Device for collecting 2-acetyl-1-pyrroline synthesized by rice root system in situ and use method

Abstract

The invention discloses in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots and a use method of the 2-acetyl-1-pyrroline in the technical field of plant biologication.The device comprises a water culture box, the upper portion of one side of the water culture box is connected with a first transverse straight pipe, and the first transverse straight pipe is communicated with a first vertical pipe through a first connecting bent pipe; the first vertical pipe is connected with the water outlet end of a water suction pump, and the water inlet end of the water suction pump is connected with the liquid inlet through a second transverse straight pipe; the lower part of the other side of the hydroponic box is connected with a third transverse straight pipe, the third transverse straight pipe is communicated with a second vertical pipe through a second connecting bent pipe, and the second vertical pipe is connected into a collecting bottle; a cultivation plate is embedded in the top of the water culture box, cultivation holes are formed in the cultivation plate, and a sealing piece for keeping the cultivation plate closed is further arranged at the joint of the cultivation plate and the top of the water culture box. The collecting device is simple in structure and easy to build, the 2-acetyl-1-pyrroline synthesized by the rice root system is successfully collected, and the problem that the 2-acetyl-1-pyrroline released by the rice root system is easy to volatilize and difficult to store is solved.

Description

Technical Field

[0001] The invention relates to the field of plant biotechnology, and in particular to 2-acetyl-1-pyrroline synthesized by in-situ collection of rice roots and a use method thereof. Background Art

[0002] As a rice variety with unique aromatic characteristics, fragrant rice is widely welcomed in the market. Its high economic value has promoted the development of agricultural economy. Among the various volatile organic compounds that make up the aroma of fragrant rice, 2-acetyl-1-pyrroline is the most important aroma component, with a popcorn-like aroma. Studies have shown that 2-acetyl-1-pyrroline is stored in rice plants in the form of a complex with amylose, and is synthesized in large quantities during the filling period and transported to the grains. 2-Acetyl-1-pyrroline is extremely volatile and is released in large quantities from the grains during the rice cooking process, becoming the main source of rice aroma.

[0003] At present, the collection methods of 2-acetyl-1-pyrroline can be divided into simultaneous distillation extraction and headspace solid phase microextraction according to the different collection materials and processes. The simultaneous distillation extraction method extracts 2-acetyl-1-pyrroline into the organic solvent by heating the organic phase and the aqueous phase at the same time and collecting them by condensation; the headspace solid phase microextraction method uses a fused silica fiber coated with a stationary phase to adsorb 2-acetyl-1-pyrroline that evaporates when the sample is heated. The 2-acetyl-1-pyrroline collected by these two methods needs to be analyzed by gas chromatography-mass spectrometry to determine its specific content. However, the application of the above collection methods in rice is mainly based on leaves and grains.

[0004] The rice root system is not only a key organ for the plant to absorb water and mineral nutrients, but also participates in signal transduction and hormone regulation, responding to environmental changes. Studies have confirmed that the content of 2-acetyl-1-pyrroline synthesis precursors and the expression of 2-acetyl-1-pyrroline synthesis-related genes in the roots are consistent with those in the leaves, and the rice roots can synthesize 2-acetyl-1-pyrroline themselves. The starch content in the root system is low, and 2-acetyl-1-pyrroline is volatile and difficult to store in the root system. The above two methods for collecting 2-acetyl-1-pyrroline are less effective when applied to the collection of 2-AP in the rice root system. When analyzed on the machine, it is often impossible to quantify it because it is below the instrument detection limit. At present, there is no systematic method for collecting 2-acetyl-1-pyrroline in the root system, so the relevant research on the synthesis of 2-acetyl-1-pyrroline in the root system has not been clarified. Research on 2-acetyl-1-pyrroline synthesized by the roots is a key part of revealing the aroma production mechanism of the whole plant, and can provide strong scientific support for enhancing the aroma of rice and improving the quality of rice. Therefore, it is particularly important to establish a collection device for 2-acetyl-1-pyrroline released by rice roots. Summary of the invention

[0005] The purpose of the present invention is to overcome the defects in the prior art and provide a device and a method for collecting 2-acetyl-1-pyrroline synthesized by rice roots in situ, thereby preventing the volatilization of 2-acetyl-1-pyrroline released by the rice roots, facilitating storage, and having a simple device structure and convenient operation.

[0006] The object of the present invention is achieved as follows: a device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots, comprising a hydroponic box, wherein a first horizontal vertical tube is connected to an upper portion of one side of the hydroponic box, the first horizontal vertical tube is connected to a first vertical tube through a first connecting elbow, the first vertical tube is connected to a water outlet of a water pump, and the water inlet of the water pump is connected to a liquid inlet through a second horizontal vertical tube; a third horizontal vertical tube is connected to a lower portion of the other side of the hydroponic box, the third horizontal vertical tube is connected to a second vertical tube through a second connecting elbow, and the second vertical tube is connected to a collection bottle; a cultivation plate is embedded on the top of the hydroponic box, a cultivation hole is opened on the cultivation plate, a sponge for fixing experimental rice is arranged in the cultivation hole, and a sealing member for maintaining airtightness is also arranged at the junction of the cultivation plate and the top of the hydroponic box.

[0007] When the present invention is working, a sealing member is arranged between the cultivation plate and the hydroponic box to ensure the air tightness of the entire device, so that 2-acetyl-1-pyrroline generated by the experimental rice root system is confined within the device, and the hydroponic box is strictly protected from light, so that interference of light on the synthesis of 2-acetyl-1-pyrroline and the starch structure is avoided. Under the sealed condition, 2-acetyl-1-pyrroline released by the experimental rice root system is complexed with starch and stored in the rice nutrient solution, and the collected rice nutrient solution is tested to determine the content of 2-acetyl-1-pyrroline therein.

[0008] Furthermore, the first horizontal straight pipe, the second horizontal straight pipe, the third horizontal straight pipe, the first vertical pipe, the second vertical pipe, the first connecting elbow pipe and the second connecting elbow pipe are all made of polyvinyl chloride.

[0009] Furthermore, a first check valve is provided between the first horizontal straight pipe and the hydroponic box, a second check valve is provided between the third horizontal straight pipe and the hydroponic box, and a flow controller is provided between the second check valve and the second connecting elbow.

[0010] Furthermore, the sponge is in a hollow circular shape that matches the cultivation hole.

[0011] Furthermore, the sealing element is a polyvinyl chloride film.

[0012] Furthermore, the hydroponic box is made of polypropylene and is filled with nutrient solution for rice growth.

[0013] A method for using a device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots, comprising the following steps: (1) Planting rice plants: soak and germinate rice seeds, evenly plant the white rice seeds in a 96-well microplate, and place them in a hydroponic box filled with rice nutrient solution to culture them until they reach the three-leaf and one-heart stage; (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour into boiling water and cook until it becomes clear and transparent, then cool in a water bath, add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g L -1 , using 2.325 mol L -1 The pH value of the nutrient solution was adjusted to 5.4 with sulfuric acid solution. High starch concentrations would form difficult-to-handle precipitates, gels, or uneven suspensions. Low starch concentrations would result in an inability to collect sufficient amounts of volatile gases. The starch concentration reached 2 g L -1 When the rice root system synthesizes 2-acetyl-1-pyrroline, it can ensure the stable complexation between the 2-acetyl-1-pyrroline and starch. (3) Transplanting rice plants: Transplant rice seedlings that have been cultured to the three-leaf and one-heart stage into a culture plate and use a hollow round sponge to fix the rice plants; (4) Adding rice nutrient solution: Open the water pump and the first check valve, and transfer the rice nutrient solution from the liquid inlet to the light-proof hydroponic box until the entire hydroponic box is filled; (5) Sealing treatment: close the first check valve and the second check valve, place the cultivation plate cover on the hydroponic box, and tightly wrap the polyvinyl chloride film around the joint between the cultivation plate and the hydroponic box to ensure the airtightness of the device; (6) Collecting 2-acetyl-1-pyrroline: placing the constructed collection device in a plant incubator or in an environment with suitable temperature for growth; (7) Collect starch solution: Open the second check valve and flow controller and collect 150 mL of starch solution into the collection bottle; (8) Extraction of 2-acetyl-1-pyrroline: using a simultaneous distillation extraction device to concentrate the volatile organic compounds in the starch solution and extract them into the organic phase of dichloromethane; (9) Determination of 2-acetyl-1-pyrroline: Analyze using gas chromatography-mass spectrometry to obtain the content of 2-acetyl-1-pyrroline.

[0014] Compared with the prior art, the present invention has the following beneficial effects: First, the collecting device of the present invention has a simple overall structure and is easy to build. The 2-acetyl-1-pyrroline synthesized by the rice root system is successfully collected through the liquid inlet, the sealing collecting device and the liquid outlet collecting device. Compared with the prior art of obtaining 2-acetyl-1-pyrroline from rice plant leaves or grains, the collecting device of the present invention solves the problem that 2-acetyl-1-pyrroline released by the rice root system is easy to volatilize and difficult to store, and fills the gap in the collection of 2-acetyl-1-pyrroline synthesized by the rice root system; through the collecting device of the present invention, the 2-acetyl-1-pyrroline synthesized by the rice root system can be qualitatively and quantitatively measured by gas chromatography-mass spectrometry, and 2-acetyl-1-pyrroline was detected in the rice nutrient solution of the aromatic rice variety Nanjing 9108, and the release rate was 6.295 ng plant -1 d -1 .

[0015] Second, the collection device of the present invention strictly protects the hydroponic box from light and seals it, which can prevent the starch structure from being broken by light, so that it still has the ability to complex with 2-acetyl-1-pyrroline; the sealing treatment allows 2-acetyl-1-pyrroline to accumulate in the hydroponic box, increasing the possibility of its complexation with starch. After 24 hours of collection, the presence of 2-acetyl-1-pyrroline can be detected in the starch nutrient solution in which the roots of the fragrant rice are immersed. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The present invention is a schematic structural diagram of a device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots.

[0017] Figure 2 This is a gas chromatogram obtained after GC-MS analysis of the starch nutrient solution collected by the device of the present invention.

[0018] Figure 3 The 2-acetyl-1-pyrroline release rate is obtained by measuring and analyzing the starch-containing nutrient solutions with different treatments collected by the device of the present invention through gas chromatography-mass spectrometry.

[0019] Among them, 1 is a liquid inlet, 2 is a second horizontal straight pipe, 3 is a water pump, 4 is a first vertical pipe, 5 is a first connecting elbow, 6 is a first horizontal straight pipe, 7 is a first check valve, 8 is a hydroponic box, 9 is a cultivation plate, 10 is a sealing member, 11 is an experimental rice, 12 is a sponge, 13 is a rice nutrient solution, 14 is a second check valve, 15 is a third horizontal straight pipe, 16 is a flow controller, 17 is a second connecting elbow, 18 is a second vertical pipe, and 19 is a collecting bottle. DETAILED DESCRIPTION Example

[0020] like Figure 1The device shown in the figure is for collecting 2-acetyl-1-pyrroline synthesized by rice roots in situ, comprising a hydroponic box 8, a first horizontal pipe 6 is connected to the upper part of one side of the hydroponic box 8, the first horizontal pipe 6 is connected to the first vertical pipe 4 through the first connecting elbow 5, the first vertical pipe 4 is connected to the water outlet of the water pump 3, and the water inlet of the water pump 3 is connected to the liquid inlet through the second horizontal pipe 2; a third horizontal pipe 15 is connected to the lower part of the other side of the hydroponic box 8, the third horizontal pipe 15 is connected to the second vertical pipe 18 through the second connecting elbow 17, and the second vertical pipe 18 is connected to the collecting bottle 19; a cultivation plate 9 is embedded on the top of the hydroponic box 8, and a cultivation hole is opened on the cultivation plate 9, and a sponge 1 for fixing the experimental rice 11 is arranged in the cultivation hole 2, the sponge 12 is a hollow round shape adapted to the cultivation hole, and a polyvinyl chloride film seal 10 is also provided at the junction of the cultivation plate 9 and the top of the hydroponic box 8 to maintain airtightness; the first horizontal straight tube 6, the second horizontal straight tube 2, the third horizontal straight tube 15, the first vertical tube 4, the second vertical tube 18, the first connecting elbow 5 and the second connecting elbow 17 are all made of polyvinyl chloride; a first check valve 7 is provided between the first horizontal straight tube 6 and the hydroponic 8, a second check valve 14 is provided between the third horizontal straight tube 15 and the hydroponic box 8, and a flow controller 16 is provided between the second check valve 14 and the second connecting elbow 17; the hydroponic box 8 is made of polypropylene, and a rice nutrient solution 13 for rice growth is poured into the hydroponic box 8, and a concentration of 2 g L is added to the nutrient solution 13 -1 High concentrations of starch will form difficult-to-handle precipitates, gels or uneven suspensions, while low concentrations of starch will result in an inability to collect sufficient amounts of volatile gases. When the starch concentration reaches 2 g L -1 At the same time, it can ensure that the 2-acetyl-1-pyrroline synthesized by the rice root system is stably complexed with starch.

[0021] The device can successfully collect 2-acetyl-1-pyrroline released by rice roots, and obtain starch nutrient solution with stably complexed 2-acetyl-1-pyrroline, which is convenient for further analysis and determination. Example

[0022] A method for using a device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots, comprising the following steps: (1) Planting rice plants: soaking and germinating rice seeds, planting the white rice seeds evenly in a 96-well microplate, placing them in a hydroponic box 8 filled with a rice nutrient solution 13 and culturing them until they reach the three-leaf and one-heart stage; (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour into boiling water and cook until it becomes clear and transparent, then cool in a water bath, add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g L-1 , using 2.325 mol L -1 The pH value of the nutrient solution was adjusted to 5.4 with sulfuric acid solution; (3) Transplanting rice plants: transplant rice seedlings that have been cultured to the three-leaf and one-heart stage to the culture plate 9, and use a hollow round sponge 12 to fix the rice plants; (4) Adding rice nutrient solution: Open the water pump 3 and the first check valve 7, and transport the rice nutrient solution 13 from the liquid inlet 1 to the light-proof hydroponic box 8 until the entire hydroponic box 8 is filled; (5) Sealing treatment: close the first check valve 7 and the second check valve 14, place the cultivation plate 9 on the hydroponic box 8, and tightly wrap the polyvinyl chloride film around the joint between the cultivation plate 9 and the hydroponic box 8 to ensure the airtightness of the device; (6) Collecting 2-acetyl-1-pyrroline: placing the constructed collection device in a plant incubator or in an environment with suitable temperature for growth; (7) Collecting starch solution: After culturing for 24 h, open the second check valve 14 and the flow controller 16, and collect 150 mL of starch solution into the collection bottle 19; (8) Extraction of 2-acetyl-1-pyrroline: using a simultaneous distillation extraction device to concentrate the volatile organic compounds in the starch solution and extract them into the organic phase of dichloromethane; (9) Determination of 2-acetyl-1-pyrroline: Analyze using gas chromatography-mass spectrometry to obtain the content of 2-acetyl-1-pyrroline.

[0023] Specifically, the rice roots were collected after soaking and growing, and the water containing 2 g L -1 The starch-containing rice nutrient solution was measured and analyzed by gas chromatography-mass spectrometry, and a blank control group and an experimental group were set up to collect and determine 2-acetyl-1-pyrroline.

[0024] After comparing the results, Figure 2 , 3 As shown, Figure 2 The gas chromatogram obtained after GC-MS analysis of the collected starch nutrient solution, in the blank control group nutrient solution without starch and plants, no 2-acetyl-1-pyrroline was detected; in the experimental group one nutrient solution with starch but no plants, no 2-acetyl-1-pyrroline was detected; in the experimental group two nutrient solution with non-fragrant rice plants and starch, no 2-acetyl-1-pyrroline was detected; in the experimental group three nutrient solution with fragrant rice plants and starch, the absorption peak of 2-acetyl-1-pyrroline was detected.

[0025] Figure 3The 2-acetyl-1-pyrroline release rate was obtained by measuring and analyzing the starch-containing nutrient solutions collected under different treatments by gas chromatography-mass spectrometry. In the blank control nutrient solution without starch and plants, the 2-acetyl-1-pyrroline content was almost 0; in the nutrient solution of the experimental group 1 with starch but no plants, the 2-acetyl-1-pyrroline content was almost 0; in the nutrient solution of the experimental group 2 with non-fragrant rice plants and starch, the 2-acetyl-1-pyrroline content was almost 0; in the nutrient solution of the experimental group 3 with fragrant rice plants and starch, the presence of 2-acetyl-1-pyrroline was detected, and the 2-acetyl-1-pyrroline release rate was 6.295 ng plant -1 d -1 .

[0026] The present invention is not limited to the above-mentioned embodiments. On the basis of the technical solution disclosed in the present invention, technicians in this field can make some substitutions and deformations to some technical features therein according to the disclosed technical content without creative labor, and these substitutions and deformations are all within the protection scope of the present invention.

Claims

1. A device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots, characterized in that: The invention comprises a hydroponic box, wherein a first horizontal vertical tube is connected to an upper part of one side of the hydroponic box, the first horizontal vertical tube is connected to a first vertical tube through a first connecting elbow, the first vertical tube is connected to a water outlet of a water pump, and a water inlet of the water pump is connected to a liquid inlet through a second horizontal vertical tube; a third horizontal vertical tube is connected to a lower part of the other side of the hydroponic box, the third horizontal vertical tube is connected to a second vertical tube through a second connecting elbow, and the second vertical tube is connected to a collecting bottle; a cultivation plate is embedded on the top of the hydroponic box, a cultivation hole is provided on the cultivation plate, a sponge for fixing experimental rice is provided in the cultivation hole, and a sealing member for maintaining airtightness is also provided at the joint between the cultivation plate and the top of the hydroponic box.

2. The device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots according to claim 1, characterized in that: The first horizontal straight pipe, the second horizontal straight pipe, the third horizontal straight pipe, the first vertical pipe, the second vertical pipe, the first connecting elbow pipe and the second connecting elbow pipe are all made of polyvinyl chloride.

3. The device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots according to claim 2, characterized in that: A first check valve is provided between the first horizontal straight pipe and the hydroponic box, a second check valve is provided between the third horizontal straight pipe and the hydroponic box, and a flow controller is provided between the second check valve and the second connecting elbow.

4. The device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots according to claim 1, characterized in that: The sponge is in a hollow circular shape that matches the cultivation hole.

5. The device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots according to claim 1, characterized in that: The sealing element is a polyvinyl chloride film.

6. The device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots according to any one of claims 1 to 5, characterized in that: The hydroponic box is made of polypropylene and is filled with nutrient solution for rice growth.

7. A method for using a device for in-situ collection of 2-acetyl-1-pyrroline synthesized by rice roots, characterized in that: The steps include: (1) Planting rice plants: soak and germinate rice seeds, evenly plant the white rice seeds in a 96-well microplate, and place them in a hydroponic box filled with rice nutrient solution to culture them until they reach the three-leaf and one-heart stage; (2) Preparation of rice nutrient solution: Add a small amount of deionized water to starch to form a suspension, pour into boiling water and cook until it becomes clear and transparent, then cool in a water bath, add N, P, K, Ca, Mg, SiO2, Mn, Mo, B, Zn, Cu, and Fe elements according to the rice nutrient solution formula determined by the International Rice Research Institute, and add deionized water until the starch concentration reaches 2 g L -1 , using 2.325 mol L -1 The pH value of the nutrient solution was adjusted to 5.4 with sulfuric acid solution; (3) Transplanting rice plants: Transplant rice seedlings that have been cultured to the three-leaf and one-heart stage into a culture plate and use a hollow round sponge to fix the rice plants; (4) Adding rice nutrient solution: Open the water pump and the first check valve, and transfer the rice nutrient solution from the liquid inlet to the light-proof hydroponic box until the entire hydroponic box is filled; (5) Sealing treatment: close the first check valve and the second check valve, place the cultivation plate cover on the hydroponic box, and tightly wrap the polyvinyl chloride film around the joint between the cultivation plate and the hydroponic box to ensure the airtightness of the device; (6) Collecting 2-acetyl-1-pyrroline: placing the constructed collection device in a plant incubator or in an environment with suitable temperature for growth; (7) Collect starch solution: Open the second check valve and flow controller and collect 150 mL of starch solution into the collection bottle; (8) Extraction of 2-acetyl-1-pyrroline: using a simultaneous distillation extraction device to concentrate the volatile organic compounds in the starch solution and extract them into the organic phase of dichloromethane; (9) Determination of 2-acetyl-1-pyrroline: Analyze using gas chromatography-mass spectrometry to obtain the content of 2-acetyl-1-pyrroline.

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