Gene, encoded protein and use of mutant nham-t11d

Abstract

The application belongs to the technical field of protein, discloses a mutant Nham-T11D, including its gene, amino acid, protein function and Na+(Li+, K+) / H+ antiporter activity detection, the application determines the important function of a mutant Nham-T11D of an amino acid site: 11 threonine (T) is mutated into aspartic acid (D) of a highly conserved Nham protein, proves that the mutant Nham-T11D protein has 0.2M NaCl and alkaline pH value resistance function.The application also points out that the gene and protein of the mutant Nham-T11D have important significance for saline-alkali soil improvement, can be used for salt-tolerant gene modification, and provides a new effective selection for the field of constructing high-efficiency salt-tolerant engineering strains, developing microbial fertilizers and improving the salt-tolerance of transgenic plants.

Description

Technical Field

[0001] This invention belongs to the field of protein technology and relates to the gene, amino acids, encoded protein, functional detection and related applications of a mutant NhaM-T11D. Background Technology

[0002] With global warming, soil salinization in arid and semi-arid regions is becoming increasingly severe. This is manifested in the continuous expansion of salinized land area, the increasing degree of salinization, the growing trend of severely salinized land, and the expansion of unusable alkaline patches, seriously affecting land productivity and national economic development. Simultaneously, due to industrial pollution, improper irrigation, and inappropriate fertilizer use, the area of ​​secondary salinized soil is also rapidly increasing. Saline-alkali land affects vegetation growth, leading to reduced or failed crop yields, indirectly causing ecological degradation, and can corrode and damage engineering facilities, resulting in losses exceeding 2.5 billion yuan annually.

[0003] It is currently believed that improving saline-alkali soil requires collaborative efforts from multiple disciplines. From a biological perspective, the main approach involves working with both microorganisms and plants simultaneously, with highly efficient salt-tolerant microorganisms and plants working synergistically. This is because many plants cannot directly access the scarce carbon and nitrogen sources in saline-alkali land, and applying only chemical fertilizers would cause even more severe environmental disasters. Studies have shown that the application of microbial fertilizers such as nitrogen-fixing bacteria and rhizobia is more effective than chemical fertilizers in promoting the improvement of saline-alkali soil by highly efficient salt-tolerant plants.

[0004] However, many microbial fertilizers and plants currently used for soil improvement lack the ability to withstand high salinity and alkalinity. Therefore, in addition to discovering highly efficient salt-tolerant microorganisms and plant resources, it is particularly important to conduct large-scale discovery of novel functional genes for highly efficient salt-tolerant microorganisms to construct corresponding genetically engineered strains, microbial fertilizers, and transgenic plants. From the perspective of molecular biology and genetic engineering technology, constructing highly efficient salt-tolerant genetically engineered strains and transgenic plants is the preferred approach to obtaining salt-tolerant microorganisms and plants. Because bacteria and plants share significant similarities in salt tolerance, they both accumulate similar compounds within their cells under hyperosmolar conditions. Therefore, the construction of a highly efficient salt-tolerant bacterial resource library and the discovery of key novel functional genes can not only solve the problem of constructing salt-tolerant bacterial genetically engineered strains, but also provide an important genetic resource reserve for constructing highly efficient salt-tolerant transgenic plants.

[0005] Cloning and functional identification of salt-tolerance genes in halophilic bacteria, and research on their molecular mechanisms, has become a hot topic in the field of bacterial salt tolerance. The study of functional genes for salt tolerance in halophilic microorganisms is of paramount importance.

[0006] Current technological shortcomings also include the fact that microbial fertilizers and plants that can be used for soil improvement do not have efficient salt and alkali tolerance capabilities, and there is a lack of theoretical basis for large-scale discovery of new functional genes with high salt and alkali tolerance to construct corresponding genetically engineered strains of microbial fertilizers and transgenic plants. Furthermore, there is a lack of effective identification methods for molecularly modifying new functional genes with high salt and alkali tolerance to improve their activity in transporting cations such as Na+ and Li+.

[0007] An unknown functional membrane protein composed of 103 amino acid residues from NEAU-ST10-40T was initially named Ump (Unknown functional membrane protein) (see the applicant's invention patent CN201810009928.4, "An Acid-Tolerant Gene ump from Bacillus halophilus and its Identification Method"). The protein encoded by the acid-tolerant gene ump does not belong to any known protein family, but it retains essential conserved charged or polar residues and aromatic residues, mainly exhibiting the structure W9-E15-H43-H80. Based on these four amino acid residues, we previously tentatively named it WEHH. Structural analysis showed that this antitransporter WEHH is the smallest known Na+ / H+ antitransporter. Therefore, using NhaM to represent the smallest Na+ / H+ antitransporter is most appropriate, and WEHHH was renamed NhaM. NhaM and its homologs form a new transporter family, named the NhaM family. NhaM has been found to have the function of a Na+ / H+ antitransporter. Summary of the Invention

[0008] To address the problems existing in the prior art, this invention provides an ump mutant gene, disclosing its nucleic acid sequence, encoded amino acids, and protein function detection. It clarifies the important function of a highly conserved amino acid site: the mutation of threonine (T) at position 11 to aspartic acid (D), forming a mutant NhaM-T11D. The invention demonstrates that the mutant NhaM-T11D protein possesses resistance to 0.2M NaCl and alkaline pH. The gene and protein of the mutant NhaM-T11D are of great significance for the improvement of saline-alkali soils and can be used in research on salt-alkali tolerance gene modification, construction of highly efficient salt-alkali tolerant engineered strains, development of microbial fertilizers, and transgenic plants to improve plant salt and alkali tolerance.

[0009] The present invention adopts the following technical solution:

[0010] The mutant NhaM-T11D has the following nucleotide sequence:

[0011] SEQ ID NO: 1.

[0012] The protein encoded by the mutant NhaM-T11D gene has the following amino acid sequence:

[0013] SEQ ID NO: 2.

[0014] The mutant NhaM-T11D gene encodes a protein that exhibits Na+ or Li+ or K+ / H+ reverse transporter activity in a fluorescence quenching assay, and this activity is pH-dependent.

[0015] NhaM has been discovered to function as a Na+ / H+ antitransporter, representing a novel class of Na+(Li+,K+) / H+ antitransporters. NhaM is a simple yet highly capable membrane protein performing Na+ / H+ antitransport. Functional analysis of key residues involved in its structure and function will undoubtedly reveal novel structure-function relationships in Na+ / H+ antitransporters. The obtained results regarding the molecular transport mechanism can also guide molecular modification of this protein to improve its activity in transporting Na+, Li+, K+, and other cations, enhancing its potential for use in constructing highly efficient salt- and alkali-tolerant genetically engineered strains. Therefore, studying mutants of this protein has significant research importance and potential application value.

[0016] The functional assay of the protein encoded by the mutant NhaM-T11D gene includes the following steps:

[0017] 1) Using pET22b-NhaM as a template, the threonine (T) at position 11 of this residue was mutated to aspartic acid (D) through site-directed amino acid mutagenesis.

[0018] 2) Escherichia coli KNabc expression mutants were cultured in LBK medium at pH 7.0 containing 0.2M NaCl or 5mM LiCl, or at pH 8.0 + 50mM NaCl. The positive control was wild-type NhaM, and the negative control was empty vector pET22b.

[0019] 3) Western blot analysis of mutant expression;

[0020] 4) The optimal Na+ / H+, Li+ / H+ and K+ / H+ reverse transporter activities were determined using E. coli KNabc expressing each mutant at pH 9.0, with wild-type NhaM as a positive control.

[0021] T11 was highly conserved among NhaM and 27 homologs, suggesting their potential involvement in NhaM function. To determine the functional or structural role of the residues, site-directed mutagenesis was performed using pET22b-NhaM as a template, replacing the threonine (Thr) residue at position 11 with aspartic acid (Asp). Each mutant was then expressed in *E. coli* KNabc in LBK medium at pH 7.0 containing 0.2 M NaCl or 5 mM LiCl, or at pH 8.0 with 50 mM NaCl. Wild-type NhaM served as the positive control, and the empty vector pET22b as the negative control. Western blot analysis was performed on the expression of all mutants that could not complement *E. coli* KNabc. Furthermore, optimal Na+ / H+, Li+ / H+, and K+ / H+ antitransporter activities were determined using *E. coli* KNabc expressing each mutant at pH 9.0, with wild-type NhaM as the positive control.

[0022] The mutant NhaM-T11D gene encodes a protein that exhibits resistance to 0.2 M NaCl and alkaline pH.

[0023] Expression of the mutant protein NhaM-T11D significantly enhances the tolerance of Escherichia coli KNabc to 0.2M NaCl or 5mM LiCl, as well as to alkaline pH 8.0.

[0024] The genes and proteins of the mutant NhaM-T11D are of great significance for the improvement of saline-alkali soils and can be used for salt-alkali tolerance gene modification, providing a new and effective option for research in the fields of constructing highly efficient salt-alkali tolerant engineered strains, developing microbial fertilizers, and improving the salt tolerance of transgenic plants.

[0025] A salt-tolerant engineered strain was constructed using the expression of the mutant NhaM-T11D gene.

[0026] Microbial fertilizer prepared using the expression of the mutant NhaM-T11D gene.

[0027] Salt-tolerant transgenic plants prepared using the NhaM-T11D mutant gene expression.

[0028] In summary, the advantages and positive effects of this invention are as follows:

[0029] 1. The gene sequence and important functions of the mutant NhaM-T11D protein were clarified. T11 is highly conserved among NhaM and its homologs, suggesting its potential involvement in NhaM function. Results showed that replacing the threonine (Thr) residue at position 11 of the highly conserved NhaM protein with aspartic acid (Asp) enabled *E. coli* KNabc to tolerate 0.2M NaCl, 5mM LiCl, and an alkaline pH of 8.0. Compared with the wild type and control, this mutant protein NhaM-T11D exhibited significant salt and alkali tolerance.

[0030] 2. Further functional verification showed that the mutant protein NhaM-T11D has Na+(Li+,K+) / H+ reverse transporter activity in the fluorescence quenching experiment, and the Na+(Li+,K+) / H+ reverse transporter activity is pH dependent.

[0031] 3. It is pointed out that the genes and proteins of the mutant NhaM-T11D are of great significance for the improvement of saline-alkali soil, and can be used for research in the fields of salt-alkali tolerance gene modification, construction of highly efficient salt-alkali tolerant engineered strains, development of microbial fertilizers, and transgenic plants that improve the salt and alkali tolerance of plants. Attached Figure Description

[0032] Figure 1 This is a growth curve of the protein encoded by the mutant NhaM-T11D described in this embodiment of the invention under 0.2M NaCl stress.

[0033] Figure 2 This is a growth curve of the protein encoded by the mutant NhaM-T11D described in this embodiment of the invention under 5mM LiCl stress.

[0034] Figure 3 This is a growth curve of the protein encoded by the mutant NhaM-T11D described in this embodiment of the invention under alkaline stress conditions of pH 8.0 + 50mM NaCl.

[0035] Figure 4 This is a schematic diagram of NhaM homology comparison provided in an embodiment of the present invention.

[0036] Figure 5 This is a detection map of the Na+(Li+,K+) / H+ reverse transporter activity of the protein encoded by the mutant NhaM-T11D described in this embodiment of the invention, with wild-type NhaM as a positive control.

[0037] Figure 6 This is a Western blot image of the protein encoded by the mutant NhaM-T11D described in this embodiment of the invention, with wild-type NhaM as a positive control. Detailed Implementation

[0038] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0039] The mutant NhaM-T11D provided in this embodiment of the invention has the following gene nucleic acid sequence: SEQ ID NO: 1.

[0040] The protein encoded by the mutant NhaM-T11D provided in this embodiment of the invention has the amino acid sequence SEQ ID NO: 2.

[0041] This invention investigated the function of the protein encoded by the mutant NhaM-T11D. The results showed that the mutant NhaM-T11D possesses 0.2M NaCl resistance. Expression of the NhaM-T11D amino acid profile enabled *E. coli* KNabc to tolerate 0.2M NaCl or 5mM LiCl, and also tolerating an alkaline pH of 8.0. Compared with the wild type and control, this mutant protein NhaM-T11D exhibits significantly greater salt and alkali tolerance. Furthermore, the mutant protein was demonstrated to possess Na+(Li+,K+) / H+ reverse transporter activity.

[0042] The detection and application principles of the present invention will be further explained below with reference to specific embodiments:

[0043] 1. Amino acid site selection and mutation

[0044] like Figure 4 As shown, T11 is highly conserved between NhaM and its homologs.

[0045] Based on the homology comparison of NhaM, one amino acid, including T11, was selected from highly conserved amino acid sites. Based on the selected site, amino acid site-directed mutagenesis primers were designed (as shown in Table 1).

[0046] Table 1: Primers for site-directed amino acid mutagenesis

[0047]

[0048] The specific steps for performing site-directed amino acid mutagenesis are as follows:

[0049] (1) Site-directed mutagenesis was performed by PCR. The 50 μL system is shown in Table 2:

[0050] Table 2: PCR reaction system and conditions

[0051]

[0052] (2) Agarose gel electrophoresis detection

[0053] Take 2 μL of the above PCR product and detect it by 0.7% agarose gel electrophoresis.

[0054] (3) Digestion of PCR products

[0055] Dilute 10 U / μL DMT enzyme 10 times. The digestion reaction system and conditions are shown in Table 3.

[0056] Table 3: DMT enzyme dilution and digestion system

[0057]

[0058] (4) Transformation

[0059] 1) Add 10 μL of DMT digestion product to 50 μL of KNabc-transformed competent cells, mix gently, and insert the centrifuge tube into crushed ice for 30 min.

[0060] 2) Place it in a preheated 42℃ water bath for 90 seconds to heat shock, then quickly remove it and place it on ice for 10 minutes.

[0061] 3) Add 300 μl of LBK liquid culture medium and incubate at 147 rpm and 37°C for 1 hour;

[0062] 4) After concentrating the bacterial culture, spread it entirely onto 0M LBK solid medium containing Amp and incubate overnight at 37°C.

[0063] (5) Positive clone plasmids were sent to BGI for sequencing. After the sequencing results were obtained, DNAMAN 6.0 software was used to check whether the mutation was successful. If the mutation was successful, the next step of functional verification was carried out.

[0064] 2. Mutant resistance physiological experiments

[0065] Physiological experiments were conducted on each mutant along with a negative control to identify the effects of mutations at different sites on salt and alkali tolerance.

[0066] (1) The (E.coli) KNabc containing the point mutation plasmid and the negative control pET22b and the positive control strain KNabc / pET22b-NhaM were activated respectively. When the OD600 value was 1.0, they were inoculated into the corresponding culture medium at a rate of 1% for testing.

[0067] (2) Na+ tolerance test: The above cultures were transferred to LBK medium containing different concentrations of NaCl, and the OD600 value was measured after 24 h of culture. The results are as follows: Figure 1 As shown.

[0068] (3) Li+ tolerance test: The above cultures were transferred to LBK medium containing different concentrations of LiCl, and the OD600 value was measured after 24 h of culture. The results are as follows: Figure 2 As shown.

[0069] (4) Alkali tolerance test: The above cultures were transferred to different LBK media with pH values ​​of 7.0, 7.5, 8.0, 8.5, and 9.0, as well as to different LBK media with pH values ​​of 7.0, 7.5, 8.0, 8.5, and 9.0 containing 50 mM NaCl. After 24 h of incubation, the OD600 values ​​were measured. The results are as follows: Figure 3 As shown.

[0070] 3. Analysis of point mutant protein function: Determination of NhaM mutant transporter activity

[0071] To verify that the physiological differences between the above mutants and the wild type are due to impaired protein function, this invention measures the activity of the mutant transport proteins and performs Western blotting. The specific determination of the transport protein activity of the NhaM mutant is as follows:

[0072] 1) Preparation of the reverse membrane

[0073] ① Cultivate and collect bacterial cells; wash the obtained bacterial cell pellet twice with pre-cooled buffer A; then resuspend the bacterial cells with the same volume of buffer A.

[0074] ② Cell disruption: When the system pressure of the high-pressure cell disruptor JG-1A reaches 2,000 psi, the cells are disrupted. After disruption, centrifuge at 4℃ and 8,000 rpm for 5 minutes. At this time, take the supernatant for ultracentrifugation. Conditions: 4℃, 100,000×g, centrifugation for 1 hour.

[0075] ③ Obtain the reverse membrane: Dissolve the precipitate obtained after ultracentrifugation in an ice bath with an appropriate amount of buffer A, and aliquot. Simultaneously, determine the protein concentration and store at low temperature.

[0076] 2) Protein concentration determination: The Lorry method was used to determine the protein concentration in the reverse membrane.

[0077] 3) Detection of the activity of mutant NhaM-T11D protein

[0078] ① Quickly add 2 μM of acridine orange (AO) fluorescent indicator and 20 μg of reverse membrane to a quartz cup containing 2.5 mL of buffer B, mix well, and measure the fluorescence using the pre-set fluorescence monitoring parameters: excitation (EX) wavelength 492 nm and emission (EM) wavelength 526 nm.

[0079] ②When the value is stable, Tris-D-lactic acid is added to the system to make the final concentration 5mM. The role of adding lactic acid is to act as a respiratory substrate, which can generate a transmembrane pH gradient, i.e., the generation of ΔpH.

[0080] ③ When the fluorescence is quenched to a constant value, immediately add Na+, K+, and Li+ to the reaction system at a final concentration of 5mM, and continue to monitor the fluorescence intensity.

[0081] ④ Estimate the activity of Na+(Li+,K+) / H+ antitransporter based on the changes in fluorescence values ​​before and after ion addition.

[0082] 4) Determination of pH tolerance range of mutant NhaM-T11D

[0083] The pH tolerance range can indicate the pH at which transporter activity is at its maximum. Using Na+, K+, and Li+ as substrates, the Na+(Li+,K+) / H+ transporter activity of the positive control, negative control, and each mutant was measured in the pH range of 7.0-9.0.

[0084] The above are as follows Figure 5 As shown.

[0085] 4. Detection of NhaM-T11D mutant protein expression

[0086] To verify whether the mutation affected protein expression, the reverse membranes of the prepared NhaM mutant, wild-type positive control, and empty vector negative control were loaded onto samples for SDS-PAGE and Western blotting experiments. The specific procedures are as follows:

[0087] (1) Take 10 μg of membrane protein and mix it with 10 μl of 5×SDS-PAGE loading buffer. Finally, incubate it in a 38℃ water bath for 30 min.

[0088] (2) Set the electrophoresis apparatus voltage to constant voltage. First, use 80V to make the bromophenol blue front pass through the stacking gel and compress it into a line. Then adjust the voltage to 120V.

[0089] (3) Transfer: Immerse the PVDF membrane in methanol for 1 min, then take it out and immerse it in 1× transfer solution along with the filter paper and sponge. Then, set up the device in a sandwich manner, with the order from bottom to top being sponge → three layers of filter paper → gel → PVDF membrane → three layers of filter paper → sponge. Transfer the membrane at 20 mA for 40 min at 4℃.

[0090] (4) Blocking: After the transfer is complete, place the protein membrane in the prepared TBST buffer, rinse for 1-2 min, add blocking solution, place on a shaker and shake slowly, block at 37℃ for 2 h;

[0091] (5) Primary antibody incubation: Refer to the instructions for the primary antibody, dilute the primary antibody at an appropriate ratio, and incubate overnight at 4°C with gentle shaking;

[0092] (6) Wash the membrane with TBST buffer, and slowly shake and rinse 3 times on a side-shaking shaker for 5-10 minutes each time; then add TBS buffer and rinse once more.

[0093] (7) Secondary antibody incubation: Refer to the instructions for the secondary antibody and dilute the horseradish peroxidase (HRP)-labeled secondary antibody at an appropriate ratio. Immediately add the diluted secondary antibody and incubate at 37°C with gentle shaking for 1 hour;

[0094] (8) Washing the membrane: Use TBST buffer to slowly shake and rinse 3 times on a side-shaking shaker for 5-10 minutes each time; then add TBS buffer to wash once more.

[0095] (9) Protein detection: Refer to the relevant instructions and use ECL reagents such as BeyoECL Plus (P0018) to detect proteins.

[0096] like Figure 6 As shown, all mutants were expressed, proving that the changes in physiological phenotype are due to alterations in protein function caused by mutations.

[0097] This invention screened out important amino acid sites, and the mutation improved the salt and alkali resistance, which is very novel among existing proteins. This invention also contributes to the understanding of the protein's mechanism of action and the targeted modification of proteins.

[0098] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. The mutant NhaM-T11D, characterized in that, Its amino acid sequence is as follows: SEQ ID NO:

2.

2. A nucleic acid molecule encoding the mutant NhaM-T11D of claim 1, characterized in that, Its nucleic acid sequence is: SEQ ID NO:

1.

3. A salt-tolerant engineered strain expressing the encoding gene of the mutant NhaM-T11D as described in claim 1.

4. The application of the mutant NhaM-T11D as described in claim 1 in the preparation of salt-tolerant microbial fertilizer.

Images