A method for analyzing the environmental adaptability of wolfberry

Abstract

The present invention proposes a method for analyzing the environmental adaptability of wolfberry, which belongs to the technical field of wolfberry environmental adaptability research. The method comprises the following steps: 1) transferring wolfberry seedlings of different varieties to a hydroponic system for hydroponic cultivation, wherein, during the hydroponic stage, the wolfberry seedlings of each variety are respectively set as a control group and a treatment group; 2) taking the leaves of the wolfberry seedlings in the control group obtained in step 1) and the leaves of the wolfberry seedlings in the treatment group obtained in step 1) and measuring the content of crude polysaccharides, flavonoids, total phenols and total saponins respectively; 3) comparing the drought treatment group of each variety with its corresponding control group to obtain the content changes of crude polysaccharides, flavonoids, total phenols and total saponins, and then comparing the obtained results between different varieties. According to the changes in the content of crude polysaccharides, flavonoids, total phenols and total saponins, the adaptability of the variety to drought, alkaline and salt environments compared with other varieties is judged. This method is mainly used for comparing the environmental adaptability of different varieties of wolfberry, and is simple to operate and has good accuracy.

Description

Technical Field

[0001] The invention belongs to the technical field of wolfberry environmental adaptability research, and in particular relates to a method for analyzing wolfberry environmental adaptability. Background Art

[0002] Goji berries have a long history of cultivation and have been known as the "Tree of Life" since ancient times. The Compendium of Materia Medica states: "In spring, goji berry leaves are harvested, known as Tianjingcao; in summer, flowers are harvested, known as Changshengcao; in autumn, seeds are harvested, known as Goji Berry Fruits; and in winter, roots are harvested, known as Digupi." Goji berry leaves not only tonify the body's essence, clear heat and relieve coughs, nourish the liver and improve eyesight, but also possess antioxidant, anti-tumor, and anti-aging properties. They can be eaten as a fresh vegetable or made into edible products such as tea. Research has shown that goji berry leaves can protect nerves, significantly improve learning and memory in experimental animals, prevent Alzheimer's disease, and mitigate the aging process of multiple organs. Therefore, goji berry leaves possess significant nutritional and health benefits.

[0003] Goji berry leaves are not only rich in nutrients such as protein, amino acids, vitamins, and trace elements, but also contain active substances such as flavonoids, terpenoids, polysaccharides, and alkaloids. Studies have shown that the nutritional and active ingredient composition of goji berry leaves is essentially the same as, or even exceeds, that of goji berries. Goji berry leaves are rich in various nutrients. For example, dried goji berry leaves contain 40% carbohydrates, 3% crude fat, and 7% crude fiber. The crude protein content is as high as 14%, higher than that of goji berry fruit, and 60% higher than that of corn, wheat, and rice, unmatched by any other plant except soybeans. Goji berry leaves are also high in flavonoids; for the same weight of dried goji berry leaves, the content of flavonoids is higher than that of the same compounds in dried goji berries.

[0004] To adapt to their environment, plants typically synthesize and accumulate secondary metabolites through metabolic regulation, thereby mitigating the effects of stress on them by preventing them from experiencing stress or enhancing their ability to tolerate it. Secondary metabolites are believed to be the result of plant adaptation to the ecological environment during their long evolutionary process. They play a crucial role in plants' ability to resist environmental stress, and their content is often affected by changes in environmental conditions.

[0005] In the existing technology, the analysis of the accumulation and changes of the effective ingredients in wolfberry leaves with the environment is usually carried out by directly analyzing the secondary metabolites of crop leaves or fruits, or the secondary metabolites at different developmental stages. However, there is no research in the existing technology that analyzes the environmental adaptability of wolfberry by simulating different growth environments through different culture conditions and then studying specific components. Summary of the Invention

[0006] The present invention provides a method for analyzing the environmental adaptability of wolfberry, and explores the adaptability of wolfberry to drought, high salt and high alkaline environments by detecting the content of characteristic functional components in wolfberry leaves.

[0007] The present invention provides a method for analyzing the environmental adaptability of different wolfberry varieties, comprising the following steps:

[0008] 1) transferring different varieties of wolfberry seedlings to a hydroponic system for hydroponics, wherein, during the hydroponic stage, each variety of wolfberry seedlings was set as a control group and a treatment group, wherein the treatment groups included a drought treatment group, a salt treatment group, and an alkali treatment group, and the control group, the drought treatment group, the salt treatment group, and the alkali treatment group were all added with Hoagland nutrient solution, the drought treatment group was also added with polyethylene glycol 6000, the salt treatment group was also added with NaCl, and the alkali treatment group was also added with NaHCO3;

[0009] 2) taking leaves of the wolfberry seedlings in the control group obtained in step 1) and leaves of the wolfberry seedlings in the treatment group obtained in step 1), and determining the contents of crude polysaccharides, flavonoids, total phenols, and total saponins, respectively;

[0010] 3) Compare the drought-treated groups of each variety with their corresponding control groups to determine changes in crude polysaccharide, flavonoid, and total phenol content. The results are then compared across varieties. The higher the percentage increase in crude polysaccharide, flavonoid, and total phenol content, the more adaptable the variety is to drought conditions.

[0011] The alkali-treated groups of each variety were compared with their corresponding control groups to obtain the changes in crude polysaccharide and total saponin content. The results were then compared between different varieties. The higher the increase in crude polysaccharide content and the higher the decrease in total saponin content, the stronger the variety's adaptability to alkaline environments compared to other varieties.

[0012] The salt-treated groups of each variety were compared with their corresponding control groups to obtain the changes in the content of crude polysaccharides and total phenols. The results were then compared between different varieties. The higher the increase in the crude polysaccharides and total phenols, the stronger the adaptability of the variety to the salt environment compared with other varieties.

[0013] Furthermore, in step 1), wolfberry seedlings are obtained by the following method: new shoots are cut from wolfberry trees, sterilized and air-dried, and then cut into culture medium, cultured in a greenhouse for 3 to 4 weeks, and healthy and uniformly growing wolfberry seedlings are selected for use.

[0014] Furthermore, in step 1), the culture medium is MS culture medium.

[0015] Furthermore, in step 1), after adding polyethylene glycol 6000, the mass concentration of polyethylene glycol 6000 in the system is 20%.

[0016] Furthermore, in step 1), after adding NaCl, the concentration of NaCl in the system is 300 mmol / L.

[0017] Furthermore, in step 1), after adding NaHCO3, the concentration of NaHCO3 in the system is 150mmol / L.

[0018] Furthermore, in step 1), the hydroponic culture time is 5 to 10 days; preferably, in step 1), the hydroponic culture time is 7 days.

[0019] The present invention has the following advantages:

[0020] The method proposed in this paper analyzes the environmental adaptability of wolfberry. This method simulates different adverse growth environments by subjecting wolfberry seedlings to different abiotic stresses (drought, high salt, and high alkalinity). The method then measures changes in the content of active ingredients (crude polysaccharides, flavonoids, total phenols, and total saponins) in the wolfberry leaves to evaluate and analyze the adaptability of different wolfberry varieties to these different environments. This method is simple to operate and produces highly accurate results, providing a new approach to analyzing the environmental adaptability of wolfberry and possessing considerable application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0022] Figure 1 Schematic diagram of the effects of high salt, high alkali and drought treatments on the crude polysaccharide content in wolfberry leaves;

[0023] Figure 2 Schematic diagram of the effects of high salt, high alkali and drought treatments on the flavonoid content in wolfberry leaves;

[0024] Figure 3 Schematic diagram of the effects of high salt, high alkali and drought treatments on the total phenolic content in wolfberry leaves;

[0025] Figure 4 Schematic diagram of the effects of high salt, high alkali and drought treatments on the total saponin content in wolfberry leaves;

[0026] Figure 5 Schematic diagram of the effects of high salt, high alkali and drought treatments on the jasmonic acid content in wolfberry leaves. DETAILED DESCRIPTION

[0027] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. The embodiments of the present invention and the features in the embodiments can be combined with each other unless there is a conflict.

[0028] An embodiment of the present invention provides a method for analyzing the environmental adaptability of different wolfberry varieties, comprising the following steps:

[0029] 1) transferring different varieties of wolfberry seedlings to a hydroponic system for hydroponics, wherein, during the hydroponic stage, each variety of wolfberry seedlings was set as a control group and a treatment group, wherein the treatment groups included a drought treatment group, a salt treatment group, and an alkali treatment group, and the control group, the drought treatment group, the salt treatment group, and the alkali treatment group were all added with Hoagland nutrient solution, the drought treatment group was also added with polyethylene glycol 6000, the salt treatment group was also added with NaCl, and the alkali treatment group was also added with NaHCO3;

[0030] 2) taking leaves of the wolfberry seedlings in the control group obtained in step 1) and leaves of the wolfberry seedlings in the treatment group obtained in step 1), and determining the contents of crude polysaccharides, flavonoids, total phenols, and total saponins, respectively;

[0031] 3) Compare the drought-treated groups of each variety with their corresponding control groups to determine changes in crude polysaccharide, flavonoid, and total phenol content. The results are then compared across varieties. The higher the percentage increase in crude polysaccharide, flavonoid, and total phenol content, the more adaptable the variety is to drought conditions.

[0032] The alkali-treated groups of each variety were compared with their corresponding control groups to obtain the changes in crude polysaccharide and total saponin content. The results were then compared between different varieties. The higher the increase in crude polysaccharide content and the higher the decrease in total saponin content, the stronger the variety's adaptability to alkaline environments compared to other varieties.

[0033] The salt-treated groups of each variety were compared with their corresponding control groups to obtain the changes in the content of crude polysaccharides and total phenols. The results were then compared between different varieties. The higher the increase in the crude polysaccharides and total phenols, the stronger the adaptability of the variety to the salt environment compared with other varieties.

[0034] The present invention simulates drought stress by adding PEG6000 to induce osmotic pressure changes, simulates alkaline stress by adding NaHCO3, and simulates salt stress by adding NaCl. The content of crude polysaccharides, flavonoids, total phenols, and total saponins in wolfberry leaves under drought, high alkalinity, and high salt conditions is analyzed. The changes in the content of these active ingredients are then used to compare and evaluate the adaptability of different wolfberry varieties to drought, high salt, and high alkalinity environments. This method is simple to operate and produces highly accurate results.

[0035] In the embodiments of the present invention, crude polysaccharides refer to complex heteropolysaccharides, mainly mucopolysaccharides, lipopolysaccharides, conjugated polysaccharides, etc. Flavonoids generally refer to flavonoid compounds, which are widely present in plants in nature and are secondary metabolites of plants. Flavonoids are a type of yellow pigment derived from flavonoids (2-phenylchromone) as the parent nucleus, including isomers of flavonoids and their hydrogenated and reduced products, that is, a series of compounds with C6-C3-C6 as the basic carbon frame. Total phenols refer to all phenolic compounds, including polyphenols and monophenols. Total saponins, also known as saponins, are a class of compounds containing glycosides such as steroidal saponins and triterpenoid saponins.

[0036] In one embodiment of the present invention, in step 1), wolfberry seedlings are obtained by the following method: new shoots are cut from wolfberry trees, sterilized and air-dried, and then cut into culture medium, cultured in a greenhouse for 3 to 4 weeks, and healthy and uniformly growing wolfberry seedlings are selected for use.

[0037] Specifically, in step 1), the culture medium is MS culture medium.

[0038] In one embodiment of the present invention, in step 1), the Hoagland nutrient solution may be NSP1020 Coolable modified Hoagland nutrient solution. Specifically, in step 1), the NSP1020 Coolable modified Hoagland nutrient solution comprises a dry powder mixture and a calcium concentrate.

[0039] In one embodiment of the present invention, in step 1), after adding Hoagland nutrient solution, the nutrients contained in the hydroponic system include 0.95gL -1 Ca(NO3)2·4H2O,0.51gL -1 KNO3,0.08gL -1 NH4NO3,0.14gL -1 KH2PO4,0.24gL -1 MgSO4,0.037gL-1 FeNaEDTA,0.001gL-1 KI,0.006gL-1 H3BO3,0.02gL -1 MnSO4·H2O,0.009gL -1 ZnSO4·7H2O,0.25mgL -1 Na2MoO4·2H2O,0.025mgL -1 CuSO4·5H2O,0.025mgL - 1 CoCl2·6H2O.

[0040] In one embodiment of the present invention, in step 1), after adding polyethylene glycol 6000, the mass concentration of polyethylene glycol 6000 in the system is 20%.

[0041] In one embodiment of the present invention, in step 1), after adding NaCl, the concentration of NaCl in the system is 300 mmol / L.

[0042] In one embodiment of the present invention, in step 1), after adding NaHCO 3 , the concentration of NaHCO 3 in the system is 150 mmol / L.

[0043] In one embodiment of the present invention, in step 1), the hydroponic culture time is 5 to 10 days; preferably, in step 1), the hydroponic culture time is 7 days.

[0044] In one embodiment of the present invention, in step 2), the crude polysaccharides, flavonoids, total phenols, and total saponins are all measured by spectrophotometry, which are all conventional methods.

[0045] In the embodiment of the present invention, in step 3), the comparison of the content changes of the effective components (crude polysaccharides, flavonoids, total phenols, and total saponins) between different wolfberry varieties can be obtained. The content change includes content increase and content decrease, wherein the higher the content increase, the higher the content increase ratio, and the higher the content decrease, the higher the content decrease ratio, thereby performing a comparison between different varieties.

[0046] The present invention will be described in detail below with reference to the embodiments.

[0047] Example 1 A method for analyzing the environmental adaptability of different wolfberry varieties (Ningqicai No. 1 C1, Ningqicai No. 1 N1), comprising:

[0048] 1) New shoots were cut from two wolfberry trees (Ningqicai No. 1 (C1) and Ningqi No. 1 (N1), sterilized and air-dried, and then planted on MS medium. The cuttings were cultured in a greenhouse for 3-4 weeks, and healthy and uniform wolfberry seedlings were selected for use.

[0049] The two varieties of wolfberry seedlings, Ningqicai No. 1 and Ningqi No. 1, were transferred to a hydroponic system for hydroponic culture (the hydroponic culture time was 7 days). In the hydroponic culture stage, the two varieties of wolfberry seedlings, Ningqicai No. 1 (C1) and Ningqi No. 1 (N1), were set up as a control group and a treatment group, respectively. The treatment groups included a drought treatment group, a salt treatment group, and an alkali treatment group. Hoagland's nutrient solution was added to the control group, the drought treatment group, the salt treatment group, and the alkali treatment group. The Hoagland's nutrient solution was NSP1020 Coolable modified Hoagland's nutrient solution. After adding the Hoagland's nutrient solution, the nutrients contained in the hydroponic system included 0.95gL -1 Ca(NO3)2·4H2O,0.51gL -1 KNO3,0.08gL -1 NH4NO3,0.14gL-1 KH2PO4,0.24gL -1 MgSO4,0.037gL-1FeNaEDTA,0.001gL-1KI,0.006gL-1H3BO3,0.02gL -1 MnSO4·H2O,0.009gL -1 ZnSO4·7H2O,0.25mgL -1 Na2MoO4·2H2O,0.025mgL -1 CuSO4·5H2O,0.025mgL -1 CoCl2·6H2O;

[0050] The drought treatment group also added polyethylene glycol 6000, and after the addition of polyethylene glycol 6000, the mass concentration of polyethylene glycol 6000 in the system was 20%; the salt treatment group added NaCl, and after the addition of NaCl, the concentration of NaCl in the system was 300mmol / L; the alkali treatment group added NaHCO3, and after the addition of NaHCO3, the concentration of NaHCO3 in the system was 150mmol / L;

[0051] 2) Taking leaves of the wolfberry seedlings in the control group obtained in step 1) and leaves of the wolfberry seedlings in the treatment group obtained in step 1), and measuring changes in the content of crude polysaccharides, flavonoids, total phenols, and total saponins, respectively; specifically as follows:

[0052] 2.1) Determination of crude polysaccharide content

[0053] Crude polysaccharides were extracted using the water extraction and alcohol precipitation method, and their content was determined using the phenol-sulfuric acid method. First, the sample was dried and pulverized. Approximately 0.05 g of sample was weighed and thoroughly homogenized with 1 mL of water. Extraction was performed in a 100°C waterbath for 2 hours (the lid must be tightly closed to prevent water loss). After cooling, the sample was centrifuged at 10,000 g for 10 minutes, and the supernatant was collected. 0.2 mL of the supernatant was slowly added to 0.8 mL of anhydrous ethanol. After mixing, the sample was allowed to stand overnight at 4°C. Centrifugation was performed at 10,000 g for 10 minutes, and the supernatant was discarded. 1 mL of water was added to the precipitate, and the mixture was thoroughly mixed to dissolve the precipitate. Separate blank and assay tubes were prepared. 400 μL of distilled water was added to the blank tube, and 400 μL of the extract solution was added to the assay tube. Then, 200 μL of 5% phenol and 1,000 μL of concentrated sulfuric acid were added, respectively. After mixing, the sample was placed in a 90°C waterbath for 20 minutes, and cooled under running water. Take 1mL of reaction solution and add it to the cuvette. Measure the absorbance value A at 490nm. Calculate △A = Adetermination - Ablank. Crude polysaccharide content (μg / g, DW) = 313.24 × (△A + 0.0037) ÷ W, W: sample mass, g. Results are shown in Figure 1 .

[0054] 2.2) Flavonoid content determination method

[0055] In alkaline nitrite solution, flavonoids and aluminum ions form a red complex with a characteristic absorption peak at 510nm. The flavonoid content of the sample can be calculated by measuring the absorbance of the sample extract at 510nm. First, the sample is fixed at 105℃ for 10-15min, dried at 80℃ to constant weight, crushed, and passed through a 40-mesh sieve. Then, about 0.05g is weighed, 1mL of extract is added, and the sample is extracted at 60℃ for 2h. Centrifuge at 12000rpm, 25℃ for 10min, take the supernatant, mix it with the reaction solution, let it stand at 25℃ for 15min, and measure the absorbance at 510nm. △A=A determination-A blank. Flavonoid content (mg / g, DW)=0.199×(△A-0.0007)÷W, W: sample mass, g. See the results. Figure 2 .

[0056] 2.3) Determination of total phenol content

[0057] Under alkaline conditions, phenolic substances reduce tungstomolybdic acid to produce a blue compound with a characteristic absorption peak at 760nm. The total phenol content of the sample can be obtained by measuring the absorbance value at 760nm.

[0058] First, the sample was sterilized at 105℃ for 10-15min, dried at 80℃ to constant weight, crushed, and passed through a 40-mesh sieve. About 0.05g was weighed, 1mL of extract was added, and the sample was extracted by shaking at 60℃ for 2h. Centrifuged at 12000rpm, 25℃ for 10min, and the supernatant was taken for detection. 50μL of supernatant, 500μL of reaction reagent, and 450μL of distilled water were mixed, and allowed to stand at 25℃ for 10min. 800μL was taken to a 1mL glass cuvette, and the absorbance of each tube at 765nm was detected, and △A=A determination-A control was calculated. Total phenol content (mg / g, DW)=0.178×(△A-0.0012)÷W, W: sample mass, g. Results are shown in Figure 3 .

[0059] 2.4) Determination of total saponin content

[0060] Use ultrasonic extraction to extract saponins from the sample, and use the vanillin-perchloric acid colorimetric system to determine the total saponin content. First, dry the sample, crush and sieve it, weigh 0.05g, add 1mL of extract, homogenize and extract by ultrasonic for 1h; centrifuge at 10000rpm and 25℃ for 10min, and take the supernatant for testing. Take 500μL of the supernatant, evaporate to dryness at 70℃, add 200μL of reagent 1 and 800μL of perchloric acid, mix well, and place in a 55℃ water bath for 20min. Take 200μL of the reaction solution in another EP tube, add 1000μL of acetic acid, mix well, take 1mL in a glass cuvette, measure the absorbance of each tube at 550nm, and calculate △A=A determination-A blank. Calculate the total saponin content (μg / g, DW)=345.79×(△A+0.0651)÷W, W: sample mass, g. See the results. Figure 4 .

[0061] 3) Compare the drought-treated groups of each variety with their corresponding control groups to determine changes in crude polysaccharide, flavonoid, and total phenol content. The results are then compared across varieties. The higher the percentage increase in crude polysaccharide, flavonoid, and total phenol content, the more adaptable the variety is to drought conditions.

[0062] The alkali-treated groups of each variety were compared with their corresponding control groups to obtain the changes in crude polysaccharide and total saponin content. The results were then compared between different varieties. The greater the increase in crude polysaccharide content and the higher the decrease in total saponin content, the stronger the variety's adaptability to alkaline environments compared to other varieties.

[0063] The salt treatment groups of each variety were compared with their corresponding control groups to obtain the changes in the content of crude polysaccharides and total phenols. The results were then compared between different varieties. The higher the increase in crude polysaccharides and total phenols, the stronger the variety's adaptability to salt environments compared to other varieties. Figures 1 to 4 , the results are shown in Table 1.

[0064] Table 1

[0065]

[0066] From Table 1, we can get:

[0067] Comparing the adaptability of N1 and C1 to drought environment, after PEG6000 treatment, the crude polysaccharide content of N1 and C1 changed by 27.06% and 67.55%, the flavonoid content of N1 and C1 changed by 23.83% and 89.10%, and the total phenol content of N1 and C1 changed by 22.82% and 33.85%. From the above comparison, it can be seen that the increase ratio of crude polysaccharides, flavonoids and total phenols in C1 is higher than that in N1, so C1 is more adaptable to drought environment than N1.

[0068] Comparing the adaptability of N1 and C1 to alkaline environment, after NaHCO3 treatment, the crude polysaccharide content of N1 and C1 changed to 54.39% and 63.32%, and the total saponin content of N1 and C1 changed to -4.87% and -14.94%. From the above comparison, it can be seen that the upward regulation ratio of crude polysaccharide of C1 is higher than that of N1, and the downward regulation ratio of total saponin of C1 is higher than that of N1. Therefore, C1 is more adaptable to alkaline environment than N1.

[0069] Comparing the adaptability of N1 and C1 to salt environment, after NaCl treatment, the crude polysaccharide content of N1 and C1 changed to 76.93% and 94.69%, and the total phenol content of N1 and C1 changed to 51.43% and 67.64%. From the above comparison, it can be seen that the increase ratio of crude polysaccharide and total phenol content of C1 is higher than that of N1, so C1 is more adaptable to salt environment than N1.

[0070] Verification Example 1 Method for determining the environmental adaptability of different wolfberry varieties based on jasmonic acid content

[0071] In order to verify the accuracy of the method described in Example 1, another existing detection method was used to compare and evaluate the adaptability of different varieties of wolfberries to the environment.

[0072] 1. The wolfberry seedlings were processed in the same manner as in step 1) of Example 1, specifically, new shoots were cut from two wolfberry trees, Ningqicai No. 1 (C1) and Ningqi No. 1 (N1), sterilized and air-dried, and then cut into MS medium. The cuttings were cultured in a greenhouse for 3 to 4 weeks, and healthy and uniformly growing wolfberry seedlings were selected for use.

[0073] Ningqicai No. 1 (C1) and Ningqi No. 1 (N1) wolfberry seedlings were set up as a control group and a treatment group, respectively. The treatment groups included a drought treatment group, a salt treatment group, and an alkali treatment group. Hoagland's nutrient solution was added to the control group, the drought treatment group, the salt treatment group, and the alkali treatment group. The Hoagland's nutrient solution was NSP1020 Coollab modified Hoagland's nutrient solution. After adding the Hoagland's nutrient solution, the nutrients contained in the hydroponic system included 0.95gL -1 Ca(NO3)2·4H2O,0.51gL -1 KNO3,0.08gL -1 NH4NO3,0.14gL -1 KH2PO4,0.24gL -1 MgSO4,0.037gL-1FeNaEDTA,0.001gL-1 KI,0.006gL-1H3BO3,0.02gL -1 MnSO4·H2O,0.009gL -1 ZnSO4·7H2O,0.25mgL -1Na2MoO4·2H2O,0.025mgL -1 CuSO4·5H2O,0.025mgL -1 CoCl2·6H2O;

[0074] The drought treatment group also added polyethylene glycol 6000. After adding polyethylene glycol 6000, the mass concentration of polyethylene glycol 6000 in the system was 20%; the salt treatment group added NaCl. After adding NaCl, the concentration of NaCl in the system was 300mmol / L; the alkali treatment group added NaHCO3. After adding NaHCO3, the concentration of NaHCO3 in the system was 150mmol / L.

[0075] 2. Processing Samples

[0076] (1) Take the wolfberry leaves obtained in step 1 and grind them into powder using a grinder (30 Hz, 1 min);

[0077] (2) Weigh 50 mg of the ground sample, add 10 μL of a 100 ng / mL internal standard solution and 1 mL of a methanol / water / formic acid (15:4:1, v / v / v) extractant, and mix well.

[0078] (3) Vortex for 10 min, centrifuge at 4°C, 12,000 rpm for 5 min, and transfer the supernatant to a new centrifuge tube for concentration;

[0079] (4) After concentration, the solution was reconstituted with 100 μL of 80% methanol / water solution, filtered through a 0.22 μm filter membrane, and placed in an injection vial for LC-MS / MS analysis.

[0080] 3. Chromatographic mass spectrometry acquisition conditions

[0081] The data acquisition instrument system mainly includes Ultra Performance Liquid Chromatography (UPLC) TM AD, https: / / sciex.com.cn / ) and tandem mass spectrometry (Tandem Mass Spectrometry, MS / MS) ( 6500+, https: / / sciex.com.cn / ).

[0082] Liquid phase conditions mainly include:

[0083] 1) Chromatographic column: Waters ACQUITY UPLC HSS T3 C18 column (1.8 μm, 100 mm × 2.1 mm id);

[0084] 2) Mobile phase: Phase A, ultrapure water (added with 0.04% acetic acid); Phase B, acetonitrile (added with 0.04% acetic acid);

[0085] 3) Gradient elution program: A / B 95:5 (V / V) at 0 min, A / B 95:5 (V / V) at 1.0 min, 5:95 (V / V) at 8.0 min, 5:95 (V / V) at 9.0 min, 95:5 (V / V) at 9.1 min, and 95:5 (V / V) at 12.0 min;

[0086] 4) Flow rate: 0.35 mL / min; column temperature: 40°C; injection volume: 2 μL.

[0087] Mass spectrometry conditions mainly include:

[0088] The electrospray ionization (ESI) source temperature was 550°C, the mass spectrometer voltage was 5500 V in positive ion mode, the mass spectrometer voltage was -4500 V in negative ion mode, and the curtain gas (CUR) was 35 psi. In a Q-Trap 6500+, each ion transition was scanned based on optimized declustering potential (DP) and collision energy (CE).

[0089] 4. Qualitative and quantitative analysis

[0090] The MWDB (Metware Database) database was constructed based on the standards to perform qualitative analysis on the mass spectrometry data.

[0091] Quantification is accomplished using the Multiple Reaction Monitoring (MRM) analysis of a triple quadrupole mass spectrometer. In the MRM mode, the quadrupole first screens the precursor ions (parent ions) of the target substance, eliminating ions corresponding to other molecular weight substances to preliminarily eliminate interference; the precursor ions are induced to ionize in the collision chamber and then break to form multiple fragment ions, which are then filtered through the triple quadrupole to select the required characteristic fragment ions, eliminating interference from non-target ions and making quantification more accurate and reproducible. After obtaining the mass spectrometry analysis data of different samples, the chromatographic peaks of all target substances are integrated and quantitative analysis is performed using the standard curve.

[0092] 5. Data Statistical Analysis

[0093] The data were analyzed using sigmaplot software, and the difference significance was analyzed by t-test (*: p < 0.05, significant difference; **: p < 0.01, extremely significant difference). Figure 5.

[0094] like Figure 5 As shown in the figure, by measuring the change ratio of jasmonic acid content, a plant stress resistance hormone, the adaptability of different wolfberry varieties to abiotic stresses such as drought, salt and alkali was compared and evaluated. Figure 5 In Ningqi 1, high salt levels induced a 29.65-fold increase in jasmonic acid expression, high alkalinity levels induced a 52.62% decrease, and drought-induced jasmonic acid decreases by 39.69%. In Ningqicai 1, high salt levels induced a 18.47% decrease in jasmonic acid expression, high alkalinity levels induced a 99.14% decrease, and drought-induced jasmonic acid decreases by 99.45%. The higher the percentage of jasmonic acid decrease, the stronger the plant's adaptability to high salt, alkalinity, and drought. Therefore, Ningqicai 1 has a stronger adaptability to high salt, alkalinity, and drought than Ningqi 1.

[0095] The above are only preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A method for analyzing the environmental adaptability of different varieties of wolfberry, characterized in that: The steps include: 1) transferring different varieties of wolfberry seedlings to a hydroponic system for hydroponics, wherein, during the hydroponic stage, each variety of wolfberry seedlings was set as a control group and a treatment group, wherein the treatment groups included a drought treatment group, a salt treatment group, and an alkali treatment group, and the control group, the drought treatment group, the salt treatment group, and the alkali treatment group were all added with Hoagland nutrient solution, the drought treatment group was also added with polyethylene glycol 6000, the salt treatment group was also added with NaCl, and the alkali treatment group was also added with NaHCO3; 2) taking leaves of the wolfberry seedlings in the control group obtained in step 1) and leaves of the wolfberry seedlings in the treatment group obtained in step 1), and determining the contents of crude polysaccharides, flavonoids, total phenols, and total saponins, respectively; 3) Compare the drought-treated groups of each variety with their corresponding control groups to determine changes in crude polysaccharide, flavonoid, and total phenol content. The results are then compared across varieties. The higher the percentage increase in crude polysaccharide, flavonoid, and total phenol content, the more adaptable the variety is to drought conditions. The alkali-treated groups of each variety were compared with their corresponding control groups to obtain the changes in crude polysaccharide and total saponin content. The results were then compared between different varieties. The higher the increase in crude polysaccharide content and the higher the decrease in total saponin content, the stronger the variety's adaptability to alkaline environments compared to other varieties. The salt-treated groups of each variety were compared with their corresponding control groups to obtain the changes in the content of crude polysaccharides and total phenols. The results were then compared between different varieties. The higher the increase in the crude polysaccharides and total phenols, the stronger the adaptability of the variety to the salt environment compared with other varieties.

2. The method according to claim 1, characterized in that In step 1), wolfberry seedlings are obtained by the following method: new shoots are cut from wolfberry trees, sterilized and air-dried, and then cut into culture medium, cultured in a greenhouse for 3 to 4 weeks, and healthy and uniformly growing wolfberry seedlings are selected for use.

3. The method according to claim 2, characterized in that In step 1), the culture medium is MS culture medium.

4. The method according to claim 1, wherein In step 1), after adding polyethylene glycol 6000, the mass concentration of polyethylene glycol 6000 in the system is 20%.

5. The method according to claim 1, characterized in that In step 1), after adding NaCl, the concentration of NaCl in the system is 300 mmol / L.

6. The method according to claim 1, characterized in that In step 1), after adding NaHCO3, the concentration of NaHCO3 in the system is 150mmol / L.

7. The method according to claim 1, characterized in that In step 1), the hydroponic culture time is 5 to 10 days.

8. The method according to claim 7, characterized in that In step 1), the hydroponic culture time is 7 days.

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