Application of burkholderia in root system microorganisms in regulation and control of ginsenoside glycosylation

Abstract

The invention discloses an application of burkholderia in root system microorganisms in regulation and control of glycosylation of ginsenoside. The invention provides application of burkholderia or a composition containing the burkholderia in any one of the following applications: 1) regulating and controlling proportions of different glycosylated saponins in ginseng; 2) increasing the proportion of monoglycosylated saponin and / or the proportion of disglycosylated saponin in ginseng; 3) preparing ginseng with a high monoglycosylated saponin ratio and / or a high disglycosylated saponin ratio; and 4) improving ginseng cultivation. In a greenhouse environment, the roots of ginseng seedlings are inoculated with burkholderia, metabolome data shows that the burkholderia regulates glycosylation formation of ginsenoside, and the proportion of monoglycosylated saponin and / or the proportion of disglycosylated saponin can be increased. Further, transcriptome sequencing data analysis shows that the burkholderia regulates and controls the expression of different glycosyl transferases, so that the diversity of glycosylation of ginsenoside is influenced. The burkholderia provides a new thought for refined improvement of a ginseng cultivation technology.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of Burkholderia in root microorganisms in regulating the glycosylation of ginsenosides. Background Art

[0002] Ginseng is a perennial herbaceous plant of the Araliaceae family and the genus Panax. It is mainly distributed in the mountainous forest areas of Changbai Mountain in Northeast China. Ginseng saponins are the main active ingredients of ginseng. They are a class of glycosylated triterpenoid compounds. In ginseng, they are mainly synthesized through two pathways. One is to use acetyl-CoA as a precursor, and the key rate-limiting enzyme 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) and other enzymes catalyze the synthesis of isopentenyl pyrophosphate (IPP) through the mevalonate pathway (MVA pathway); the other pathway is to use 5-phospho-deoxyxylulose (DXP) as a precursor, and catalyze the synthesis of IPP through the methylerythritol phosphate pathway (MEP pathway). The IPP synthesized by the above two pathways is then subjected to a series of enzymatic reactions to produce protopanaxadiols (PDD), protopanaxadiols (PPT) and low-content oleanolic acid (OA), and then glycosylated by different glycosyltransferases (UDP-glycosyltransferases, UGTs), ultimately forming ginsenosides with different glycosylation.

[0003] The higher the degree of glycosylation in ginseng plants, the easier it is to accumulate. For example, PPD-type 4-glycosylated saponins Rb1 and Rc, and PPT-type 3-glycosylated saponins Re, etc., are relatively high in ginseng roots. Saponins with low glycosylation, such as PPD-type monoglycosylated saponins CK and Rh2, disaccharide saponins Rg3, etc., and PPT-type monoglycosylated saponins F1 and Rh1, disaccharide saponins Rf and Rg1, etc., are very low in ginseng roots. However, in the human body, polyglycosylated ginsenosides cannot enter the blood directly. Most of them need to be decomposed by intestinal microorganisms before they can be absorbed, and their bioavailability is relatively low. Monoglycosylated and disaccharide saponins are easy to enter the blood and have relatively high bioavailability. Ginseng has a wide range of effects, and its main active ingredient is ginsenosides. Monoglycosylated saponins are widely used in anti-tumor, etc., and polyglycosylated saponins are mainly used in the prevention and treatment of chronic diseases such as neurodegenerative diseases.

[0004] Therefore, establishing a method to regulate the glycosylation of ginsenosides will help to refine the ginseng cultivation technology. Summary of the invention

[0005] The technical problem solved by the present invention is how to obtain ginseng with increased content of monosaccharide and disaccharide ginsenosides that are easily absorbed by human body.

[0006] In order to solve the above technical problems, the first aspect of the present invention provides the use of Burkholderia or a composition containing the same in any of the following: 1) Regulate the ratio and / or content of different glycosylated saponins in ginseng; 2) increasing the content of monoglycosylated saponins and / or disaccharidated saponins in ginseng; 3) Increase the proportion of monoglycosylated saponins and / or disaccharidated saponins in ginseng; 4) preparing ginseng with a high monoglycosylated saponin ratio and / or a high diglycosylated saponin ratio; 5) preparing ginseng with high monoglycosylated saponin content and / or high diglycosylated saponin content; 6) Improve ginseng cultivation.

[0007] In the above application, the monosaccharide saponin is PPD type CK, PPD type Rh2, PPT type F1, and / or PPT type Rh1.

[0008] In the above application, the disaccharidized saponin is PPD type Rg3, PPT type Rf and / or PPT type Rg1.

[0009] In the above application, the monosaccharide saponin ratio is the mass ratio of monosaccharide saponins to total ginseng saponins.

[0010] In the above application, the ratio of disaccharidized saponins is the mass ratio of disaccharidized saponins to total ginseng saponins.

[0011] Furthermore, the total ginsenosides are composed of CK, Rh2, F1, Rh1, Rg3, Rf, Rg1, Re, Ro, Rb1 and Rc.

[0012] In the above application, the cultivation of ginseng is improved to obtain ginseng having a high ratio of monoglycosylated saponins and / or a high ratio of disaccharidated saponins.

[0013] In the above application, the composition is a culture obtained by culturing the Burkholderia in a microbial culture medium.

[0014] Specifically, the composition can be prepared by culturing Burkholderia in Burkholderia culture solution (each liter of culture solution contains 10 g of peptone, 3 g of beef powder, 5 g of sodium chloride, and the balance is distilled water, pH=7.0) (30°C, 200 rpm, 16 hours) to obtain Burkholderia culture solution.

[0015] In a second aspect, the present invention provides a method for regulating the ratio or content of different glycosylated saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same to achieve regulation of the ratio or content of different glycosylated saponins in ginseng.

[0016] In the above, the regulation of the ratio of different glycosylated saponins in ginseng is to increase the ratio or content of monoglycosylated saponins and / or the ratio or content of disaccharidized saponins in ginseng.

[0017] In a third aspect, the present invention provides a method for increasing the proportion of monoglycosylated saponins and / or disaccharidized saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, thereby increasing the proportion of monoglycosylated saponins and / or disaccharidized saponins in ginseng.

[0018] In a fourth aspect, the present invention provides a method for increasing the content of monoglycosylated saponins and / or disaccharidized saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, thereby increasing the content of monoglycosylated saponins and / or disaccharidized saponins in ginseng.

[0019] In a fifth aspect, the present invention provides a method for preparing ginseng with a high ratio of monoglycosylated saponins and / or a high ratio of disaccharidized saponins, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, and culturing to obtain the target ginseng with a high ratio of monoglycosylated saponins and / or a high ratio of disaccharidized saponins.

[0020] In a sixth aspect, the present invention provides a method for preparing ginseng with a high content of monoglycosylated saponins and / or a high content of disaccharidized saponins, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, and culturing to obtain the target ginseng with a high content of monoglycosylated saponins and / or a high content of disaccharidized saponins.

[0021] In the above method, the contacting is watering the ginseng seedlings with the composition containing the same.

[0022] In an embodiment of the present invention, watering ginseng with a composition containing the same, specifically watering ginseng seedlings with a culture of Burkholderia spp., can inoculate each ginseng seedling with about 3.2 × 10 9 cfu Burkholderia. Before watering, the Burkholderia culture solution can be diluted with Hoagland culture solution to make its OD600 absorbance value uniformly 0.2 (1.6 × 10 8 cfu / ml).

[0023] In the above, the high monoglycosylated saponin ratio and / or high diglycosylated saponin ratio means that the monoglycosylated saponin ratio and / or high diglycosylated saponin ratio is increased compared to ginseng seedlings not inoculated with Burkholderia or a composition containing the same.

[0024] In the above, the high monoglycosylated saponin content and / or high diglycosylated saponin content means that the monoglycosylated saponin content and / or high diglycosylated saponin content is increased compared to ginseng seedlings not inoculated with Burkholderia or a composition containing the same.

[0025] In the above, Burkholderia in the embodiments of the present invention may be Burkholderia ( Burkholderia cepacia , BNCC337012) as an example.

[0026] The present invention found in the identification of ginseng root microbial system that Burkholderia is one of the most enriched bacteria in ginseng root system. In a greenhouse environment, Burkholderia was inoculated on the roots of ginseng seedlings. Metabolomics data showed that Burkholderia regulated the glycosylation formation of ginsenosides, and could increase the proportion of monoglycosylated saponins and / or disaccharidized saponins. Further analysis of transcriptome sequencing data showed that Burkholderia regulated the expression of different glycosyltransferases, thereby affecting the diversity of glycosylation of ginsenosides. Burkholderia will provide new ideas for the refined improvement of ginseng cultivation technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 Identification of bacterial microorganisms in the soil and roots of cultivated ginseng; A, schematic diagram of bacterial microorganism identification; B, analysis of the bacterial microorganism content in the soil (Soil) and root (Root) at the target classification level.

[0028] Figure 2 Phenotypic analysis of ginseng after inoculation with Rhizobium and Burkholderia; A, schematic diagram of bacterial inoculation; B, growth of ginseng 30 days after inoculation; C, determination of chlorophyll and carotenoid content 30 days after inoculation; D, determination of chlorophyll a / b.

[0029] Figure 3 Determination of saponin content after ginseng inoculation; A, comparison of total ginseng saponin content; B, classification of the number of glycosylation of ginsenosides (1 sugar group is marked as 1G, 2 sugar groups are marked as 2G, and 3 or more sugar groups are marked as nG), and calculation of the proportion in the total saponins; C, heat map analysis of monomer saponin content.

[0030] Figure 4 Expression analysis of glycosyltransferases after ginseng inoculation; A, schematic diagram of transcriptome sequencing 30 days after ginseng inoculation; B, heat map analysis of the expression of glycosyltransferase UGT in ginseng. DETAILED DESCRIPTION

[0031] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0032] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0033] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0034] Chlorophyll content determination in the following examples: After removing the midrib, the fresh ginseng leaves were cut into pieces and extracted using the Solebao chlorophyll extraction kit (BC0990). The entire process was carried out in the dark. Chlorophyll a, chlorophyll b and carotenoids have maximum absorption peaks at 663 nm, 645 nm and 470 nm, respectively. The absorbance values ​​at the corresponding wavelengths were measured using a spectrophotometer, and their contents were calculated according to the kit instructions.

[0035] Example 1: Application of Burkholderia in regulating the glycosylation of different saponins 1. Burkholderia is significantly enriched in ginseng roots The rhizosphere soil (Soil) and root system (Root) of ginseng growing in the forest of Changbai Mountain in Northeast China for 5 years were collected, and the genomic DNA was extracted using the genome extraction kit (DP336) of Tiangen Biochemical Technology Co., Ltd., and 16S amplicon sequencing was performed ( Figure 1 A), sequencing data were analyzed by the company for bacterial microbial species content.

[0036] Sequencing data analysis showed that Rhizobiales and Burkholderiales were significantly enriched in the root system, such as Figure 1 B The arrows point to two types of bacteria.

[0037] Next, the regulation of ginsenoside synthesis by these two bacteria was studied in a greenhouse environment.

[0038] 2. The influence of Burkholderia on the normal growth of ginseng Representative strains of these two targets, Rhizobium leguminosarum ( Rhizobium leguminosarum , CGMCC1.87) and Burkholderia spp. ( Burkholderia cepacia , BNCC337012), were inoculated into the roots of ginseng seedlings grown in the greenhouse ( Figure 2 A).

[0039] The details are as follows: The experimental material grown in the greenhouse is 5-year-old ginseng ( Panax ginsengCA Meyer), ginseng seedlings were transplanted into pots containing sterilized vermiculite and regularly watered with Hoagland culture medium (Coolable, NSP1020), with a temperature and photoperiod of 16 h (light, 23 °C) / 8 h (dark, 19 °C).

[0040] Rhizobium ( Rhizobium leguminosarum , CGMCC 1.87) and Burkholderia spp. ( Burkholderia cepacia , BNCC337012) were purchased from China General Microbiological Culture Collection and Beina Biotechnology, respectively.

[0041] Rhizobium CGMCC 1.87 was cultured in YMA culture medium (containing 0.5 g of potassium hydrogen phosphate, 10 g of mannitol, 1 g of yeast extract, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of sodium chloride per liter of culture medium, and the balance was distilled water, pH = 7.0) (28°C, 200 rpm, 16 hours) to obtain a rhizobium culture medium; Burkholderia BNCC337012 was cultured in a Burkholderia culture solution (containing 10 g of peptone, 3 g of beef powder, 5 g of sodium chloride, and the balance being distilled water per liter of culture solution, pH=7.0) (30°C, 200 rpm, 16 hours) to obtain a Burkholderia culture solution.

[0042] Dilute the culture medium of Rhizobium and Burkholderia with Hoagland culture medium to make the OD600 absorbance value of both of them 0.2 (1.6 × 10 8 cfu / ml) to obtain Rhizobium and Burkholderia bacterial solutions.

[0043] The above-mentioned Rhizobium solution (referred to as Rle group) and Burkholderia solution (referred to as Bce group) were used to water the ginseng seedlings for inoculation. Each ginseng seedling was watered with 20 ml, about 3.2 × 10 9 cfu. Hoagland culture medium without bacteria was used as the control Mock group. Samples were taken 30 days after inoculation (irrigation).

[0044] The growth of ginseng seedlings was observed 30 days after inoculation. Compared with the control group Mock, there was no significant difference in the growth of the Bce group and the Rle group ( Figure 2 B).

[0045] The chlorophyll content of fresh leaves was measured 30 days after inoculation. It was found that compared with the control group Mock, the chlorophyll a, b and carotenoids in the Bce group and the Rle group increased to a certain extent after inoculation ( Figure 2 C), but the chlorophyll a / b ratio, which characterizes photosynthetic efficiency, was not significantly different ( Figure 2D), indicating that the two bacteria had no significant effect on the growth of ginseng 30 days after inoculation.

[0046] 3. Burkholderia regulates the glycosylation of different saponins The root materials of ginseng in each group after 30 days of inoculation were sliced ​​and dried, then ground into powder using a mortar and pestle, and then passed through a 40-mesh sieve. 50 mg of the powder was taken into a 15 ml centrifuge tube, and 3 ml of 70% methanol aqueous solution was added to dissolve the sample. The sample was ultrasonicated for one hour (temperature <40°C), centrifuged at 4000 rpm for 10 minutes, and the supernatant was taken into a 5 ml volumetric flask, and the volume was fixed to the scale line with 70% methanol aqueous solution. After mixing, 1 ml was pipetted into a 1.5 ml EP tube with a pipette. After centrifugation at 14000 rpm for ten minutes, the supernatant was taken into an injection vial to obtain a sample with a concentration of 10 mg / ml.

[0047] UPLC-QTOF-MS was used for detection under the following conditions. Column temperature: 35 °C, mobile phase: A [water + 0.1% formic acid (v / v)], solvent B [acetonitrile + 0.1% formic acid (v / v)]. Elution gradient was as follows: 0~0.5 min, B 15%; 0.5~7 min, B 20~28%; 7~8 min, B 28~30%; 8~16 min, B 30~35%; 16~21 min, B 35~60%; 21~22.8 min, B 60~98%; 22.8~25 min, B 98%; 25~25.2 min, B 85~15%; 25.2~28 min, B 15%. Injection volume: 5 µL, flow rate: 300 µL / min.

[0048] Standard products of ginsenoside monomers were purchased from Shanghai Yuanye Biotechnology Co., Ltd. with the catalog numbers CK (ginsenoside CK, B21045), Rh2 (R-type ginsenoside Rh2, B21729), F1 (ginsenoside F1, A10273), Rh1 (R-type ginsenoside Rh1, B21728), Rg3 (20R-type ginsenoside Rg3, A10389), Rf (ginsenoside Rf, B21056), Rg1 (ginsenoside Rg1, A10015), Re (ginsenoside Re, A10036), Ro (ginsenoside Ro, A10306), Rb1 (ginsenoside Rb1, A10021), and Rc (ginsenoside Rc, B21053).

[0049] The mass spectrometry conditions were set as follows: source temperature, 120 °C; desolvation temperature, 500 °C; capillary voltage: 1.0 kV; cone voltage, 20 V; cone gas flow, 50 L / h; desolvation gas flow, 800 L / h. Scan settings m / z: 100-1500; scan time: 0.3 s; collision energy: low energy: 6.00 ev high energy: 35.00 ev-50.00 ev.

[0050] Preparation of QC samples: Evenly measure appropriate amounts of all sample extracts to be analyzed, mix them evenly, and then divide them equally into injection vials. One QC sample is injected for every 8 samples.

[0051] The content of ginsenoside monomers was determined by LC / MS using a Waters ACQUITY UPLC-QTOF instrument. The contents of 11 ginsenoside monomers, including CK, Rh2, F1, Rh1, Rg3, Rf, Rg1, Re, Ro, Rb1 and Rc, were determined, and their sum was taken as the total ginsenoside content.

[0052] Ginsenoside content Figure 3 As shown in A, it can be seen that compared with the control Mock group, the group inoculated with Rhizobium Rle can increase the content of total ginsenosides in ginseng, while the group inoculated with Burkholderia Bce has no significant change in the content of total ginsenosides in ginseng.

[0053] Ginsenosides are classified according to the number of glycosylation on the ginsenoside monomers detected above. One glycosyl group is marked as 1G (including CK, Rh2, F1 and Rh1), two glycosyl groups are marked as 2G (including Rg3, Rf and Rg1), and three or more glycosyl groups are marked as nG (including Re, Ro, Rb1 and Rc), and their respective proportions in the total ginsenosides are calculated. Gs-1G represents monoglycosylated saponins; Gs-2G represents diglycosylated saponins; and Gs-nG represents n-glycosylated saponins.

[0054] The results are as follows Figure 3As shown in Figure 3B and 3C, it can be seen that compared with the control Mock group, both the Rle group and the Bce group regulated the proportions of different glycosylated saponins. Specifically, compared with the control Mock group, the proportions of monoglycosylated saponins Gs-1G (such as PPD-type CK and Rh2, PPT-type F1 and Rh1) and disaccharidized saponins Gs-2G (such as PPD-type Rg3, PPT-type Rf and Rg1) in the total ginsenosides in the Bce group were increased, and the proportions of Gs-nG in the total ginsenosides in the compared with the control Mock group, the proportion of monosaccharidized saponin Gs-1G (such as PPD type Rh2, PPT type F1 and Rh1) and disaccharidized saponin Gs-2G (such as PPD type F2 and Rg3, PPT type Rf and Rg1) in total ginsenosides in the Rle group was reduced, and the proportion of polysaccharidized saponin Gs-nG (such as PPD type Rb1, Rb2, Rb3 and Rc, OA type Ro) in total ginsenosides was increased.

[0055] For the group inoculated with Burkholderia Bce, compared with the control Mock group, the content of Rh1 monomer increased by 160%, the content of F1 monomer increased by 70%, the content of Rf monomer increased by 80%, and the content of Rg3 monomer increased by 40%.

[0056] Therefore, Burkholderia can increase the ratio of monoglycosylated saponins and disaccharidated saponins in the total saponins, thereby obtaining ginseng with a high ratio of monoglycosylated and disaccharidated ginsenosides.

[0057] 4. Rhizobium and Burkholderia regulate the expression of different glycosyltransferases to affect the diversity of ginsenosides The ginseng root materials of each group after 30 days of inoculation were first ground into powder using a mortar and pestle, and then total RNA was extracted using TRIgent (MF034) from Polymer. The extracted total RNA was sequenced by transcriptome sequencing at Shenzhen BGI ( Figure 4 A). Sequencing data were used to identify differentially expressed genes using DESeq2.

[0058] Sequencing data analysis Figure 4 As shown in B, it can be seen that the glycosyltransferase UGT genes required for ginsenoside glycosylation are regulated differently. Inoculation with Burkholderia (Bce group) mainly induced the expression of UGT71 and UGT74 glycosyltransferase genes, which are mainly responsible for the formation of single glycosylation. Inoculation with Rhizobium (Rle group) mainly induced the expression of UGT94 glycosyltransferase genes (mainly responsible for the formation of polysaccharides), as well as some UGT71 and UGT74.

[0059] In summary, the rhizobia in the root microorganisms induced the synthesis and accumulation of polyglycosylated saponins by regulating the expression of multiple types of glycosyltransferases such as UGT94, UGT71 and UGT74, while the Burkholderia in the root microorganisms mainly regulated the expression of glycosyltransferases such as UGT71 and UGT74, inducing the synthesis of monoglycosylated saponins and disaccharides. This discovery provides new ideas for the refinement of ginseng cultivation technology, such as improving the preparation of ginseng with a high proportion of monoglycosylated and / or disaccharide saponins.

[0060] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Use of Burkholderia or a composition containing the same in any of the following: 1) Regulate the ratio and / or content of different glycosylated saponins in ginseng; 2) increasing the content of monoglycosylated saponins and / or disaccharidated saponins in ginseng; 3) Increase the proportion of monoglycosylated saponins and / or disaccharidated saponins in ginseng; 4) preparing ginseng with a high monoglycosylated saponin ratio and / or a high diglycosylated saponin ratio; 5) preparing ginseng with high monoglycosylated saponin content and / or high diglycosylated saponin content; 6) Improve ginseng cultivation.

2. The use according to claim 1, characterized in that: The composition is a culture obtained by culturing the Burkholderia in a microbial culture medium.

3. A method for regulating the ratio or content of different glycosylated saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same to achieve regulation of the ratio or content of different glycosylated saponins in ginseng.

4. A method for increasing the proportion of monoglycosylated saponins and / or disaccharidized saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, thereby increasing the proportion of monoglycosylated saponins and / or disaccharidized saponins in ginseng.

5. A method for increasing the content of monoglycosylated saponins and / or disaccharidized saponins in ginseng, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, thereby increasing the content of monoglycosylated saponins and / or disaccharidized saponins in ginseng.

6. A method for preparing ginseng with a high monoglycosylated saponin ratio and / or a high disaccharylated saponin ratio, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, and culturing to obtain the target ginseng with a high monoglycosylated saponin ratio and / or a high disaccharylated saponin ratio.

7. A method for preparing ginseng with a high content of monoglycosylated saponins and / or a high content of disaccharidized saponins, comprising the following steps: contacting ginseng seedlings with Burkholderia or a composition containing the same, and culturing to obtain the target ginseng with a high content of monoglycosylated saponins and / or a high content of disaccharidized saponins.

8. The method according to any one of claims 3 to 7, characterized in that: The contacting is watering the ginseng seedlings with the composition containing the ginseng seedlings.

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