Application of low-temperature pretreatment in induction of ginsenoside accumulation

Abstract

The invention discloses application of low-temperature pretreatment in induction of ginsenoside accumulation. The invention provides a method for cultivating ginseng with high ginsenoside content, which comprises the following steps of: firstly carrying out low-temperature pretreatment culture on ginseng seedlings, and then recovering normal culture to obtain target ginseng with high ginsenoside content. According to the ginseng subjected to long-time low-temperature pretreatment (simulating winter of a ginseng planting place), after the temperature is recovered, the ginsenoside content of the ginseng is remarkably increased, and an MVA pathway synthesized by saponin is remarkably activated. The action mechanism of low temperature in winter in saponin accumulation in four-season change is clarified, and a new thought is provided for ginseng cultivation.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to the application of low-temperature pretreatment in inducing the accumulation 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 high-latitude mountain forest areas with distinct four seasons in the eastern part of the Asian continent. Ginseng has a wide range of effects. Its main active ingredients are ginsenosides of triterpenoids, which are secondary metabolites of ginseng. They have significant therapeutic effects in improving immunity and preventing cardiovascular diseases. Ginsenosides are mainly synthesized in ginseng 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 key 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 then undergoes a series of enzymatic reactions to produce protopanaxadiol (PDD), protopanaxadiol (PPT) and oleanolic acid (OA) ginsenosides.

[0003] A variety of stress factors, such as salicylic acid, jasmonic acid, osmotic stress, salt stress and other adverse conditions can induce the expression of specific transcription factors, thereby promoting the transcription of MVA or MEP pathway genes, and thus increasing the content of ginsenosides. However, most of the stress factor treatments are aimed at ginseng hairy roots cultured in vitro, and are often short-term transient treatments. The accumulation of saponins often disappears with the removal of stress factors. Summary of the invention

[0004] The technical problem solved by the present invention is how to induce the accumulation of ginsenosides.

[0005] In order to solve the above technical problems, the first aspect of the present invention provides a method for cultivating ginseng with high ginsenoside content, comprising the following steps: The ginseng seedlings are first subjected to low temperature pretreatment and culture, and then restored to normal culture, so as to obtain target ginseng with high ginsenoside content.

[0006] The target ginseng with a high ginsenoside content as mentioned above has a high ginsenoside content compared to normally cultured ginseng.

[0007] In a second aspect, the present invention provides a method for increasing the content of ginsenosides, comprising the following steps: pre-culturing ginseng seedlings at low temperature, and then restoring normal culture to increase the content of ginsenosides.

[0008] In a third aspect, the present invention provides a method for inducing ginsenoside accumulation, comprising the following steps: pre-culturing ginseng seedlings at low temperature, and then restoring normal culture to achieve inducing ginsenoside accumulation.

[0009] In the above methods, the temperature of the low-temperature pretreatment culture is 4-10 degrees.

[0010] In the above methods, the low-temperature pretreatment culture time is 1 day to 4 weeks.

[0011] Furthermore, the low-temperature pretreatment culture time may be 1 week to 4 weeks.

[0012] In the above methods, the temperature of the low-temperature pretreatment culture is 4 degrees and the time is 1 week.

[0013] In the methods described above, the time for resuming normal culture is greater than or equal to 1 week.

[0014] Furthermore, the time for resuming normal culture may be greater than or equal to 1 month.

[0015] In a third aspect, the present invention provides the use of the low-temperature pretreatment culture described in any one of the first to third aspects in any of the following: 1) Inducing the accumulation of ginsenosides; 2) Increase the content of ginsenosides; 3) Ginseng cultivation and breeding.

[0016] In the above, the inducing ginsenoside accumulation or increasing the ginsenoside content is achieved by activating the MVA pathway required for ginsenoside synthesis; The application is to first perform low temperature pretreatment culture on ginseng and then resume normal culture.

[0017] The above-mentioned ginseng cultivation and breeding can specifically be the cultivation of ginseng with high ginsenoside content.

[0018] In the application described above, the object of the low-temperature pretreatment culture is ginseng seedlings.

[0019] The ginseng of the present invention that has been pretreated with low temperature for a long time (simulating the winter in the place where ginseng is planted) has a significantly increased ginsenoside content after the temperature is restored, and the MVA synthesized by saponins is significantly activated. The present invention clarifies the mechanism of action of low temperature in winter in the accumulation of saponins during the change of seasons, and provides a new idea for ginseng cultivation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1Phenotypic analysis of ginseng after low temperature pretreatment; A, schematic diagram of low temperature pretreatment; B, ginseng seedlings grown for one month after temperature recovery; C, determination of chlorophyll and carotenoid contents; D, chlorophyll a / b ratio.

[0021] Figure 2 Determination of ginsenoside content; A, schematic diagram of the determination of ginsenoside content by the vanillin-perchloric acid method; B, reaction solution of total ginsenosides of ginseng; C, relative content of total ginsenosides of ginseng; D, relative content of monomeric ginsenosides of ginseng.

[0022] Figure 3 Transcriptome sequencing analysis of ginseng root materials; A, schematic diagram of transcriptome sequencing of ginseng root materials; B, PCA analysis of transcriptome sequencing data; C, cluster analysis of transcriptome sequencing data; D, low temperature pretreatment inhibited expression, and after temperature recovery, the expression level exceeded that of the Ctrl group and its GO enrichment analysis; E, heat map analysis of gene expression in the MVA pathway that regulates ginsenoside synthesis.

[0023] Figure 4 Effects of temperature recovery time on ginsenoside accumulation and ginsenoside biosynthesis gene expression; A, relative content of total ginsenosides; B, heat map analysis of gene expression in the MVA pathway that regulates ginsenoside biosynthesis.

[0024] Figure 5 Effect of low-temperature pretreatment time on saponin content; A, Schematic diagram of low-temperature pretreatment for different time periods; B, Total ginsenoside content of ginseng determined by vanillin-perchloric acid method. DETAILED DESCRIPTION

[0025] 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.

[0026] 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.

[0027] Unless otherwise specified, the quantitative tests in the following examples were performed three times and the results were averaged.

[0028] The greenhouse cultivation of ginseng in the following embodiments is as follows: The experimental material of this study is 3-year-old ginseng (Panax ginseng CA Meyer). The ginseng seedlings are transplanted into pots containing nutrient soil and vermiculite (ratio 1:2), and the pots are placed in a temperature-controlled incubator for growth. The normal growth temperature is 23°C, and the photoperiod is 16 hours (light) / 8 hours (darkness). The temperature during low temperature treatment is 4°C, and the photoperiod is consistent with normal growth.

[0029] The chlorophyll content determination in the following examples is as follows: After removing the midrib, the fresh ginseng leaves are cut into pieces and extracted using the Solebao chlorophyll extraction kit (BC0990). The entire process is carried out under light protection. 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 are measured using a spectrophotometer, and their contents are calculated according to the kit instructions.

[0030] The determination of ginsenoside content in the following examples is as follows: the ginseng root material is first ground into powder using a mortar, and then the saponin content is determined using the total saponin extraction kit (AK370V) of Boosun. At the same time, the standard curve is drawn using the total ginsenoside purchased from Yuanye Biological as the standard (S25997) (y=131.31x-6.6334, R 2 = 0.9996, x is the absorbance value of OD560, y is the content of ginsenosides). The non-targeted metabolomics of ginseng root materials was completed by Shenzhen BGI, and the detected ginsenoside monomer components were selected for content analysis.

[0031] The total RNA extraction and transcriptome sequencing in the following examples are as follows: the ginseng root material was first ground into powder using a mortar, and then total RNA was extracted using TRIgent (MF034) from Polymer, and the extracted total RNA was subjected to transcriptome sequencing at Shenzhen BGI. The sequencing data was used to identify differentially expressed genes using DESeq2, and the differentially expressed genes were subjected to functional cluster analysis using easyGSEA.

[0032] Example 1: Low temperature pretreatment induces accumulation of ginsenoside content in ginseng 1. Pre-treat ginseng at 4℃ The three-year-old ginseng seedlings were transplanted into nutrient pots, which contained nutrient soil and vermiculite (mass ratio of 1:2). The pots were placed in a temperature-controlled incubator for growth and divided into the following two groups for cultivation (e.g. Figure 1 A): Control group (Ctrl): grown at normal temperature (23°C, 16 h light / 8 h dark) for 2 months; Low temperature treatment group (T4-23-1M): grown in a 4°C incubator (16 hours light / 8 hours dark), after one month of low temperature treatment, moved to a normal temperature incubator and resumed growth for another month.

[0033] After the low-temperature treatment group resumed growth for one month, samples of the two groups of cultivated ginseng were collected for the following chlorophyll a, b and carotenoid content tests and total saponin content determination.

[0034] 2. Effect of low temperature treatment on the survival of ginseng The growth status, chlorophyll a, b, carotenoid content and chlorophyll a / b of ginseng after treatment with the T4-23-1M group and the Ctrl group in the above 1 were tested.

[0035] Growth status results such as Figure 1 As shown in B, it can be seen that there is no significant difference between the T4-23-1M group and the Ctrl group; there is no significant difference in the content of chlorophyll a, b and carotenoids in the leaves ( Figure 1 C), chlorophyll a / b, which represents photosynthetic efficiency, has no significant difference ( Figure 1 D). These results indicate that pre-treatment of ginseng at 4°C for 1 month and then growing it for 1 month after the temperature is restored will not significantly affect the normal growth of ginseng.

[0036] 3. Low temperature pretreatment of ginseng significantly increases its saponin content The total saponin content of ginseng root samples treated with Ctrl and T4-23-1M was determined by the vanillin-perchloric acid method described above ( Figure 2 A).

[0037] The results are as follows Figure 2 As shown in B, it was found that the T4-23-1M group had a higher OD560 absorbance value, the absorbance value of the Ctrl group was 0.414±0.020, and the T4-23-1M group had a larger absorbance value, which was 0.630±0.039.

[0038] The total saponin content of each group was calculated based on the standard curve prepared with the aforementioned ginsenoside standard.

[0039] The results are as follows Figure 2 As shown in C, the total saponins in the T4-23-1M group were significantly increased compared with the Ctrl group.

[0040] Non-targeted metabolome analysis of ginseng root samples after treatment with the Ctrl and T4-23-1M groups also revealed that most ginsenoside monomers were significantly upregulated in the T4-23-1M group ( Figure 2 D).

[0041] 4. Low temperature pretreatment promotes the expression of ginsenoside synthesis genes In order to explore the mechanism by which low-temperature pretreatment increases saponin content, whole-genome transcriptome sequencing analysis was carried out.

[0042] Low temperature treatment group T4: grown in a 4°C incubator (16 hours light / 8 hours dark), and samples were directly collected after one month of low temperature treatment.

[0043] Total RNA was extracted from the ginseng roots of the two groups (control group Ctrl and low temperature pretreatment group T4-23-1M) treated with the above 1 and the ginseng roots of the low temperature treatment group T4, and transcriptome sequencing was performed ( Figure 3 A).

[0044] PCA analysis of sequencing data revealed that gene expression in the three groups of samples was significantly different ( Figure 3 B). Cluster analysis found that the gene expression of T4-23-1M in the low temperature pretreatment group was closer to that of the Ctrl group ( Figure 3 C), indicating that the gene expression of ginseng in the low temperature pretreatment T4-23-1M group also partially recovered to the level of the Ctrl group. Further analysis found that 595 genes were inhibited by low temperature and their expression levels exceeded Ctrl after temperature recovery ( Figure 3 D, T4 vs Ctrl =<2, p =<0.05; T2-23-1M vs Ctrl>= 1.5, p =<0.05), these genes were mainly enriched in pathways related to ginsenoside synthesis, such as Figure 3 The functional entries marked in red in D indicate that after low-temperature pretreatment and temperature recovery, the ginsenoside synthesis genes showed an up-regulated expression trend, resulting in a significant increase in the ginsenoside content after temperature recovery.

[0045] In summary, after ginseng was pretreated with low temperature and the temperature was restored, the genes related to ginsenoside synthesis were significantly activated, such as the MVA pathway required for ginsenoside synthesis ( Figure 3 E), resulting in the accumulation of ginsenosides.

[0046] 5. Effect of growth time after temperature recovery on the expression of ginsenoside synthesis genes In order to explore the regulation of growth time on ginsenoside accumulation and ginsenoside synthesis genes after temperature recovery, the growth of ginseng in the low-temperature pretreatment group was extended to 2 months after temperature recovery. Then, the total saponin content of ginseng root samples in the Ctrl group, the temperature recovery one month group (T4-23-1M) and the temperature recovery two months group (T4-23-2M) was determined by the aforementioned vanillin-perchloric acid method.

[0047] The total saponin content of each group was calculated using the standard curve prepared using the aforementioned ginsenoside standard.

[0048] As mentioned above, compared with the Ctrl group, the total saponins in the T4-23-1M group increased significantly, while the T4-23-2M group maintained the same level as the T4-23-1M group and did not increase further ( Figure 4 A).

[0049] Furthermore, the materials of the T4-23-2M group were also subjected to whole-genome transcriptome sequencing analysis together with the aforementioned materials (Ctrl group, T4 group, and T4-23-1M group). As mentioned above, after low-temperature pretreatment and temperature recovery, the ginsenoside synthesis genes in the T4-23-1M group showed an up-regulated expression trend, but in the T4-23-2M group, the levels of these genes decreased and returned to the level of Ctrl ( Figure 4 B), so that the ginsenoside content did not continue to increase after the temperature was restored for 2 months.

[0050] In summary, after ginseng was pretreated with low temperature, the genes related to ginsenoside synthesis were significantly activated one month after the temperature was restored, but two months after the temperature was restored, the genes related to ginsenoside synthesis were inhibited and ginsenosides did not continue to accumulate.

[0051] 6. The duration of low-temperature pretreatment affects the degree of saponin accumulation To further explore the effect of low-temperature pretreatment duration on saponin accumulation, ginseng was pretreated with low temperature for 1 day (1d), 1 week (1w), 2 weeks (2w) and 3 weeks (3w), and then returned to room temperature for 1 week. These four groups of materials were marked as T4-1d-23, T4-1w-23, T4-2w-23 and T4-3w-23, respectively. Figure 5 A). The cells without cold pretreatment were used as the control (Ctrl group).

[0052] The total saponin content of the ginseng roots of the above groups was determined by the above-mentioned vanillin-perchloric acid method.

[0053] The results are as follows Figure 5 B. The total ginsenoside content of the 4 groups of materials was significantly higher than that of the Ctrl group. The saponin content of the materials treated with low temperature for 1 day increased less, while the saponin content of the materials treated for 1 week, 2 weeks and 3 weeks increased more. After 1 week of low temperature treatment, the saponin content would not increase with the increase of low temperature treatment time.

[0054] In summary, it was found that after ginseng was pretreated with low temperature, the genes related to ginsenoside synthesis were significantly activated and the content of ginsenosides was significantly increased one week after the temperature was restored. The saponin accumulation was significant after low temperature treatment for no less than one week, and the saponin accumulation was even more significant after low temperature treatment for one week.

[0055] 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. A method for cultivating ginseng with a high ginsenoside content, comprising the following steps: pre-culturing ginseng seedlings at low temperature, and then restoring normal culture to obtain target ginseng with a high ginsenoside content.

2. A method for increasing the content of ginsenosides, comprising the following steps: pre-culturing ginseng seedlings at low temperature, and then restoring normal culture to increase the content of ginsenosides.

3. A method for inducing ginsenoside accumulation, comprising the following steps: pre-culturing ginseng seedlings at low temperature, and then restoring normal culture to achieve inducing ginsenoside accumulation.

4. The method according to any one of claims 1 to 3, characterized in that: The temperature of the low temperature pretreatment culture is 4-10 degrees.

5. The method according to any one of claims 1 to 4, characterized in that: The low temperature pretreatment culture time is 1 day to 4 weeks.

6. The method according to any one of claims 1 to 5, characterized in that: The temperature of the low-temperature pretreatment culture is 4 degrees and the time is 1 week.

7. Use of the low temperature pretreatment culture according to any one of claims 1 to 6 in any of the following: 1) Inducing the accumulation of ginsenosides; 2) Increase the content of ginsenosides; 3) Ginseng cultivation and breeding.

8. The use according to claim 7, characterized in that: The object of the low-temperature pretreatment culture is ginseng seedlings.

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