Method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root exudates

Abstract

The invention relates to the field of chemical analysis, and discloses a method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root exudates, which comprises the following steps: (1) preparing a series of mixed standard mother liquor of 3-indole-acetic acid and 3-indole-formaldehyde with concentration gradient; (2) drawing a standard curve; (3) preparing a root exudate extracting solution; (4) detecting the root exudate extracting solution in the step (3) by adopting a method which is the same as that in the step (2); and (5) comparing the detection result in the step (4) with the standard sample in the step (2), and determining the content of 3-indole-acetic acid and 3-indole-formaldehyde in the root exudates. The method disclosed by the invention has the advantages of simplicity in operation, high detection speed, short time consumption, small amount of required samples, large linear range of determination, good repeatability and good quantitative effect, can be suitable for samples with large concentration fluctuation range, and is particularly suitable for quantitative detection of root exudates.

Description

Technical Field

[0001] The invention relates to the field of chemical analysis, and in particular to a method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root secretions. Background Art

[0002] Plant hormones are a class of small molecule compounds that participate in regulating plant growth and development. 3-Indole-acetic acid and 3-Indole-formaldehyde are important plant hormones that not only play a significant role in the growth and development of plants, but also play an important role in signal transmission between plants. Therefore, in the study of plant-to-plant interactions, the quantitative detection of such auxins secreted by plants is a key step in revealing the mechanism of signal transmission between plants.

[0003] Patent CN112229928A discloses a method for simultaneously quantifying multiple plant hormones in plant samples, which includes the following steps: grinding the sample with liquid nitrogen, adding methanol, water, and formic acid in a volume ratio of (12-15): (4-6): 1 to prepare an extractant for extraction, and using LC-MS / MS detection and analysis under specific chromatographic conditions and mass spectrometry conditions. Although this method can also be applied to the detection of plant hormones in plant samples, it has problems such as a small linear range and a long detection time, and it only targets the detection of plant tissue samples, and does not involve the extraction and detection of effluxed plant hormones. There are still certain limitations in practical applications. Summary of the invention

[0004] The purpose of the present invention is to overcome the problems of the prior art, such as small linear range, long detection time, and unsuitability for extraction and detection of effluxed plant hormones, and to provide a method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant roots. The method has the advantages of simple operation, fast detection speed and short time consumption, and requires a small amount of sample, a large linear range of determination, good repeatability and quantitative effect, and is applicable to samples with a large concentration fluctuation range, thereby providing a reliable method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde for the study of effluxed plant hormones.

[0005] In order to achieve the above object, the present invention provides a method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root exudates, the method comprising the following steps:

[0006] (1) preparing a mixed mother solution of 3-indole-acetic acid and 3-indole-formaldehyde standard substances, and performing gradient dilution to prepare a series of standard solutions with concentration gradients;

[0007] (2) The standard solution is determined under the multiple reaction monitoring mode of a high performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. A standard curve is plotted with the peak area ratio of the target substance measured as the ordinate and the corresponding concentration as the abscissa.

[0008] (3) Add grinding zirconium beads, freeze-dried root exudate powder and extraction solvent into a centrifuge tube in sequence. Then place the centrifuge tube in a grinder for vibration, followed by ultrasonic treatment, standing, and then centrifugation to obtain the supernatant, thus obtaining the root exudate extract.

[0009] (4) The root exudate extract prepared in step (3) is detected by the same method as in step (2).

[0010] (5) Compare the multiple reaction monitoring chromatogram of the root exudate extract obtained in step (4) with the multiple reaction monitoring chromatogram of the standard sample obtained in step (2). Identify 3-indoleacetic acid and 3-indoleformaldehyde according to the retention time and characteristic ions obtained by HPLC-MS. Determine the contents of 3-indoleacetic acid and 3-indoleformaldehyde in the root exudate powder according to the chromatographic peak area of the sample to be measured and the standard curve.

[0011] Preferably, in step (2), gradient elution is carried out with an aqueous formic acid solution and methanol as the mobile phase.

[0012] Preferably, the concentration of the aqueous formic acid solution is 0.05 - 0.3 wt%.

[0013] Preferably, taking the total volume of the mobile phase as 100%, the elution time is t, and the elution conditions include:

[0014] When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95 - 100%, and the content of methanol is 0 - 5%;

[0015] When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%;

[0016] When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 40 - 60%, and the content of methanol is 40 - 60%;

[0017] When 3 < t ≤ 5 min, the content of the aqueous formic acid solution is 10 - 60%, and the content of methanol is 40 - 90%;

[0018] When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 5 - 20%, and the content of methanol is 80 - 95%;

[0019] When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 5 - 95%, and the content of methanol is 5 - 95%;

[0020] When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 80 - 100%, and the content of methanol is 0 - 20%.

[0021] More preferably, based on the total volume of the mobile phase being 100%, the elution time is t, and the elution conditions include:

[0022] When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95%, and the content of methanol is 5%;

[0023] When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%;

[0024] When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 50 - 60%, and the content of methanol is 40 - 50%;

[0025] When 3 < t ≤ 5 min, the content of the aqueous formic acid solution is 10 - 50%, and the content of methanol is 50 - 90%;

[0026] When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 10%, and the content of methanol is 90%;

[0027] When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 10 - 95%, and the content of methanol is 5 - 90%;

[0028] When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 95%, and the content of methanol is 5%.

[0029] Preferably, in step (2), the detection conditions of the liquid chromatography - mass spectrometry include: the chromatographic column is 3μm NX - C18, the flow rate of the mobile phase is 0.1 - 0.5 mL / min, the column temperature is 30 - 50 °C, and the injection volume is 0.5 - 1.5 μL.

[0030] Preferably, in step (3), the size of the grinding zircon beads is 0.5 - 3 mm.

[0031] Preferably, in step (3), two or more kinds of grinding zircon beads with different sizes are used.

[0032] Preferably, in step (3), the extraction solvent is a mixed solution of methanol and water.

[0033] Preferably, in step (3), the volume ratio of methanol to water is (1 - 6):1.

[0034] Preferably, in step (3), the mass - to - volume ratio of the freeze - dried root exudate powder to the extraction solvent is 10 - 30 mg: 300 μL.

[0035] Preferably, in step (3), the centrifuge tube is placed in a grinder and vibrated at 1000-1500 RPM for 5-10 min, then ultrasonicated at 2-6°C for 20-40 min, then allowed to stand at -25 to -15°C for 20-40 min, and then centrifuged at 0-5°C for 10-20 min.

[0036] The method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root secretions of the present invention is simple to operate, has the advantages of fast detection speed and short time consumption, requires a small amount of sample, has a large linear range of measurement, good repeatability, and good quantitative effect, and can be applied to samples with a large concentration fluctuation range, providing a reliable method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde for the study of efflux plant hormones. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the extracted ion chromatogram (XIC) of 3-indole-acetic acid and 3-indole-carboxaldehyde in the standard sample;

[0038] Figure 2 The extracted ion current chromatogram (XIC) of standard samples 3-indole-acetic acid and 3-indole-carboxaldehyde based on 5 mmol / L ammonium acetate as mobile phase A. DETAILED DESCRIPTION

[0039] The specific embodiments of the present invention are described in detail below. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0040] The endpoints and any values ​​of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0041] The method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root exudates of the present invention comprises the following steps:

[0042] (1) preparing a mixed mother solution of 3-indole-acetic acid and 3-indole-formaldehyde standard substances, and performing gradient dilution to prepare a series of standard solutions with concentration gradients;

[0043] (2) measuring the standard solution in a multiple reaction monitoring mode of a high performance liquid chromatography-mass spectrometer, and drawing a standard curve with the peak area ratio of the measured target substance as the ordinate and the corresponding concentration as the abscissa;

[0044] (3) Sequentially add grinding zirconium beads, freeze-dried root exudate powder, and extraction solvent into a centrifuge tube, then place the centrifuge tube into a grinder for vibration, followed by ultrasonic treatment, standing, and then centrifuging to obtain the supernatant, thereby obtaining the root exudate extract;

[0045] (4) Detect the root exudate extract prepared in step (3) using the same method as in step (2);

[0046] (5) Compare the multiple reaction monitoring chromatogram of the root exudate extract obtained in step (4) with the multiple reaction monitoring chromatogram of the standard sample obtained in step (2), identify 3-indole-acetic acid and 3-indole-carboxaldehyde based on the retention time and characteristic ions obtained by high performance liquid chromatography-mass spectrometry, and determine the contents of 3-indole-acetic acid and 3-indole-carboxaldehyde in the root exudate powder according to the chromatographic peak area of the sample to be tested in combination with the standard curve.

[0047] In step (1) of the present invention, the steps for preparing the standard solution include: separately adding 3-indole-acetic acid and 3-indole-carboxaldehyde standards into a methanol solution to prepare a mixed stock solution with a concentration of 1 - 3 μg / mL, and then performing gradient dilution on the stock solution at a 2-fold gradient; wherein, the diluent is a mixture of methanol and water in a ratio of (3 - 5):1.

[0048] In step (2) of the present invention, gradient elution is performed using a formic acid aqueous solution and methanol as the mobile phase; preferably, the concentration of the formic acid aqueous solution is 0.05 - 0.3 wt%; more preferably, the concentration of the formic acid aqueous solution is 0.05 - 0.15 wt%. Using a formic acid aqueous solution with a concentration of 0.05 - 0.15 wt% and methanol as the mobile phase provides better separation effect, can separate 3-indole-acetic acid and 3-indole-carboxaldehyde faster, and shortens the detection time.

[0049] In step (2) of the present invention, based on the total volume of the mobile phase being 100%, the elution time is t, and the elution conditions include:

[0050] When 0 ≤ t ≤ 1 min, the content of the formic acid aqueous solution is 95 - 100%, and the content of methanol is 0 - 5%;

[0051] When 1 < t ≤ 2.5 min, the content of the formic acid aqueous solution is 60 - 95%, and the content of methanol is 5 - 40%;

[0052] When 2.5 < t ≤ 3 min, the content of the formic acid aqueous solution is 40 - 60%, and the content of methanol is 40 - 60%;

[0053] When 3 < t ≤ 5 min, the content of the formic acid aqueous solution is 10 - 60%, and the content of methanol is 40 - 90%;

[0054] When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 5 - 20%, and the content of methanol is 80 - 95%;

[0055] When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 5 - 95%, and the content of methanol is 5 - 95%;

[0056] When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 80 - 100%, and the content of methanol is 0 - 20%.

[0057] In a preferred embodiment, based on the total volume of the mobile phase being 100%, the elution time is t, and the elution conditions include:

[0058] When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95 - 100%, and the content of methanol is 0 - 5%;

[0059] When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%;

[0060] When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 50 - 60%, and the content of methanol is 40 - 50%;

[0061] When 3 < t ≤ 5 min, the content of the aqueous formic acid solution is 10 - 50%, and the content of methanol is 50 - 90%;

[0062] When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 5 - 15%, and the content of methanol is 85 - 95%;

[0063] When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 10 - 95%, and the content of methanol is 5 - 90%;

[0064] When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 90 - 100%, and the content of methanol is 0 - 10%.

[0065] In a further preferred embodiment, based on the total volume of the mobile phase being 100%, the elution time is t, and the elution conditions include:

[0066] When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95%, and the content of methanol is 5%;

[0067] When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%;

[0068] When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 50 - 60%, and the content of methanol is 40 - 50%;

[0069] When 3 < t ≤ 5 min, the content of formic acid aqueous solution is 10 - 50%, and the content of methanol is 50 - 90%;

[0070] When 5 < t ≤ 6 min, the content of formic acid aqueous solution is 10%, and the content of methanol is 90%;

[0071] When 6 < t ≤ 6.5 min, the content of formic acid aqueous solution is 10 - 95%, and the content of methanol is 5 - 90%;

[0072] When 6.5 < t ≤ 7.5 min, the content of formic acid aqueous solution is 95%, and the content of methanol is 5%.

[0073] By adopting the above - preferred elution conditions, better separation effect and faster separation speed can be obtained, making the method for quantitatively detecting 3 - indole - acetic acid and 3 - indole - formaldehyde in plant root exudates have the advantages of short detection time and good quantitative effect.

[0074] In step (2) of the present invention, the detection conditions of the liquid chromatography - mass spectrometry include: the chromatographic column is 3μm NX - C18, the flow rate of the mobile phase is 0.1 - 0.5 mL / min, the column temperature is 30 - 50 °C, and the injection volume is 0.5 - 1.5 μL; preferably, the flow rate of the mobile phase is 0.2 - 0.4 mL / min, the column temperature is 35 - 45 °C, and the injection volume is 0.8 - 1.2 μL.

[0075] In step (2) of the present invention, the detection conditions of the liquid chromatography - mass spectrometry further include: an electrospray ion source is used in the mass spectrometry, the ion source temperature is 450 - 550 °C, the voltage is negative (4300 - 4600) V, and data collection is carried out in the negative ion mode.

[0076] In step (2) of the present invention, the detected ion conditions of the liquid chromatography - mass spectrometry are shown in Table 1.

[0077] Table 1

[0078]

[0079] Note: * is the quantitative ion.

[0080] In a specific embodiment, the formic acid aqueous solution is mobile phase A, the methanol is mobile phase B, the flow rate of the mobile phase is 0.3 mL / min, the chromatographic column is Phenomenex, 3μm NX - C18, 50×2 mm, the column temperature is 40 °C, the injection volume is 1 μL, and the chromatographic detection gradient elution program is set as shown in Table 2:

[0081] Table 2

[0082] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 0 95 5 0.3 1.00 95 5 0.3 2.50 60 40 0.3 3.00 50 50 0.3 5.00 10 90 0.3 6.00 10 90 0.3 6.50 95 5 0.3 7.50 95 5 0.3

[0083] In step (3) of the present invention, the size of the grinding zirconium beads is 0.5-3 mm; preferably, two or more grinding zirconium beads of different sizes are used. Preferably, the centrifuge tube is a sterile and pure centrifuge tube. Using grinding zirconium beads of different particle sizes in the same centrifuge tube can increase the intensity of grinding oscillation, making the sample powder more fully ground and facilitating subsequent extraction operations; using a sterile and pure centrifuge tube can avoid the introduction of contaminants and ensure the accuracy of the detection.

[0084] In step (3) of the present invention, the extraction solvent is a mixed solution of methanol and water; preferably, the volume ratio of methanol to water is (1-6):1; more preferably, the volume ratio of methanol to water is (3-5):1.

[0085] In step (3) of the present invention, the mass volume ratio of the freeze-dried root exudate powder to the extraction solvent is 10-30 mg:300 μL; preferably, the mass volume ratio of the freeze-dried root exudate powder to the extraction solvent is 15-25 mg:300 μL.

[0086] In step (3) of the present invention, the centrifuge tube is placed in a grinder and vibrated at 1000-1500 RPM for 5-10 minutes, then ultrasonicated at 2-6° C. for 20-40 minutes, then allowed to stand at -25 to -15° C. for 20-40 minutes, and then centrifuged at 0-5° C. for 10-20 minutes. By extracting the root secretions by high-speed shaking and low-temperature ultrasonication, plant hormones in the secretions can be fully extracted, especially auxins such as 3-indole-acetic acid and 3-indole-formaldehyde.

[0087] The following examples further illustrate the method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root secretions of the present invention. The examples are implemented based on the technical solution of the present invention, and detailed implementation methods and specific operation processes are given, but the protection scope of the present invention is not limited to the following examples.

[0088] The experimental methods in the following examples, unless otherwise specified, are all conventional methods in the art. The experimental materials used in the following examples, unless otherwise specified, are all commercially available.

[0089] Example 1

[0090] The 3-indole-acetic acid and 3-indole-formaldehyde standard samples used in this example are both liquids with a concentration of 1000 μg / mL, purchased from Tianjin Alta Technology Co., Ltd.

[0091] (1) Preparation of standard solution

[0092] Take 10μL of each of the two standards, add 80μL of diluent to make a 100μg / mL mixed stock solution. Take 10μL of the mixed stock solution, add 90μL of diluent to make a 10μg / mL mixed standard solution; then take 20μL of the mixed standard solution, add 80μL of diluent to make a 2μg / mL mixed standard solution. Take 50 μL of 2 μg / mL mixed standard solution and add 50 μL of diluent to prepare 1 μg / mL mixed standard solution; according to this method, the mother solution is diluted by 2 times gradient to obtain mixed standard solutions with concentrations of 500 ng / mL, 250 ng / mL, 125 ng / mL, 62.5 ng / mL, 31.25 ng / mL, 15.625 ng / mL, 7.8125 ng / mL, 3.90625 ng / mL, 1.953125 ng / mL, 0.9765625 ng / mL and 0.48828125 ng / mL, respectively. The diluent is methanol and water, mixed in a ratio of 4:1.

[0093] (2) Draw a standard curve

[0094] Liquid chromatography conditions:

[0095] Chromatographic column: Phenomenex, 3μm NX-C18, 50×2mm;

[0096] Mobile phase: Phase A is 0.1% formic acid aqueous solution; Phase B is pure methanol solution;

[0097] Flow rate: 0.3 mL / min, column temperature 40 °C, injection volume 1 μL

[0098] Gradient elution program:

[0099] Time (min) Mobile phase A (%) Mobile phase B (%) Flow rate (mL / min) 0 95 5 0.3 1.00 95 5 0.3 2.50 60 40 0.3 3.00 50 50 0.3 5.00 10 90 0.3 6.00 10 90 0.3 6.50 95 5 0.3 7.50 95 5 0.3

[0100] Mass spectrometry conditions:

[0101] ESI source, data collection was performed in negative ion mode, voltage was −4500 V, and ion source temperature was 500 °C;

[0102] Detected ion conditions:

[0103]

[0104] Note: * indicates quantitative ion.

[0105] Take standard samples for multiple reaction monitoring chromatographic detection. Figure 1It is the extracted ion chromatogram (XIC) of standard samples 3-indole-acetic acid and 3-indole-carboxaldehyde;

[0106] The standard solutions of a series of concentrations described in step (1) are measured under the multiple reaction monitoring mode of a high performance liquid chromatography-mass spectrometer, and a standard curve is drawn with the peak area ratio of the measured target as the ordinate and the corresponding concentration as the abscissa. The results are shown in Table 3. It can be seen from the data in Table 3 that the method of the present invention has a large linear range and is applicable to samples with a large concentration fluctuation range.

[0107] Table 3

[0108] Target substance Linear range (ng / mL) Correlation coefficient Linear equations 3-Indole-acetic acid 4-2000 0.99708 y=689.54155x+3529.34125 3-Indole-carboxaldehyde 0.5-250 0.99543 y=2516.27789x+1219.93151

[0109] According to the above method, standard samples were taken for 6 tests every day for three consecutive days. The precision data of the method are shown in Table 4. It can be seen from the data in Table 4 that the method of the present invention has high precision and excellent quantitative detection effect.

[0110] Table 4

[0111] Target substance Intra-day precision (RSD%, N=6) Inter-day precision (RSD%, N=3) 3-Indole-acetic acid 3.73％ 3.14％ 3-Indole-carboxaldehyde 9.65％ 5.44％

[0112] (3) Preparation of root exudate extract

[0113] In a 2mL sterile, pure centrifuge tube, add 0.5mm, 1mm, and 3mm grinding zirconium beads in sequence, then add 20mg of freeze-dried soybean root exudate powder, 240μL of methanol, and 60μL of water, then put the centrifuge tube into a grinder, vibrate at 1200RPM for 6 minutes, then move it to an ultrasonic instrument, and perform ultrasonic treatment at 4°C for 30 minutes, then take out the centrifuge tube, let it stand at -20°C for 30 minutes, and finally centrifuge it at 4°C. After centrifugation for 15 minutes, take the supernatant in the centrifuge tube to obtain the soybean root exudate extract.

[0114] (4) Detecting the root exudate extract prepared in step (3) using the same method as step (2).

[0115] (5) The multiple reaction monitoring chromatogram of the root exudate extract obtained in step (4) is compared with the multiple reaction monitoring chromatogram of the standard sample obtained in step (2), and 3-indole-acetic acid and 3-indole-formaldehyde are identified according to the retention time and characteristic ions obtained by high performance liquid chromatography-mass spectrometry. The contents of 3-indole-acetic acid and 3-indole-formaldehyde in the root exudate powder are determined according to the chromatographic peak area of ​​the sample to be tested combined with the standard curve. The data obtained by the detection are shown in Table 5.

[0116] Verification of spiked recovery rate: A mixed standard was added to the soybean root secretion powder in step (3), and extraction and detection were performed according to the methods of steps (1) to (5) above. The recoveries of 3-indoleacetic acid and 3-indoleformaldehyde are shown in Table 5. From the data in Table 5, it can be seen that the method of the present invention has a good extraction effect on the target substance, thereby making the detection result more accurate.

[0117] Table 5

[0118]

[0119] Repeatability verification: From the same soybean root secretion sample in step (3), 6 portions, each weighing 20 mg, were weighed as 6 parallel samples, and then extracted and tested according to the methods of steps (1) to (5) above. The test data are shown in Table 6. It can be seen from the data in Table 6 that the method of the present invention has good repeatability, and the RSD is less than 10%.

[0120] Table 6

[0121] Indole-3-acetic acid (ng / mL) Indole-3-carboxaldehyde (ng / mL) Parallel sample 1 1502.07 12.18 Parallel sample 2 1631.83 13.85 Parallel sample 3 1583.16 14.03 Parallel sample 4 1643.34 13.91 Parallel sample 5 1290.23 11.75 Parallel sample 6 1607.58 13.24 Repeatability (RSD%, N=6) 8.66 7.41

[0122] Embodiment 2-6

[0123] The test was performed according to the method of Example 1, except that the samples were 6 randomly selected soybean root secretions. The test results are shown in Table 7.

[0124] Table 7

[0125] Example No. 3-Indole-acetic acid (ng / mL) 3-Indole-carboxaldehyde (ng / mL) Example 1 264.12 7.68 Example 2 1527.12 29.88 Example 3 111.69 19.17 Example 4 429.68 20.59 Example 5 523.88 21.69 Example 6 392.23 28.89

[0126] Comparative Example 1

[0127] The detection was carried out according to the method of Example 1, except that in step (2), the mobile phase A was changed to 5 mmol / L ammonium acetate aqueous solution. The detection results are as follows: Figure 2 shown. Figure 2 The extracted ion current chromatogram (XIC) of standard samples 3-indole-acetic acid and 3-indole-formaldehyde is based on 5 mM ammonium acetate as mobile phase A. It can be seen from the figure that the peak shape of 3-indole-acetic acid deteriorates and the peak width increases to 0.7 minutes. Compared with the detection method of the present invention, the use of this mobile phase will make the response of the target substance lower and the peak area accuracy worse, thereby affecting the sensitivity and accuracy of quantification.

[0128] Comparative Examples 2-3

[0129] A sample was randomly selected, and 3 portions of 20 mg of root secretion were accurately weighed, and processed in the following three ways. Control sample: Extraction and detection were performed according to the method of Example 1; Comparative Example 2: Detection was performed according to the method of Example 1, except that in step (3), no ground zirconium beads were added; Comparative Example 3: Detection was performed according to the method of Example 1, except that in step (3), 20 mg of freeze-dried soybean root secretion powder, 240 μL of methanol, and 60 μL of water were directly added to a centrifuge tube, and then centrifuged at 4°C for 15 minutes, and the supernatant in the centrifuge tube was taken to obtain a soybean root secretion extract. The test results are shown in Table 8.

[0130] Table 8

[0131] Control samples Comparative Example 2 Comparative Example 3 3-Indole-acetic acid (ng / mL) 766.01 710.46* 719.42* 3-Indole-carboxaldehyde (ng / mL) 10.87 10.20 10.05*

[0132] Note: * indicates that there is a significant difference between this comparison ratio and the control sample (t.test, p<0.05)

[0133] It can be seen from the data in Table 3-8 that the root exudate powder is ground using two or more grinding zirconium beads of different sizes, and then 3-indole-acetic acid and 3-indole-formaldehyde in the root exudates are extracted by high-speed shaking, low-temperature ultrasound, etc. The operation is simple and the recovery efficiency is high, which is more suitable for the subsequent detection of 3-indole-acetic acid and 3-indole-formaldehyde in plant root secretions.

[0134] In summary, the method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde in plant root secretions according to the present invention has the advantages of fast detection speed and short time consumption, and requires a small amount of sample, a large linear range of measurement, good repeatability, and excellent quantitative effect. It can be applied to samples with a large concentration fluctuation range, and provides a reliable method for quantitatively detecting 3-indole-acetic acid and 3-indole-formaldehyde for the study of efflux plant hormones.

[0135] The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims

1. A method for quantitatively detecting 3-indoleacetic acid and 3-indolecarboxaldehyde in plant root exudates, characterized in that, the method comprises the following steps: (1) Prepare a mixed stock solution of 3-indoleacetic acid and 3-indolecarboxaldehyde standard substances, and gradient dilute to prepare a series of standard solutions with concentration gradients; (2) Measure the standard solutions in the multiple reaction monitoring mode of a high performance liquid chromatography-mass spectrometry (HPLC-MS) instrument. Plot a standard curve with the peak area ratio of the measured target as the ordinate and the corresponding concentration as the abscissa; (3) Sequentially add grinding zirconium beads, freeze-dried root exudate powder, and extraction solvent into a centrifuge tube. Then place the centrifuge tube in a grinder to vibrate, followed by ultrasonic treatment, standing, and then centrifuging to obtain the supernatant to get the root exudate extract; (4) Detect the root exudate extract prepared in step (3) using the same method as in step (2); (5) Compare the multiple reaction monitoring chromatogram of the root exudate extract obtained in step (4) with the multiple reaction monitoring chromatogram of the standard sample obtained in step (2). Identify 3-indoleacetic acid and 3-indolecarboxaldehyde based on the retention time and characteristic ions obtained by HPLC-MS. Determine the contents of 3-indoleacetic acid and 3-indolecarboxaldehyde in the root exudate powder according to the chromatographic peak area of the sample to be tested and the standard curve.

2. The method according to claim 1, characterized in that, in step (2), gradient elution is carried out using an aqueous formic acid solution and methanol as the mobile phase.

3. The method according to claim 2, characterized in that, the concentration of the aqueous formic acid solution is 0.05 - 0.3 wt%.

4. The method according to claim 2 or 3, characterized in that, taking the total volume of the mobile phase as 100%, and the elution time is t. The elution conditions include: When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95 - 100%, and the content of methanol is 0 - 5%; When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%; When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 40 - 60%, and the content of methanol is 40 - 60%; When 3 < t ≤ 5 min, the content of the aqueous formic acid solution is 10 - 60%, and the content of methanol is 40 - 90%; When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 5 - 20%, and the content of methanol is 80 - 95%; When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 5 - 95%, and the content of methanol is 5 - 95%; When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 80 - 100%, and the content of methanol is 0 - 20%.

5. The method according to claim 4, characterized in that, taking the total volume of the mobile phase as 100%, and the elution time is t. The elution conditions include: When 0 ≤ t ≤ 1 min, the content of the aqueous formic acid solution is 95%, and the content of methanol is 5%; When 1 < t ≤ 2.5 min, the content of the aqueous formic acid solution is 60 - 95%, and the content of methanol is 5 - 40%; When 2.5 < t ≤ 3 min, the content of the aqueous formic acid solution is 50 - 60%, and the content of methanol is 40 - 50%; When 3 < t ≤ 5 min, the content of the aqueous formic acid solution is 10 - 50%, and the content of methanol is 50 - 90%; When 5 < t ≤ 6 min, the content of the aqueous formic acid solution is 10%, and the content of methanol is 90%; When 6 < t ≤ 6.5 min, the content of the aqueous formic acid solution is 10 - 95%, and the content of methanol is 5 - 90%; When 6.5 < t ≤ 7.5 min, the content of the aqueous formic acid solution is 95%, and the content of methanol is 5%.

6. The method according to claim 2 or 3, characterized in that In step (2), the detection conditions of the liquid chromatography-mass spectrometry include: the chromatographic column is 3μm NX-C18, the flow rate of the mobile phase is 0.1-0.5mL / min, the column temperature is 30-50℃, and the injection volume is 0.5-1.5μL.

7. The method according to claim 1, characterized in that in step (3), the size of the grinding zirconia beads is 0.5 - 3 mm; Preferably, two or more kinds of grinding zirconia beads with different sizes are used.

8. The method according to claim 1 or 7, characterized in that in step (3), the extraction solvent is a mixed solution of methanol and water; Preferably, the volume ratio of methanol to water is (1 - 6):

1.

9. The method according to claim 1, 7 or 8, characterized in that in step (3), the mass - to - volume ratio of the freeze - dried root exudate powder to the extraction solvent is 10 - 30 mg:300 μL.

10. The method according to claim 1, 7 or 8, characterized in that in step (3), the centrifuge tube is placed in a grinder and vibrated at 1000 - 1500 RPM for 5 - 10 min, then sonicated at 2 - 6 °C for 20 - 40 min, then left standing at - 25 to - 15 °C for 20 - 40 min, and then centrifuged at 0 - 5 °C for 10 - 20 min.

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