Gene, encoded protein and use of mutant nham-s67t

Abstract

This invention belongs to the field of protein technology and discloses a mutant NhaM-S67T, including its gene, amino acids, protein function, and the detection of Na+(Li+,K+) / H+ reverse transporter activity. This invention clarifies the important function of a highly conserved amino acid site in the NhaM protein: the mutation of serine (S) at position 67 to threonine (T) in the mutant NhaM-S67T. It demonstrates that the mutant NhaM-S67T protein possesses resistance to 0.2M NaCl and alkaline pH. This invention also indicates that the gene and protein of the mutant NhaM-S67T 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.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of proteins, and relates to a mutant gene, amino acid, encoded protein, function detection and related application of a mutant NhaM-S67T. BACKGROUND

[0002] Various stress factors such as salinity, temperature and moisture may exist in the environment of bacterial growth. Among them, salinity is considered to be one of the most harmful abiotic stress factors. High salinity environment not only has a great toxic effect on bacterial cells itself, but also produces high concentrations of osmotic pressure and ionic pressure, which seriously affects the normal physiological functions of bacteria. However, the salt-tolerant ability of bacteria with important economic value in agricultural production, such as rhizobium, nitrogen-fixing bacteria, phosphate bacteria, potassium bacteria and plant growth-promoting bacteria, is very limited, which seriously restricts their application prospect in agricultural production.

[0003] In contrast, halophilic bacteria are a kind of microorganisms that can grow only in salt-containing conditions, and their salt-tolerant range is 0.5-32.5% (w / v) NaCl, and many halophilic bacteria also have high-efficiency alkali-tolerant ability. High-efficiency salt-tolerant and alkali-tolerant ability means that halophilic bacteria have more salt-tolerant and alkali-tolerant genes, and the salt-tolerant and alkali-tolerant molecular mechanisms are more complex. At present, the cloning and functional identification of halophilic bacteria salt-tolerant and alkali-tolerant genes and the study of their salt-tolerant and alkali-tolerant molecular mechanisms have become a research hotspot in the field of bacterial salt-tolerant and alkali-tolerant. The research on salt-tolerant and alkali-tolerant functional genes in salt-tolerant microorganisms is of great significance.

[0004] The current technical defects are that the microbial fertilizers and plants used for soil improvement do not have high-efficiency salt-tolerant and alkali-tolerant ability, and there is a lack of theoretical basis in large-scale exploration of new functional genes with high-efficiency salt-tolerant and alkali-tolerant ability for constructing corresponding genetically engineered strain microbial fertilizers and transgenic plants, and there is a lack of effective identification method in molecular modification of new functional genes with high-efficiency salt-tolerant and alkali-tolerant ability to improve the activity of transporting Na+, Li+ and other cations.

[0005] A functional unknown membrane protein consisting of 103 amino acid residues from NEAU-ST10-40T, which is originally named as Ump (Unknown functional membrane protein) (see the patent application CN201810009928.4 of the applicant for a salt-tolerant and alkali-tolerant gene ump of Andakia halophila and a method for identifying the same). It is proved to have the function of Na+ / H+ antiporter. It represents a new type of Na+(Li+, K+) / H+ antiporter. SUMMARY

[0006] In view of the problems in the prior art, the application provides a mutant gene of UMP, discloses a nucleic acid sequence, amino acid sequence of coding expression and protein function detection, and determines a highly conserved amino acid site: 67th serine (S) is mutated into threonine (T) to form a mutant NhaM-S67T important function, and it is proved that the mutant NhaM-S67T protein has 0.2M NaCl and alkaline pH value resistance function.The gene and protein of the mutant NhaM-S67T have important significance for saline-alkali soil improvement, and can be used in the fields of salt-tolerant gene modification, construction of high-efficiency salt-tolerant engineering strains, development of microbial fertilizers and transgenic plants for improving plant salt-tolerance.

[0007] The application adopts the following technical solutions:

[0008] The mutant NhaM-S67T has a gene nucleic acid sequence of SEQ ID NO:1.

[0009] The protein coded by the mutant NhaM-S67T gene has an amino acid sequence of SEQ ID NO:2.

[0010] The salt-tolerant gene UMP coded protein does not belong to any known protein family, but it retains necessary conservative charged or polar residues and aromatic residues, mainly W9-E15-H43-H80, according to the four amino acid residues, we previously tentatively named it as WEHH.

[0011] Structural analysis shows that the reverse transporter WEHH is the smallest one among the known Na+ / H+ reverse transporters, therefore, it is most appropriate to represent the smallest Na+ / H+ reverse transporter as NhaM, and WEHH is renamed as NhaM, and NhaM and its homologues constitute a new transporter family, which is named as NhaM family.

[0012] NhaM is a novel membrane protein which is so simple but can perform Na+ / H+ reverse transport. The functional analysis of important residues related to structure or function will reveal new structure-function relationship of Na+ / H+ reverse transporter. The obtained results related to molecular transport mechanism can also guide us to perform molecular modification on the protein to improve the activity of transporting Na+, Li+, K+ and other cations, and to improve the potential of the gene for constructing high-efficiency salt-tolerant genetic engineering strains. Therefore, the research on the protein mutant has important research significance and potential application value.

[0013] S67 (serine (S) at position 67) is highly conserved among NhaM and 27 homologues. This indicates that this site can be involved in the function of NhaM. The residue serine (S) at position 67 was mutated to threonine (T) or other corresponding charged or polar residues by site-directed mutagenesis using pET22b-NhaM as template. To determine the functional or structural role of this amino acid residue, the inventors performed:

[0014] Functional detection of the protein encoded by the mutant NhaM-S67T gene:

[0015] 1) The residue serine (S) at position 67 was mutated to threonine (T) by site-directed mutagenesis using pET22b-NhaM as template.

[0016] 2) The mutant was expressed in E. coli KNabc in LBK medium at pH 7.0 containing 0.2 M NaCl or 5 mM LiCl, or pH 8.0 + 50 mM NaCl, with wild-type NhaM as positive control and empty vector pET22b as negative control.

[0017] 3) The expression of the mutant was detected by western blot.

[0018] 4) The optimal Na+ / H+, Li+ / H+ and K+ / H+ antiport activities were determined using E. coli KNabc expressing each mutant at pH 9.0, with wild-type NhaM as positive control.

[0019] The tests showed that the expression of NhaM-S67T made the salt-sensitive deficient strain KNabc of E. coli resistant to 0.2 M NaCl or 5 mM LiCl, and to alkaline pH 8.0.

[0020] The protein encoded by the mutant NhaM-S67T gene has Na+ or Li+ or K+ / H+ antiport activity in the fluorescence quenching experiment, and the Na+ or Li+ or K+ / H+ antiport activity has pH dependence.

[0021] The protein having Na+ or Li+ or K+ / H+ antiport activity expressed by the mutant NhaM-S67T gene is utilized.

[0022] Further functional verification showed that the mutant protein NhaM-S67T has Na+(Li+, K+) / H+ antiport activity in the fluorescence quenching experiment, and the Na+(Li+, K+) / H+ antiport activity has pH dependence.

[0023] The mutant gene of NhaM-S67T and the protein thereof have important significance for saline-alkali soil improvement.

[0024] The salt-tolerant and alkali-tolerant engineering strain prepared by the mutant gene expression of NhaM-S67T.

[0025] The microbial fertilizer prepared by the mutant gene expression of NhaM-S67T.

[0026] The transgenic plant with salt-tolerant and alkali-tolerant prepared by the mutant gene expression of NhaM-S67T.

[0027] Therefore, the mutant gene of NhaM-S67T and the protein thereof have important significance for saline-alkali soil improvement.

[0028] 1. The important function of the mutant gene of NhaM-S67T and the protein thereof is determined, and S67 is highly conserved between NhaM and homologues, which indicates that S67 may be involved in the function of NhaM.

[0029] 2. Further functional verification shows that the mutant protein of NhaM-S67T has Na+(Li+, K+) / H+ antiporter activity in the fluorescence quenching experiment, and the Na+(Li+, K+) / H+ antiporter activity has pH dependence.

[0030] 3. The mutant gene of NhaM-S67T and the protein thereof have important significance for saline-alkali soil improvement, and can be used in the fields of salt-tolerant and alkali-tolerant gene modification, construction of high-efficiency salt-tolerant and alkali-tolerant engineering strains, development of microbial fertilizers and improvement of salt-tolerant and alkali-tolerant transgenic plants. BRIEF DESCRIPTION OF DRAWINGS

[0031] Figure 1 is a growth curve diagram of the protein encoded by the mutant NhaM-S67T under the stress condition of 0.2M NaCl.

[0032] Figure 2 is a growth curve diagram of the protein encoded by the mutant NhaM-S67T under the stress condition of 5mM LiCl.

[0033] Figure 3 is a growth curve diagram of the protein encoded by the mutant NhaM-S67T under the stress condition of pH8.0+50mM NaCl.

[0034] Figure 4This is a schematic diagram of NhaM homology comparison provided in an embodiment of the present invention.

[0035] Figure 5 This is a detection map of the Na+(Li+,K+) / H+ reverse transporter activity of the protein encoded by the mutant NhaM-S67T described in this embodiment of the invention, with wild-type NhaM as a positive control.

[0036] Figure 6 This is a Western blot image of the protein encoded by the mutant NhaM-S67T described in this embodiment of the invention, with wild-type NhaM as a positive control. Detailed Implementation

[0037] 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.

[0038] The mutant NhaM-S67T provided in this embodiment of the invention has the following gene nucleic acid sequence: SEQ ID NO: 1.

[0039] The protein encoded by the mutant NhaM-S67T provided in this embodiment of the invention has the amino acid sequence SEQ ID NO: 2.

[0040] This invention investigated the function of the protein encoded by the mutant NhaM-S67T. The results showed that the mutant NhaM-S67T exhibited significant resistance to 0.2M NaCl. Expression of the NhaM-S67T amino acid profile enabled *E. coli* KNabc to tolerate 0.2M NaCl or 5mM LiCl, and also to tolerate an alkaline pH of 8.0. Furthermore, the mutant protein was demonstrated to possess Na+(Li+,K+) / H+ antitransporter activity.

[0041] The detection and application principles of the present invention will be further explained below with reference to specific embodiments:

[0042] 1. Amino acid site selection and mutation

[0043] like Figure 4 As shown, S67 is highly conserved between NhaM and its homologs.

[0044] Based on homology alignment with NhaM, one amino acid, S67, was selected from highly conserved amino acid sites. Based on this selected site, site-directed mutagenesis primers were designed (as shown in Table 1).

[0045] Table 1: Primers for site-directed amino acid mutagenesis

[0046]

[0047] Site-directed mutagenesis of amino acids was performed according to the following steps:

[0048] (1) PCR for site-directed mutagenesis, 50 μL system as shown in Table 2:

[0049] Table 2: PCR reaction system and conditions

[0050]

[0051]

[0052] (2) Agarose gel electrophoresis detection

[0053] Take 2 μL of the above PCR product, 0.7% agarose gel electrophoresis electrophoresis detection.

[0054] (3) Digestion of PCR product

[0055] Dilute 10 U / μL DMTase 10 times, and the digestion reaction system and conditions are shown in Table 3:

[0056] Table 3: DMTase dilution and digestion system

[0057]

[0058] (4) Transformation

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

[0060] 2) Put into a preheated 42°C water bath for 90 s, and quickly take out and place on ice for 10 min.

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

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

[0063] (5) Positive clone plasmid, send to Huada for sequencing. After the sequencing results are obtained, use DNAMAN6.0 software to compare whether the mutation is successful, and if the mutation is successful, proceed to the next step of function verification.

[0064] 2. Physiological experiment of mutant resistance

[0065] Each mutant was subjected to physiological experiment together with negative control to identify the effect of mutation at different sites on salt-tolerant function.

[0066] (1) Respectively activate (E. coli) KNabc containing point mutation plasmid and negative control pET22b and positive control strain KNabc / pET22b-NhaM, when OD600 value is 1.0, inoculate into corresponding medium at 1% inoculation amount for testing.

[0067] (2) Na+ resistance experiment: inoculate the above culture into LBK medium containing different concentrations of NaCl, measure OD600 value after 24h culture, and the result is shown in Table 2. Figure 1

[0068] (3) Li+ resistance experiment: inoculate the above culture into LBK medium containing different concentrations of LiCl, measure OD600 value after 24h culture, and the result is shown in Table 3. Figure 2

[0069] (4) Alkali resistance experiment: inoculate the above culture into 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 50mM NaCl, measure OD600 value after 24h culture, and the result is shown in Table 4. Figure 3

[0070] 3. Analysis of function of point mutation protein: determination of activity of mutant transport protein

[0071] To verify that the physiological results of the above mutants are different from the wild type due to the influence on protein function, the present application determines the activity of mutant transport protein and performs Western Blot detection, and the determination of activity of mutant transport protein is as follows:

[0072] 1) Preparation of inverted membrane

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

[0074] ② Break the cell wall: when the system pressure of high-pressure cell crusher JG-1A reaches 2,000psi, the effect of breaking the cell wall is achieved, and after breaking, centrifuge at 4℃, 8,000rpm for 5min, at this time, pay attention to take the supernatant for ultracentrifugation, condition: 4℃, 100,000xg, centrifuge for 1h.

[0075] ③ Obtain inverted membrane: dissolve the obtained precipitate after ultracentrifugation in an appropriate amount of buffer A in an ice bath, and perform protein concentration determination, and store at low temperature.

[0076] 2) Protein concentration determination: determine the protein concentration in the inverted membrane by Lorry method​​​

[0077] 3) Detection of mutant NhaM-S67T protein activity

[0078] ① 2 μM of fluorescent indicator acridine orange (AO) and 20 μg of inverted membrane were rapidly added into a quartz cuvette containing 2.5 mL of buffer B, mixed, and measured under the set fluorescent monitoring parameters: excitation light (EX) wavelength 492 nm, emission light (EM) wavelength 526 nm.

[0079] ② When the value was stable, Tris-D-lactic acid was added to the system to a final concentration of 5 mM. The lactic acid served as a respiratory substrate and generated a transmembrane pH gradient, i.e., ΔpH.

[0080] ③ When the fluorescence quenching reached a constant value, Na+, K+, Li+ were added to the reaction system to a final concentration of 5 mM, and the fluorescence intensity was continuously monitored.

[0081] ④ The activity of Na+(Li+, K+) / H+ antiporter was estimated according to the change in fluorescence value before and after the addition of ions.

[0082] 4) Determination of the pH tolerance range of mutant NhaM-S67T

[0083] The pH tolerance range can show the pH at which the activity of the transporter is the highest. The activities of Na+(Li+, K+) / H+ transporters of the positive and negative controls and each mutant were determined at pH 7.0-9.0 using Na+, K+, Li+ as substrates.

[0084] The above is shown in Table 1. Figure 5

[0085] 4. Detection of mutant NhaM-S67T protein expression

[0086] To verify whether the mutation affects the expression of the protein, the inverted membranes of the above-prepared NhaM mutants, wild type positive control, and empty vector negative control were loaded and subjected to SDS-PAGE and Western blotting. The specific operation method is as follows:

[0087] (1) 10 μg of membrane protein was mixed with 10 μl of 5×SDS-PAGE loading buffer, and finally subjected to 38°C water bath for 30 min;

[0088] (2) The voltage of the electrophoresis apparatus was set to constant voltage, and first 80 V was used to make the front of bromophenol blue condensed into a line, and then the voltage was adjusted to 120 V;

[0089] ​(3) Transferring membrane: the PVDF membrane is soaked in methanol for 1 min, and after being taken out, the filter paper and the sponge are soaked in 1x transferring membrane liquid, and then the device is arranged in a sandwich manner, and the sequence from bottom to top is sponge -> three layers of filter paper -> glue -> PVDF membrane -> three layers of filter paper -> sponge, and the transferring membrane is performed at 4 DEG C for 40 min at 20 mA;

[0090] (4) Blocking: after the transferring membrane is completed, the protein membrane is placed in the prepared TBST buffer, rinsed for 1-2 min, and the blocking liquid is added, and slowly shaken on a shaker, and blocked at 37 DEG C for 2h;

[0091] (5) Primary antibody incubation: according to the instructions of the primary antibody, the primary antibody is diluted according to the appropriate proportion, and slowly shaken at 4 DEG C overnight;

[0092] (6) The membrane is washed with TBST buffer, and slowly shaken on a side swing shaker for 3 times, each time for 5-10 min; and then washed with TBS buffer for 1 time;

[0093] (7) Secondary antibody incubation: according to the instructions of the secondary antibody, the secondary antibody labeled with horseradish peroxidase (HRP) is diluted according to the appropriate proportion, and the diluted secondary antibody is immediately added, and incubated at 37 DEG C on a shaker for 1h;

[0094] (8) Washing membrane: TBST buffer is used, and the membrane is slowly shaken on a side swing shaker for 3 times, each time for 5-10 min; and then washed with TBS buffer for 1 time;

[0095] (9) Protein detection: according to the related instructions, ECL reagents such as BeyoECL Plus (P0018) are used to detect the protein.

[0096] As shown in Figure 6 , all the mutants have been expressed, proving that the change of physiological phenotype is caused by the change of protein function caused by mutation.

[0097] The present application screens important amino acid sites, and the salt and alkali resistance is improved after mutation, which is very novel in the existing proteins, and the present application is also helpful for understanding the mechanism of the protein and the directed modification of the protein.

[0098] The above only describes the preferred embodiments of the present application, and is not used to limit the present application, and any modification, equivalent replacement and improvement made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. Mutant NhaM-S67T, characterized in that, The amino acid sequence thereof is: SEQ ID NO:

2.

2. A nucleic acid molecule encoding the mutant NhaM-S67T of claim 1, characterized in that, The nucleic acid sequence thereof is: SEQ ID NO:

1.

3. A salt-tolerant and alkali-tolerant engineering strain expressing the gene encoding the mutant NhaM-S67T of claim 1.

4. Use of the mutant NhaM-S67T of claim 1 in the preparation of a salt-tolerant and alkali-tolerant microbial fertilizer.

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