Salt-tolerant active substance separated from metabolite of rhizosphere growth-promoting PANNI and application of salt-tolerant active substance
By isolating and purifying the salt-tolerant active substances of Pan_ST in Panroniacea, the problem of insufficient research on salt tolerance in rice rhizosphere was solved, and a significant enhancement effect of rice growth under salt stress was achieved.
Patent Information
- Application Number
- CN202411069152.7
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-08-06
- Publication Date
- 2025-07-11
AI Technical Summary
In the prior art, there are few studies on the active substances of Panronia in the rice rhizosphere for enhancing salt tolerance, and their specific substances are not clear, resulting in limited growth of saline-alkali crops.
By isolating and purifying the salt-resistant active substances of Pan_ST of Panronia, using Landy culture medium fermentation, ethyl acetate extraction and column chromatography, benzene ring salt-resistant biomass C40H44O18 was prepared, which was used to verify its activity in promoting rice growth during the rice seedling stage hydroponic experiment.
The plant height, root length, fresh weight of rice seedlings under salt stress was significantly improved, and the rice's salt stress tolerance was enhanced.
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Figure CN120289544A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the field of microorganisms, and specifically relates to a salt-tolerant growth-promoting substance produced by Pannonibacter Pan_ST and its application. Background Art
[0002] Soil salinization not only leads to soil compaction and decreased fertility, but also affects the root development of crops, is not conducive to the absorption of nutrients by crops, and causes a decrease in crop yield and quality. It has become one of the global problems affecting crop yields and endangering soil productivity. Providing a healthy food source for the growing global population has become one of the greatest challenges of this century. Therefore, the development of new technologies and products with low energy consumption and low pollution is crucial for the green and sustainable development of agriculture.
[0003] Pannonibacter was first discovered in 2003. It is a novel and independent genus belonging to the Stappia-Roseibium cluster in Alphaproteobacteria. So far, there are only a few existing species in this genus, and their natural habitats are soda lakes and hot springs, indicating that the bacteria in this genus belong to extremophiles and can survive in harsh environments. Currently, the research on the identification and purification of active substances of Pannonibacter mostly focuses on wastewater treatment and the promotion of the absorption of Cr(VI) ions. There is still little research on the identification of active substances of Pannonibacter in the rice rhizosphere. In previous laboratory studies, a strain of Pannonibacter Pan_ST was screened from the rhizosphere of rice plants growing in saline-alkali land. This strain can significantly improve the germination rate of rice seeds and plant growth indexes under salt stress when directly applied or made into seed coatings. However, the substances in the strain that enhance the salt tolerance of rice are still unclear. Developing functional microbial agents that enhance the salt stress tolerance of crops and improve the growth of crops in saline-alkali land using secondary metabolites produced by the strain has strong application prospects. Summary of the Invention
[0004] The purpose of the present invention is to provide a salt-tolerant active substance fermented by Pannonibacter Pan_ST, and to provide a method for extracting and separating the salt-tolerant active substance and its application.
[0005] The strain provided by the present invention has been deposited with the China General Microbiological Culture Collection Center (abbreviation: CGMCC; address: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences; postal code: 100101). The preservation number of Pannonibacter Pan_ST is CGMCC No. 28063.
[0006] To solve the above technical problems, the present invention first provides a benzene-ring type salt-tolerant growth-promoting substance isolated from Pannonibacter Pan_ST, and the structure is as follows:
[0007]
[0008] The salt-tolerant active substance described in the present invention is prepared by the following method: fermenting Pantoea alkaliphila Pan_ST with Landy medium and collecting the supernatant of the fermentation broth. After the supernatant is extracted with ethyl acetate, the ethyl acetate organic phase is collected and the solvent is removed by rotary evaporation. Then, column chromatography is used for separation, and elution separation is carried out successively with a petroleum ether-ethyl acetate mixed system and an ethyl acetate-methanol mixed system. Finally, the salt-tolerant active substance is prepared under the conditions of acetonitrile: water = 35:65, detection wavelength of 278 nm, flow rate of 160 ml / min, and injection volume of 20 ml.
[0009] Each liter of the described Landy medium contains 5 g of L-glutamic acid, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 1 g of potassium dihydrogen phosphate, 0.15 mg of ferrous sulfate heptahydrate, 5 mg of manganese sulfate tetrahydrate, 0.16 mg of copper sulfate heptahydrate, 1 g of yeast powder, 2 mg of L-phenylalanine, and 20 g of glucose. After adjusting the pH value to 7.0, it is sterilized at 115 °C for 30 min.
[0010] The preparation method of the fermentation broth of Pantoea alkaliphila Pan_ST is to transfer the Pan_ST seed liquid to a conical flask containing fresh Landy medium at an inoculation amount of 1%, and culture it at 30 °C and 170 rpm for 2 d; then inoculate the cultured bacterial liquid into a 100 L fermenter filled with 70 L of Landy medium. After the bacterial liquid enters the stationary phase, a large amount of secondary metabolites are produced, and fermentation continues for 3 d.
[0011] The method also includes tracking the bioactive functions of the separated active components, and finally obtaining the salt-tolerant active substance.
[0012] Beneficial effects:
[0013] Through the seedling hydroponic experiment of the present invention, it is verified that the fermentation broth of Pantoea alkaliphila Pan_ST improves the salt tolerance activity of rice, explores the optimal medium and culture conditions for the fermentation of secondary metabolites, and isolates and purifies a benzene ring type salt-tolerant growth-promoting active substance C 40 H 44 O 18 , and the molecular structure of the substance is identified. It is verified that the salt-tolerant active substance can promote the plant height, root length, fresh weight of the above-ground part, and fresh weight of the underground part of rice. Description of the drawings
[0014] Figure 1 For the salt-tolerant growth-promoting activity of the fermentation broth of strain Pan_ST on rice seedlings under salt stress
[0015] A is the effect of the fermentation broth of strain Pan_ST on the growth phenotype of rice seedlings under salt stress
[0016] B is the effect of the fermentation broth of strain Pan_ST on the plant height of rice seedlings under salt stress
[0017] C is the effect of the fermentation broth of strain Pan_ST on the root length of rice seedlings under salt stress
[0018] D is the effect of the fermentation broth of strain Pan_ST on the fresh weight of the above-ground parts of rice seedlings under salt stress
[0019] E is the effect of the fermentation broth of strain Pan_ST on the fresh weight of the underground parts of rice seedlings under salt stress Figure 2 is the salt tolerance and growth-promoting activity of each extract of the fermentation broth of strain Pan_ST on rice seedlings under salt stress
[0020] A is the effect of each extract of the fermentation broth of strain Pan_ST on the growth phenotype of rice seedlings under salt stress
[0021] B is the effect of each extract of the fermentation broth of strain Pan_ST on the plant height of rice seedlings under salt stress
[0022] C is the effect of each extract of the fermentation broth of strain Pan_ST on the root length of rice seedlings under salt stress
[0023] D is the effect of each extract of the fermentation broth of strain Pan_ST on the fresh weight of the above-ground parts of rice seedlings under salt stress
[0024] E is the effect of each extract of the fermentation broth of strain Pan_ST on the fresh weight of the underground parts of rice seedlings under salt stress
[0025] Figure 3 is the salt tolerance and growth-promoting activity of the third segment of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on rice seedlings under salt stress
[0026] A is the effect of the third segment of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the growth phenotype of rice seedlings under salt stress
[0027] B is the effect of the third segment of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the plant height of rice seedlings under salt stress
[0028] C is the effect of the third segment of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the root length of rice seedlings under salt stress
[0029] D is the effect of the third segment of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the fresh weight of the above-ground parts of rice seedlings under salt stress
[0030] Effect of the third fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the fresh weight of the underground part of rice seedlings under salt stress
[0031] Figure 4 For the salt tolerance and growth-promoting activity of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on rice seedlings under salt stress
[0032] A shows the effect of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the growth phenotype of rice seedlings under salt stress
[0033] B shows the effect of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the plant height of rice seedlings under salt stress
[0034] C shows the effect of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the root length of rice seedlings under salt stress
[0035] D shows the effect of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the fresh weight of the aboveground part of rice seedlings under salt stress
[0036] E shows the effect of the 3-2 fraction of the ethyl acetate phase separation of the fermentation broth of strain Pan_ST on the fresh weight of the underground part of rice seedlings under salt stress
[0037] Table 1 shows the analytical results of the hydrogen spectrum, carbon spectrum, 1 H- 1 H COSY spectrum, HSQC spectrum, and HMBC spectrum tests of the salt-tolerant active substance
[0038] Biological sample preservation information
[0039] Pan_ST, classified as Pannonibacter phragmitetus, is preserved in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms. The preservation address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The preservation time is July 28, 2023, and the preservation number is CGMCC No. 28063. Detailed implementation methods
[0040] The present invention discloses an active substance for promoting rice salt tolerance. Those skilled in the art can refer to the content of this article and appropriately modify the parameters to implement it. To enable those skilled in the art to better understand the technical solution of the present invention, the following further detailed description of the present invention is provided in combination with specific implementation cases.
[0041] The tested rice variety is Nanjing 46, a salt-sensitive rice variety.
[0042] Example 1: Verifying the salt tolerance and plant growth promoting activity of the fermentation broth of Pantoea ananatis Pan_ST of the present invention
[0043] The Pan_ST seed liquid was transferred to a conical flask containing fresh Landy medium at an inoculation amount of 1%, and cultured at 30 °C and 170 rpm for 2 days; then the cultured bacterial liquid was inoculated into a 100 L fermenter filled with 70 L of Landy medium. After the bacterial liquid entered the stationary phase, secondary metabolites began to be produced in large quantities, and fermentation continued for 3 days. The cells were removed by centrifugation at 7000 rpm and 4 °C for 10 min, and the supernatant was collected and stored at 4 °C in a refrigerator for later use.
[0044] After surface disinfection and germination promotion of Nanjing 46 rice seeds, seedlings with consistent germination status were selected and transplanted into a hydroponic box containing 1 L of Hoagland nutrient solution. Each liter of the Hoagland nutrient solution contains 0.115 g of ammonium dihydrogen phosphate, 0.242 g of magnesium sulfate heptahydrate, 0.605 g of potassium nitrate, 1.359 g of calcium nitrate tetrahydrate, 0.181 g of calcium chloride dihydrate, iron salts (30.78 mg of disodium ethylenediaminetetraacetate and 27.75 mg of ferrous sulfate heptahydrate), 7.2 mg of boric acid, 0.2 mg of copper chloride dihydrate, 4.5 mg of manganese chloride tetrahydrate, 0.6 mg of zinc chloride, and 0.098 mg of ammonium molybdate monohydrate. The pH value of the Hoagland nutrient solution was 6.0. The nutrient solution was changed every three days, and the hydroponic growth test under salt stress was carried out until the three-leaf and one-heart stage.
[0045] Rice seedlings with consistent growth vigor were selected and their roots were immersed in the supernatant for 24 h. The control treatment was immersion of roots only in Hoagland nutrient solution. The seedlings after root immersion were transplanted into Hoagland nutrient solution containing 120 mM NaCl for salt stress growth test. The nutrient solution was changed every 3 days, and various indexes of rice plants were detected after 1 week of culture.
[0046] The detection indexes included plant height, fresh weight of the above-ground part and the underground part. The plant height was measured with a tape measure, and the fresh weight of the above-ground part and the underground part was measured with an electronic balance.
[0047] Results and analysis
[0048] As Figure 1 shown, Mock Control was the treatment without adding salt and the fermentation broth of the strain, and Salt Control was the treatment with adding salt and without adding the fermentation broth of the strain.
[0049] As Figure 1 shown in Figure A of
[0050] As Figure 1As shown in Figure B, the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings with roots soaked in the fermentation broth of strain Pan_ST is significantly higher than that of the control treatment with salt added but without the addition of the strain fermentation broth.
[0051] As Figure 1 As shown in Figure C, the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings with roots soaked in the fermentation broth of strain Pan_ST is significantly higher than that of the control treatment with salt added but without the addition of the strain fermentation broth.
[0052] As Figure 1 As shown in Figure D, the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The fresh weight of the above-ground part of rice seedlings with roots soaked in the fermentation broth of strain Pan_ST is significantly higher than that of the control treatment with salt added but without the addition of the strain fermentation broth.
[0053] As Figure 1 As shown in Figure E, the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings with roots soaked in the fermentation broth of strain Pan_ST is significantly higher than that of the control treatment with salt added but without the addition of the strain fermentation broth.
[0054] Example 2: Verification of the salt tolerance-promoting activity of different extracts of the strain fermentation broth of the present invention
[0055] The germination of rice seeds and the cultivation of three-leaf-stage seedlings were the same as in Example 1.
[0056] The collection of the strain supernatant was the same as in Example 1.
[0057] Pour the strain supernatant into a separatory funnel, add ethyl acetate and shake well up and down. After standing for 30 minutes, collect the ethyl acetate organic phase; then mix the separated aqueous phase with n-hexane evenly, and collect the n-hexane organic phase after standing for 30 minutes; continue to mix the separated aqueous phase with petroleum ether evenly and stand for 30 minutes, and collect the petroleum ether organic phase; finally, mix the separated aqueous phase with chloroform evenly and stand for 30 minutes, and collect the chloroform organic phase. After that, dry the collected organic phases and aqueous phase by rotary evaporation, and store them in a -80°C refrigerator for later use.
[0058] Select rice seedlings with consistent growth for salt stress growth experiments in a mixed solution of Hoagland solution containing 100 mM NaCl and a dimethyl sulfoxide (DMSO) solution of each component. DMSO was used as the control group. The mixed solution was changed every 3 days, and after culturing for 1 week, various indexes of rice plants were detected. The detection indexes included plant height, fresh weight of the above-ground part and underground part. The plant height was measured with a tape measure, and the fresh weight of the above-ground part and underground part was measured with an electronic balance.
[0059] Results and analysis
[0060] As shown Figure 2 in the figure, Mock Control is the treatment of the extraction phase substance without adding salt and bacterial strain fermentation broth, SaltControl is the treatment of the extraction phase substance with added salt and without adding bacterial strain fermentation broth, and 0.1% DMSO is the treatment with only DMSO solution added.
[0061] As shown Figure 2 in Figure A, adding the ethyl acetate extraction phase substance of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The growth of rice seedlings treated with the ethyl acetate extraction phase of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with added salt and without adding the fermentation broth of the strain, and is better than that of rice seedlings treated with other extraction phase substances.
[0062] As shown Figure 2 in Figure B, adding the ethyl acetate extraction phase substance of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings treated with the ethyl acetate extraction phase of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with added salt and without adding the fermentation broth of the strain, and is longer than that of rice seedlings treated with other extraction phase substances.
[0063] As shown Figure 2 in Figure C, adding the ethyl acetate extraction phase substance of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the ethyl acetate extraction phase of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with added salt and without adding the fermentation broth of the strain, and is longer than that of rice seedlings treated with other extraction phase substances.
[0064] As shown Figure 2 in Figure D, adding the ethyl acetate extraction phase substance of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The fresh weight of the above-ground part of rice seedlings treated with the ethyl acetate extraction phase of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with added salt and without adding the fermentation broth of the strain, and is better than that of rice seedlings treated with other extraction phase substances.
[0065] As shown Figure 2 in Figure E, adding the ethyl acetate extraction phase substance of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The fresh weight of the underground part of rice seedlings treated with the ethyl acetate extraction phase of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with added salt and without adding the fermentation broth of the strain, and is better than that of rice seedlings treated with other extraction phase substances.
[0066] Example 3: Preparation and isolation and purification of the salt-tolerant active substance of the present invention
[0067] The collection of the supernatant of the strain was the same as in Example 1. The supernatant was extracted in the order of ethyl acetate, n-hexane, petroleum ether, and chloroform. Each extraction phase was collected and the solvent was removed by rotary evaporation. The substance in the ethyl acetate extraction phase was taken, and a glass chromatography column of 4.0×70 cm was used. The crude product was mixed with silica gel (100-200 mesh) for column chromatography separation. A mixed system of petroleum ether-ethyl acetate was selected, and the mixing ratio was gradient eluted from 10:1 to 1:1. One bottle was received every 250 ml. After HPLC detection, similar fractions were combined, divided into 5 segments, and the growth-promoting activities on rice development were verified for each segment in turn (the method was referred to Example 2). The third segment had strong activity. The substance in the third segment was taken, and a glass chromatography column of 2.5×60 cm was used. The crude product was mixed with silica gel (100-200 mesh) for column chromatography separation. A mixed system of ethyl acetate-methanol was selected, and the mixing ratio was gradient eluted from 20:1 to 10:1. One bottle was received every 100 ml and detected by HPLC. The HPLC conditions were as follows: mobile phase: acetonitrile: water = 35:65 (V:V), detection wavelength was 278 nm, flow rate was 160 ml / min, and injection volume was 20 ml. Similar fractions were combined and divided into 2 segments again. The 3-2 segment had good growth-promoting activity on rice development (the method was referred to Example 2), and finally the pure product of the active component was obtained. The active component was tested by mass spectrometry and NMR (including two-dimensional NMR structure analysis), and finally the salt-tolerant active substance was obtained. The molecular structure is shown as follows:
[0068]
[0069] The collected components were lyophilized to obtain the active substance powder, and then dissolved in 50 ml of 10% DMSO solvent as the mother liquor of the active substance, and further diluted by gradient to obtain 10 2 、10 4 、10 6 and 10 8 times dilution solutions.
[0070] Results and Analysis
[0071] As Figure 3 shown, Mock Control was the treatment without adding salt and the third segment of the ethyl acetate phase separation of the strain fermentation broth; Salt Control was the treatment with adding salt and without adding the third segment of the ethyl acetate phase separation of the strain fermentation broth; the 10 -2 treatment group, that is, the dilution multiple of the added substance was 10 2 times; the 10 -4 treatment group, that is, the dilution multiple of the added substance was 10 4 times; the 10 -6 treatment group, that is, the dilution multiple of the added substance was 10 6 times, the 10 -8 treatment group, that is, the dilution multiple of the added substance was 108 times
[0072] As Figure 3 shown in Figure A, adding the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The growth of rice seedlings treated with the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with salt added but without the addition of the strain fermentation broth, and is better than that of rice seedlings treated with other extraction fraction substances.
[0073] As Figure 3 shown in Figure B, adding the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings treated with the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST at 10 -6 is significantly better than that of the control treatment with salt added but without the addition of the strain fermentation broth, and is longer than that of rice seedlings treated with other treatments.
[0074] As Figure 3 shown in Figure C, adding the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST at 10 -4 is significantly better than that of the control treatment with salt added but without the addition of the strain fermentation broth, and is longer than that of rice seedlings treated with other treatments.
[0075] As Figure 3 shown in Figure D, adding the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The above-ground fresh weight of rice seedlings treated with the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST at 10 -6 is significantly better than that of the control treatment with salt added but without the addition of the strain fermentation broth, and is better than that of rice seedlings treated with other treatments.
[0076] As Figure 3 shown in Figure E, adding the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The below-ground fresh weight of rice seedlings treated with the third fraction of the ethyl acetate extract of the fermentation broth of strain Pan_ST at 10 -6 is significantly better than that of the control treatment with salt added but without the addition of the strain fermentation broth, and is better than that of rice seedlings treated with other treatments.
[0077] As Figure 4As shown, Mock Control is the treatment of the ethyl acetate phase separation of the 3-2nd paragraph of the fermentation broth without adding salt and strains, and Salt Control is the treatment of the ethyl acetate phase separation of the 3-2nd paragraph of the fermentation broth with adding salt but without adding strains. 10 -2 treatment groups, that is, the dilution factor of the added substance is 10 2 times; 10 -4 treatment groups, that is, the dilution factor of the added substance is 10 4 times; 10 -6 treatment groups, that is, the dilution factor of the added substance is 10 6 times, 10 -8 treatment groups, that is, the dilution factor of the added substance is 10 8 times.
[0078] As Figure 4 shown in Figure A of, the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The growth of rice seedlings treated with the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with salt added but without adding the fermentation broth of the strain, and is better than that of rice seedlings treated with other substances in each extraction phase.
[0079] As Figure 4 shown in Figure B of, the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The plant height of rice seedlings treated with the 10 -4 treatment group of the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with salt added but without adding the fermentation broth of the strain, and is longer than that of rice seedlings treated with other treatments.
[0080] As Figure 4 shown in Figure C of, the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The root length of rice seedlings treated with the 10 -2 treatment group of the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST is significantly better than that of the control treatment with salt added but without adding the fermentation broth of the strain, and is longer than that of rice seedlings treated with other treatments.
[0081] As Figure 4 shown in Figure D of, the substance in the 3-2nd paragraph of the ethyl acetate extraction phase separation of the fermentation broth of strain Pan_ST can significantly improve the hydroponic growth of rice seedlings under salt stress. The 10 -4The above-ground fresh weight of rice seedlings treated in the treatment group was significantly better than that of the control treatment with salt added but without the fermentation broth of the strain, and was better than that of the above-ground fresh weight of rice seedlings in other treatments.
[0082] As Figure 4 shown in Figure E of [], the substance in the 3-2 section separated by ethyl acetate extraction of the fermentation broth of strain Pan_ST can significantly improve the growth of rice seedlings hydroponically under salt stress. Using 10 of the 3-2 section separated by ethyl acetate extraction of the fermentation broth of strain Pan_ST -4 The underground fresh weight of rice seedlings treated in the treatment group was significantly better than that of the control treatment with salt added but without the fermentation broth of the strain, and was better than that of the underground fresh weight of rice seedlings in other treatments.
[0083] As shown in Table 1, through 1H-NMR coupling information and 1H-1H COSY spectra, the couplings between H-1 to H-6 in the structure can be determined. Combining with the HSQC spectrum, the chemical shifts of the carbon spectra of C-1, C-2, C-3, C-4, C-5, and C-6 are found to be δC 100.5, 73.7, 77.1, 70.2, 76.8, and 61.2 respectively. Similarly, the signals and assignments of H-8 to H-9, H-11 to H-12, H-20 to H-21, H-23 to H-24, H-25 to H-30, H-32 to H-33, and H-37 to H-39 can be determined. In the HMBC spectrum, the long-range correlation points of H-1 with C-2 and 7, H-8 / 12 with C-7, 9, and 11, and H-9 / 11 with C-7, 10, and 12 can be used to determine that the chemical shifts of the relevant quaternary carbons C-7 and C-10 in the upper benzene ring structure are δC 157.5 and 130.2 respectively. At the same time, it can be determined that this glucosyl group is attached to C-7. In addition, the HMBC correlation points of H-13 with C-10 and 14 confirm that the above benzene ring is connected by an ester bond between C-13 and C-14. In the left benzene glycoside fragment below, through the long-range correlation points of H-25 with C-22 and 27, H-20 / 24 with C-19, and H-21 / 23 with C-19 and 22, the chemical shifts of the relevant quaternary carbons C-19 and C-22 in the fragment structure are found to be δC 129.6 and 157.8 respectively. At the same time, it can be determined that the 25th position of this glucose is attached to C-22. In addition, the HMBC correlation points of H-18 with C-17 and 19 confirm that the above benzene ring is connected by an ester bond between C-18 and C-17. In the right phenylpropenoate fragment, through the long-range correlation points of H-32 with C-22 and 27, H-33 with C-19, H-35 with C-19 and 22, H-37 with C-19 and 22, and H-40 with C-35 and 36, the chemical shifts of the relevant carbons C-31, C-34, C-35, C-36, and C-40 in the fragment structure are found to be δC 166.5, 134.4, 131.0, 136.1, and 44.8 respectively. At the same time, in the HMBC spectrum, the significant correlation of H-29 with C-31 can confirm that C-30 and C-31 are connected by an ester bond. In the HMBC spectrum, through the long-range correlation points of H-16 with C-14, 15, 17, and 40, and H-40 with C-14, 15, and 16, the structure of the glutaric acid diester fragment in the structure and the chemical shifts of its relevant carbons C-15 and C-16 are found to be δC 75.8 and 43.1 respectively. Finally, through separation, purification, and identification, the molecular formula of the salt-tolerant active substance is determined to be C 40 H 44 O 18 .
[0084] Table 1: Mass spectrometry and NMR analysis results
[0085]
[0086]
Claims
1. A salt-tolerant growth-promoting substance isolated from Alkalibacterium panonicum Pan_ST, characterized in that, The structural formula is as follows:
2. A method for isolating the salt-tolerant growth-promoting substance according to claim 1 from Pantoea ananatis Pan_ST, characterized in that, Collect the supernatant of the fermentation broth of Pantoea alkaliphila Pan_ST with the fermentation preservation number of CGMCC No. 28063. After the supernatant is extracted with ethyl acetate, collect the ethyl acetate organic phase and rotary evaporate to remove the solvent. Then, separate it by silica gel column chromatography, and elute and separate it successively with a mixed system of petroleum ether - ethyl acetate and a mixed system of ethyl acetate - methanol. Finally, prepare the salt - tolerant active substance by HPLC chromatography under the conditions that the mobile phase is acetonitrile: water = 35:65, the detection wavelength is 278 nm, the flow rate is 160 ml / min, and the injection volume is 20 ml.
3. The method according to claim 2, characterized in that The fermentation medium of Pantoea alkaliphila Pan_ST with the fermentation preservation number of CGMCC No. 28063 is Landy medium, and its components are 5 g of L - glutamic acid, 0.5 g of magnesium sulfate heptahydrate, 0.5 g of potassium chloride, 1 g of potassium dihydrogen phosphate, 0.15 mg of ferrous sulfate heptahydrate, 5 mg of manganese sulfate tetrahydrate, 0.16 mg of copper sulfate heptahydrate, 1 g of yeast powder, 2 mg of L - phenylalanine, 20 g of glucose per liter. After adjusting the pH value to 7.0, sterilize it at 115 °C for 30 min.
4. The method according to claim 2, wherein The fermentation method of Pantoea alkaliphila Pan_ST includes: transfer the Pan_ST seed liquid to a conical flask containing fresh Landy medium at an inoculation amount of 1%, culture it at 30 °C and 170 rpm for 2 d; then inoculate the cultured bacterial liquid into a 100 L fermenter filled with 70 L of Landy medium. After the bacterial liquid enters the stationary phase, start to produce a large amount of secondary metabolites and continue to ferment for 3 d.
5. Use of the salt - tolerant growth - promoting substance described in claim 1 in promoting the growth of rice under salt stress.
6. Use of the salt - tolerant growth - promoting substance described in claim 1 in the preparation of a preparation for promoting the growth of rice under salt stress.