived from alfalfa and its encoding gene, and provides a method for cultivating transgenic nodulating plants with high nodulation ability and / or high stress resistance using the nucleic acid molecule encoding MtWRKY76—these methods suffer from drawbacks such as being time-consuming, costly, lacking versatility, and having low levels of automation. Integrating exogenous stress-resistance elements and constructing artificial stress-resistance modules using synthetic biology techniques represents a new strategy and approach to improve the stress resistance of nitrogen-fixing microorganisms and enable their widespread application in agriculture. Currently, while publicly disclosed nitrogen-fixing bacteria obtained through synthetic biology methods can effectively improve nitrogen fixation in plants, their tolerance to adverse conditions is poor or they only exhibit tolerance to a single condition.

[0004] Therefore, it is necessary to use synthetic biology techniques to construct artificial composite modules in the nitrogen-fixing microbial chassis that can enhance the stress resistance of nitrogen-fixing microorganisms, in order to build an efficient stress-resistant nitrogen-fixing system. Summary of the Invention

[0005] The purpose of this invention is to construct an artificial composite module in the nitrogen-fixing microbial chassis using synthetic biology techniques, which can enhance the stress resistance of nitrogen-fixing microorganisms, for the construction of an efficient stress-resistant nitrogen-fixing system.

[0006] To achieve the above objectives, the present invention provides a combined module and its application in improving the stress resistance of nitrogen-fixing microorganisms. Specifically, it provides a combined module, a recombinant expression vector, a transformant, and a method for improving the high-temperature and drought resistance of crops.

[0007] The first aspect of the present invention provides a combined module comprising, in a direction from upstream to downstream, a constitutive promoter element Pc and an RNA polymerase σ factor selected from the genus *Pseudomonas*. AlgUand molecular chaperone proteins selected from the genus *Anomala* DosH .

[0008] Optionally, the nucleotide sequence of the combined module is shown in SEQ ID NO: 1.

[0009] Optionally, the nucleotide sequence of the constitutive promoter element Pc is as shown in nucleotides 1-99 of SEQ ID NO: 1;

[0010] The functional element AlgU The nucleotide sequence is shown as nucleotides 100-699 in SEQ ID NO: 1;

[0011] The functional element DosH The nucleotide sequence is shown as nucleotides 700-1596 in SEQ ID NO: 1.

[0012] The present invention also provides a recombinant expression vector, wherein the recombinant expression vector is inserted with the combined module and forms an expression cassette for expressing stress tolerance protein.

[0013] Another aspect of the present invention provides the application of the combined module in improving the stress resistance of nitrogen-fixing microorganisms, wherein the combined module is introduced into nitrogen-fixing microorganisms to obtain recombinant strains.

[0014] The present invention also provides a transformant in which the recombinant expression vector is introduced.

[0015] Optionally, the host cell of the transformant is a nitrogen-fixing bacterium.

[0016] Optionally, the nitrogen-fixing bacteria include: *Pseudomonas aeruginosa* A1501 (… Pseudomonas stutzeri ), brown azotocin ( Azotobacter vinelandii ) or Rhizobium fischeri ( Sinorhizobium fredii ).

[0017] The present invention also provides a method for improving the heat resistance and drought resistance of crops by applying the transformant to the crops.

[0018] Optionally, the crop includes at least one of rice, corn, and soybeans.

[0019] Through the above technical solution, this invention obtains Pc- via biosynthesis. algU-dosH The combined module was constructed by seamlessly cloning and then introducing it into an expression vector to create the recombinant expression vector pLAFR3-Pc-. algU-dosH The recombinant vector was then transferred into nitrogen-fixing bacteria, resulting in three nitrogen-fixing recombinant engineered strains. P. stutzeri (Pc- algU-dosH ), A.vinelandii (Pc- algU-dosH )and S. fredii (Pc- algU-dosH It has been demonstrated that, compared with a single stress-resistance module, the artificial stress-resistance combination module of the present invention can significantly improve the stress resistance of chassis nitrogen-fixing microorganisms under high temperature and drought stress conditions. Under high temperature and drought conditions, the artificial stress-resistance combination module Pc- of the present invention... algU-dosH It can significantly improve the nitrogenase activity and survival ability of chassis microorganisms. Furthermore, the artificial Pc- of this invention... algU-dosH The modular design strategy can be applied to the construction of efficient stress-resistant nitrogen fixation systems, providing a high-temperature-resistant and drought-resistant plant rhizosphere nitrogen-fixing microbial system in the plant rhizosphere nitrogen fixation system, thereby improving nitrogen fixation efficiency.

[0020] Other features and advantages of the present invention will be described in detail in the following detailed description section. Attached Figure Description

[0021] The accompanying drawings are provided to further illustrate the invention and form part of the specification. They are used together with the following detailed description to explain the invention, but do not constitute a limitation thereof. In the drawings:

[0022] Figure 1 For artificial Pc- algU-dosH A schematic diagram of the construction of the combined module expression vector, with the insertion site being... BamH I and Hin d Ⅲ.

[0023] Figure 2 For the recombinant expression vector Pc- algU-dosH The positive colony PCR verification results.

[0024] Figure 3 The figure shows the results of the survival test of the chassis nitrogen-fixing strain and the recombinant engineered strain under high temperature conditions.

[0025] Figure 4 The figure shows the results of the survival test of the chassis nitrogen-fixing strain and the recombinant engineered strain under drought conditions. Detailed Implementation

[0026] The following provides a detailed description of specific embodiments of the present invention. It should be understood that the specific embodiments described herein are for illustrative and explanatory purposes only and are not intended to limit the scope of the invention.

[0027] Unless otherwise specified, all experimental conditions were performed according to conventional conditions well-known to those skilled in the art, such as those described in the "Sambrook" molecular cloning manual (New York: Cold Spring Harbor Laboratory Press, 1989), or as recommended by the manufacturer. The terms "upstream" and "downstream" in this invention refer to the direction of gene expression.

[0028] The first aspect of the present invention provides a combined module comprising, in a direction from upstream to downstream, a constitutive promoter element Pc and an RNA polymerase σ factor selected from the genus *Pseudomonas*. AlgU and molecular chaperone proteins selected from the genus *Anomala* DosH .

[0029] In this preferred embodiment, the AlgU It is a key protein regulating the abiotic stress tolerance of bacterial strains, including osmotic stress, oxidative stress, desiccation, and biofilm formation, and can achieve rapid physiological adaptation in most bacteria; DosH It plays an important role in the resistance to oxidation and drying stress in radiation-resistant abnormal cocci, and is transferred to E. coli When combined, it can significantly enhance its antioxidant capacity; these two single genes have strong stress resistance and application effects, and are ideal functional elements for constructing a combined module. When these two functional elements are combined, their expression is controlled by the constitutive promoter element Pc; the combined module has the effect of improving the stress resistance of nitrogen-fixing microorganisms.

[0030] In a preferred embodiment, the nucleotide sequence of the combinatorial module is shown in SEQ ID NO: 1. Preferably, the combinatorial module is Pc- algU-dosH The obtained artificially combined module fragment has a total length of 1596bp.

[0031] In a preferred embodiment, the nucleotide sequence of the constitutive promoter element Pc is as shown in nucleotides 1-99 of SEQ ID NO: 1;

[0032] The functional element AlgU The nucleotide sequence is shown as nucleotides 100-699 in SEQ ID NO: 1;

[0033] The functional element DosH The nucleotide sequence is shown as nucleotides 700-1596 in SEQ ID NO: 1.

[0034] In this preferred embodiment, the artificial assembly module comprises the following components:

[0035] (1) Composed of 600bp and 897bp respectively AlgU , DosH Two functional components are combined and synthesized.

[0036] (2) The constitutive promoter element Pc expressed by the anti-reverse module is activated, and its fragment size is 99 bp.

[0037] This invention also provides a recombinant expression vector, in which the combined module is inserted to form an expression cassette for stress tolerance proteins. Preferably, the expression vector and the combined module are connected in a seamless cloning manner. The ends of the expression vector and the primer ends of the combined module should have 15-20 homologous bases. The resulting PCR product will then have homologous arms composed of 15-20 bases homologous to the vector sequence at both ends. These arms form a loop through complementary base pairing, eliminating the need for enzyme ligation and allowing direct transformation of host bacteria. The recombinant plasmid entering the host bacteria repairs the gaps using its own enzyme system. A schematic diagram of the recombinant expression vector construction is shown below. Figure 1 As shown, the positive colony amplification results of the combined module are as follows: Figure 2 As shown.

[0038] In this preferred embodiment, the expression vector is preferably pLAFR3, and the resulting recombinant vector is pLA-Pc- algU-dosH The expression vector pLAFR3 was subjected to... BamH I and Hindi III double enzyme digestion, followed by seamless cloning technology to integrate artificially synthesized Pc- algU-dos The H fragment is inserted at its multiple cloning site to obtain the artificial combination module Pc- of this invention. algU-dosH The recombinant vector pLA-Pc- algU-dosH like Figure 1 As shown.

[0039] Another aspect of the present invention provides an application of a combined module in improving the stress resistance of nitrogen-fixing microorganisms. This application involves introducing the combined module into nitrogen-fixing microorganisms to obtain recombinant strains. Preferably, the nitrogen-fixing microorganisms are bacteria involved in nitrogen fixation in the rhizosphere environment of plants, including rice, corn, and soybean.

[0040] The present invention also provides a transformant in which the recombinant expression vector is introduced.

[0041] In a preferred embodiment, the host cell of the transformant is a nitrogen-fixing bacterium.

[0042] In a preferred embodiment, the nitrogen-fixing bacteria include: *Pseudomonas aeruginosa* A1501 (… Pseudomonas stutzeri ), brown azotocin ( Azotobacter vinelandii) or Rhizobium fischeri ( Sinorhizobium fredii ).

[0043] In the preferred embodiment described above, three nitrogen-fixing recombinant engineered strains were obtained. P. stutzeri (Pc- algU- dosh ), A.vinelandii (Pc- algU-dosH )and S. fredii (Pc- algU-dosH The recombinant expression vector can be transferred into nitrogen-fixing biological chassis via triparental conjugation or electroporation, respectively.

[0044] The present invention also provides a method for improving the heat resistance and drought resistance of crops by applying the transformant to the crops.

[0045] In a preferred embodiment, the crop includes at least one of rice, corn, and soybean. In this preferred embodiment, the survival ability of the recombinant strain under high-temperature and drought stress was measured, comparing the survival ability of chassis nitrogen-fixing microorganisms with that of recombinant nitrogen-fixing engineered bacteria under these conditions. The results showed that, compared to a single stress-resistant module, the introduction of the stress-resistant combination module significantly improved the survival rate of chassis nitrogen-fixing microorganisms under adverse conditions, increasing the survival rate by approximately two orders of magnitude or more. Figure 3 and Figure 4 As shown.

[0046] The present invention will be further described in detail below through examples, but the present invention is not limited thereto.

[0047] All raw materials used in the following examples can be obtained through commercial purchase.

[0048] Example 1

[0049] Constructing Pc- algU-dosH Recombinant strains of the module.

[0050] This embodiment includes the following methods:

[0051] 1) A 1565bp full-length gene fragment sequence was synthesized using artificial chemical synthesis methods. algU-dosH Its expression is controlled by a compositional initiation element Pc with a size of 99 bp, and the above three functional elements constitute a combined module Pc- algU-dosH The sequence is shown in SEQ ID NO: 1;

[0052] 2) The broad-host expression vector pLAFR3 was used... BamH I and Hindi III was subjected to double enzyme digestion, and the combinatorial module Pc- was cloned using a seamless cloning method. algU-dosHThe recombinant vector was obtained by ligating it into a linearized pLAFR3 vector; and the sequence was verified to be correct by PCR sequencing, resulting in the expression vector pLA-Pc-. algU-dosH ;

[0053] 3) The expression vector was transformed into three different nitrogen-fixing microbial substrates (Pseudomonas aeruginosa) via triphilic conjugation or electroporation. P. stutzeri Brown nitrogen-fixing bacteria A. vinelandii and Rhizobium fischeri Sinorhizobium fredii Three recombinant nitrogen-fixing engineered strains were obtained from the study.

[0054] In this embodiment, an artificial stress-resistance module Pc- was obtained using a method of artificial chemical synthesis. algae - dosh The full-length nucleic acid sequence was successfully constructed to express artificial Pc- algU-dosH The module contains a recombinant expression vector and recombinant engineered bacterial strain. The recombinant expression vector sequence was verified to be correct by PCR sequencing and named pLA-Pc-. algU-dosH Contains pLA-Pc- algU-dosH The three recombinant engineered strains were *Pseudomonas aeruginosa*. P. stutzeri (Pc- algU-dosH ), brown nitrogen-fixing bacteria A. vinelandii (Pc- algU-dosH ) and Rhizobium fischeri S. fredii (Pc- algU-dosH ).

[0055] Example 2

[0056] Survival ability of recombinant strains under high temperature stress was determined.

[0057] This embodiment includes the following methods:

[0058] 1) Select nitrogen-fixing Pseudomonas stearothermia P. stutzerii、P. stutzerii (Pc- algU-dosH ), P. Stutzer (Pc- algae )and P. stutzeri (Pc- dosh Brown nitrogen-fixing bacteria A. vinelandii , A. vinelandii (Pc- algU-dosH ), A. vinelandii (Pc- algae )and A. vinelandii (Pc- dosh Rhizobium fischeri S. fredii、S. fredii (Pc- algU-dosH ), S. fredii (Pc- algae )and S. fredii(Pc- dosh Single colonies were inoculated into LB, Buik's and YT liquid media respectively (the recombinant strain can be tetracycline resistant (Tc), and the wild type can be non-resistant). The culture conditions can be 30°C, 200 rpm shaker overnight.

[0059] 2) Transfer the overnight cultured bacterial suspension to fresh 20mL LB, Buik's, and YT liquid media at a 1% inoculum for expansion culture. Culture until the early logarithmic growth phase, OD... 600 The value can be 0.6. Take 1 mL of bacterial solution and shock it at 48℃ for 1 h; perform serial dilutions on the treated bacterial solution to achieve a final concentration of 10. -1 10 -2 10 -3 10 -4 and 10 -5 For each dilution, 8 µL of bacterial culture was inoculated onto the corresponding solid culture medium plate. After incubation at a constant temperature for 1 day, the growth status of the strain was observed and photographed.

[0060] This embodiment demonstrates that, compared to a single stress-resistance module, the combined stress-resistance module significantly improves the survival ability of chassis nitrogen-fixing bacteria under high-temperature conditions. The artificial stress-resistance combined module Pc-, induced to express under high-temperature conditions... algU-dosH It can significantly improve the survival rate of nitrogen-fixing bacteria in the chassis.

[0061] Example 3

[0062] Survival assessment of recombinant strains under drought stress.

[0063] 1) Select nitrogen-fixing Pseudomonas stearothermia P. stutzerii、P. stutzerii (Pc- algU-dosH ), P. Stutzer (Pc- algae )and P. stutzeri (Pc- dosh Brown nitrogen-fixing bacteria A. vinelandii、A. vinelandii (Pc- algU-dosH ), A. vinelandii (Pc- algae )and A. vinelandii (Pc- dosh Rhizobium fischeri S. fredii、S. fredii (Pc- algU-dosH ), S. fredii (Pc- algae )and S. fredii (Pc- doshSingle colonies were inoculated into LB, Buik's and YT liquid media (recombinant strains can be tetracycline resistant (Tc), wild type without resistance), and cultured overnight at 30°C and 200 rpm on a shaker.

[0064] 2) Transfer the overnight cultured bacterial suspension to fresh 20mL LB, Buik's, and YT liquid media at a 1% inoculation rate for further culture. Take 1mL of OD from each medium for further culture. 600 After centrifuging the bacterial culture with a pH of 0.6 at 5000 rpm for 10 minutes, the cells were washed twice with PBS buffer, the supernatant was discarded, and the cells were collected in 2 mL centrifuge tubes. Each centrifuge tube was then placed open in a desiccator for drying. Every 5 days, the treated cells were removed, added to liquid culture, and incubated on a shaker at the corresponding temperature for 15 minutes. Then, serial dilutions were performed to achieve a final concentration of 10-6. -1 10 -2 10 -3 10 -4 and 10 -5 For each dilution, 8 µL of bacterial culture was inoculated onto the corresponding solid culture medium plate. After incubation at a constant temperature for 1 day, the growth status of the strain was observed and photographed.

[0065] This embodiment demonstrates that, compared to a single stress-resistance module, the combined stress-resistance module significantly improves the survival ability of chassis nitrogen-fixing bacteria under drought conditions. The artificial stress-resistance combined module Pc-, induced to express under drought conditions, is also demonstrated. salary-dosH It can significantly improve the survival rate of chassis strains.

[0066] The preferred embodiments of the present invention have been described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, various simple modifications can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

[0067] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not describe the various possible combinations separately.

[0068] Furthermore, various different embodiments of the present invention can be combined in any way, as long as they do not violate the spirit of the present invention, they should also be regarded as the content disclosed by the present invention.

Claims

1. A combined module, characterized in that, This combined module, from upstream to downstream, includes a constitutive promoter element Pc, an RNA polymerase σ factor selected from the genus Pseudomonas, and so on. AlgU and molecular chaperone proteins selected from the genus *Anomala* DosH ; The nucleotide sequence of the combined module is shown in SEQ ID NO:

1.

2. The combined module according to claim 1, wherein, The nucleotide sequence of the constitutive promoter element Pc is shown as the nucleotides from position 1 to 99 in SEQ ID NO: 1; The AlgU The nucleotide sequence is shown as nucleotides 100-699 in SEQ ID NO: 1; The DosH The nucleotide sequence is shown as nucleotides 700-1596 in SEQ ID NO:

1.

3. A recombinant expression vector, characterized in that, The recombinant expression vector is inserted with the combination module as described in any one of claims 1-2.

4. The application of the combined module according to claim 1 in improving the stress resistance of nitrogen-fixing microorganisms, characterized in that, The combined module described in claim 1 is introduced into nitrogen-fixing microorganisms to obtain recombinant strains; The nitrogen-fixing microorganism is *Pseudomonas aeruginosa* (Nitrobacter schistosomiasis). Pseudomonas stutzeri ), brown azotocin ( Azotobacter vinelandii ) or Rhizobium fischeri ( Sinorhizobium fredii ); The stress resistance is described as being resistant to high temperatures and drought.

5. A transformant, characterized in that, The transformant contains the recombinant expression vector as described in claim 3; The host cells of the transformant are nitrogen-fixing bacteria.

6. The transformant according to claim 5, wherein, The nitrogen-fixing bacteria is *Pseudomonas aeruginosa* (Nitrobacter schistosomiasis). Pseudomonas stutzeri ), brown azotocin ( Azotobacter vinelandii ) or Rhizobium fischeri ( Sinorhizobium fredii ).

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