Method for detecting endogenous strigolactones in cotton root exudates

Abstract

The invention relates to the technical field of chemical analysis, in particular to a method for detecting endogenous strigolactones in cotton root exudates. The method comprises the following steps: (1) soaking cotton seeds in water, germinating in a dark place, transferring the cotton seeds into a culture container which is filled with a culture medium and is provided with an empty bottom, culturing the cotton seeds by using a half Hoagland's nutrient solution, culturing the cotton seeds by using a half phosphorus-deficient Hoagland's nutrient solution, and finally collecting cotton root exudates; (2) extracting cotton strigolactones from the cotton root exudates collected in the step (1) by adopting an SPE C18 column; and (3) firstly carrying out liquid chromatography separation on the cotton strigolactones obtained in the step (2), and then carrying out mass spectrometry detection. The method can realize high-content collection of endogenous strigolactones of cotton, and has the advantages of high detection accuracy, high sensitivity, simplicity, convenience, rapidness and good selectivity.

Description

Technical Field

[0001] The present invention relates to the technical field of chemical analysis, and in particular to a method for detecting endogenous strigolactones in cotton root exudates. Background Art

[0002] Strigolactones (SLs) are a new class of plant hormones that play an important role in regulating plant growth and development. They also serve as rhizosphere communication signaling molecules, stimulating seed germination in root-parasitic plants and promoting the establishment of beneficial symbioses between arbuscular mycorrhizal fungi and plants. To date, over forty natural SLs have been isolated and identified, and they can be divided into two main categories based on their structural characteristics: typical and atypical. Typical strigolactones have four rings, ABCD, and are further divided into "orobanchol" and "strigol" types based on the conformation of the C ring. Atypical strigolactones possess only a conserved, characteristic D ring structure, while the structures of the groups in other parts vary greatly. Strigol was first extracted from cotton root secretions. Compared with traditional hormones, the physiological concentration of strigolactone in plants is lower, resulting in even smaller amounts secreted by plants. Moreover, the chemical structure of strigolactone is unstable and it is easily degraded in vitro. In addition, due to their similar structures, it is difficult to distinguish between strigolactones with different structures, which restricts in-depth research on the metabolic mechanism of strigolactone in plants and its molecular mechanism of action.

[0003] For ultra-low levels of SLs, efficient collection and detection methods are the key to analysis. Plants will produce more strigolactones under phosphorus deficiency conditions.

[0004] Early quantitative analysis methods for SLs primarily relied on bioactivity assays, based on their physiological functions of promoting parasitic plant seed germination and branching of arbuscular mycorrhizal fungal hyphae. Both of these functions operate within a relatively low concentration range. Some researchers have used the growth of arbuscular symbiotic fungal hyphae or the germination rate of parasitic plant seeds as indicators to measure the relative content of SLs. While this assay can provide a certain degree of accuracy, its sensitivity falls far short of meeting the needs of scientists for quantitative SL detection and is currently used only for preliminary qualitative analysis. Liquid chromatography (LC) has been applied to the quantitative analysis of SLs due to its superior separation and detection performance. However, SLs lack a large conjugated structure, resulting in weak absorption within the UV-visible range, making PDA detection less sensitive. In recent years, with the advancement of mass spectrometry and the maturity of interfacing technologies, liquid chromatography coupled to mass spectrometry has become the most powerful technique for the quantitative analysis of SLs. Mass spectrometry offers unparalleled advantages in quantitative analysis. During mass spectrometry, the target compound is first ionized, and then the ion fragments are separated by a mass analyzer based on their mass-to-charge ratio (m / z) for detection. Detection in multiple reaction monitoring (MRM) mode can largely eliminate impurity interference and improve sensitivity, which is particularly helpful for analyzing SLs with small sample amounts and high structural similarity. Summary of the Invention

[0005] Currently, there is no prior art for the collection and detection of strigolactones in cotton root secretions. The present invention relates to a method for collecting endogenous strigolactones in cotton root secretions, and in particular to a method for increasing the content of collected strigolactones by using stress treatment; the present invention relates to a method for detecting endogenous strigolactones in cotton root secretions, and in particular to a method for detecting strigolactones with different structures in cotton.

[0006] In order to collect and detect low-content SLs in cotton root exudates, the present invention provides the following technical solutions:

[0007] A method for detecting endogenous strigolactones in cotton root exudates, the method comprising the following steps:

[0008] (1) Material culture and collection of cotton root exudates:

[0009] The cotton seeds were soaked in water and germinated in the dark, and then transferred to a culture container with a bottom hole filled with a culture medium. They were first cultured with half Hoagland's nutrient solution, and then cultured with half Hoagland's nutrient solution with phosphorus deficiency. Finally, the cotton root exudates were collected.

[0010] (2) Extraction of cotton strigolactone:

[0011] Extracting cotton strigolactones from the cotton root exudates collected in step (1) using an SPE C18 column;

[0012] (3) Detection of strigolactone in cotton:

[0013] The cotton strigolactone obtained in step (2) is first separated by liquid chromatography and then detected by mass spectrometry.

[0014] Preferably, in step (1), the cotton seeds are soaked in water for 12 hours, and the dark-protected germination time is 2 days.

[0015] Preferably, in step (1), the culture time of the half Hoagland's nutrient solution culture is 14 days; the culture time of the half phosphorus-deficient Hoagland's nutrient solution culture is 7 days.

[0016] Preferably, in step (2), the specific method for extracting cotton strigolactone from the cotton root exudates in step (1) using an SPE C18 column is as follows: first, the SPE C18 column is activated with methanol, and then the methanol is rinsed with ultrapure water, and vacuum filtered with a vacuum pump at a pressure of -20 MPa; then, the cotton root exudates are added to the SPE C18 column and filtered, and ultrapure water is added for rinsing, and then chromatographic grade acetone is added for infiltration, and the filtrate is collected and concentrated to obtain a concentrated solution; and then the concentrated solution is dissolved in acetonitrile and filtered to remove impurities to obtain the cotton strigolactone.

[0017] Preferably, the conditions for liquid chromatography separation in step (3) are:

[0018] The chromatographic column is ACQUITY BEH C18;

[0019] Column temperature: 45°C;

[0020] The mobile phase consists of A and B, where A is a 1‰ formic acid aqueous solution and B is a 1‰ formic acid acetonitrile solution.

[0021] The elution method is gradient elution, as follows:

[0022]

[0023] Preferably, the specific method of mass spectrometry detection is: using multiple reaction monitoring mode for detection in ESI source positive ion mode, the ion source parameters are: capillary voltage 3.1KV; source temperature 150°C; ion source gas 1 50psi; ion source gas 2 50psi; CAD gas 9; using 10 -7The parent ions and daughter ions of Strigone and Strigol were determined respectively, wherein the Strigone selected 345.1>231.1, 345.1>97.0, 345.1>203.0 and 345.1>215.2 with higher responses as qualitative MRM ion channels; the Strigol selected 347.1>233.1, 347.1>97, 347.1>329.1 and 347.1>215.1 with higher responses as qualitative MRM ion channels. The results showed that both the standard and the actual sample could detect the two cotton endogenous SLs, Strigone and Strigol, very well and could be effectively distinguished (Strigone retention time was 5.65 min, Strigol retention time was 5.77 min).

[0024] Beneficial effects of the present invention:

[0025] The advantages of the detection method of the present invention are specifically reflected in: (1) stress treatment is used to increase the content of the collected strigolactones. (2) cotton is cultured in sand culture, and a homemade 50ml perforated centrifuge tube can be used as a culture container and can filter sand to collect root secretions, which has fewer impurities than culture in nutrient soil. No enrichment is required, and a single plant can be tested, solving the problem that it is difficult to obtain a large amount of precious genetically modified plant materials. (3) The precise and efficient liquid phase elution procedure can accurately separate and detect strigolactones of different structures in cotton, and the results are stable. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a front view of a 50ml perforated centrifuge tube according to the present invention;

[0027] Figure 2 for Figure 1 side view;

[0028] Figure 3 The intensity of cotton strigolactones was detected by the present invention through phosphorus deficiency culture, and normal phosphorus was used as a control;

[0029] Figure 4 The TIC and MRM diagrams of the present invention for detecting strigolactones in standard substances such as Strigone and Strigol and actual cotton root exudate samples;

[0030] Figure 5 To compare the TIC and MRM patterns of strigolactones in the patented detection standards such as Strigone and Strigol with those in actual cotton root exudate samples;

[0031] Figure 6To compare the TIC and MRM patterns of Strigone, Strigol and other standard substances detected in the literature and actual cotton root exudate samples. DETAILED DESCRIPTION

[0032] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0033] Example

[0034] 1. Material cultivation and collection of cotton root secretions:

[0035] Cotton seeds were soaked in water for 12 h, then germinated on filter paper in the dark. Two days later, they were transferred to a 50 ml centrifuge tube filled with sand. The bottom of the 50 ml centrifuge tube had four 1.2 mm holes ( Figure 1-2 ) to ensure that the nutrient solution can seep through the holes while the sand does not. After 14 days of incubation with half Hoagland's nutrient solution, switch to half phosphorus-deficient Hoagland's nutrient solution. During this period, water the nutrient solution every two days until the holes seep out. On the fifth day of phosphorus-deficient incubation, flush the nutrient solution in the 50ml centrifuge tube to ensure that the tube is filled with fresh nutrient solution. After another two days of incubation, rinse the centrifuge tube with pure water on the seventh day. Place a new 50ml centrifuge tube at the bottom to collect the flushed root exudates, collecting 25-30ml.

[0036] 2. Extraction of cotton strigolactone:

[0037] Cotton strigolactones were extracted from collected cotton root exudates using an SPE C18 column. 3 ml of methanol was added to the column to activate it, followed by rinsing with 3 ml of ultrapure water. The column was then vacuum-filtered at -20 MPa. 25-30 ml of root exudates were added, filtered, and rinsed with 3 ml of ultrapure water. 2.5 ml of chromatographic-grade acetone was added to the column to infiltrate the mixture, and a collection flask was placed at the bottom to collect the filtrate. The flask was then placed in a vacuum freeze concentrator at 1500 rpm for 2 hours. Once the filtrate was concentrated, 200 μL of 25% acetonitrile was added and shaken to dissolve. The solution was then pipetted through a 0.22 μm filter tube to remove impurities. The solution was then transferred to a sample injection vial and stored at -20°C until testing.

[0038] 3. Detection of strigolactone in cotton

[0039] Liquid chromatography separation was performed using Waters ACQUITY The chromatographic analysis was performed on a BEH C18 column (2.1×150 mm, 1.7 μm). The mobile phase and gradient are shown in Table 1. The column temperature was set to 45°C. Detection was performed in multiple reaction monitoring (MRM) mode using the ESI source in positive ion mode. The ion source parameters were: capillary voltage 3.1 kV; source temperature 150°C; ion source gas 1 at 50 psi; ion source gas 2 at 50 psi; and CAD gas 9. -7 Strigone and Strigol standards of M were used to determine their parent ions and daughter ions, respectively. For Strigone, 345.1>231.1, 345.1>97.0, 345.1>203.0 and 345.1>215.2 with higher responses were selected as qualitative MRM ion channels. For Strigol, 347.1>233.1, 347.1>97, 347.1>329.1 and 347.1>215.1 with higher responses were selected as qualitative MRM ion channels. The results are shown in the figure below. Figure 3-4 As shown in the results, Strigone and Strigol can be detected in both the standard and cotton samples, with good separation effect (Strigone retention time is 5.65min, Strigol retention time is 5.77min) and detection intensity, which can accurately identify and distinguish the different structures of Strigolactone in cotton root secretions.

[0040] The liquid chromatography tandem mass spectrometer was SCIEX QTRAP 5500+;

[0041] The chromatographic column is ACQUITY BEH C18, 2.1×150mm, 1.7um;

[0042] The mobile phase consists of A and B, where A is a 1‰ formic acid aqueous solution and B is a 1‰ formic acid acetonitrile solution.

[0043] The elution method is gradient elution as shown in Table 1:

[0044] Table 1 Gradient elution conditions

[0045]

[0046] Ionization mode: ESI source positive ion mode, scanning mode: MRM;

[0047] The ion source parameters were as follows: capillary voltage 3.1 kV; source temperature 150°C; desolvation gas temperature 250°C; desolvation gas flow rate 1000 L / h; cone gas flow 150 L / h; collision gas (argon) 0.15 ml / min.

[0048] Comparative Example 1: Method described in patent (CN109374765A)

[0049] Column temperature: 30°C

[0050] Liquid phase: The mobile phase consists of A and B, where A is a 0.05% by volume aqueous acetic acid solution and B is a 0.05% by volume acetic acid acetonitrile solution. Table 2 shows the elution conditions.

[0051] Table 2 Elution conditions

[0052]

[0053] Mass spectrometry part: ion source parameters are: capillary voltage 3.3 kV; source temperature 120°C; desolvation gas temperature 400°C; desolvation gas flow rate 800 L / h; cone gas flow 50 L / h; multiplier voltage 650 V; collision gas (argon) 0.18 mL / min.

[0054] Comparative Example 2: Method described in the literature (Confirmation and Quantification of Strigolactones, Germination Stimulants for Root Parasitic Plants Striga and Orobanche, Produced by Cotton)

[0055] Column temperature: 30°C

[0056] Liquid phase: 50-100% MeOH in H2O (0-20 min, linear gradient); flow rate: 0.2 ml min -1

[0057] Mass spectrometry: capillary voltage 3 kV, source temperature 120 ° C, desolvation temperature 350 ° C. Nebulizing gas and desolvation nitrogen flow rates 50 and 600 L / h, respectively

[0058] The present invention establishes a method for collecting and detecting endogenous strigolactones in cotton root secretions. The method can achieve high-content collection of endogenous strigolactones in cotton, has high detection accuracy and sensitivity, and is simple, fast, and has good selectivity. It is of great significance for studying the physiological functions and molecular mechanisms of action of strigolactone compounds. The advantages of the method are specifically reflected in: (1) using stress treatment to increase the content of collected strigolactones. (2) cotton is cultured in sand culture, and a homemade 50ml perforated centrifuge tube can be used as a culture container and to filter sand to collect root secretions, which has fewer impurities than nutrient soil culture. No enrichment is required, and a single plant can be tested, solving the problem that it is difficult to obtain a large amount of precious transgenic plant materials. (3) The precise and efficient liquid phase elution procedure can accurately separate and detect strigolactones of different structures in cotton, and the results are stable.

[0059] Patent (CN 109374765 A) method cannot effectively separate Strigone and other SL (results Figure 5 The literature method (Confirmation and Quantification of Strigolactones, Germination Stimulants for Root Parasitic Plants Striga and Orobanche, Produced by Cotton) had low detection intensity and no SL was detected in the sample (results Figure 6 shown).

Claims

1. A method for detecting endogenous strigolactones in cotton root exudates, characterized in that: The method comprises the following steps: (1) Material culture and collection of cotton root exudates: The cotton seeds were soaked in water and germinated in the dark, and then transferred to a culture container with a bottom hole filled with a culture medium. They were first cultured with half Hoagland's nutrient solution, and then cultured with half Hoagland's nutrient solution with phosphorus deficiency. Finally, the cotton root exudates were collected. (2) Extraction of cotton strigolactone: Extracting cotton strigolactones from the cotton root exudates collected in step (1) using an SPE C18 column; (3) Detection of strigolactone in cotton: The cotton strigolactone obtained in step (2) is first separated by liquid chromatography and then detected by mass spectrometry.

2. The detection method according to claim 1, wherein In step (1), the cotton seeds are soaked in water for 12 hours and germinated in the dark for 2 days.

3. The detection method according to claim 1, wherein In step (1), the culture time of the half Hoagland's nutrient solution culture is 14 days; the culture time of the half phosphorus-deficient Hoagland's nutrient solution culture is 7 days.

4. The detection method according to claim 1, wherein The specific method for extracting cotton strigolactone from the cotton root exudates in step (1) using an SPE C18 column in step (2) is as follows: first, the SPE C18 column is activated with methanol, and then the methanol is rinsed with ultrapure water, and vacuum filtered with a vacuum pump at a pressure of -20 MPa; then, the cotton root exudates are added to the SPE C18 column, filtered, and rinsed with ultrapure water, and then chromatographic grade acetone is added for infiltration, the filtrate is collected, and then concentrated to obtain a concentrated solution; and then the concentrated solution is dissolved in acetonitrile, filtered, and impurities are removed to obtain the cotton strigolactone.

5. The detection method according to claim 1, wherein The conditions for liquid chromatography separation in step (3) are: The chromatographic column is ACQUITY BEH C18; Column temperature: 45°C; The mobile phase consists of A and B, where A is a 1‰ formic acid aqueous solution and B is a 1‰ formic acid acetonitrile solution. The elution method is gradient elution, as follows:

6. The detection method according to claim 1, characterized in that The specific method of mass spectrometry detection is: using multiple reaction monitoring mode for detection in ESI source positive ion mode, the ion source parameters are: capillary voltage 3.1KV; source temperature 150°C; ion source gas 1 50psi; ion source gas 2 50psi; CAD gas 9; using 10 -7 M Strigone and Strigol standards were prepared, and the parent ions and daughter ions of the Strigone and Strigol were determined respectively. Among them, for the Strigone, 345.1>231.1, 345.1>97.0, 345.1>203.0 and 345.1>215.2 with higher responses were selected as qualitative MRM ion channels; for the Strigol, 347.1>233.1, 347.1>97, 347.1>329.1 and 347.1>215.1 with higher responses were selected as qualitative MRM ion channels.

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