Preparation method and application of beta-aminobutyric acid for improving saline-alkaline tolerance of crops

Abstract

The invention discloses a preparation method and application of beta-aminobutyric acid for improving saline-alkaline tolerance of crops, and relates to the technical field of biochemistry. The invention provides a preparation method of beta-aminobutyric acid, which comprises the following steps: synthesizing a desoxyribonucleic acid sequence SEQ ID NO: 2 for coding transaminase according to an amino acid sequence shown in SEQ ID NO: 1, connecting the sequence shown in SEQ ID NO: 2 with a vector by using ligase, constructing a recombinant vector, introducing the recombinant vector into host bacteria to obtain recombinant bacteria, culturing, adding an inducer, and carrying out freeze-drying to obtain beta-aminobutyric acid. And collecting thalli to obtain the transaminase. The prepared beta-aminobutyric acid can improve the saline-alkali resistance of crops, increase the relative water content in saline-alkali stress plants, improve the lipid peroxidation capacity of the plants and effectively relieve the influence of saline-alkali stress on plant growth.

Description

Technical Field

[0001] The invention relates to the technical field of biochemistry, and in particular to a preparation method of beta-aminobutyric acid for improving the salt-alkali tolerance of crops and application thereof. Background Art

[0002] Saline-alkali soil is a type of soil salinization. High salt and alkali content causes soil compaction and deterioration of soil structure, which seriously affects crop yield and quality. The main ways to improve saline-alkali land are water conservancy measures, physical improvement, chemical improvement, and biological improvement. However, the improvement cycle of saline-alkali land is long and the effect is slow. The preparation of functional substances that can improve the tolerance of crops to saline-alkali soil and enable crops to grow normally under saline-alkali conditions is currently an effective means to increase the cultivated area of ​​crops in saline-alkali soil.

[0003] β-aminobutyric acid (BABA), also known as 3-aminobutyric acid, contains two functional groups, amino and carboxyl, and its chemical formula is C4H9NO2. β-aminobutyric acid has two isomers due to the different positions of the functional groups, namely α-aminobutyric acid (AABA) and γ-aminobutyric acid (GABA). According to the difference in optical rotation, β-aminobutyric acid also has two enantiomers, R-β-aminobutyric acid and S-β-aminobutyric acid.

[0004] β-Aminobutyric acid can be absorbed and transported by plants and has a protective effect on plants, making them defensive and immune to viruses, bacteria, etc., and resisting oocytes, fungi, nematodes, arthropods, and improving tolerance to some non-biological stimuli, such as water shortage, high temperature, high salt and other stresses.

[0005] The synthesis of β-aminobutyric acid mainly includes chemical method and enzyme catalysis method. The chemical method uses formaldehyde as raw material, obtains tert-butyl 2-butenoate through Horner-Wadsworth-Emmons reaction, and then further uses methyl crotonate to synthesize (R)-3-aminobutyric acid, and uses ammonia and butenoic acid to react under pressure to synthesize β-aminobutyric acid. However, the metal or organic catalysts used in the chemical synthesis method are not environmentally friendly, and protective agents are usually required during the reaction, and the reaction conditions are complicated. The important synthase for the enzyme-catalyzed synthesis of β-aminobutyric acid is aspartase, which has a definite specific structure, does not require auxiliary factors, has high reaction specificity, and has high enzyme activity.

[0006] Currently, there is little research on the synthesis of β-aminobutyric acid, and there is an urgent need to provide a preparation method of β-aminobutyric acid that can effectively improve the salt-alkali resistance of crops. Summary of the invention

[0007] The purpose of the present invention is to provide a method for preparing beta-aminobutyric acid for improving the salt-alkali tolerance of crops.

[0008] To achieve the above object, the technical solution of the present invention is as follows: On the one hand, the present invention provides a method for preparing β-aminobutyric acid, comprising: synthesizing a deoxyribonucleic acid sequence SEQ ID NO: 2 encoding a transaminase according to the amino acid sequence shown in SEQ ID NO: 1, connecting the sequence shown in SEQ ID NO: 2 with a vector using a ligase, constructing a recombinant vector, introducing the recombinant vector into a host bacterium to obtain a recombinant bacterium, culturing, adding an inducer, collecting the bacteria, and obtaining the transaminase.

[0009] Specifically, the ligase is T4 DNA ligase.

[0010] Specifically, the vector is selected from one or more of pET28a, pEZZ18, pTA1529, pUB110, pE194, pMK3, pPIC9, pPIC9K, and pHIL-S1.

[0011] Furthermore, the vector is pET28a.

[0012] Specifically, the host bacteria is the host Escherichia coli BL21.

[0013] Specifically, the culturing is to inoculate the strain into TB medium for culturing; Further, the culture conditions are 35-38°C, 180-220rpm; Furthermore, the culture conditions are 37° C. and 200 rpm.

[0014] Specifically, the culturing is to culture the bacteria until the OD600 value is 4.0.

[0015] Specifically, the inducing agent is IPTG; further, the final concentration of the IPTG is 0.1-1 mM; Furthermore, the final concentration of IPTG is 0.2-0.6 mM.

[0016] Specifically, the induction conditions are 25-30°C and induction for 10-14h; Furthermore, the induction conditions are 28° C. and 12 hours of induction.

[0017] Specifically, after induction, the bacteria were collected by centrifugation at 4°C and 3000-5000 rpm for 10-20 min; Furthermore, the centrifugation conditions are 4°C, 4000 rpm for 15 min.

[0018] Specifically, after collecting the bacterial cells, the step of resuspending the cells with a buffer is also included; Furthermore, the buffer is a phosphate buffer, the pH of the phosphate buffer is 6.0-8.0, and the concentration is 40-60mM; Furthermore, the pH of the phosphate buffer is 7.0 and the concentration is 50 mM.

[0019] Specifically, the preparation method of β-aminobutyric acid includes taking crotonic acid as a substrate, adding MgCl2 and ammonium sulfate, adjusting the pH, and then adding a transaminase enzyme solution to react, thereby obtaining β-aminobutyric acid.

[0020] Furthermore, the mass volume ratio of the crotonic acid to the transaminase enzyme solution is (1-3): (2-5); Furthermore, the mass volume ratio of the crotonic acid to the transaminase enzyme solution is 2:3, 3:5 or 1:2; Furthermore, the mass volume ratio of the crotonic acid to the transaminase enzyme solution is 2:3.

[0021] Furthermore, the final concentration of MgCl2 is 0.5-1 mM; further, the final concentration of MgCl2 is 0.5 mM.

[0022] Furthermore, the reaction temperature is 45-55°C; furthermore, the reaction temperature is 50°C.

[0023] Furthermore, the final concentration of the ammonium sulfate is 80-120 mM; further, the final concentration of the ammonium sulfate is 100 mM.

[0024] Further, the pH of the reaction is 9.0-10.0; further, the pH of the reaction is 9.0.

[0025] In another aspect, the present invention provides β-aminobutyric acid prepared by the above preparation method.

[0026] In yet another aspect, the present invention provides the use of the above-mentioned β-aminobutyric acid in improving the salt-alkali stress tolerance of crops.

[0027] Specifically, the crops include but are not limited to tobacco, cucumber, rice, corn, sorghum, watermelon, soybean, cotton, peanut, and wheat.

[0028] Furthermore, the crop is tobacco or corn.

[0029] The beneficial effects of the present invention are: The invention provides a preparation method of beta-aminobutyric acid. The prepared beta-aminobutyric acid can improve the salt-alkali resistance of crops, increase the relative water content in salt-alkali stress plants, increase the lipid peroxidation capacity, enhance the MDA content in crops, effectively alleviate the influence of salt-alkali stress on plant growth, and improve the germination rate and germination potential of crop seeds. DETAILED DESCRIPTION

[0030] The present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation mode, other embodiments obtained by those skilled in the art without creative work all belong to the protection scope of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional methods, the equipment used are all conventional equipment, and the equipment materials used in each example are the same.

[0031] SEQ ID NO: 1: MNKPQSWEARAETYSLYGFTDMPSLHQRGTVVVTHGEGPYVVDVNGRRYLDANSGLWNMVAGFDHKGLIDAAKAQYERFPGYHAFFGRMSDQTVMLSEKLVEVSPFDSGRVFY TNSGSEANDTMVKMLWYLHAAEGKPQKRKILTRWNAYHGVTAVSASMTGKPYNSVFGLPLPGFVHLTEPHYWRYGEEGETEEQFVARLARELEETIQREGADTIAGFFAEPVM GAGGVIPPAKGYFQAILPILRKYDIPVISDEVICGFGRTGNTWGCVTYDFTPDAIISSKNLTAGFFPMGAVILGPELSKRLETAIEAIEEFPHGFDASGHPVGCAIALKAIDV VMNEGLAENVRRLAPRFEERLKHIAERPNIGEYRGIGFMYALEATKDKASKTPFDGNLSVSERIANTCTDLGLICSPIGQSVVLCPPFILTEAQMDEMFDKLEMALDKVFQEVA SEQ ID NO:2: Example 1 Preparation of β-aminobutyric acid The preparation method of β-aminobutyric acid comprises the following steps: (1) Synthesize the deoxyribonucleic acid sequence SEQ ID NO: 2 encoding the transaminase according to the amino acid sequence shown in SEQ ID NO: 1, connect the pET28a plasmid using a ligase, the restriction sites are composed of BamH I and Xho I, and add the enzyme-linked vector to the host Escherichia coli BL21 competent cells for transformation.

[0032] The strain was inoculated in TB medium and cultured at 37°C, 200 rpm until the OD600 value was 4.0, IPTG with a final concentration of 0.2 mM was added for induction, and the induction was carried out at 28°C for 12 hours. The bacteria were collected by centrifugation at 4°C, 4000 rpm for 15 min.

[0033] The cells were resuspended in phosphate buffer (50 mM, pH 7.0) and disrupted by ultrasound to obtain transaminase enzyme solution.

[0034] (2) In a 100 ml reaction system, add 20 g of crotonic acid, 0.5 mM MgCl2, 100 mM ammonium sulfate, ammonia water to adjust the pH to 9.0, and 30 ml of transaminase solution at 50 °C.

[0035] Example 2 Preparation of β-aminobutyric acid The preparation method of β-aminobutyric acid comprises the following steps: (1) Synthesize the deoxyribonucleic acid sequence SEQ ID NO: 2 encoding the transaminase according to the amino acid sequence shown in SEQ ID NO: 1, link the enzyme to the pET28a plasmid, the restriction sites are composed of BamH I and Xho I, and add the enzyme-linked vector to the host Escherichia coli BL21 competent cells for transformation.

[0036] The strain was inoculated in TB medium and cultured at 37°C, 200 rpm until the OD600 value was 4.0, IPTG with a final concentration of 0.5 mM was added for induction, and the induction was carried out at 28°C for 10 hours. The bacteria were collected by centrifugation at 4°C, 4000 rpm for 15 min.

[0037] The cells were resuspended in phosphate buffer (50 mM, pH 7.0) and disrupted by ultrasound to obtain transaminase enzyme solution.

[0038] (2) In a 100 ml reaction system, add 30 g of crotonic acid, 0.6 mM MgCl2, 110 mM ammonium sulfate, ammonia water to adjust the pH to 10.0, and 50 ml of transaminase solution at 55 °C.

[0039] Example 3 Preparation of β-aminobutyric acid The preparation method of β-aminobutyric acid comprises the following steps: (1) Synthesize the deoxyribonucleic acid sequence SEQ ID NO: 2 encoding the transaminase according to the amino acid sequence shown in SEQ ID NO: 1, link the enzyme to the pET28a plasmid, the restriction sites are composed of BamH I and Xho I, and add the enzyme-linked vector to the host Escherichia coli BL21 competent cells for transformation.

[0040] The strain was inoculated in TB medium and cultured at 37°C, 200 rpm until the OD600 value was 4.0, IPTG with a final concentration of 0.6 mM was added for induction, and the induction was carried out at 28°C for 12 hours. The bacteria were collected by centrifugation at 4°C, 4000 rpm for 15 min.

[0041] The cells were resuspended in phosphate buffer (60 mM, pH 7.0) and disrupted by ultrasound to obtain transaminase enzyme solution.

[0042] (2) In a 100 ml reaction system, add 10 g of crotonic acid, 0.8 mM MgCl2, 100 mM ammonium sulfate, ammonia water to adjust the pH to 9.0, and 20 ml of transaminase solution at 50 °C.

[0043] Comparative Example 1 Compared with Example 1, the only difference is that the mass volume ratio of crotonic acid to the transaminase enzyme solution is different, and the rest are the same. In Comparative Example 1, the mass volume ratio of crotonic acid to the transaminase enzyme solution is 1:6.

[0044] Comparative Example 2 Compared with Example 1, the only difference is that magnesium sulfate is used to replace MgCl2, and ammonium chloride is used to replace ammonium sulfate, and the rest is the same.

[0045] Comparative Example 3 Compared with Example 1, the only difference is that the concentrations of MgCl2 and ammonium sulfate in the reaction system are different, and the rest are the same.

[0046] In the 100 ml reaction system of Comparative Example 3, 20 g of crotonic acid, 2 mM MgCl2, 150 mM ammonium sulfate, ammonia water were added at 50°C to adjust the pH to 9.0, and 30 ml of transaminase solution were added.

[0047] Experimental Example 1 Study on the Alkali Resistance of β-Aminobutyric Acid 1 Experimental methods 1.1 Experimental groups and treatments Tobacco seedlings with consistent growth were selected and transplanted into a seedling box containing the same components and dosages, and were divided into 8 groups, namely, a control group, an alkali stress group, an embodiment 1-3 group (respectively, the β-aminobutyric acid prepared by embodiments 1-3) and a comparative example 1-3 group (respectively, the β-aminobutyric acid prepared by comparative examples 1-3), and each group of tobacco seedlings was pretreated at a temperature of 28±2°C and a light of 2500LX for 16 hours per day. The pretreatment operation is shown in Table 1 below, and then the alkali stress group, the embodiment 1-3 group and the comparative example 1-3 group were respectively transferred to a 1 / 2 nutrient solution containing 10mM Na2CO3, and the control group replaced the same nutrient solution. On the 5th day after the alkali stress, samples were taken to measure the relevant physiological indicators of the tobacco seedlings, and the experiment was repeated 3 times.

[0048] Table 1 Treatment conditions

[0049] 1.2 Detection indicators 1.2.1 Relative water content detection (1) Weigh the fresh weight of tobacco leaves and record it as Wf; (2) Place the fresh weight of the tobacco leaves in an oven at 105°C for drying. After drying to a constant weight, weigh the dry weight, which is recorded as Wd. (3) At the same time, place the weighed tobacco leaves in a beaker of double distilled water and soak for 24 hours to allow the tissue to absorb water to saturation. Use filter paper to absorb excess water on the surface of the leaves and weigh them. Repeat the operation several times until a constant weight is reached. The saturated water weight of the tobacco leaves is obtained and recorded as Wt.

[0050] Relative water content (%) = (Wf-Wd) / (Wt-Wd).

[0051] 1.2.2 MDA content detection Take 0.2g of fresh tobacco leaves, add 1mL 10% TCA and grind them. After fully crushing, add 1mL 10% TCA and centrifuge for 10min. Take 1.7mL of supernatant from the experimental group and take an equal amount of 10% TCA from the control group. Then add 1.7mL 0.6% TBA respectively. After fully mixing, place in a water bath at 100℃ for 15min, centrifuge at room temperature, take the supernatant and measure its OD value at wavelengths of 532nm, 600nm and 450nm, and calculate the MDA content according to the following formula.

[0052] Cz=6.45(D 532 -D 600 )-0.56D 450 ; MDA content (μmol / g) = Cz*V / W; Where: Cz---MDA concentration (μmol / L); V---extraction volume (mL); W---Fresh weight of plant tissue (g).

[0053] 2 Experimental Results 2.1 Relative water content Table 2 Relative water content

[0054] As can be seen from Table 2 above, when tobacco was subjected to alkali stress (alkali stress group), its relative water content decreased significantly, by 15.2% relative to the control group. After being treated with the β-aminobutyric acid prepared in Examples 1-3, its relative water content increased significantly, by 17.3%, 13.3% and 14.2% respectively relative to the alkali stress group. However, the β-aminobutyric acid prepared in Comparative Examples 1-3 had a smaller effect on improving the relative water content of tobacco subjected to alkali stress.

[0055] 2.2 MDA content Table 3 MDA content

[0056] Note: Compared with the control group, **p<0.01; compared with the alkaline stress group, #p<0.05, ##p<0.01.

[0057] As can be seen from the table above, compared with the control group, the MDA content of tobacco in the alkali stress group increased significantly (p < 0.01), and after the administration of β-aminobutyric acid prepared in Example 1-3, the MDA content decreased significantly ( p <0.05, p <0.01). However, the effect of the β-aminobutyric acid prepared in Comparative Examples 1-3 on reducing the MDA content in tobacco was not significantly different from that in the alkaline stress group.

[0058] Experimental Example 2 Study on the salt resistance of β-aminobutyric acid 1 Experimental methods 1.1 Experimental groups and treatments Take corn seeds of the same size and shape, disinfect the seed surface with 70% alcohol for 5 minutes, rinse the seed surface repeatedly with distilled water for 3 times, then dry with absorbent paper, soak the seeds in clean water for 24 hours, sterilize the culture dish, put 2 layers of filter paper in the culture dish, divide the corn seeds into 8 groups, namely the control group, salt stress group, Example 1-3 group (respectively β-aminobutyric acid prepared in Example 1-3) and Comparative Example 1-3 group (respectively β-aminobutyric acid prepared in Comparative Example 1-3), add 100mM NaCl to the salt stress group, add 1mM β-aminobutyric acid and 100mM NaCl prepared in Example 1-3 group and Comparative Example 1-3 group, respectively, and add clean water to the control group. 100 seeds are placed in each group, and the seeds are kept moist during germination, the temperature is controlled at 26±2°C, the light time is 12h / 12h, the relative humidity is 60±2%, and the light intensity is 4000lux. The germination standard was a sprout length greater than 2 mm. After the first seed germinated, the seeds were observed for 7 consecutive days, and the number of sprouts was counted at the same time every day. The observation ended when no sprouts occurred for 3 consecutive days.

[0059] 1.2 Detection indicators 1.1 Seed germination rate Seed germination rate (%) = (total number of seeds germinated / total number of tested seeds) × 100%.

[0060] Germination potential (%) = (number of seeds germinated on the second day / total number of seeds tested) × 100%.

[0061] 2 Experimental Results Table 4

[0062] It can be seen from the above table that the seed germination rate and germination potential will be significantly reduced under salt stress. Treatment with the β-aminobutyric acid prepared in Examples 1-3 of the present invention can effectively increase the seed germination rate and germination potential, and improve the salt resistance of corn seeds.

[0063] 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, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing β-aminobutyric acid, characterized in that: The method comprises the following steps: synthesizing a deoxyribonucleic acid sequence SEQ ID NO: 2 encoding a transaminase according to the amino acid sequence shown in SEQ ID NO: 1, connecting the sequence shown in SEQ ID NO: 2 with a vector using a ligase, constructing a recombinant vector, introducing the recombinant vector into a host bacterium to obtain a recombinant bacterium, culturing, adding an inducer, collecting the bacterium, and obtaining the transaminase.

2. The preparation method according to claim 1, characterized in that: The preparation method of β-aminobutyric acid comprises the following steps: Use crotonic acid as a substrate, add MgCl2 and ammonium sulfate, adjust the pH, and then add transaminase solution to react to obtain β-aminobutyric acid.

3. The preparation method according to claim 2, characterized in that: The mass volume ratio of the crotonic acid to the transaminase enzyme solution is (1-3): (2-5).

4. The preparation method according to claim 3, characterized in that: The mass volume ratio of the crotonic acid to the transaminase enzyme solution is 2:3, 3:5 or 1:

2.

5. The preparation method according to claim 2, characterized in that: The final concentration of MgCl2 is 0.5-1 mM.

6. The preparation method according to claim 2, characterized in that: The final concentration of ammonium sulfate is 80-120 mM.

7. The preparation method according to claim 2, characterized in that: The pH of the reaction was 9.0-10.

0.

8. β-aminobutyric acid prepared by the preparation method according to any one of claims 1 to 7.

9. Use of the β-aminobutyric acid according to claim 8 in improving the salt-alkali stress tolerance of crops.

10. The use according to claim 9, characterized in that: The crops include tobacco, cucumber, rice, corn, sorghum, watermelon, soybean, cotton, peanut and wheat.

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