Rhizobium polypeptide and application thereof in panax notoginseng planting

Abstract

The invention discloses a rhizobium polypeptide and application thereof in panax notoginseng planting, and belongs to the field of polypeptides and application thereof. According to the invention, three polypeptides are separated and purified from rhizobium, and the amino acid sequences of the polypeptides are Arg-Val-Pro-Ala-Thr-Ser-Arg (RVPATSR), Arg-Gly-Thr-Ala-Pro-Ser-Arg (RGTAPSR) or Arg-Val-Glu-Ala-Thr-Ser-Arg (RVEATSR), and the amino acid sequences of the polypeptides are as shown in the specification. The polypeptide is used for planting pseudo-ginseng, the quality of the pseudo-ginseng can be improved, and especially the content of ginsenoside Rg1 and Rb1 and notoginsenoside R1 in the pseudo-ginseng can be improved. When the polypeptide is applied to planting of the pseudo-ginseng, the quality of the pseudo-ginseng can be improved, pollution to the environment is avoided, and a new way is provided for a sustainable pseudo-ginseng planting mode.

Description

Technical Field

[0001] The invention relates to a rhizobium polypeptide and application thereof in Panax notoginseng planting, belonging to the field of polypeptides and applications thereof. Background Art

[0002] Traditional Chinese medicine has a long history in China, and its theoretical system and accumulated practical experience have made it an important part of the global medical field. In recent years, with the recognition of the efficacy of traditional Chinese medicine, the treatment methods of traditional Chinese medicine have attracted widespread attention around the world.

[0003] Panax notoginseng (Panax notoginseng) is a leader in Chinese medicinal materials. Its rhizomes are widely used in the treatment of cardiovascular and cerebrovascular diseases. For this reason, it is particularly important to ensure the quality of Panax notoginseng cultivation and the improvement of its effective ingredients. However, Panax notoginseng cultivation today faces severe challenges. In pursuit of high yields, farmers generally rely on a large amount of fertilizers and pesticides to increase the growth rate of crops and prevent and control pests and diseases. Although the yield of Panax notoginseng has been improved in the short term, it has also caused serious damage to land resources and an imbalance in soil microbial communities, ultimately affecting the medicinal value and overall quality of Panax notoginseng. Therefore, exploring a sustainable ecological planting model has become a top priority. Summary of the invention

[0004] In order to obtain a sustainable Panax notoginseng planting model, one of the objects of the present invention is to provide a rhizobium polypeptide, the amino acid sequence of the rhizobium polypeptide is Arg-Val-Pro-Ala-Thr-Ser-Arg (RVPATSR), Arg-Gly-Thr-Ala-Pro-Ser-Arg (RGTAPSR) or Arg-Val-Glu-Ala-Thr-Ser-Arg (RVEATSR).

[0005] Another object of the present invention is to provide an application of rhizobium polypeptide in Panax notoginseng planting.

[0006] Preferably, the rhizobium polypeptide is prepared into a solution with a mass concentration of 0.3% to 0.9% and sprayed on the soil where Panax notoginseng is planted.

[0007] The rhizobium polypeptide can be obtained by synthesis or extracted from rhizobia.

[0008] Preferably, the method for extracting the rhizobium polypeptide from rhizobia is as follows:

[0009] (1) Inoculate rhizobia into nutrient broth medium and culture until the logarithmic growth phase.

[0010] (2) After the culture is completed, the bacterial liquid is centrifuged to remove the culture medium and retain the bacterial cells, and the bacterial weight is recorded.

[0011] (3) Crush the bacteria.

[0012] (4) Mix the disrupted bacterial suspension with PBS, shake at 4°C for 1 hour, then centrifuge to remove the bacteria and retain the supernatant.

[0013] (5) The supernatant is placed into an ultrafiltration membrane for ultrafiltration to obtain a polypeptide mixture.

[0014] (6) Separating the peptide mixture by reverse phase high performance liquid chromatography to obtain different peptide eluates.

[0015] (7) The purity of different peptide eluates was tested by HPLC to ensure the uniqueness of the peptides with a purity higher than 95%.

[0016] (8) The different peptide eluates obtained are analyzed using mass spectrometry (MS) technology to confirm their molecular weight and amino acid sequence.

[0017] (9) The polypeptides with the amino acid sequences of Arg-Val-Pro-Ala-Thr-Ser-Arg (RVPATSR), Arg-Gly-Thr-Ala-Pro-Ser-Arg (RGTAPSR), and Arg-Val-Glu-Ala-Thr-Ser-Arg (RVEATSR) were freeze-dried for subsequent use.

[0018] Preferably, the pH of the nutrient agar medium in step (1) is 6.8-7.0, and the culture temperature is 28°C.

[0019] Preferably, the centrifugation condition in step (2) is: centrifugation at a rate of 4000 rpm for 10 minutes.

[0020] Preferably, there are two methods for disrupting the bacteria in step (3): the first method is to use a lysis buffer (containing SDS) and gently mix at room temperature for 15 to 30 minutes; the second method is to use ultrasonic treatment.

[0021] Preferably, in step (4), the volume ratio of the bacterial suspension to PBS is 1:1; and the centrifugation conditions are: centrifugation at a rate of 8000 rpm for 10 to 15 minutes.

[0022] Preferably, the molecular weight cutoff of the ultrafiltration membrane in step (5) is 3 kDa.

[0023] Beneficial effects of the present invention

[0024] (1) The application of the rhizobium polypeptide obtained in the present invention to the cultivation of Panax notoginseng can not only improve the quality of Panax notoginseng, but also has no impact on the environment, thus realizing a sustainable development of Panax notoginseng cultivation mode.

[0025] (2) The rhizobium polypeptide of the present invention can also increase the ginsenoside Rg in Panax notoginseng. 1 , Rb 1 and notoginsenoside R 1 The content. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Plant height and stem diameter of Panax notoginseng grown in dilutions of different types and concentrations of rhizobium peptides.

[0027] Figure 2 The number of leaves and single leaf area of ​​Panax notoginseng grown in different types and concentrations of rhizobium peptide dilutions.

[0028] Figure 3 Relative chlorophyll content of different species grown under different concentrations of rhizobium peptide dilutions.

[0029] Figure 4 The concentration of Arg-Val-Pro-Ala-Thr-Ser-Arg (RVPATSR) peptide solution and the active ingredient of Panax notoginseng, ginsenoside Rg 1 , Rb 1 and notoginsenoside R 1 Content correlation analysis. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following embodiments are merely simple examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0031] Unless otherwise specified, the methods used in the examples are conventional methods well known to those skilled in the art;

[0032] Unless otherwise specified, the reagents used in the examples are conventional commercially available reagents.

[0033] Example 1

[0034] The preparation of rhizobium polypeptide specifically comprises the following steps:

[0035] (1) Rhizobium leguminosarum was inoculated into 10 mL of a nutrient agar medium having a pH of 7 and cultured at 28° C. until the logarithmic growth phase to obtain a bacterial solution.

[0036] (2) The bacterial solution obtained in step (1) was centrifuged at 4000 rpm for 10 minutes, the supernatant was removed, and the bacterial cells were retained. The bacterial cell weight was 0.7 g / L.

[0037] (3) The bacterial cells obtained in step (2) are broken by ultrasonication at an ultrasonic power of 80% for a total of 15 minutes to fully release the substances in the bacterial cells.

[0038] (4) The disrupted bacteria were mixed with PBS buffer at a volume ratio of 1:1, shaken at 4°C for 1 hour, and centrifuged at 8000 rpm for 15 minutes. The bacteria were discarded and the supernatant was retained.

[0039] (5) The supernatant obtained in step (4) is ultrafiltered using an ultrafiltration membrane with a molecular cutoff of 3 kDa.

[0040] (6) The ultrafiltrated liquid was subjected to reverse phase high performance liquid chromatography (HPLC). C18 chromatographic column: AngilentEclipse XDB-C18 column (4×250mm, 5μm). Mobile phase configuration: aqueous phase (use deionized water to ensure that the water quality is ultrapure water during the preparation process to reduce background noise); organic phase (the configured acetonitrile is chromatographic grade, ensuring that its purity is higher than 99.9%); HPLC operation settings: flow rate (1mL / min) and temperature (25°C); gradient program: 0-5 minutes to maintain 100% water; 5-15 minutes to gradually increase acetonitrile to 25%; 15-25 minutes to maintain 25% acetonitrile; the last 5 minutes to restore to 100% water to elute the impurities that are not separated. Monitoring and data acquisition: absorbance monitoring (use UV detector and set at 308nm wavelength, calibrate the instrument in advance to ensure signal stability). Collect different fractionation tubes (3mL per fractionation tube), and collect a total of 3 tubes of different peptides. Detection of polypeptide purity: The collected polypeptide samples in each tube were subjected to HPLC detection, and the purity of each polypeptide was higher than 95%.

[0041] (7) Mass spectrometry (MS) analysis. C18 chromatographic column: Angilent Eclipse XDB-C18 column (4×250 mm, 5 μm). Mobile phase: Liquid A (ultrapure deionized water + 0.1% formic acid); Liquid B (acetonitrile + 0.1% formic acid). Flow rate, 0.6 mL / min, temperature, 25°C, detection wavelength 308 nm. The time setting of the mobile phase in mass spectrometry analysis refers to the following program: 0-5 minutes, phase A 100%, phase B 0%; 5-15 minutes, phase A 75%, phase B 25%; 15-20 minutes, phase A 50%, phase B 50%; 20-30 minutes, phase A 25%, phase B 75%; 30-35 minutes, phase A 100%, phase B 0%. Mass spectrometry conditions: Resolution is set to FullMS: 40000, ddMS2: 20000.

[0042] Mass spectrometry detection was performed in positive ion mode, covering a mass range of 150-2200 m / z. The resulting liquid chromatography mass spectrometry data were analyzed by DeNovo TMThe software automatically identified and extracted the peptide chain sequence and its molecular weight, and screened out peptides with an average local confidence (ALC) > 90% to ensure the accuracy and reliability of the data. The amino acid sequences of the three peptides were: Arg-Val-Pro-Ala-Thr-Ser-Arg (RVPATSR), Arg-Gly-Thr-Ala-Pro-Ser-Arg (RGTAPSR), Arg-Val-Glu-Ala-Thr-Ser-Arg (RVEATSR), and the peptides were freeze-dried for easy storage and subsequent use.

[0043] Example 2

[0044] The Panax notoginseng variety selected in this experiment was “Miaoxiang Panax notoginseng No. 1”, and the soil for planting Panax notoginseng was red soil without Panax notoginseng planting. The soil pH was 6.02 and the organic matter content was 15.67 g·kg -1 , total nitrogen is 0.0196%, alkaline nitrogen is 54.78 mg kg -1 , total phosphorus is 0.126%, total potassium is 0.85%, and available potassium is 25.33 mg·kg -1 , available phosphorus 0.2330 mg kg -1 The light transmittance of the rain shelter for growing Panax notoginseng is set at about 10%. In the actual production of Panax notoginseng, the planting density of Panax notoginseng is 35,000 trees / hectare, and the fertilizer ratio of nitrogen, phosphorus and potassium is 1:1:2. The fertilizer in this experiment is 225kg·hm2 of nitrogen fertilizer. -2 (Compound fertilizer: N:P:K=32:0:4), phosphate fertilizer 225kg·hm -2 (Potassium dihydrogen phosphate: P 2 O 5 >51.5%、K 2 O>34%) and potassium fertilizer 450kg·hm -2 (Potassium sulfate: K 2 O>51%), and fertilizer was applied four times in February, May, July and August.

[0045] The application of the three polypeptides prepared in Example 1 in the cultivation of Panax notoginseng specifically comprises the following steps:

[0046] (1) The three freeze-dried polypeptide powders obtained in Example 1 were prepared into polypeptide solutions with mass concentrations of 0.3%, 0.6%, 0.9% and 1.2% with sterile distilled water. The obtained polypeptide solutions were divided and stored in a refrigerator at 4°C to avoid polypeptide degradation.

[0047] (2) Three-year-old Panax notoginseng was selected for the experiment and divided into five groups. Group 1 was the control group, in which distilled water was used instead of the polypeptide solution. Groups 2 to 5 were experimental groups. Group 2 was sprayed with a polypeptide solution with a mass concentration of 0.3%, Group 3 was sprayed with a polypeptide solution with a mass concentration of 0.6%, Group 4 was sprayed with a polypeptide solution with a mass concentration of 0.9%, and Group 5 was sprayed with a polypeptide solution with a mass concentration of 1.2%. Ten pots were set for each treatment, and three trees were planted in each pot. After calculation, the fixed application of P in each pot was 1.2%. 2 O 5 :1.8g and K 2 O: 3.6g, nitrogen fertilizer 1.8g. 1 / 4 of the total fertilizer amount is applied each time, that is, P is applied at a fixed rate each time. 2 O 5 :0.45g / pot, K 2 O: 0.9g / pot, nitrogen fertilizer 0.45g / pot. Peptide solution (experimental group) or distilled water (control group) was applied regularly during the seedling and leafing stage, bud stage, flowering stage, fruiting stage, green seed stage and fruit ripening stage of Panax notoginseng. Apply once at each growth stage. Apply 100mL of polypeptide solution (experimental group) or distilled water (control group) to the roots of each Panax notoginseng plant each time to ensure that the plants are evenly covered. During the application process, pay attention to keep the soil moisture at 25%. During the planting process, standardized prevention and control of pests and diseases were carried out.

[0048] Related index detection and result analysis

[0049] During the fruit maturity period of Panax notoginseng, the stem diameter (mm) and plant height of Panax notoginseng treated with different types and concentrations of peptide solutions and Panax notoginseng without peptide application were measured. The results are as follows: Figure 1 As shown in the figure, it can be seen that with the increase of polypeptide liquid concentration, the plant height and stem thickness of Panax notoginseng under different treatments first increased and then decreased, and both had maximum values ​​at 0.6%. The stem thickness and plant height under 0.6% RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) dilution had relatively large values, which increased by 71.7% and 27.2% respectively compared with the RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) control (CK), increased by 6.4% and 16.3% respectively compared with 0.6% RGTAPSR (Arg-Gly-Thr-Ala-Pro-Ser-Arg), and increased by 9.8% and 9.1% respectively compared with 0.6% RVEATSR (Arg-Val-Glu-Ala-Thr-Ser-Arg).

[0050] During the fruit ripening period of Panax notoginseng, the single leaf area and the number of leaves per plant of Panax notoginseng treated with different types and concentrations of peptide solutions and Panax notoginseng without peptide application were measured. The results are as follows: Figure 2 As shown in the figure, it can be seen that:

[0051] There was no significant difference in the number of leaves per plant of Panax notoginseng under different treatments. Under the RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) treatment, with the increase of the dilution concentration of the polypeptide solution, the single leaf area first increased and then decreased. The single leaf area of ​​Panax notoginseng under 0.6% RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) was significantly higher than that of other treatments.

[0052] During the budding and flowering stages of Panax notoginseng, the net photosynthetic rate of the gas exchange parameters of the leaves of each treatment was measured using a Li-6400XT photosynthesis-fluorescence recorder (Li-Cor, USA) (the instrument was equipped with a 6400-40 chlorophyll fluorescence leaf chamber and a 6400-10 CO 2 Injection system), use open air path, air flow rate 300ml min -1 , temperature 25℃, relative humidity 65%±5%. The results of net photosynthetic rate determination are shown in Table 1. It can be seen from the table that the net photosynthetic rate of Panax notoginseng treated with the same type of rhizobium peptides all reached the maximum value at a concentration of 0.6%. The net photosynthetic rate of 0.6% RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) reached the maximum value, which was 19% and 18.5% higher than that of 0.6% RGTAPSR (Arg-Gly-Thr-Ala-Pro-Ser-Arg) and 0.6% RVEATSR (Arg-Val-Glu-Ala-Thr-Ser-Arg), respectively.

[0053] Table 1 Net photosynthetic rate of Panax notoginseng treated with different types and concentrations of rhizobium peptide dilutions

[0054] (μmolCO 2 ·m -2 ·s -1 )

[0055] CK 0.3％ 0.6％ 0.9％ 1.2％ RVPATSR 4.56±0.21Ad 536±0.15Ac 7.57±0.27Aa 6.25±0.73Ab 6.29±0.12Ab RGTAPSR 4.32±0.36Ad 5.31±0.57Ac 6.364±0.67Ba 5.81±0.62Bb 5.92±0.26Bb RVEATSR 4.16±0.37Ac 5.32±0.48Ab 6.394±0.26Ba 6.434±0.39Aa 6.37±0.35Aa

[0056] The results of chlorophyll relative content determination are as follows Figure 3 As shown, from Figure 3It can be seen that except for the relative chlorophyll content (SPAD) of different types of rhizobium peptides at a concentration of 1.2%, there were significant differences among other treatments. The SPAD of Panax notoginseng treated with different types of rhizobium peptides reached the maximum value at a concentration of 0.6%. The SPAD of 0.6% RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) reached the maximum value, which was 12.2% and 6.4% higher than that of 0.6% RGTAPSR (Arg-Gly-Thr-Ala-Pro-Ser-Arg) and 0.6% RVEATSR (Arg-Val-Glu-Ala-Thr-Ser-Arg), respectively.

[0057] Determination of ginsenoside Rg, the active ingredient of Panax notoginseng, with reference to the Chinese Pharmacopoeia (2020) 1 , Rb 1 The concentration of RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) polypeptide solution and the active ingredient of Panax notoginseng, ginsenoside Rg 1 , Rb 1 and notoginsenoside R 1 Content correlation analysis Figure 4 As shown in the figure, it can be seen that the concentration of RVPATSR polypeptide solution is related to ginsenoside Rg 1 , Rb 1 and notoginsenoside R 1 The contents showed a significant curvilinear relationship, and the best RVPATSR polypeptide solution concentration (i.e., the vertex of the fitted parabola) was 0.55%.

[0058] Ginsenoside Rg treated with different types and concentrations of rhizobium peptide dilutions 1 , Rb 1 and notoginsenoside R 1 The changes in the total content are shown in Table 2. It can be seen from Table 2 that the Rg content of Panax notoginseng treated with different types of rhizobium peptides, RVPATSR (Arg-Val-Pro-Ala-Thr-Ser-Arg) and RGTAPSR (Arg-Gly-Thr-Ala-Pro-Ser-Arg) 1 +Rb 1 +R 1 The total amount reached the maximum value at 0.6% concentration. 1 +Rb 1 +R 1 The total amount reached the maximum value, which was increased by 38.8% compared with 0.6% RGTAPSR (Arg-Gly-Thr-Ala-Pro-Ser-Arg).

[0059] Table 2 Ginsenoside Rg treated with different types and concentrations of rhizobium peptide dilutions 1 , Rb 1 and notoginsenoside R 1 Content Total content (%)

[0060] CK 0.3％ 0.6％ 0.9％ 1.2％ RVPATSR 5.12±0.31Ac 6.54±1.18Ab 7.58±0.57Aa 6.05±0.63Ab 5.21±0.49Ac RGTAPSR 5.32±0.43Aa 5.24±0.26Ba 5.46±0.54Ba 5.05±0.17Ca 5.11±0.38Aa RVEATSR 5.93±0.28Ab 6.14±0.21Aa 5.25±0.43Bc 5.83±0.25Bb 5.28±0.15Ac

[0061] In summary, the three rhizobium polypeptides described in the present invention can improve the quality of Panax notoginseng, including the plant height, stem thickness, single leaf area, net photosynthetic rate and relative chlorophyll content of Panax notoginseng, and the three rhizobium polypeptides can increase the content of ginsenoside Rg in Panax notoginseng. 1 , Rb 1 and notoginsenoside R 1 content.

Claims

1. A rhizobium polypeptide, characterized in that: The amino acid sequence of the rhizobium polypeptide is Arg-Val-Pro-Ala-Thr-Ser-Arg, Arg-Gly-Thr-Ala-Pro-Ser-Arg or Arg-Val-Glu-Ala-Thr-Ser-Arg.

2. Use of the rhizobium polypeptide according to claim 1 in Panax notoginseng cultivation.

3. The use according to claim 2, characterized in that: The rhizobium polypeptide is prepared into a solution with a mass concentration of 0.3% to 1.2% and sprayed into the soil where Panax notoginseng is planted.

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