Recombinant plant lactobacillus gasseri overexpressing adenosine triphosphate-binding cassette transporter protein gene and application thereof

Abstract

The present invention discloses a recombinant Lactobacillus plantarum that overexpresses an ATP-binding cassette transporter protein gene and its application. This relates to the field of bioengineering technology. The present invention aims to improve the cold-resistant growth ability of Lactobacillus plantarum, increase its ability to produce lactic acid and degrade nitrite, and improve the integrity and permeability of Lactobacillus plantarum cells. Lactiplantibacillus plantarum ), using Lactobacillus plantarum as the starting strain, overexpressing the nucleic acid molecule shown in SEQ ID NO. 1. It is used for fermentation products in the food industry.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of bioengineering, and particularly relates to a recombinant Lactobacillus plantarum overexpressing an ATP-binding cassette transporter protein gene and application thereof. BACKGROUND

[0002] Low-temperature fermented foods have higher quality, softer and more delicious taste, and no odor. However, the microorganisms in the fermentation system are often affected by low-temperature stress, which reduces the fermentation performance of the microorganisms and further affects the product quality. Therefore, the application provides a construction method of a recombinant Lactobacillus plantarum overexpressing an ATP-binding cassette transporter protein gene, which has good cold tolerance in a low-temperature environment and is used for low-temperature fermentation to produce acid and degrade nitrite, so as to improve the flavor and quality of low-temperature fermented foods such as pickled vegetables and shorten the fermentation time.

[0003] In the food industry, many fermented products (such as yogurt, fermented milk, pickles, etc.) need to be stored and transported under low-temperature conditions to prolong the shelf life and inhibit the growth of harmful microorganisms. If the Lactobacillus plantarum has good cold tolerance and can maintain activity at low temperature, it can help maintain the fermentation characteristics, flavor and safety of the product. The cold-tolerant Lactobacillus plantarum can be applied to more fermentation or health food production processes that require low-temperature environments, such as cold-fermented foods, low-temperature preserved probiotic preparations, etc., to expand its industrial application space.

[0004] The accumulation of lactic acid in the fermentation process of Lactobacillus plantarum not only reduces the pH value, but also helps to improve the quality and consistency of fermented foods by increasing the yield of lactic acid. In addition, it can also efficiently produce industrial chemicals such as lactic acid through microbial fermentation, thereby reducing environmental pollution and resource waste, and facilitating industrial application.

[0005] The structure of the cell membrane and cell wall of Lactobacillus plantarum cells is complete, which can ensure the stability of the intracellular environment, make various organelles function normally, and ensure the smooth progress of basic life activities such as metabolism and reproduction. Maintaining the normal physiological function of the cell is crucial for improving the growth and metabolism of Lactobacillus plantarum in a low-temperature environment and exerting its fermentation characteristics and functions. For example, a complete cell membrane can maintain appropriate ion concentration and pH value in the cell, provide suitable conditions for enzyme activity, and ensure the normal progress of processes such as energy metabolism and material synthesis of the cell. There is an urgent need for Lactobacillus plantarum with the above-mentioned capabilities. SUMMARY

[0006] The purpose of the application is to improve the cold-tolerant growth ability of Lactobacillus plantarum, increase the ability to produce lactic acid and degrade nitrite, and improve the integrity and permeability of Lactobacillus plantarum cells.

[0007] The application provides a recombinant Lactobacillus plantarum (Lactiplantibacillus plantarum ), the plant Lactobacillus rhamnosus SC-MDJ is used as a starting strain, and the gene shown in SEQ ID NO. 1 is overexpressed; the plant Lactobacillus rhamnosus SC-MDJ is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC NO. 28112, and is preserved on August 7, 2023, and is located at No. 1, Xibaheyi, Beijing, China Institute of Microbiology, Chinese Academy of Sciences.

[0008] The application provides a microbial preparation containing the recombinant plant Lactobacillus rhamnosus SC-MDJ.

[0009] The application provides a cold-resistant Lactobacillus rhamnosus breeding method or a method for improving the cold resistance of Lactobacillus rhamnosus, in which the gene shown in SEQ ID NO. 1 is overexpressed in the plant Lactobacillus rhamnosus SC-MDJ.

[0010] The application provides a breeding method for improving the lactic acid yield of Lactobacillus rhamnosus, in which the gene shown in SEQ ID NO. 1 is overexpressed in the plant Lactobacillus rhamnosus SC-MDJ.

[0011] The application provides a breeding method for improving the nitrite degradation capacity of Lactobacillus rhamnosus, in which the gene shown in SEQ ID NO. 1 is overexpressed in the plant Lactobacillus rhamnosus SC-MDJ.

[0012] The application provides an application of the above-mentioned recombinant plant Lactobacillus rhamnosus in cold-resistant fermentation, lactic acid fermentation or nitrite degradation fermentation.

[0013] Beneficial effects: inoculation of the Lactobacillus rhamnosus SC-MDJ-NC8_RS00340 overexpressing the ATP-binding cassette transporter protein gene can improve the lactic acid yield of the strain by 32.68% and reduce the pH of the fermentation system by 10.24% under low-temperature environmental conditions. L. plantarum SC-MDJ-NC8_RS00340 can improve the OD of the strain to 1.884 under low-temperature environmental conditions, improve the lactic acid yield of the strain by 32.68%, and reduce the pH of the fermentation system to 4.47, reducing the pH of the fermentation system by 10.24%. 600nm The cell membrane integrity and the fluorescence intensity are inversely proportional, the fluorescence intensity is reduced to 135.35, and the cell membrane integrity is improved by 74.39% compared with the control group; and the cell membrane permeability and the conductivity are proportional, the conductivity is improved to 65.10%, and the cell membrane permeability is improved by 181% compared with the control group.

[0014] Inoculation of the Lactobacillus rhamnosus SC-MDJ-NC8_RS00340 overexpressing the ATP-binding cassette transporter protein gene can improve the lactic acid yield of the strain by 32.68% and reduce the pH of the fermentation system by 10.24% under low-temperature environmental conditions. L. plantarum SC-MDJ-NC8_RS00340 can improve the OD of the strain to 1.884 under low-temperature environmental conditions, improve the lactic acid yield of the strain by 32.68%, and reduce the pH of the fermentation system to 4.47, reducing the pH of the fermentation system by 10.24%.

[0015] Lactiplantibacillus plantarum overexpressing the ATP-binding cassette transporter protein gene L. plantarum The fermentation of SC-MDJ-NC8_RS00340 under 15 °C environmental conditions can increase the lactic acid content in the fermentation system to 11.50 mg / mL, which is 39.73% higher than the control group. The increase of lactic acid content can increase the unique sour taste of food, thereby helping to promote the formation of good flavor of food.

[0016]

Biological preservation information

[0017] Figure 1 The construction process of the ATP-binding cassette transporter protein gene overexpression plasmid;

[0018] Figure 2 Lactiplantibacillus plantarum overexpressing the ATP-binding cassette transporter protein gene L. plantarum PCR identification results of

[0019] Figure 3 The NC8_RS00340 gene mRNA relative expression level result graph. DETAILED DESCRIPTION

[0020] MRS broth medium: proteose peptone 10.0 g, beef infusion powder 8.0 g, yeast extract powder 4.0 g, glucose 20.0 g, potassium phosphate dibasic 2.0 g, ammonium citrate dibasic 2.0 g, sodium acetate 5.0 g, magnesium sulfate 0.2 g, manganese sulfate 0.04 g, Tween 80 1.0 g, distilled water 1000 mL, pH = 5.7 ± 0.2.

[0021] LB broth medium: tryptone 10.0 g, yeast extract powder 5.0 g, sodium chloride 10.0 g, distilled water 1000 mL, pH = 7.0 ± 0.1.

[0022] Example 1. Method for constructing recombinant Lactiplantibacillus plantarum

[0023] (1) Extraction of genomic DNA and acquisition of ATP-binding cassette transporter protein gene

[0024] Lactiplantibacillus plantarum (L. plantarum) is a Gram-positive, rod-shaped, catalase-positive, and endospore-forming lactic acid bacteria. It is a member of the family Lactiplantibacillaceae and is widely distributed in nature. It is a common food spoilage bacteria and is also used in food fermentation. It is a kind of bacteria that can produce lactic acid and has a wide range of applications in food industry. Lactiplantibacillus plantarum) SC-MDJ was inoculated into MRS broth medium at an inoculation amount of 2%, cultured at 30 °C, 200 rpm to the logarithmic growth phase and subcultured twice, and then strain genomic DNA was extracted using a bacterial genomic DNA extraction kit. Primers were designed according to the reference gene L. plantarum sequences in the National Center for Biotechnology Information (NCBI) database NC8_RS00340 PCR amplification. After the PCR product was detected by 1% agarose gel electrophoresis, the target gene fragment was cut off, the target gene fragment was recovered using a centrifugal column type ordinary agarose gel DNA recovery kit, and was stored in a -20 °C refrigerator for use. Adenine triphosphate binding cassette transporter protein gene (apt) NC8_RS00340

[0025] Table 1 Primers and sequences used

[0026]

[0027] Table 2 PCR amplification reaction system

[0028]

[0029] Table 3 PCR amplification reaction program

[0030]

[0031] (2) Plasmid extraction and linearization

[0032] E. coli containing pMG36e plasmid was inoculated into LB broth containing a final concentration of 600 pg / mL erythromycin at a 2% inoculation amount and cultured at 37 °C, 200 rpm until the logarithmic growth phase and subcultured twice, and then the plasmid was extracted using a plasmid mini extraction kit. The pMG36e plasmid was double-digested using restriction endonucleases I and III, and the digestion reaction was performed at 37 °C for 20 min. After the reaction, 1% agarose gel electrophoresis was performed for detection, and the target fragment was recovered using a centrifugal column type ordinary agarose gel DNA recovery kit and stored at -20 °C for use. The double digestion reaction system is shown in Table 4. Escherichia coli Xba I and Hind III restriction endonucleases, and the digestion reaction was performed at 37 °C for 20 min. After the reaction, 1% agarose gel electrophoresis was performed for detection, and the target fragment was recovered using a centrifugal column type ordinary agarose gel DNA recovery kit and stored at -20 °C for use. The double digestion reaction system is shown in Table 4.

[0033] Table 4 Double digestion reaction system

[0034]

[0035] (3) Connection of target gene and plasmid and transformation

[0036] The purified target gene and linearized pMG36e plasmid were connected using a one-step cloning kit, and the reaction was performed at 37 °C for 30 min, after which the reaction was immediately placed on ice. Then, the DH5a competent cells were placed on ice for rehydration, 100 uL of the competent cells were gently mixed with 10 uL of the plasmid and placed on ice for 30 min, and the connected plasmid was transformed into the DH5a competent cells using a heat shock transformation method, and finally plated on LB agar medium containing 600 pg / mL erythromycin, and after the plate grew colonies, the transformant selection and verification were performed. E. coli E. coli (3) Connection of target gene and plasmid and transformation

[0037] (4) Selection and verification of E. coli transformants

[0038] ​​After picking individual colonies with a sterile pipette tip, transformants were verified by PCR using primers pMG36e-F and pMG36e-R. Correctly identified colonies were inoculated into LB broth containing a final concentration of 600 µg / mL erythromycin and cultured at 37°C, 200 rpm, until the logarithmic growth phase. The bacterial suspension was aspirated and washed twice with sterile saline (0.85% NaCl, w / v) before DNA sequencing. The E. coli transformant suspension was stored in a glycerol freezer at -80°C, completing the construction of the recombinant plasmid pMG36e-NC8_RS00340. The PCR verification reaction system for transformants is shown in Table 5, and the amplification reaction procedure is shown in Table 6.

[0039] Table 5 Transformant PCR verification reaction system

[0040]

[0041] Table 6 PCR amplification reaction program

[0042]

[0043] (5) Extraction and transformation of recombinant plasmid

[0044] The recombinant plasmid pMG36e-NC8_RS00340 was E. coli DH5α strain was inoculated into LB broth with a final concentration of 600 μg / mL erythromycin at a 2% inoculum volume and cultured at 37 °C, 200 rpm until the logarithmic growth phase. The recombinant plasmid was extracted using a plasmid extraction kit. 10 μL of plasmid was mixed with 100 μL of L. plantarum Gently mix the SC-MDJ competent cells and incubate on ice for 5 minutes. Then, use an electroporator to transform the plasmid into the competent cells. Using a 1 mm gap cuvette, adjust the voltage to 1.25 kV and perform two consecutive electroporations. Immediately add 890 μL of pre-chilled MRS broth and incubate at 30°C for 2.5 hours. After completion, concentrate the culture solution by centrifugation and spread it on MRS agar containing 100 μg / mL erythromycin. Incubate at 30°C until colonies emerge for screening and verification.

[0045] (6) Screening and verification of gene overexpression strains

[0046] The single colony was inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin using a sterile gun head, cultured at 30 °C, 200 rpm to the logarithmic growth phase, and then the bacterial liquid was aspirated and the bacterial cells were washed twice with sterile normal saline (0.85% NaCl, w / v). PCR verification was performed using pMG36e-F and pMG36e-R primers. The correct verification bacterial liquid was inoculated again into MRS broth containing a final concentration of 10 μg / mL erythromycin, cultured at 30 °C, 200 rpm to the logarithmic growth phase, and then the bacterial liquid was preserved in a -80 °C refrigerator by a glycerol preservation method. The overexpression of the ATP-binding cassette transporter protein gene L. plantarum was completed and named L. plantarum SC-MDJ-NC8_RS00340. The PCR verification reaction system was the same as Table 5, and the amplification reaction program was the same as Table 6.

[0047] Figure 1 The construction process of the overexpression plasmid of the ATP-binding cassette transporter protein gene. Figure 2 The overexpression of the ATP-binding cassette transporter protein gene L. plantarum The PCR identification results of the overexpression of the ATP-binding cassette transporter protein gene. The successfully constructed gene recombinant plasmid pMG36e-NC8_RS00340 was extracted from E. coli DH5α using a plasmid DNA extraction kit, and transformed into L. plantarum SC-MDJ by an electroporation instrument. Single colonies were picked from erythromycin-resistant MRS agar for PCR verification of the transformants to test whether the recombinant plasmid was successfully transformed into L. plantarum SC-MDJ, thereby obtaining the ATP-binding cassette transporter protein gene overexpression strain. The 1% agarose gel electrophoresis detection results are shown in Figure 2 , and a single bright band was present at about 2505 bp, indicating that the recombinant plasmid pMG36e-NC8_RS00340 was successfully transformed into L. plantarum SC-MDJ, and the ATP-binding cassette transporter protein gene overexpression strain L. plantarum SC-MDJ-NC8_RS00340 was constructed. In addition, L. plantarum the NC8_RS00340 mRNA relative expression of the gene in SC-MDJ-NC8_RS00340 was increased by 26.41 times, indicating that the gene overexpression was successful, and the results are shown in Figure 3 .

[0048] Example 2. Determination method for improving the cold growth ability of L. plantarum SC-MDJ-NC8_RS00340

[0049] (1) Preparation of starter culture

[0050] Overexpression strains L. plantarum SC-MDJ-NC8_RS00340 (experimental group) and control strains L. plantarum SC-MDJ were inoculated into MRS broth containing erythromycin at a final concentration of 10 μg / mL at a 2% inoculum volume, cultured at 30 °C, 200 rpm until the logarithmic growth phase, subcultured twice, and the cultures were collected.

[0051] (2) Establishment of fermentation system

[0052] The culture was inoculated into MRS broth containing erythromycin at a final concentration of 10 µg / mL to inoculate L. plantarum The SC-MDJ fermentation system was used as the control group and inoculated L. plantarum The fermentation system of SC-MDJ-NC8_RS00340 was used as the experimental group and fermented at 15 °C and 200 rpm for 6 days.

[0053] (3) Determination of fermentation culture growth, pH, and cell membrane characteristics

[0054] After the culture was collected, the OD was measured using a spectrophotometer. 600nm To evaluate the growth ability of the strain, the pH of the fermentation broth was measured using a pH meter to evaluate the acid production ability of the strain. The strain (OD 600nm =0.8) and the cell membrane integrity (fluorescence intensity) of the strains were measured using a conductivity meter (representing cell membrane permeability) to evaluate the cell membrane properties.

[0055] Table 7 L. plantarum OD of SC-MDJ-NC8_RS00340 600nm , pH, cell membrane integrity and permeability

[0056]

[0057] Note: Different lowercase letters in the vertical row indicate significant differences among treatments ( P <0.05).

[0058] Table 7 is L. plantarum OD of SC-MDJ-NC8_RS00340 600nm , pH, cell membrane integrity and permeability test results. The test results showed that the cells overexpressing the ATP-binding cassette transporter gene L. plantarum Fermentation of SC-MDJ-NC8_RS00340 under low temperature conditions can increase the OD600nm The total acid content was increased to 1.884, which was increased by 32.68% compared with the control group, and the pH of the fermentation system was reduced to 4.47, which was reduced by 10.24% compared with the control group. In addition, the cell membrane integrity was inversely proportional to the fluorescence intensity, and the fluorescence intensity was reduced to 135.35, and the cell membrane integrity was increased by 74.39% compared with the control group; while the cell membrane permeability was proportional to the conductivity, and the conductivity was increased to 65.10%, and the cell membrane permeability was increased by 181% compared with the control group. Therefore, the above results show that overexpression of the ATP-binding cassette transporter protein gene can significantly improve the ability of SC-MDJ to grow, produce acid and cell membrane characteristics in low temperature environment, which endows the starter with unique physiological functions, provides favorable conditions for the strain to restore normal physiological metabolism, which not only helps to improve the fermentation speed of low-temperature fermented vegetables such as pickled cabbage, but also improves the flavor, quality and safety of the product, and reduces the waste of resources in the food production process. L. plantarum The ability of SC-MDJ to grow, produce acid and cell membrane characteristics in low temperature environment endows the starter with unique physiological functions, provides favorable conditions for the strain to restore normal physiological metabolism, which not only helps to improve the fermentation speed of low-temperature fermented vegetables such as pickled cabbage, but also improves the flavor, quality and safety of the product, and reduces the waste of resources in the food production process.

[0059] Example 3. L. plantarum Determination of the total acid and degradation of nitrite levels of SC-MDJ-NC8_RS00340 fermentation

[0060] L. plantarum Determination of the total acid produced by SC-MDJ-NC8_RS00340 fermentation:

[0061] (1) Establishment of fermentation system

[0062] In the MRS broth containing a final concentration of 10 μg / mL erythromycin, 1 mg / mL of sodium nitrite was added, and the culture medium without inoculation of starter was used as a control. The overexpression strain L. plantarum SC-MDJ-NC8_RS00340 was inoculated with starter group, and the total acid content in the fermentation system was determined after fermentation at 15 °C, 200 rpm for 4 days.

[0063] (2) Determination of total acid content

[0064] 10.0 mL of fermentation broth was taken and diluted to 100 mL with pure water, and the impurities were filtered with gauze. 50 mL of filtrate was collected and titrated with 0.1 mol / L NaOH, while 2 drops of 10 g / L phenolphthalein indicator were added, and the volume of NaOH solution when the solution was titrated to pink color (V1) and the volume of NaOH solution consumed when pure water was used instead of sample solution (V2) were recorded.

[0065] (3) The calculation formula of total acid content (mg / mL) is as follows:

[0066]

[0067] Note: c: concentration of sodium hydroxide standard titration solution, unit: mol / L; V1: volume of sodium hydroxide standard titration solution consumed when titrating the test solution, unit: mL; V2: volume of sodium hydroxide standard titration solution consumed when titrating the blank test, unit: mL; k: conversion coefficient of acid (calculated based on lactic acid, 0.090); F: dilution multiple of the solution; m: mass of the sample, unit: mL; 1000: conversion coefficient.

[0068] L. plantarum Determination of nitrite degradation by SC-MDJ-NC8_RS00340 fermentation:

[0069] (1) Establishment of fermentation system

[0070] In MRS broth containing a final concentration of 10 µg / mL erythromycin, 1 mg / mL of sodium nitrite was added, and the culture medium without inoculation of starter culture was used as a control. The overexpression strain L. plantarum SC-MDJ-NC8_RS00340 was inoculated into the starter culture group, and the fermentation was carried out at 15 °C and 200 rpm for 4 days. The content of sodium nitrite in the fermentation system was determined.

[0071] (2) Determination of nitrite content

[0072] The content of nitrite was determined using a nitrite content determination kit (Gexisi, Suzhou, China).

[0073] (3) The calculation formula of nitrite degradation rate is as follows:

[0074]

[0075] Note: X1 is the content of sodium nitrite in the control medium, unit: mg / mL; X2 is the content of sodium nitrite in the medium inoculated with starter culture, unit: mg / mL.

[0076] Table 8 L. plantarum Total acid and sodium nitrite content in SC-MDJ-NC8_RS00340 fermentation system

[0077]

[0078] Note: Different lowercase letters in the vertical column indicate significant differences between samples P <0.05).

[0079] Table 8 is L. plantarum The results of the determination of total acid and sodium nitrite content in SC-MDJ-NC8_RS00340 fermentation system. The test results show that the overexpression of the adenosine triphosphate-binding cassette transporter protein gene in the inoculated starter cultureL. plantarum SC-MDJ-NC8_RS00340 can increase the total acid content in the fermentation system to 15.70 mg / mL under 15 °C environmental conditions, while the sodium nitrite content can be reduced to 0.013 mg / mL, and the nitrite degradation rate can reach 98.70%. The increase in total acid content can increase the taste and flavor of food, thereby helping to promote the formation of good flavor of food. Therefore, L. plantarum The SC-MDJ-NC8_RS00340 fermenting agent also has a role in improving the flavor of fermented food. Compared with medium-temperature fermentation, it can significantly improve the acid-producing capacity and nitrite degradation capacity of the fermenting agent under lower temperature conditions, not only helping to improve the quality and safety of low-temperature fermented vegetable foods such as pickled cabbage, but also greatly improving the fermentation speed of the product and reducing resource waste in the production process of the product.

[0080] Example 4. L. plantarum Determination of lactic acid production capacity of SC-MDJ-NC8_RS00340

[0081] (1) Preparation of fermenting agent

[0082] The overexpression strain L. plantarum SC-MDJ-NC8_RS00340 and the control strain L. plantarum SC-MDJ was inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin at an inoculation amount of 2%, and cultured at 30 °C, 200 rpm until the logarithmic growth phase and subcultured twice, and the culture was collected.

[0083] (2) Establishment of fermentation system

[0084] The culture was inoculated into MRS broth containing a final concentration of 10 μg / mL erythromycin, and the inoculation amount was L. plantarum The fermentation system of SC-MDJ was used as a control group, and the inoculation amount was L. plantarum The fermentation system of SC-MDJ-NC8_RS00340 was used as the test group, and was fermented at 15 °C, 200 rpm for 6 days.

[0085] (3) Determination of lactic acid production capacity

[0086] After the fermentation broth was collected, the lactic acid content in the fermentation broth was determined using a high-performance liquid chromatograph, and the lactic acid production capacity of the strain under low-temperature conditions was evaluated.

[0087] Table 9 L. plantarum Lactic acid production capacity of SC-MDJ-NC8_RS00340

[0088]

[0089] Note: Different lowercase letters in the vertical column indicate significant differences between treatments P <0.05).

[0090] Table 9 is L. plantarum The results of lactic acid content determination in SC-MDJ-NC8_RS00340 fermentation system showed that the fermentation of SC-MDJ-NC8_RS00340, which was inoculated with the strain overexpressing the gene of ATP-binding cassette transporter protein, could increase the lactic acid content in the fermentation system to 11.50 mg / mL, which was 39.73% higher than that of the control group. L. plantarum The increase of lactic acid content could increase the unique sour taste of food, thereby helping to promote the formation of good flavor of food. Therefore, L. plantarum The SC-MDJ-NC8_RS00340 fermenting agent also had a promoting effect on the flavor of fermented food, which not only helped to improve the quality and safety of low-temperature fermented vegetable food such as pickled Chinese cabbage, but also promoted the fermentation maturity of the product. In addition, since microbial fermentation method could efficiently produce industrial chemical lactic acid, it could also reduce environmental pollution and resource waste, thus being helpful for industrial application.

Claims

1. A recombinant Lactobacillus plantarum ( Lactiplantibacillus plantarum ), characterized in that, Lactobacillus plantarum SC-MDJ was used as the starting strain to overexpress the gene shown in SEQ ID NO.1; the Lactobacillus plantarum SC-MDJ had a deposit number of CGMCC NO.28112 and was deposited in the China General Microorganism Collection Center on August 7, 2023. The deposit address was the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

2. A microbial preparation containing the recombinant Lactobacillus plantarum according to claim 1.

3. A breeding method for improving the cold tolerance of Lactobacillus plantarum, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

4. A breeding method for increasing lactic acid production of Lactobacillus plantarum, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

5. A breeding method for improving the ability of Lactobacillus plantarum to degrade nitrite, characterized in that: The gene shown in SEQ ID NO. 1 was overexpressed in Lactobacillus plantarum SC-MDJ.

6. Use of the recombinant plant lactobacillus according to claim 1 in cold-resistant fermentation, lactic acid-producing fermentation or nitrite-degrading fermentation.

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