Detection and analysis method for endogenous RGFs small peptide in plant sample

Abstract

The invention discloses a method for detecting and analyzing endogenous RGFs small peptides in a plant sample. The detection and analysis method of the endogenous RGFs small peptide in the plant sample comprises the following steps: (1) pretreating a plant sample to be detected; (2) enriching and purifying the pretreated to-be-detected plant sample in the step (1) by adopting a solid-phase extraction technology to obtain an endogenous RGFs small peptide analysis solution of the to-be-detected plant; and (3) detecting and analyzing the to-be-detected plant endogenous RGFs small peptide analysis solution by adopting a liquid chromatography-tandem mass spectrometry method to realize qualitative and quantitative analysis of the plant sample endogenous RGFs small peptide. According to the method, the sample pretreatment process is optimized to efficiently remove impurities, and the advantages of accuracy and high sensitivity of the high-resolution mass spectrum are utilized, so that accurate qualitative and quantitative analysis of trace RGFs small peptide is finally realized, and a reliable technical support is provided for analyzing a molecular mechanism of the RGFs small peptide for regulating and controlling plant growth and development.

Description

Technical Field

[0001] This invention belongs to the fields of biotechnology and analytical chemistry, and relates to a method for detecting and analyzing endogenous RGFs small peptides in plant samples. Background Technology

[0002] Plant peptides, as an important new class of signaling molecules, mediate intercellular communication, which is one of the core mechanisms regulating plant growth, development, morphogenesis, and environmental responses. Root meristem growth factor (RGF), an indispensable small peptide hormone, together with other family members, constitutes a key part of the plant signal transduction network. Studies have shown that in Arabidopsis thaliana... RGF RGF peptides are widely expressed in multiple organs, including roots, stems, leaves, and flowers. Through a sophisticated molecular network, they regulate root tip meristem activity, maintain root tip stem cell homeostasis, and influence root geotropism and lateral root development. Their broad tissue expression also suggests they may play a crucial role in stem and leaf growth regulation, floral organ development, and reproduction. In-depth research on these peptides will not only systematically elucidate the regulatory network of multi-organ growth and development in plants but also promises potential applications in agricultural production through dynamic regulation of stem cell activity, enhanced environmental adaptability, and improved nutrient utilization efficiency.

[0003] According to bioinformatics, members of the RGF family have been reported in species such as Arabidopsis thaliana, rice, and tomato. These small peptides typically consist of 13-18 amino acid residues and exhibit highly conserved domain characteristics: the N-terminus generally contains a conserved DY motif, while the C-terminus carries a PXHN motif. Post-translational modifications in some family members also show high conservation. Accurate detection and analysis of endogenous RGF peptides in plants are indispensable for elucidating the molecular mechanisms by which RGF peptides regulate plant growth and development, and for comprehensively exploring their diverse biological functions. Highly sensitive liquid chromatography-tandem mass spectrometry (LC-MS / MS) has opened up broad prospects for the qualitative and quantitative analysis of endogenous plant peptides. Although some researchers have used nanoliter LC-MS / MS in overexpression... AtRGF AtRGF peptides have been identified in Arabidopsis thaliana immersion culture medium. However, due to the extremely low content of endogenous plant RGF peptides and the severe interference caused by complex plant matrices, it is currently impossible to directly quantify RGF peptides in plant tissue materials, which seriously hinders the research progress on the mechanism of action and function of plant peptides. Therefore, establishing an efficient method for the enrichment, purification, and highly sensitive detection of endogenous plant RGF peptides will provide key technical support for revealing the biological functions of RGF peptides. Summary of the Invention

[0004] The purpose of this invention is to provide a method for detecting and analyzing endogenous RGFs small peptides in plant samples.

[0005] This invention optimizes the sample pretreatment process for efficient impurity removal and utilizes the accuracy and sensitivity of high-resolution mass spectrometry to achieve precise qualitative and quantitative analysis of trace RGF peptides, providing reliable technical support for elucidating their molecular mechanisms of regulating plant growth and development.

[0006] This invention provides a method for detecting and analyzing endogenous RGFs small peptides in plant samples, comprising the following steps:

[0007] (1) Pre-treat the plant samples to be tested; (2) The pretreated plant samples in step (1) were enriched and purified by solid phase extraction to obtain the endogenous RGFs small peptide analysis solution of the plant samples. (3) The endogenous RGFs small peptide analysis solution of the plant to be tested was detected and analyzed by liquid chromatography-tandem mass spectrometry to achieve qualitative and quantitative analysis of endogenous RGFs small peptides in plant samples.

[0008] In the above method, the pretreatment method of the plant sample to be tested in step (1) is as follows: take the plant sample to be tested and process it, use solvent to extract the plant endogenous RGFs small peptides, and remove macromolecular interfering substances at the same time.

[0009] In the above method, the plant sample to be tested is selected from at least one of Arabidopsis thaliana, rice, corn, tomato, sorghum and soybean; The plant sample to be tested is selected from plant tissue material, which is selected from at least one tissue of the root, stem, leaf and flower of the plant.

[0010] In this invention, the plant endogenous RGFs peptides can be currently discovered plant endogenous RGFs peptides, specifically including RGFs peptides of different species predicted by bioinformatics or other methods and / or undiscovered plant endogenous RGFs peptides.

[0011] In the above method, the tissue material of the plant sample to be tested is processed by grinding the plant tissue material into powder in liquid nitrogen; The solvent is selected from at least one of the following: water, a mixed solution of an organic solvent and water, an aqueous solution containing an acidic additive, and a mixed solution of an organic solvent and water containing an acidic additive; wherein the organic solvent is selected from at least one of methanol, acetonitrile, and acetone, and the acidic additive is selected from at least one of formic acid, acetic acid, and trifluoroacetic acid, preferably a formic acid-water mixed solution with a volume fraction of 0.1%. The extraction method is ultrasonic, oscillation and / or low temperature overnight extraction at 4℃~10℃; specifically, it can be ultrasonic extraction, and the ultrasonic time can be 10~60 min, specifically 10 min; The macromolecular interference includes proteins, and the macromolecular interference is removed by at least one of high-temperature denaturation, low-temperature sedimentation, organic solvent sedimentation and ultrafiltration; specifically, it may be high-temperature denaturation followed by low-temperature sedimentation, centrifugation to remove plant residues, collection of supernatant, and organic solvent sedimentation or ultrafiltration. The heating method for high-temperature denaturation is a water bath, oil bath, or metal bath, and the temperature can be 70℃~100℃. The aforementioned low-temperature sedimentation can be at a temperature of -80℃ to 10℃, specifically 4℃; The solvent used for organic solvent sedimentation is at least one of methanol, acetonitrile and acetone, or a mixed solution containing 40% to 90% by volume of the above-mentioned solvent and water. The organic solvent sedimentation temperature can be -80℃ to 10℃, and the sedimentation time can be 5 h to 16 h.

[0012] In the above method, step (2) involves enrichment and purification using solid-phase extraction technology as follows: 1) salt is removed by a first solid-phase extraction, 2) the target peptide eluent is collected by a second solid-phase extraction, and after concentration, the endogenous RGFs small peptide analysis solution of the plant to be tested is obtained.

[0013] In the above method, in step (2)-1), the first solid phase extraction uses a polar or reversed phase solid phase extraction column; based on the hydrophilic and hydrophobic properties of RGFs small peptides, solid phase extraction columns such as C18, C8 and Waters HLB that separate according to the degree of hydrophobicity are selected, with Waters Oasis HLB solid phase extraction column being preferred. In step (2)-2), the second solid-phase extraction uses a mixed-mode weak cation exchange column, specifically a Waters Oasis WCX solid-phase extraction column.

[0014] In the above method, the treatment of the polar or reversed-phase solid-phase extraction column during the first solid-phase extraction includes activation equilibration, sample loading, rinsing, and elution steps; in the first solid-phase extraction (preferably a Waters Oasis HLB solid-phase extraction column), the sample loading solvent is a mixture of water or formic acid and water, specifically a mixture of formic acid and water with a volume fraction of 0.1%; the rinsing solvent is a mixture of water, formic acid, or acetic acid and water, specifically a mixture of formic acid and water with a volume fraction of 0.1%; the elution solvent is at least one organic solvent selected from methanol, acetonitrile, or acetone, or a mixture of the above organic solvents and water, specifically a methanol-water mixture with a volume fraction of 90%. The second solid-phase extraction process for the mixed-mode weak cation exchange column includes activation equilibration, sample loading, rinsing, and elution steps. In the second solid-phase extraction, the sample loading solvent can be a mixture of water, formic acid, or aqueous acetic acid, or a mixture of methanol, acetonitrile, and water (specifically, a mixture of methanol, acetonitrile, and water with a volume fraction of less than 50%, or a mixture containing formic acid and acetic acid additives, specifically a mixture of 0.01% formic acid and water). The rinsing solvent can be a mixture of water, formic acid, or aqueous acetic acid, methanol, or acetonitrile, specifically water followed by methanol. The elution solvent is a mixture of acid and organic solvent; the acid can be at least one of formic acid, acetic acid, and trifluoroacetic acid; the organic solvent is at least one of methanol, acetonitrile, and acetone; the volume fraction of the acid in the elution solvent can be 0.1% to 10%, specifically a 90% methanol aqueous solution containing 2% formic acid.

[0015] In the above method, step (2) further includes drying the analytical liquid of the plant endogenous RGFs small peptides under test with nitrogen gas, and reconstituted and concentrated using a reconstituted solvent to improve the detection sensitivity; The resolution solvent is an aqueous solution of acetonitrile containing acid, an aqueous solution of methanol containing acid, and an aqueous solution containing acid; wherein the acid is formic acid, acetic acid, or trifluoroacetic acid; the volume fraction of the acid in the resolution solvent can be 0-5%; the volume fraction of acetonitrile in the aqueous solution of acetonitrile containing acid can be 0-50%; the volume fraction of methanol in the aqueous solution of methanol containing acid can be 0-50%, and the resolution solvent can specifically be an aqueous solution of formic acid with a volume fraction of 0.1%.

[0016] In the above method, the liquid chromatography-tandem mass spectrometry method uses high performance liquid chromatography, ultra-high performance liquid chromatography, or microliter liquid chromatography, and the mass spectrometry uses quadrupole mass spectrometry, time-of-flight high-resolution mass spectrometry, ion trap mass spectrometry, or ion cyclotron resonance mass spectrometry, specifically Waters ultra-high performance liquid chromatography tandem with SELECT SERIES Cyclic IMS high-resolution mass spectrometry.

[0017] In this invention, the chromatographic column of the liquid chromatography is a column capable of separating target peptides, such as a reversed-phase matrix, a reversed-phase matrix with surface-charged hybridization, a high-strength silica gel, or an amide matrix.

[0018] In this invention, the chromatographic conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry system are as follows: The chromatographic column can be a reversed-phase matrix, a reversed-phase matrix with charged surface hybridization, a high-strength silica gel, or an amide matrix, etc., which can separate the target peptide. Specifically, it can be a Waters Peptide CSH column with a size of 2.1 mm × 100 mm, 1.7 μm, and a column temperature of 35℃. The mobile phase consists of A and B, where A is a 0.1% (v / v) aqueous solution of formic acid and B is a 0.1% (v / v) formic acid-acetonitrile solution. Gradient elution is used. The mass spectrometry conditions in the ultra-high performance liquid chromatography-tandem mass spectrometry system are as follows: flow rate 0.30 mL / min; 0 min, A 99.0%, B 1.0%; 1 min, A 99.0%, B 1.0%; 10 min, A 85.0%, B 15.0%; 12 min, A 0%, B 100.0%; 13 min, A 0%, B 100.0%; 14 min, A 99.0%, B 1.0%; 15 min, A 99.0%, B 1.0%. An ESI source in positive ion mode with a resolution of 60,000 was used, and leucine enkephalin was used as the calibration mass to ensure the quality accuracy of mass spectrometry data acquisition within ±1 ppm. Ion source parameters: capillary voltage: 2.5 kV; cone voltage: 40 V; source temperature: 120℃; desolvation temperature: 350℃; cone gas flow rate: 30 L / h; desolvation gas flow rate: 800 L / h; nebulizer gas pressure: 6 bar.

[0019] In the above method, step (3) uses the external standard method for detection and analysis. The standard used in the external standard method is the SlRGFs small peptide standard, which includes at least one of small peptide standard 1, small peptide standard 2, small peptide standard 3 and small peptide standard 4. The amino acid sequence of peptide standard 1 is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 2 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 3 is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 4 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated.

[0020] In this invention, the proline is modified by hydroxylation so that the hydroxyl group is attached to C4; the tyrosine is modified by sulfonation so that the phenolic hydroxyl group of tyrosine is sulfonated (i.e., tyrosine-O-sulfonation).

[0021] The present invention has the following beneficial effects: This invention establishes a sample pretreatment method for the efficient enrichment and purification of plant endogenous RGFs small peptides. Combined with high-sensitivity liquid chromatography-tandem mass spectrometry detection technology, it provides a reliable solution for the accurate qualitative and quantitative determination of plant endogenous RGFs small peptides in complex plant matrices, and solves the bottleneck problem that plant endogenous small peptides are difficult to detect due to their extremely low abundance and severe matrix interference. Attached Figure Description

[0022] Figure 1 Comparison of enrichment and purification effects under different pretreatment conditions.

[0023] Figure 2 Chromatogram showing the detection limit of SlRGF in plant matrix.

[0024] Figure 3 Chromatograms of endogenous SlRGF1-1 in tomatoes were compared with those of standard samples.

[0025] Figure 4 Chromatograms of tomato endogenous SlRGF1-2 were compared with those of standard samples.

[0026] Figure 5 Chromatograms of endogenous SlRGF2-1 in tomatoes were compared with those of standard samples.

[0027] Figure 6 Chromatograms of endogenous SlRGF2-2 in tomatoes were compared with those of standard samples. Detailed Implementation

[0028] The present invention will be described below through specific embodiments, but the present invention is not limited thereto.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0031] Unless otherwise specified, all concentrations mentioned in the following examples are volume fractions.

[0032] The amino acid sequence of peptide standard 1 in the following examples is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the proline at position 10 is hydroxylated and denoted as SlRGF1-1; the amino acid sequence of peptide standard 2 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the proline at position 10 is hydroxylated and denoted as SlRGF2-1; the amino acid sequence of peptide standard 3 is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated and denoted as SlRGF1-2; the amino acid sequence of peptide standard 4 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated and denoted as SlRGF2-2; all the above SlRGF standards were chemically synthesized by Nanjing Genscript Biotech Co., Ltd.

[0033] The overexpression used in the following examples SlRGF Tomato plants transmit the full-length coding sequence (gene number as follows: SlRGF1 is...) to the plant. Solyc01g090960.1.1 SlRGF2 is Solyc04g051420.2.1 The recombinant plasmid was fused with the CaMV 35S promoter to construct an overexpression vector. After the recombinant plasmid was introduced into Agrobacterium, tomato materials were transformed by Agrobacterium infection to obtain overexpressing plants.

[0034] The liquid chromatography-tandem mass spectrometry system used in the following examples is an ultra-high performance liquid chromatography-tandem SELECTSERIES Cyclic IMS mass spectrometry system (Waters Corporation, USA).

[0035] Example 1: Spiked recovery experiment of SlRGFs small peptides in daylily sample matrix 1) Sample pretreatment of plant tissue materials: To ensure that the selected materials did not contain the target peptide, daylily samples with significant species differences were chosen as the plant matrix. The plant tissue was ground into powder under liquid nitrogen. 200 mg of daylily pollen powder was weighed into a centrifuge tube, and 1 mL of 0.1% formic acid aqueous solution was added. Then, SlRGFs small peptide standards (SlRGF1-1, SlRGF2-1, SlRGF1-2, SlRGF2-2) were added, vortexed to mix thoroughly, sonicated for 10 min, heated at 90℃ for 30 min, and cooled with ice water for 30 min. The mixture was centrifuged at 10,000 g for 15 min at 4℃. 1.5 mL of methanol was added to the supernatant, and the mixture was allowed to settle at 4℃ for 12 h. After settling, the mixture was centrifuged at 10,000 g for 15 min at 4℃, and the supernatant was dried.

[0036] 2) Solid-phase extraction enrichment and purification: The dried extract was reconstituted with 1 mL of 0.1% formic acid aqueous solution and loaded onto an Oasis HLB solid-phase extraction column. After elution with 0.1% formic acid aqueous solution, the eluent was eluted with 90% methanol aqueous solution. The eluent was collected, dried under nitrogen, reconstituted with 1 mL of 0.01% formic acid aqueous solution, and loaded onto an Oasis WCX solid-phase extraction column. After elution with water and methanol, the eluent was eluted with 90% methanol aqueous solution containing 2% formic acid. The eluent was collected, dried under nitrogen, and reconstituted with 100 μL of 0.1% formic acid aqueous solution for analysis by liquid chromatography-tandem mass spectrometry.

[0037] 3) Liquid chromatography methods and mass spectrometry conditions: The chromatographic conditions were as follows: Waters Peptide CSH column, 2.1 mm × 100 mm, 1.7 μm, column temperature 35℃; mobile phase consisted of A and B, where A was a 0.1% (v / v) formic acid aqueous solution and B was a 0.1% (v / v) formic acid acetonitrile solution; gradient elution was used, the elution flow rate was 0.3 mL / min, and the elution sequence was: 0-1-10-12-13-14-15 min, 1%B-1%B-15%B-100%B-100%B-1%B-1%B.

[0038] The mass spectrometry conditions were as follows: ESI source in positive ion mode with a resolution of 60,000 ppm was used, and leucine enkephalin was used as the calibration mass to ensure the mass accuracy of the acquired mass spectrometry data was within ±1 ppm. Ion source parameters: capillary voltage: 2.5 kV; cone voltage: 40 V; source temperature: 120 °C; desolvation temperature: 350 °C; cone gas flow rate: 30 L / h; desolvation gas flow rate: 800 L / h; nebulizer gas pressure: 6 bar. The mass-to-charge ratio information is as follows: [M+4H] of SlRGF1-1 4+ The value is 383.9460, [M+3H] of SlRGF1-2. 3+ The value is 538.2443, [M+4H] of SlRGF2-1. 4+ The value is 388.9616, [M+4H] of SlRGF2-2. 4+ The value is 408.9508.

[0039] 4) Calculation of recovery rate: The peak area of ​​the untreated SlRGF peptide is denoted as P1, and the peak area of ​​the standard of the same concentration after sample pretreatment is denoted as P2. The recovery rate of this method is calculated according to Formula 1: Recovery rate = P1 / P2 (Formula 1). The recovery rate of SlRGF1-1 is 77%, the recovery rate of SlRGF1-2 is 61%, the recovery rate of SlRGF2-1 is 68%, and the recovery rate of SlRGF2-2 is 47%.

[0040] Example 2: Comparison of enrichment and purification effects under different pretreatment conditions To verify the effectiveness of the established pretreatment method, this embodiment conducted a single-factor experiment based on the steps of Example 1. By comparing the detection effects of different pretreatment combinations (protein removal-HLB-WCX, protein removal-WCX, and no protein removal-HLB-WCX), and using the peak area and signal-to-noise ratio of SlRGF1-2 as indicators, the role of protein removal and HLB solid-phase extraction for desalting and impurity removal in the SlRGF detection process was investigated. The results are as follows: Figure 1 As shown. (Summary) Figure 1 The results of peak area and signal-to-noise ratio show that the protein removal-HLB-WCX pretreatment combination can retain the target analyte to the greatest extent (represented by the highest peak area) and effectively remove proteins, salts and other interfering impurities from the sample (represented by the highest signal-to-noise ratio), significantly improving the accuracy and sensitivity of the detection. It is the best combination among the three pretreatment methods. On the other hand, the absence of the protein removal step or the HLB solid-phase extraction desalting and impurity removal step will lead to a decrease in the target analyte response value and increased interference, thereby affecting the detection effect.

[0041] Example 3: Determination of the detection limit under plant substrate method The limit of detection (LOD) is an important indicator for evaluating the sensitivity of an analytical method. The LOD of this method was investigated by following the steps in Example 1. To ensure that the selected material did not contain the target peptide, daylily samples were selected as the plant matrix. The daylily sample material was ground into powder in liquid nitrogen. 200 mg of the plant material powder was accurately weighed and added to 1 mL of 0.1% formic acid aqueous solution, followed by the addition of 10 ng of SlRGFs small peptide. The remaining steps were performed according to the steps in Example 1. The LOD was calculated based on a signal-to-noise ratio of 3. The LOD for SlRGF1-1 was 0.91 ng / μL, for SlRGF1-2 it was 0.84 ng / μL, for SlRGF2-1 it was 0.87 ng / μL, and for SlRGF2-2 it was 0.68 ng / μL. Figure 2 As shown.

[0042] Example 4, Overexpression SlRGF1 Detection and analysis of SlRGF1 in tomato samples with genetic predisposition Following the steps in Example 1, the overexpression SlRGF1Tomato samples containing the SlRGF1 gene were analyzed for SlRGF1 detection. The tomato sample material was ground into powder in liquid nitrogen. 500 mg of the plant material powder was accurately weighed and added to 2 mL of 0.1% formic acid aqueous solution. The mixture was vortexed until homogeneous, sonicated for 10 min, heated at 90℃ for 30 min, and cooled with ice water for 30 min. The mixture was centrifuged at 10,000 g for 15 min at 4℃. 3.0 mL of methanol was added to the supernatant, and the mixture was allowed to settle at 4℃ for 12 h. The mixture was then centrifuged at 10,000 g for 15 min at 4℃, and the supernatant was dried. The remaining conditions were performed according to Example 1. The peak area of ​​SlRGFs in the sample was recorded as P3, and the peak area of ​​the standard at concentration C after sample pretreatment was recorded as P4. The amount of plant sample weighed was W. The content of endogenous SlRGFs in the plant was calculated as (P3 × 100 × C) / (P4 × W) (Formula 2). The detected SlRGFs were quantified according to Formula 2, and the overexpression level was calculated. SlRGF1 The content of SlRGF1-1 in the tomato samples containing the gene was 2.85 ± 0.10 ng / g FW; the content of SlRGF1-2 was 1.40 ± 0.11 ng / g FW. Figure 3-4 As shown.

[0043] Example 5, Overexpression SlRGF2 Detection and analysis of SlRGF2 in tomato samples with genetic predisposition Following the steps in Example 1, the overexpression SlRGF2 Tomato samples containing the gene were analyzed for SlRGF2. The remaining steps were performed according to the procedures in Example 3. The detected SlRGFs were quantified according to Formula 2, and the overexpression was calculated. SlRGF2 The content of -SlRGF2-1 in the tomato sample containing the gene was 12.07 ± 1.43 ng / g FW; the content of SlRGF2-2 was 2.00 ± 0.24 ng / g FW. Figure 5-6 As shown.

Claims

1. A method for detecting and analyzing endogenous RGFs small peptides in plant samples, comprising the following steps: (1) Pre-treat the plant samples to be tested; (2) The pretreated plant samples in step (1) were enriched and purified by solid phase extraction to obtain the endogenous RGFs small peptide analysis solution of the plant samples. (3) The endogenous RGFs small peptide analysis solution of the plant to be tested was detected and analyzed by liquid chromatography-tandem mass spectrometry to achieve qualitative and quantitative analysis of endogenous RGFs small peptides in plant samples.

2. The method according to claim 1, characterized in that, The method for pretreatment of the plant sample to be tested in step (1) is as follows: take the plant sample to be tested and process it by extracting the plant endogenous RGFs small peptides with solvent, while removing macromolecular interfering substances.

3. The method according to claim 1 or 2, characterized in that, The plant samples to be tested are selected from at least one of Arabidopsis thaliana, rice, corn, tomato, sorghum and soybean; The plant sample to be tested is selected from plant tissue material, which is selected from at least one tissue of the root, stem, leaf and flower of the plant.

4. The method according to claim 3, characterized in that, The tissue material of the plant sample to be tested is processed by grinding the plant tissue material into powder in liquid nitrogen; The solvent is selected from at least one of the following: water, a mixed solution of an organic solvent and water, an aqueous solution containing an acidic additive, and a mixed solution of an organic solvent and water containing an acidic additive; wherein the organic solvent is selected from at least one of methanol, acetonitrile, and acetone, and the acidic additive is selected from at least one of formic acid, acetic acid, and trifluoroacetic acid. The extraction method is ultrasonic, vibration and / or low temperature overnight extraction at 4℃~0℃; The macromolecular interfering substances include proteins, and the macromolecular interfering substances are removed by at least one of high-temperature denaturation, low-temperature sedimentation, organic solvent sedimentation, and ultrafiltration.

5. The method according to claim 4, characterized in that, The heating method for high-temperature denaturation is a water bath, oil bath, or metal bath, with a temperature of 70℃~100℃. The temperature of the low-temperature sedimentation is -80℃ to 10℃; The solvent used for organic solvent sedimentation is at least one of methanol, acetonitrile and acetone, or a mixed solution containing 40% to 90% by volume of the above-mentioned solvent and water. The organic solvent sedimentation temperature is -80℃ to 10℃, and the sedimentation time is 5 h to 16 h.

6. The method according to claim 1, characterized in that, In step (2), the steps of enrichment and purification using solid phase extraction technology are as follows: 1) salt is removed by the first solid phase extraction, 2) the target peptide eluent is collected by the second solid phase extraction, and after concentration, the analysis solution of the endogenous RGFs small peptides of the plant to be tested is obtained.

7. The method according to claim 6, characterized in that, In step (2)-1), the first solid-phase extraction uses a polar or reversed-phase solid-phase extraction column; In step (2)-2), the second solid-phase extraction uses a mixed-mode weak cation exchange column.

8. The method according to claim 7, characterized in that, The treatment of the polar or reversed-phase solid-phase extraction column during the first solid-phase extraction includes activation equilibration, sample loading, rinsing, and elution steps. In the first solid-phase extraction, the sample loading solvent is a mixture of water or formic acid and water, the rinsing solvent is a mixture of water, formic acid, or acetic acid and water, and the elution solvent is at least one organic solvent selected from methanol, acetonitrile, or acetone, or a mixture of the above organic solvents and water. The second solid-phase extraction process for the mixed-mode weak cation exchange column includes activation equilibration, sample loading, rinsing, and elution steps. In the second solid-phase extraction, the sample loading solvent is a mixture of water, formic acid, or aqueous acetic acid, or a mixture of methanol or acetonitrile and water. The elution solvent is a mixture of an acid and an organic solvent, wherein the acid is at least one of formic acid, acetic acid, and trifluoroacetic acid, and the organic solvent is at least one of methanol, acetonitrile, and acetone. The volume fraction of the acid in the elution solvent is 0.1% to 10%.

9. The method according to claim 1 or 5, characterized in that, Step (2) also includes drying the analytical liquid of the plant endogenous RGFs small peptides under nitrogen and reconstituted and concentrated using a reconstituted solvent; The resolution solvent is an aqueous solution of acetonitrile containing acid, an aqueous solution of methanol containing acid, and an aqueous solution containing acid; wherein the acid is formic acid, acetic acid, or trifluoroacetic acid; the volume fraction of the acid in the resolution solvent is 0-5%; the volume fraction of acetonitrile in the aqueous solution of acetonitrile containing acid is 0-50%; and the volume fraction of methanol in the aqueous solution of methanol containing acid is 0-50%.

10. The method according to claim 1, characterized in that, In the liquid chromatography-tandem mass spectrometry method, the liquid chromatography is high performance liquid chromatography, ultra-high performance liquid chromatography or microliter liquid chromatography, and the mass spectrometry is quadrupole mass spectrometry, time-of-flight high resolution mass spectrometry, ion trap mass spectrometry or ion cyclotron resonance mass spectrometry. In step (3), the external standard method is used for detection and analysis. The standard used in the external standard method is SlRGFs small peptide standard, which includes at least one of small peptide standard 1, small peptide standard 2, small peptide standard 3 and small peptide standard 4. The amino acid sequence of peptide standard 1 is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 2 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 3 is shown in SEQ ID NO:1: DYLPARTHPPVHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated; the amino acid sequence of peptide standard 4 is shown in SEQ ID NO:2: DYTPARKKPPIHN, wherein the tyrosine at position 2 is sulfonated and the proline at position 10 is hydroxylated.

Images