A method for determining the content of suberin monomers in the tuberous rhizome suberized tissue of dioscorea

Abstract

This invention discloses a method for determining the content of suberin monomers in the suberized tissue of yam tubers. Suberin is a macromolecular polyester, with monomers mainly covalently linked by oxygen-ester bonds. Its aliphatic compounds are linked to the polysaccharide components in the cell wall by glycosidic bonds and located on the inner side of the cell wall. Therefore, to determine the suberin monomer content, it is necessary to first destroy the plant cell structure, and then hydrolyze the oxygen-ester bonds between the suberin monomers to release the suberin monomer components. This invention first uses ultrasonic pretreatment to destroy plant cells, and then uses lipase hydrolysis combined with ultrasonic assistance and acid hydrolysis (sulfuric acid / methanol) to fully and efficiently hydrolyze the suberin into monomers. The reagents used are relatively safe and non-toxic, and the operation is relatively simple, solving the technical problem of difficult determination of suberin monomer content in the study of suberin metabolism after yam tuber injury healing.

Description

Technical Field

[0001] This invention belongs to the field of plant secondary metabolite determination technology, specifically relating to a method for determining the content of suberin monomers in the suberized tissue of yam tubers. Technical Background

[0002] Suberin, acting as an external barrier in plants, is deposited on the inner side of cell walls and plays a crucial role in responding to mechanical damage. Plant tissue healing is a suberization process involving the extensive deposition of aliphatic compounds and phenolic substances, the main components of suberin, in the cell walls. Therefore, the degree of suberization in the cell walls of damaged tissues is critical to plant wound healing.

[0003] The determination of suberin content in suberized tissues is a crucial foundation for studying suberin synthesis and metabolism during plant wound healing, and it is also an important indicator of the degree of wound healing. However, suberin is a macromolecular polymer in which aliphatic compounds are covalently cross-linked by oxygen-ester bonds. When determining suberin content, chemical methods are usually used to depolymerize suberin, and then the depolymerized monomers are derivatized. The content of suberin monomers is then determined by gas chromatography-mass spectrometry (GC-MS). Therefore, the method of suberin depolymerization is particularly important for accurately understanding the suberin content and monomer types in tissues with different degrees of suberization.Currently, the main methods for depolymerizing suberin are as follows: (1) Depolymerizing Arabidopsis seed coat at 70℃ for 16 h using boron trifluoride and methanol solution (Franke R, Briesen I, Wojciechowski T, Faust A, Yephremov A, Nawrath C, Schreiber L. Apoplastic polyesters in Arabidopsis surface tissues-a typical suberin and a particular cutin[J]. Phytochemistry, 2005, 66(22):2643-2658); (2) Depolymerizing Arabidopsis seed coat at 60℃ for 2 h using sodium methoxide, methanol and methyl acetate solution (Jenkin S, Molina I. Isolation and Compositional Analysis of Plant Cuticle Lipid Polyester Monomers[J]. J Vis Exp, 2015, 22(105):53386); (3) Using sulfuric acid and methanol solution to depolymerize the suberized tissue of kiwifruit fruit injury for 3 h at 85℃ (Wei XP, Lu WJ, Mao LC, Han XY, Wei XB, Zhao XX, Xia M, Xu CJ. ABF2 and MYBtranscription factors regulate feruloyl transferase FHT involved in ABA-mediated wound suberization of kiwifruit[J]. J Exp Bot, 2020, 71(1):305-317); (4) In addition, in order to fully depolymerize the suberin in the suberized tissue of potato tuber injury, Jin et al. (Jin L, Cai Q, Huang W, Dastmalchi K, Rigau J, Molinas M, Figueras M, Serra O, Stark RE. Potato native and wound periderms are differently affected by down-regulationof FHT, a suberin feruloyltransferase[J]. Phytochemistry, (2018, 147:30-48) First, cell walls are hydrolyzed using cellulase and pectinase, and then depolymerized using boron trifluoride and methanol solution.

[0004] In summary, current reports indicate that boron trifluoride and sodium methoxide are flammable, explosive, and toxic, posing a high risk during experimental procedures. Enzyme-assisted hydrolysis methods are time-consuming (requiring 6 days). However, most importantly, when using the above four methods to determine the suberin monomers in the suberized tissue of yam tubers, the important suberin monomers ω-hydroxy acid and α,ω-diacid were not completely detected. Summary of the Invention

[0005] To address the problems existing in the prior art, this invention provides a safe and efficient method for determining the content of suberin monomers in the suberized tissue of yam tubers. The specific steps are as follows:

[0006] (1) Treatment of yam tubers and acquisition of corky tissue

[0007] a) Plant material selection and pretreatment:

[0008] Select uniformly sized, disease-free Chinese yam tubers. Wash the tubers with sterile distilled water and then soak them in a 0.5% sodium hypochlorite solution for 1 minute for sterilization. Rinse them with sterile distilled water and air dry at room temperature. Then, use a sterile scalpel to cut the tubers into 5 cm sections in a clean bench. The cut surfaces at both ends will be the wounds. Immediately place them in a sterile incubator at 20°C and 85% relative humidity to allow the wounds to heal in the dark.

[0009] b) Obtaining cork tissue:

[0010] Two-mm thick tuber discs (peeled) were cut from both ends of the damaged tuber using a sterile scalpel at 0 days and 7 days respectively. These discs were the corky tissue. The 2-mm thick tuber discs obtained at 0 days were taken immediately after the tuber was damaged.

[0011] (2) Determination of suberin accumulation in tissue samples

[0012] To determine whether the obtained samples contained suberin, toluidine blue staining was first used to observe suberin accumulation, and the expression levels of genes related to the synthesis of the main suberin monomers (ω-hydroxy acid and α,ω-dicarboxylic acid) were measured.

[0013] (3) Determination of suberin monomer content in tissue samples

[0014] a) Ultrasonic pretreatment:

[0015] Weigh 0.2 g of tissue sample and grind it into powder under liquid nitrogen. Add 8 mL of acetate buffer solution (pH 5.5, 100 mM sodium acetate) to the sample and sonicate for 30 min to 2 h at a power of 300 to 700 W.

[0016] b) Enzymatic hydrolysis:

[0017] Add 10-50 mg of lipase (Aladdin Biochemical Technology Co., Ltd., derived from Aspergillus niger, ≥120000 U / g) to the buffer solution, stir magnetically at 30℃-50℃ for 24-96 h, then centrifuge at 9000 r / min for 20 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, centrifuge at 9000 r / min for 20 min, repeat the washing 3 times, and blow the precipitate dry with nitrogen.

[0018] c) Ultrasonic-assisted depolymerization:

[0019] Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the dried sample, and add methyl heptadecanoate as an internal standard. Sonicate for 30 min to 2 h at a power of 300 to 700 W, then incubate at 75°C to 85°C for 2 to 4 h. Add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix thoroughly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove moisture from the organic phase, and dry with nitrogen.

[0020] d) Derivatization:

[0021] Add 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine to the depolymerized and dried sample, and incubate at 70°C–110°C for 15–40 min. After cooling to room temperature, dry the derivative under a nitrogen stream, and finally dissolve the dried derivative in 0.5 mL of dichloromethane.

[0022] e) Gas chromatography-mass spectrometry conditions:

[0023] 7890B-5977A GC-MS (Agilent)

[0024] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0025] Injection mode: Splitless

[0026] Carrier gas: High-purity helium

[0027] Flow rate: 1.5 mL / min

[0028] Inlet temperature: 280℃

[0029] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0030] Electron collision energy: 70 Ev

[0031] f) Data Analysis:

[0032] The suberin monomers were identified by matching with the NIST14 database, and the monomer content was calculated using the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0033] The beneficial effects of this invention: Suberin is a macromolecular polyester, with monomers mainly covalently linked by oxygen-ester bonds. Its aliphatic compounds are linked to the polysaccharide components in the cell wall by glycosidic bonds and are located on the inner side of the cell wall. Therefore, to determine the monomeric components of suberin, it is necessary to first destroy the plant cell structure, and then hydrolyze the oxygen-ester bonds between the suberin monomers to release the suberin monomer components. This invention first uses ultrasonic pretreatment to destroy plant cells, and then uses lipase hydrolysis combined with ultrasonic assistance and acid hydrolysis (sulfuric acid / methanol) to fully and efficiently hydrolyze suberin into monomers. The reagents used are relatively safe and non-toxic, and the operation is relatively simple, solving the technical problem of difficult determination of suberin monomer content in the study of suberin metabolism in yam tuber injury healing. Attached Figure Description

[0034] Figure 1 Images of toluidine blue staining of suberized tissue from damaged yam tubers in Example 1. Note: 0d represents staining immediately after tuber damage; 7d represents staining of suberized tissue 7 days after tuber damage; the blue-green substance in the cell wall is suberin.

[0035] Figure 2 In Example 1, the suberized tissue of a damaged yam tuber CYP86A1 (A) and CYP86B1 (B) Gene expression levels. Note: CYP86A1 and CYP86B1 are cytochrome P450 fatty acid ω-hydroxylases, which catalyze the hydroxylation of fatty acids to produce ω-hydroxy acids and α,ω-diacids.

[0036] Figure 3 The values ​​represent the content of ω-hydroxy acid (A) and α,ω-diacid (B) in the suberized tissue of yam tubers in Example 1. Note: ω-hydroxy acid and α,ω-diacid contain different carbon chain lengths, C16, C18, C22, and C24.

[0037] Figure 4The values ​​represent the content of ω-hydroxy acid (A) and α,ω-diacid (B) in the suberized tissue of yam tubers in Example 2. Note: ω-hydroxy acid and α,ω-diacid contain different carbon chain lengths, C16, C18, C22, and C24.

[0038] Figure 5 The content of hexadecane and octadecane ω-hydroxy acids in the corky tissue of yam tubers in Comparative Example 1.

[0039] Figure 6 The content of hexadecane ω-hydroxy acid in the corky tissue of yam tubers in Comparative Example 2.

[0040] Figure 7 The content of hexadecane and octadecane ω-hydroxy acids in the corky tissue of yam tubers in Comparative Example 3 was determined.

[0041] Figure 8 The content of hexadecane, octadecane, and 22-carbon ω-hydroxy acids in the corky tissue of yam tubers in Comparative Example 4 was determined. Detailed Implementation

[0042] The present invention will be further described below with reference to the accompanying drawings and specific embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Those skilled in the art can make some non-essential improvements and adjustments based on the above description of the invention.

[0043] Example 1

[0044] The method for determining the content of suberin monomers in the suberized tissue of yam tubers in this embodiment includes the following steps:

[0045] (1) Treatment of yam tubers and acquisition of corky tissue

[0046] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0047] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0048] (2) The method for determining the accumulation of suberin in tissue samples is as follows: To determine whether the obtained sample contains suberin, toluidine blue staining is first used to observe the accumulation of suberin. Figure 1 ), and determined the expression levels of genes related to the synthesis of major suberin monomers (ω-hydroxy acids and α,ω-dicarboxylic acids). Figure 2 ).

[0049] (3) Determination of suberin monomer content in tissue samples

[0050] a) Ultrasonic pretreatment: Weigh 0.2 g of the cork sample and grind it into powder under liquid nitrogen. Add the sample to 8 mL of acetic acid buffer solution (pH 5.5, 100 mM sodium acetate) and sonicate for 1 h at an ultrasonic power of 700 W.

[0051] b) Enzymatic hydrolysis: Add 40 mg of lipase (Aladdin Biochemical Technology Co., Ltd., derived from Aspergillus niger, ≥120000 U / g) to the buffer solution, stir magnetically at 37℃ for 96 h, then centrifuge at 9000 r / min for 20 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, centrifuge at 9000 r / min for 20 min, repeat the washing 3 times, and blow the precipitate dry with nitrogen.

[0052] c) Ultrasonic-assisted depolymerization: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanate as an internal standard. Ultrasonicate for 1 h at 700 W, then incubate at 75°C for 3 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and dry with nitrogen.

[0053] d) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and incubated at 85 °C for 20 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally dissolved in 0.5 mL of dichloromethane.

[0054] e) GC-MS conditions:

[0055] 7890B-5977A GC-MS (Agilent)

[0056] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0057] Injection mode: Splitless

[0058] Carrier gas: High-purity helium

[0059] Flow rate: 1.5 mL / min

[0060] Inlet temperature: 280℃

[0061] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0062] Electron collision energy: 70 Ev

[0063] f) Data analysis: The main monomers ω-hydroxy acid and α,ω-diacid were identified by matching with the NIST14 database. The monomer content was calculated by the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0064] Results analysis: The above method can detect ω-hydroxy acids and α,ω-diacids with different carbon chain lengths. Figure 3 ).

[0065] Example 2

[0066] The method for determining the content of suberin monomers in the suberized tissue of yam tubers in this embodiment includes the following steps:

[0067] (1) Treatment of yam tubers and acquisition of corky tissue

[0068] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0069] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0070] (2) Determination of suberin monomer content in tissue samples

[0071] a) Ultrasonic pretreatment: Weigh 0.2g of the cork sample and grind it into powder under liquid nitrogen. Add the sample to 8mL of acetate buffer solution (pH 5.5, 100mM sodium acetate) and sonicate for 2 hours at an ultrasonic power of 600W.

[0072] b) Enzymatic hydrolysis: Add 50 mg of lipase (Aladdin Biochemical Technology Co., Ltd., derived from Aspergillus niger, ≥120000 U / g) to the buffer solution, stir magnetically at 45℃ for 48 h, then centrifuge at 9000 r / min for 20 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, centrifuge at 9000 r / min for 20 min, repeat the washing 3 times, and blow the precipitate dry with nitrogen.

[0073] c) Ultrasonic-assisted depolymerization: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanate as an internal standard. Ultrasonicate for 2 h at 600 W, then incubate at 85°C for 2 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and dry with nitrogen.

[0074] d) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and incubated at 70 °C for 40 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally dissolved in 0.5 mL of dichloromethane.

[0075] e) GC-MS conditions:

[0076] 7890B-5977A GC-MS (Agilent)

[0077] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0078] Injection mode: Splitless

[0079] Carrier gas: High-purity helium

[0080] Flow rate: 1.5 mL / min

[0081] Inlet temperature: 280℃

[0082] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0083] Electron collision energy: 70 Ev

[0084] f) Data Analysis:

[0085] The main monomers of suberin, ω-hydroxy acid and α,ω-diacid, were identified by matching with the NIST14 database. The monomer content was calculated based on the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0086] Results analysis: The above method can detect ω-hydroxy acids and α,ω-diacids with different carbon chain lengths. Figure 4 ).

[0087] Comparative Example 1 (without enzyme treatment)

[0088] (1) Treatment of yam tubers and acquisition of corky tissue

[0089] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0090] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0091] (2) Determination of suberin monomer content in tissue samples

[0092] a) Ultrasonic pretreatment: Weigh 0.2 g of the cork sample and grind it into powder under liquid nitrogen. Add the sample to 8 mL of acetate buffer solution (pH 5.5, 100 mM sodium acetate), sonicate for 1 h at an ultrasonic power of 700 W, then centrifuge at 9000 r / min for 20 min. After discarding the supernatant, add 5 mL of ultrapure water, vortex for 2 min, then centrifuge at 9000 r / min for 20 min. Repeat the washing process 3 times and blow the precipitate dry with nitrogen.

[0093] b) Ultrasonic-assisted depolymerization: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanate as an internal standard. Ultrasonicate for 1 h at 700 W, then incubate at 75°C for 3 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and dry with nitrogen.

[0094] c) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and incubated at 85 °C for 20 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally the dried derivative was dissolved in 0.5 mL of dichloromethane.

[0095] d) GC-MS conditions: 7890B-5977A GC-MS (Agilent)

[0096] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0097] Injection mode: Splitless

[0098] Carrier gas: High-purity helium

[0099] Flow rate: 1.5 mL / min

[0100] Inlet temperature: 280℃

[0101] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0102] Electron collision energy: 70 Ev

[0103] e) Data Analysis:

[0104] The main monomers of suberin, ω-hydroxy acid and α,ω-diacid, were identified by matching with the NIST14 database. The monomer content was calculated based on the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0105] Results analysis: The above method can detect hexadecane and octadecane ω-hydroxy acids, while C22 and C24 ω-hydroxy acids and α,ω-diacids were not detected. Figure 5 ).

[0106] Comparative Example 2 (without ultrasound pretreatment)

[0107] (1) Treatment of yam tubers and acquisition of corky tissue

[0108] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0109] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0110] (2) Determination of suberin monomer content in tissue samples

[0111] a) Enzymatic hydrolysis (lipase): Weigh 0.2 g of sample and add it to 8 mL of acetate buffer solution (pH 5.0, 100 mM sodium acetate), then add 40 mg of lipase (Aladdin Biochemical Technology Co., Ltd., derived from Aspergillus niger, ≥120000 U / g), stir magnetically at 37℃ for 96 h, then centrifuge at 9000 r / min for 20 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, then centrifuge at 9000 r / min for 20 min, repeat the washing 3 times, and blow the precipitate dry with nitrogen.

[0112] b) Ultrasonic depolymerization treatment: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanate as an internal standard. Ultrasonicate for 1 h at 700 W, then incubate at 75°C for 3 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and blow dry with nitrogen.

[0113] c) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and incubated at 85 °C for 20 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally the dried derivative was dissolved in 0.5 mL of dichloromethane.

[0114] d) GC-MS conditions:

[0115] 7890B-5977A GC-MS (Agilent)

[0116] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0117] Injection mode: Splitless

[0118] Carrier gas: High-purity helium

[0119] Flow rate: 1.5 mL / min

[0120] Inlet temperature: 280℃

[0121] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0122] Electron collision energy: 70 Ev

[0123] e) Data Analysis:

[0124] The main monomers of suberin, ω-hydroxy acid and α,ω-diacid, were identified by matching with the NIST14 database. The monomer content was calculated based on the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0125] Results analysis: The above method can only detect hexadecane ω-hydroxy acids, while C18, C22, and C24 ω-hydroxy acids and α,ω-diacids were not detected. Figure 6 ).

[0126] Comparative Example 3 (no ultrasound during depolymerization treatment)

[0127] (1) Treatment of yam tubers and acquisition of corky tissue

[0128] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0129] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0130] (2) The method for determining the accumulation of suberin in tissue samples is as follows: To determine whether the obtained sample contains suberin, toluidine blue staining is first used to observe the accumulation of suberin. Figure 1 ), and determined the expression levels of genes related to the synthesis of major suberin monomers (ω-hydroxy acids and α,ω-dicarboxylic acids). Figure 2 ).

[0131] (3) Determination of suberin monomer content in tissue samples

[0132] a) Ultrasonic pretreatment: Weigh 0.2 g of the cork sample and grind it into powder under liquid nitrogen. Add the sample to 8 mL of acetic acid buffer solution (pH 5.5, 100 mM sodium acetate) and sonicate for 1 h at an ultrasonic power of 700 W.

[0133] b) Enzymatic hydrolysis: Add 40 mg of lipase (Aladdin Biochemical Technology Co., Ltd., derived from Aspergillus niger, ≥120000 U / g) to the buffer solution, stir magnetically at 37℃ for 96 h, then centrifuge at 9000 r / min for 20 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, centrifuge at 9000 r / min for 20 min, repeat the washing 3 times, and blow the precipitate dry with nitrogen.

[0134] c) Depolymerization: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanoate as an internal standard. Then incubate at 75°C for 3 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and blow dry with nitrogen.

[0135] d) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and incubated at 85 °C for 20 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally dissolved in 0.5 mL of dichloromethane.

[0136] e) GC-MS conditions:

[0137] 7890B-5977A GC-MS (Agilent)

[0138] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0139] Injection mode: Splitless

[0140] Carrier gas: High-purity helium

[0141] Flow rate: 1.5 mL / min

[0142] Inlet temperature: 280℃

[0143] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0144] Electron collision energy: 70 Ev

[0145] f) Data analysis: The main monomers ω-hydroxy acid and α,ω-diacid were identified by matching with the NIST14 database. The monomer content was calculated by the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0146] Results analysis: The above method can only detect hexadecane and octadecane ω-hydroxy acids, and α,ω-diacids were not detected. Figure 7 ).

[0147] Comparative Example 4 (Treatment with cellulase and pectinase)

[0148] (1) Treatment of yam tubers and acquisition of corky tissue

[0149] a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% sodium hypochlorite solution for 1 minute for sterilization, rinse with sterile distilled water, and air dry at room temperature. Then, use a sterile scalpel to cut the Chinese yam tubers into 5 cm sections in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity in the dark to heal the damage.

[0150] b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

[0151] (2) The method for determining the accumulation of suberin in tissue samples is as follows: To determine whether the obtained sample contains suberin, toluidine blue staining is first used to observe the accumulation of suberin. Figure 1 ), and determined the expression levels of genes related to the synthesis of major suberin monomers (ω-hydroxy acids and α,ω-dicarboxylic acids). Figure 2 ).

[0152] (3) Determination of suberin monomer content in tissue samples

[0153] a) Ultrasonic pretreatment: Weigh 0.2 g of the cork sample and grind it into powder under liquid nitrogen. Add the sample to 8 mL of acetate buffer solution (pH 5.5, 100 mM sodium acetate) and sonicate for 1 h at an ultrasonic power of 700 W.

[0154] b) Enzymatic hydrolysis (cellulase and pectinase): Weigh 0.2 g of sample and add it to 8 mL of acetate buffer (pH 5.0, 100 mM sodium acetate), then add 40 mg of cellulase. Stir magnetically at 37°C for 48 h, then at 44°C for 48 h. Centrifuge at 9000 r / min for 10 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, then centrifuge at 9000 r / min for 10 min. Repeat washing 3 times. Add 8 mL of pectinase buffer (pH = 4.0, 100 mM acetate buffer, 40 mg pectinase) to the washed sample. Stir magnetically at 28°C for 24 h, then at 31°C for 24 h. Centrifuge at 9000 r / min for 10 min, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, then centrifuge at 9000 r / min for 10 min. Repeat washing 3 times. Dry the precipitate with nitrogen.

[0155] c) Ultrasonic-assisted depolymerization: Add 4 mL of sulfuric acid / methanol (5:95, v / v) solution to the precipitate, and add methyl heptadecanate as an internal standard. Ultrasonicate for 1 h at 700 W, then incubate at 75°C for 3 h. Then add 3 mL of 0.9% sodium chloride solution and 8 mL of dichloromethane, vortex to mix evenly, centrifuge at 9000 r / min for 20 min, collect the organic phase, wash twice with 3 mL of 0.5 mol / L sodium chloride solution, add 1 g of anhydrous sodium sulfate to remove water from the organic phase, and dry with nitrogen.

[0156] d) Derivatization: 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine were added to the depolymerized and dried sample, and the mixture was incubated at 85°C for 20 min. After cooling to room temperature, the derivative was dried under a nitrogen stream, and finally the dried derivative was dissolved in 0.5 mL of dichloromethane.

[0157] e) GC-MS conditions:

[0158] 7890B-5977A GC-MS (Agilent)

[0159] Chromatographic column: HP-5MS column (30 m × 0.25 mm × 0.25 μm)

[0160] Injection mode: Splitless

[0161] Carrier gas: High-purity helium

[0162] Flow rate: 1.5 mL / min

[0163] Inlet temperature: 280℃

[0164] Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min.

[0165] Electron collision energy: 70 Ev

[0166] f) Data analysis: The main monomers ω-hydroxy acid and α,ω-diacid were identified by matching with the NIST14 database. The monomer content was calculated by the peak area of ​​the monomers and the peak area of ​​the internal standard (methyl heptadecanoate).

[0167] Results analysis: The above method can only detect ω-hydroxy acids with carbon-16, carbon-18, and carbon-22 atoms; α,ω-diacids were not detected. Figure 8 ).

[0168] The foregoing has shown and described the basic principles and main features of the present invention, as well as its advantages. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A method for determining the content of suberin monomers in the suberized tissue of yam tubers, characterized in that... Includes the following steps: (1) Processing of yam tubers and acquisition of corky tissue; (2) Determination of suberin accumulation in tissue samples; (3) Determination of suberin monomer content in tissue samples, including: a) Ultrasonic pretreatment: Weigh 0.2 g of tissue sample and grind it into powder under liquid nitrogen, then add 8 mL of acetic acid buffer solution and perform ultrasonic treatment; b) Enzymatic hydrolysis: Add 10-50 mg of lipase to the sonicated acetic acid buffer solution system, stir magnetically at 30℃-50℃ for 24-96 h, then centrifuge, discard the supernatant, add 5 mL of ultrapure water, vortex for 2 min, centrifuge again, repeat washing 3 times, and blow the precipitate with nitrogen to obtain a dry sample. c) Ultrasonic-assisted depolymerization: Add 4 mL of a mixed solution of sulfuric acid and methanol to the dry sample, and add methyl heptadecanoate as an internal standard. After ultrasonic treatment and incubation, add 3 mL of 0.9%wt sodium chloride solution and 8 mL of dichloromethane. Vortex and mix evenly. Centrifuge, collect the organic phase, and wash twice with 3 mL of 0.5 mol / L sodium chloride solution. Add 1 g of anhydrous sodium sulfate to remove moisture from the organic phase. Dry with nitrogen to obtain the depolymerized and dried sample. d) Derivatization: Add 100 μL of N,O-bis(trimethylsilyl)trifluoroacetamide (BSTFA) and 100 μL of pyridine to the depolymerized and dried sample, and incubate at 70°C~110°C for 15-40 min. After cooling to room temperature, dry the derivative under a nitrogen stream, and finally dissolve the dried derivative in 0.5 mL of dichloromethane. e) Perform gas chromatography-mass spectrometry analysis; f) Data analysis.

2. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: Step (1), the treatment of yam tubers and the acquisition of corky tissue, includes the following steps: a) Plant material selection and pretreatment: Select uniform-sized, disease-free Chinese yam tubers, wash the tubers with sterile distilled water, soak them in 0.5% wt sodium hypochlorite solution for 1 min for sterilization, rinse with sterile distilled water, air dry at room temperature, and then use a sterile scalpel to cut the Chinese yam tubers into 5 cm stem segments in a clean bench. The cut surfaces at both ends are the wounds. Immediately place them in a sterile incubator at 20℃ and 85% relative humidity to heal the damage in the dark. b) Obtaining corky tissue: 2 mm thick tuber discs were cut from both ends of the damaged tuber using a sterile scalpel at 0 d and 7 d respectively and the skin was removed. These are the corky tissues. The 2 mm thick tuber discs obtained at 0 d were obtained immediately after the tuber was damaged.

3. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The method for determining the accumulation of suberin in the tissue sample in step (2) is as follows: In order to determine whether the obtained sample contains suberin, toluidine blue staining is first used to observe the accumulation of suberin, and the expression levels of genes related to the synthesis of the main monomers of suberin, ω-hydroxy acid and α,ω-dicarboxylic acid, are measured.

4. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The gas chromatography-mass spectrometry conditions for step (3) are as follows: Agilent 7890B-5977A GC-MS (Gas Chromatograph-Mass Spectrometer) Chromatographic column: HP-5MS column, 30 m × 0.25 mm × 0.25 μm Injection mode: Splitless Carrier gas: High-purity helium Flow rate: 1.5 mL / min Inlet temperature: 280℃ Column oven conditions: maintain 80℃ for 1 min, then increase to 180℃ at 10℃ / min, maintain for 0.5 min, then increase to 250℃ at 2℃ / min, maintain for 0.5 min, then increase to 300℃ at 20℃ / min, and maintain for 10 min; Electron collision energy: 70 Ev.

5. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The data analysis method for step (3) is as follows: the suberin monomers are identified by matching with the NIST14 database, and the monomer content is calculated by using the peak area of ​​the monomers and the peak area of ​​the internal standard methyl heptadecanoate.

6. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The acetate buffer solution in step (3) is a 100mM sodium acetate solution with pH=5.

5.

7. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The ultrasonic time during the ultrasonic pretreatment in step (3) is 30 min to 2 h, and the ultrasonic power is 300 to 700 W.

8. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: In step (3), the ultrasonic time for ultrasonic-assisted depolymerization is 30 min to 2 h, the ultrasonic power is 300 to 700 W, and the incubation is carried out at 75°C to 85°C for 2 to 4 h.

9. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: In step (3), during ultrasonic-assisted depolymerization, the volume ratio of sulfuric acid to methanol in the mixed solution of sulfuric acid and methanol is 5:

95.

10. The method for determining the content of suberin monomers in the suberized tissue of yam tubers according to claim 1, characterized in that: The centrifugation conditions were 9000 r / min for 20 min.

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