Gene of mutant NhaM-T11D, encoded protein and application

Abstract

The invention belongs to the technical field of protein, and discloses a mutant NhaM-T11D, which comprises gene, amino acid, protein function and Na < + > (Li < + >, K < + >) / H < + > reverse transport protein activity detection, and the invention clarifies a highly conservative amino acid site of NhaM protein: the important function of the mutant NhaM-T11D of which 11-site threonine (T) is mutated into aspartic acid (D), it is proved that the mutant NhaM-T11D protein has a 0.2 M NaCl and alkaline pH value resistance function. The invention also indicates that the gene and protein of the mutant NhaM-T11D have important significance on saline-alkali soil improvement, can be used for saline-alkali tolerant gene modification, and provides a new effective choice for research in the fields of construction of efficient saline-alkali tolerant engineering strains, development of microbial fertilizers and improvement of transgenic plants for saline-alkali tolerance of plants.

Description

Technical Field

[0001] The present invention belongs to the technical field of proteins, and relates to a gene, amino acids, encoded protein, functional detection and related applications of a mutant NhaM-T11D. Background Art

[0002] With global warming, soil salinization in arid and semi-arid regions is becoming increasingly serious. Specifically, the area of ​​salinized land is expanding, the degree of salinization is increasing, the area of ​​severely salinized land is increasing, and the area of ​​unusable alkaline spots is expanding, which seriously affects the improvement of land productivity and the development of the national economy. At the same time, due to industrial pollution, unreasonable irrigation and improper use of fertilizers, the area of ​​secondary salinized soil is also increasing rapidly. Salt-alkali land affects vegetation growth, reduces or even fails to produce crops, indirectly causes ecological environment deterioration, and can corrode and damage engineering facilities, causing losses of more than 2.5 billion yuan each year.

[0003] It is currently believed that the improvement of saline-alkali soil requires the joint efforts of multiple disciplines. From a biological perspective, the main method is to start with both microorganisms and plants, with efficient salt-tolerant microorganisms and plants working synergistically. This is because many plants cannot directly obtain the poor carbon and nitrogen sources in saline-alkali land, and if only chemical fertilizers are applied, it will cause more serious 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 efficient salt-alkali-tolerant plants to improve saline-alkali soil.

[0004] However, many microbial fertilizers and plants that can be used for soil improvement do not have the ability to resist high salinity and alkali. Therefore, in addition to exploring efficient salt-alkali tolerant microorganisms and plant resources, it is particularly important to explore efficient salt-alkali tolerant new functional genes on a large scale to construct corresponding genetically engineered strains, microbial fertilizers and transgenic plants. From the perspective of molecular biology and genetic engineering technology, constructing efficient salt-alkali tolerant genetically engineered strains and transgenic plants is the preferred way to obtain salt-alkali tolerant microorganisms and plants. Since bacteria and plants have great similarities in salt-alkali tolerance, they accumulate similar compounds in cells under hypertonic conditions. Therefore, the construction of an efficient salt-alkali tolerant bacterial resource library and the exploration of key new functional genes can not only solve the problem of constructing salt-alkali tolerant bacterial genetically engineered strains, but also excellent efficient salt-alkali tolerant bacterial genes can provide important gene resource reserves for constructing efficient salt-alkali tolerant transgenic plants.

[0005] The cloning and functional identification of salt-alkali tolerance genes in halophilic bacteria and the study of their molecular mechanisms have become a hot topic in the field of salt-alkali tolerance in bacteria. The study of salt-alkali tolerance functional genes in salt-tolerant microorganisms is of great significance.

[0006] The current technical defects also lie in the fact that the microbial fertilizers and plants that can be used for soil improvement do not have the ability to efficiently tolerate salt and alkali, and there is a lack of theoretical basis for the large-scale discovery of new functional genes that are highly resistant to salt and alkali for the construction of corresponding genetically engineered strains of microbial fertilizers and transgenic plants. There is also a lack of effective identification methods for molecular modification of new functional genes that are highly resistant to salt and alkali to improve their activity in transporting cations such as Na+ and Li+.

[0007] A membrane protein of unknown function composed of 103 amino acid residues from NEAU-ST10-40T was originally named Ump (Unknown functional membrane protein) (see the applicant's invention patent CN201810009928.4 A salt-alkali tolerant gene ump of Halobacillus andrae and its identification method). The protein encoded by the salt-alkali tolerant gene ump does not belong to any known protein family, but it retains the necessary conservative charged or polar residues and aromatic residues, mainly W9-E15-H43-H80. Based on these four amino acid residues, we have tentatively named it WEHH. Structural analysis shows that this antiporter WEHH is the smallest of the known Na+ / H+ antiporters, so it is most appropriate to use NhaM to represent the smallest Na+ / H+ antiporter, and WEHH is renamed NhaM. NhaM and its homologs form a new transporter family named NhaM family. NhaM was found to have the function of Na+ / H+ antiporter. Summary of the invention

[0008] In view of the problems existing in the prior art, the present invention provides an ump mutant gene, discloses its nucleic acid sequence, the amino acids encoded and expressed, and the protein function detection, clarifies the important function of a highly conserved amino acid site: a mutant NhaM-T11D formed by the mutation of threonine (T) at position 11 to aspartic acid (D), and proves that the mutant NhaM-T11D protein has 0.2M NaCl and alkaline pH resistance. The gene and protein of the mutant NhaM-T11D are of great significance to the improvement of saline-alkali soil, and can be used for the research in the field of salt-alkali tolerant gene modification, the construction of efficient salt-alkali tolerant engineering strains, the development of microbial fertilizers, and the improvement of plant salt-alkali tolerance transgenic plants.

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

[0010] The mutant NhaM-T11D has a gene nucleic acid sequence of:

[0011] SEQ ID NO: 1.

[0012] The mutant NhaM-T11D gene encodes a protein whose amino acid sequence is:

[0013] SEQ ID NO:2.

[0014] The mutant NhaM-T11D gene encodes a protein having Na+, Li+ or K+ / H+ antiporter activity in a fluorescence quenching experiment, and the Na+, Li+ or K+ / H+ antiporter activity is pH-dependent.

[0015] NhaM was found to have the function of a Na+ / H+ antiporter. It represents a new type of Na+(Li+,K+) / H+ antiporter. NhaM is a novel membrane protein that is so simple yet capable of Na+ / H+ antiporter. Functional analysis of important residues involved in structure or function will inevitably reveal a new structure-function relationship of Na+ / H+ antiporter. The results obtained on the molecular transport mechanism can also guide us to molecularly modify the protein to improve its activity in transporting cations such as Na+, Li+, and K+, and enhance the potential of this gene to be used as a genetically engineered strain for the construction of efficient salt-alkali tolerance. Therefore, studying this protein mutant has important research significance and potential application value.

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

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

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

[0019] 3) Western blot was used to detect the expression of mutants;

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

[0021] T11 is highly conserved between NhaM and 27 homologs. This suggests that they may be involved in the function of NhaM. To determine the functional or structural role of the residues, the threonine (Thr) residue at position 11 was replaced with aspartic acid (Asp) by amino acid site-directed mutagenesis using pET22b-NhaM as a template. Each mutant was then expressed in E. coli KNabc and cultured in LBK medium at pH 7.0 containing 0.2 M NaCl or 5 mM LiCl, or pH 8.0 + 50 mM NaCl. The positive control was wild-type NhaM, and the negative control was the empty vector pET22b. The expression of all mutants that could not be complemented by E. coli KNabc was detected by western blot. In addition, we also determined the optimal Na+ / H+, Li+ / H+, and K+ / H+ antiporter activities using E. coli KNabc expressing each mutant at pH 9.0, with wild-type NhaM as a positive control.

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

[0023] The expression of the mutant protein NhaM-T11D apparently enabled E. coli KNabc to tolerate 0.2 M NaCl or 5 mM LiCl, as well as an alkaline pH of 8.0.

[0024] The genes and proteins of the mutant NhaM-T11D are of great significance to the improvement of saline-alkali soil and can be used for salt-alkali tolerance gene modification, providing a new and effective choice for the construction of efficient salt-alkali tolerant engineering strains, the development of microbial fertilizers and the research in the field of transgenic plants to improve the salt-alkali tolerance of plants.

[0025] The salt-alkali tolerant engineering strain is constructed by expressing the mutant NhaM-T11D gene.

[0026] The microbial fertilizer is prepared by expressing the mutant NhaM-T11D gene.

[0027] The salt-alkali tolerant transgenic plant is prepared by expressing the mutant NhaM-T11D gene.

[0028] In summary, the advantages and positive effects of the present invention are:

[0029] 1. The gene sequence of the mutant NhaM-T11D and the important functions of the encoded protein were clarified. T11 is highly conserved between NhaM and homologs, indicating that it may be involved in the function of NhaM. The results showed that a highly conserved amino acid site in the NhaM protein: the replacement of the threonine (Thr) residue at position 11 with aspartic acid (Asp) can make E. coli KNabc tolerate 0.2MNaCl, 5mM LiCl, and alkaline pH 8.0. Compared with the wild type and blank, this mutant protein NhaM-T11D has obvious salt and alkali tolerance.

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

[0031] 3. It is pointed out that the genes and proteins of the mutant NhaM-T11D are of great significance to the improvement of saline-alkali soil, and can be used for salt-alkali tolerance gene modification, construction of efficient salt-alkali tolerance engineering strains, development of microbial fertilizers and research in the field of transgenic plants to improve plant salt-alkali tolerance. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a growth curve diagram of the protein encoded by the mutant NhaM-T11D described in the example of the present invention under 0.2M NaCl stress conditions.

[0033] Figure 2 It is a growth curve diagram of the protein encoded by the mutant NhaM-T11D described in the example of the present invention under 5mM LiCl stress conditions.

[0034] Figure 3 It is a growth curve diagram of the protein encoded by the mutant NhaM-T11D described in the example of the present invention under the alkaline stress condition of pH 8.0+50mM NaCl.

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

[0036] Figure 5 This is a diagram of the Na+(Li+, K+) / H+ antiporter activity detection of the protein encoded by the mutant NhaM-T11D described in the examples of the present invention, and the wild-type NhaM is used as a positive control.

[0037] Figure 6 This is a Western Blot detection diagram of the protein encoded by the mutant NhaM-T11D described in the examples of the present invention, and the wild-type NhaM is used as a positive control. DETAILED DESCRIPTION

[0038] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

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

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

[0041] The present invention embodiment detects the function of the protein encoded by the mutant NhaM-T11D, and the results show that the mutant NhaM-T11D has 0.2M NaCl resistance function, and the expression of NhaM-T11D amino acids can make Escherichia coli KNabc tolerate 0.2M NaCl or 5mM LiCl, and can also tolerate an alkaline pH of 8.0. Compared with the wild type and the blank, this mutant protein NhaM-T11D has a more obvious salt-alkali tolerance. In addition, the mutant protein was shown to have Na+(Li+,K+) / H+ antiporter activity.

[0042] The detection and application principle of the present invention will be further described below in conjunction with specific embodiments:

[0043] 1. Amino acid selection and mutation

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

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

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

[0047]

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

[0049] (1) PCR for site-directed mutagenesis, 50 μL system as 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 10U / 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 competent cells, mix gently, and place the centrifuge tube in crushed ice for 30 minutes.

[0060] 2) Heat shock the tube in a preheated 42°C water bath for 90 seconds, then quickly remove and place on ice for 10 minutes.

[0061] 3) Add 300 μl LBK liquid medium and resuscitate at 147 rpm and 37°C for 1 h;

[0062] 4) After the bacterial solution is concentrated, spread it all on 0M LBK solid culture medium containing Amp and culture it at 37°C overnight.

[0063] (5) The positive clone plasmid was sent to BGI for sequencing. After the sequencing results came out, DNAMAN6.0 software was used to compare whether the mutation was successful. If the mutation was successful, the next step of functional verification was carried out.

[0064] 2. Mutant resistance physiological experiment

[0065] Physiological experiments were performed on each mutant together with the negative control to identify the effects of mutations at different sites on salt-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, and when the OD600 value was 1.0, they were inoculated at 1% into the corresponding culture medium for testing.

[0067] (2) Na+ resistance experiment: The above cultures were transferred to LBK medium containing different concentrations of NaCl. After culturing for 24 h, the OD600 value was measured. The results were as follows: Figure 1 shown.

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

[0069] (4) Alkali resistance test: The above culture was transferred to LBK medium with different pH values ​​of 7.0, 7.5, 8.0, 8.5 and 9.0 and LBK medium with different pH values ​​of 7.0, 7.5, 8.0, 8.5 and 9.0 containing 50 mM NaCl, and the OD600 value was measured after culturing for 24 h. The results are as follows: Figure 3 shown.

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

[0071] In order to verify that the physiological results of the above mutants are different from those of the wild type due to the influence on the protein function, the present invention performs the determination of the activity of the mutant transporter and Western Blot detection. The determination of the activity of the NhaM mutant transporter is as follows:

[0072] 1) Preparation of inversion membrane

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

[0074] ② Break the cell wall: When the system pressure of the high-pressure cell crusher JG-1A reaches 2,000psi, the cell is broken. After breaking, centrifuge at 4℃, 8,000rpm for 5min. At this time, take the supernatant for ultracentrifugation. The conditions are: 4℃, 100,000×g, and centrifuge for 1h.

[0075] ③ Obtain inversion membrane: Dissolve the precipitate obtained after ultracentrifugation in an ice bath with an appropriate amount of buffer A and dispense. At the same time, measure the protein concentration and store at low temperature.

[0076] 2) Determination of protein concentration: 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 Rapidly add 2 μM fluorescent indicator acridine orange (AO) and 20 μg of reverse membrane into a quartz cup containing 2.5 mL of buffer B, mix well, and measure under the set fluorescence monitoring parameters: excitation light (EX) wavelength 492 nm, emission light (EM) wavelength 526 nm.

[0079] ② When the value is stable, add Tris-D-lactic acid to the system to a final concentration of 5 mM. The role of adding lactic acid is to serve as a respiratory substrate, which can produce a transmembrane pH gradient, that is, the production of ΔpH.

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

[0081] ④ Estimate the activity of Na+(Li+,K+) / H+ antiporter 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 shows the pH at which the transporter activity is 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 Figure 5 shown.

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

[0086] In order to verify whether the mutation affects protein expression, the NhaM mutant prepared above, the wild-type positive control and the empty vector negative control were loaded on the reverse membrane and subjected to SDS-PAGE and protein immunoblotting experiments. The specific operation method is as follows:

[0087] (1) Take 10 μg of membrane protein and mix it with 10 μl of 5× SDS-PAGE loading buffer, and then place it in a 38°C water bath for 30 min;

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

[0089] (3) Transfer: Soak the PVDF membrane in methanol for 1 min, take it out and soak it in 1× transfer solution together with the filter paper and sponge, and then install it in a sandwich manner. The order from bottom to top is sponge → three layers of filter paper → glue → PVDF membrane → three layers of filter paper → sponge. Transfer the membrane at 20 mA for 40 min at 4°C.

[0090] (4) Blocking: After the transfer is completed, place the protein membrane in the pre-prepared TBST buffer, rinse for 1-2 minutes, add the blocking solution, place it on a shaker and shake it slowly, and block it at 37°C for 2 hours;

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

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

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

[0094] (8) Wash the membrane: Use TBST buffer to slowly shake on a side-to-side shaker and rinse three times for 5-10 min each time; then wash once with TBS buffer;

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

[0096] like Figure 6 As shown, all mutants were expressed, demonstrating that the changes in physiological phenotypes were due to altered protein functions caused by the mutations.

[0097] The present invention screens out important amino acid sites, and the salt-alkali resistance is improved after mutation, which is very novel in existing proteins. The present invention also helps to understand the mechanism of action of proteins and the directional 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 in the protection scope of the present invention.

Claims

1. A mutant NhaM-T11D, characterized in that Its gene nucleic acid sequence is: SEQ ID NO:

1.

2. The protein encoded by the mutant NhaM-T11D gene according to claim 1, characterized in that: The amino acid sequence of the protein is: SEQ ID NO:

2.

3. The protein encoded by the mutant NhaM-T11D gene according to claim 2, characterized in that: The protein has Na+, Li+ or K+ / H+ antiporter activity in a fluorescence quenching experiment, and the Na+, Li+ or K+ / H+ antiporter activity is pH-dependent.

4. The functional detection of the protein encoded by the mutant NhaM-T11D gene according to claim 2, characterized in that: The steps include: 1) Using pET22b-NhaM as a template, the threonine (T) at position 11 of the residue was mutated to aspartic acid (D) by site-directed amino acid mutagenesis; 2) E. coli KNabc mutants were cultured in LBK medium at pH 7.0 containing 0.2 M NaCl or 5 mM LiCl, or pH 8.0 + 50 mM NaCl, with the wild-type NhaM as the positive control and the empty vector pET22b as the negative control; 3) Western blot was used to detect the expression of mutants; 4) The optimal Na+ / H+, Li+ / H+, and K+ / H+ antiporter activities were determined using E. coli KNabc expressing each mutant at pH 9.0, and wild-type NhaM was used as a positive control.

5. The protein encoded by the mutant NhaM-T11D gene according to claim 2, characterized in that: The protein has 0.2M NaCl and alkaline pH resistance functions.

6. A salt-alkali tolerant engineered strain constructed by expressing the mutant NhaM-T11D gene according to claim 1.

7. A microbial fertilizer prepared by expressing the mutant NhaM-T11D gene according to claim 1.

8. A salt- and alkaline-tolerant transgenic plant prepared by expressing the mutant NhaM-T11D gene according to claim 1.

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