Plant leaf original state rapid freezing sampling device, sampling system and sampling method

Abstract

The present invention provides a device and a sampling system and a sampling method for rapidly freezing and sampling plant leaves in their original state. The sampling device includes at least one device main body, a control system and a device peripheral gas circuit. The device main bodies are connected in series in the device peripheral gas circuit, and the device peripheral gas circuit is connected to the control system; each device main body includes a device bracket, and a semi-open leaf chamber assembly, a freezing hammer, a freezing box and a pneumatic driving structure mounted on the device bracket. The freezing hammer is located directly above the semi-open leaf chamber assembly, the freezing box is installed below the semi-open leaf chamber assembly, and the pneumatic driving structure is connected to the freezing hammer to drive the freezing hammer to sample into the semi-open leaf chamber assembly to obtain a sample, and the sample falls downward into the freezing box for rapid freezing. The present invention greatly reduces the sampling time, improves the sampling accuracy and freezing reliability, and the device structure is compactly designed and has strong scalability. Multiple machines can be connected in parallel or in series according to experimental needs, so as to achieve high-throughput labeled sampling.

Description

Technical Field

[0001] The present invention relates to the field of plant leaf sampling, and particularly relates to a device, a system and a method for quickly freezing and sampling plant leaves in their original state. Background Art

[0002] Metabolism is the basis for organisms to carry out life activities. Metabolic profiling analysis refers to an experimental method that uses chromatography-mass spectrometry coupling technology to collect the metabolic profiles of samples, compare the contents of metabolites in different groups of samples, identify differentially expressed metabolites, and further explore the metabolic pathways among the differentially expressed metabolites. The metabolic flux analysis technology refers to using isotope labeling ( 13 C, 15 N or 2 H, etc.) means to quantitatively track the dynamic flow direction and change law of heavy isotopes in cells over time, so as to elaborate the flux distribution of the intracellular metabolic network and biological problems related to functions.

[0003] At present, these two types of technologies have been widely applied to the research of plant basic metabolism. For the photosynthetic metabolic flux analysis of plant leaves, usually 13 CO2 is used as the isotope-labeled gas, multiple labeled samples are taken according to the length of the labeling time, and then the abundance of 13 C in various photosynthesis-related intermediate metabolites is analyzed by a high-resolution mass spectrometer to obtain the change pattern with the increase of the labeling time. Finally, the reaction rate of each metabolic reaction is fitted by relying on the metabolic pathway model, which is the metabolic flux.

[0004] Different from animals and microorganisms, the basic metabolism of plant leaves is very sensitive to environmental changes. The isotope labeling experiment of plant leaves has high requirements for environmental stability and the sampling process, especially for the photosynthesis-related intermediate metabolites. Taking the light condition as an example, within 100 ms when the light intensity changes, the contents of energy substrates for photosynthesis, such as ATP and NADPH, will change. Within 2 s when the light intensity changes, the concentrations of intermediate metabolites participating in the Calvin-Benson cycle will be affected (Stitt and Zhu, 2014). When the light intensity changes for more than one minute, the photosystem of chloroplasts will undergo state transitions, and the enzyme activities of functional enzymes in the Calvin-Benson cycle will also show adaptive changes (Eberhard, et al., 2008). Therefore, for the research of plant leaf basic metabolism, whether it is metabolic profiling or metabolic flux experiments, there are high time and error requirements for sampling.

[0005] Currently, the completion of such experiments mainly relies on manual operation with some simple assembly tools. The manual sampling method mainly depends on some simple assembly tools, and each step of the labeling experiment is completed manually (Heise, et al., 2014; Ma, et al., 2014; Ma, et al., 2017). There are disadvantages such as long sampling time, difficult homogenization in sample processing, and large systematic errors, which are not conducive to the analysis of fine plant basic metabolic fluxes, especially photosynthetic metabolic fluxes. In addition, some existing sampling instruments (Arrivault, et al., 2017; Badger, et al., 1984; Caemmerer and Edmondson, 1986) have problems such as cumbersome operation, low sampling throughput, and incomplete freezing during sampling, making it difficult to meet the requirements of realistic isotope labeling experiments.

[0006] In the prior art, the sampling time of the plant leaf original state sampling device and sampling method cannot achieve a better short-time effect. However, for the sampling of plant leaf original state, there is a qualitative difference between long and short sampling times. The main reasons are as follows:

[0007] The metabolic state of plant leaves responds quickly to environmental changes. However, in actual operation, the time required for sampling cannot be infinitesimal. We need to determine the supremum of this time for how short a time sampling can be completed without significantly affecting the metabolic state of the plant. Using the analysis method of linear time-invariant systems, we can calculate the theoretical upper limit value of this technical index (sampling time, τs). According to our experimental results, the response characteristic time (τ p ) of rice leaves to light intensity changes is about 10 seconds. According to the Nyquist-Shannon sampling theorem, in order to obtain all the information carried by the system, the sampling time interval (τ i ) should not exceed 1 / 2 of the characteristic time of the system.

[0008]

[0009] That is to say, for a leaf with a metabolically changing state, the sampling interval needs to be less than 5 seconds for the sampling result to truly reflect the changes of the leaf metabolism system. Similarly, according to the sampling theorem, the system change process within the required sampling interval can be reasonably approximated as several linear processes. In this linear process, the proportion of the sampling process (τ s ) itself in the sampling interval (τ i ) is the proportion of the systematic error caused by the sampling process in the true metabolic change of the leaf in response to the experimental conditions in the total measured metabolic change. We require that the influence of the systematic error should not affect the statistical significance of the experimental results. Therefore, the sampling process (τ s) should be at most 5% of the sampling interval (τ i )。

[0010]

[0011] From this, the acceptable upper limit of the sampling time (τ s ) can be calculated to be 0.25 seconds. Although the above analysis is based on the dynamic changes of the system, it is also applicable to the steady-state situation.

[0012] In previous literature reports, the sampling of metabolic profiles was usually done manually (Arrivault, et al., 2019; Mallmann, et al., 2014; Sommer, et al., 2012; Turner, et al., 2016; Wang, et al., 2014). According to the above analysis, it can be seen that the time consumed by manual sampling is much greater than the acceptable time upper limit (0.25 seconds), which means that the experimental data obtained by manual sampling will inevitably introduce systematic errors and cause the experimental results to deviate more or less from the true situation. And previous metabolic flux experiments were all carried out through some relatively simple self-made leaf chambers to complete isotope labeling and sampling (Heise, et al., 2014; Ma, et al., 2017; Szecowka, et al., 2013; Xu, et al., 2021). For the sampling process, some used manual sampling and some used some mechanical-assisted sampling (the sampling time was about 0.1 - 0.5 seconds). For simple leaf chambers, each time only one leaf could be labeled and sampled, which would seriously affect the experimental progress and the repeatability of the experiment for metabolic flux experiments that require a large number of labeling time points and biological samples. This device can achieve high-throughput labeling and sampling with multi-machine expansion for the first time while ensuring the sampling time.

[0013] In view of this, those skilled in the art have designed a device, a sampling system, and a sampling method for rapid cryogenic sampling of plant leaves in their original state in order to overcome the above technical problems. Summary of the Invention

[0014] The technical problem to be solved by the present invention is to provide a device, a sampling system, and a sampling method for rapid cryogenic sampling of plant leaves in their original state in order to overcome the defects in the prior art that the sampling of plant leaves has high time and error requirements, is cumbersome to operate, has low sampling throughput, and is not thoroughly frozen during sampling.

[0015] The present invention solves the above technical problems through the following technical solutions:

[0016] A rapid cryogenic sampling device for the original state of plant leaves, characterized in that the rapid cryogenic sampling device for the original state of plant leaves includes at least one device main body, a control system and a device peripheral air circuit, the device main bodies are connected in series in the device peripheral air circuit, and the device peripheral air circuit is connected to the control system;

[0017] Each of the device main bodies includes a device bracket, and a semi-open leaf chamber assembly, a freezing hammer, a freezing box and a pneumatic driving structure installed on the device bracket. The freezing hammer is located directly above the semi-open leaf chamber assembly, the freezing box is installed below the semi-open leaf chamber assembly, and the pneumatic driving structure is connected to the freezing hammer to drive the freezing hammer to sample into the semi-open leaf chamber assembly to obtain a sample, and the sample falls downward into the freezing box for rapid freezing.

[0018] According to an embodiment of the present invention, the semi-open leaf chamber assembly includes an upper leaf chamber with a through hole in the middle, a lower leaf chamber with a through hole in the middle and a sealing ring. The upper leaf chamber and the lower leaf chamber are stacked up and down, the sealing ring is clamped between the upper leaf chamber and the lower leaf chamber, a plant leaf is clamped in the through hole, and a transparent film is covered on the upper surface of the upper leaf chamber and the lower surface of the lower leaf chamber;

[0019] Air inlet holes are provided on the side parts of the same side of the upper leaf chamber and the lower leaf chamber, air flow enters the leaf chamber from the air inlet holes, a first air outlet hole is opened on the side part of the other side of the lower leaf chamber, and a second air outlet hole is opened on the side part of the other side of the upper leaf chamber, and the first air outlet hole and the second air outlet hole are communicated with each other.

[0020] According to an embodiment of the present invention, an installation platform is provided on the device bracket, the semi-open leaf chamber assembly is fixed on the installation platform, the pneumatic driving structure includes a high-speed cylinder and a connecting rod, the high-speed cylinder is installed at the lower part of the device bracket, and the semi-open leaf chamber assembly and the freezing hammer are connected through a precision guide post;

[0021] The connecting rod passes through the semi-open leaf chamber assembly, is connected to the freezing hammer at one end, and is connected to the high-speed cylinder at the other end, and the high-speed cylinder is used to drive the freezing hammer to move up and down.

[0022] According to an embodiment of the present invention, the designed stroke of the high-speed cylinder is 10 cm, and the time taken for a one-way stroke is less than or equal to 0.05 seconds.

[0023] According to an embodiment of the present invention, the freezing hammer is in the shape of a hollow cylinder, the outer edge of the lower part of the freezing hammer is the same size as the through hole, and the outer edge of the lower part of the freezing hammer is in the shape of a cutting edge.

[0024] According to an embodiment of the present invention, the rapid cryogenic sampling device for plant leaf in its original state further includes an LED light source, and the LED light source is arranged on two symmetric sides diagonally above the semi-open leaf chamber assembly.

[0025] According to an embodiment of the present invention, the light transmittance of the transparent film is greater than 90%, and the elastic strength is less than 1 kg·mm -2 .

[0026] According to an embodiment of the present invention, the peripheral gas circuit of the device includes a carrier gas source, a mass flow controller and a humidifier connected in sequence, and a normal gas source, a first gas path switching valve and a second mass flow controller connected in sequence. The carbon dioxide sensor and the second mass flow controller are respectively connected to a gas mixer;

[0027] The gas mixer is connected to a second gas path switching valve, the second gas path switching valve is connected to the device main body, and then comes out from the device main body and is connected to a second mass flow measuring device, and the waste gas is discharged. A first check valve is arranged between the device main body and the second mass flow measuring device.

[0028] According to an embodiment of the present invention, a temperature and humidity sensor and a carbon dioxide sensor are further arranged between the humidifier and the gas mixer.

[0029] According to an embodiment of the present invention, the humidifier is connected in parallel with a throttle valve.

[0030] According to an embodiment of the present invention, a first mass flow measuring device is further arranged between the gas mixer and the second gas path switching valve.

[0031] According to an embodiment of the present invention, a bypass branch is further arranged between the second gas path switching valve and the second mass flow measuring device, and a second check valve is arranged in the bypass branch.

[0032] According to an embodiment of the present invention, the peripheral gas circuit of the device further includes an isotope gas source and a third mass flow measuring device, and the isotope gas source and the third mass flow measuring device are sequentially connected to the first gas path switching valve.

[0033] According to an embodiment of the present invention, the control system is directly controlled by a programmable logic controller, and the programmable logic controller works according to the instructions issued by the upper computer.

[0034] The present invention also provides a rapid cryogenic sampling system for plant leaf in its original state, characterized in that the rapid cryogenic sampling system for plant leaf in its original state includes at least one rapid cryogenic sampling device for plant leaf in its original state as described above.

[0035] The present invention also provides a method for quickly freezing and sampling plant leaves in their original state, characterized in that the method for quickly freezing and sampling plant leaves in their original state uses the device for quickly freezing and sampling plant leaves in their original state as described above, and it includes the following steps:

[0036] S1. Assemble the transparent film onto the semi-open leaf chamber assembly;

[0037] S2. Configure the gas source for the device for quickly freezing and sampling plant leaves in their original state;

[0038] S3. Place the leaf in the semi-open leaf chamber assembly;

[0039] S4. Set the parameters of each instrument;

[0040] S5. After the environmental parameters in the semi-open leaf chamber assembly are stable, allow for an adaptation period to keep the metabolism of the leaf in a steady state;

[0041] S6. Pre-cool the freezing box and the freezing hammer;

[0042] S7. After the freezing box is placed in position, perform freezing sampling;

[0043] S8. Take out the freezing box and extract the sample;

[0044] S9. End of sampling.

[0045] According to an embodiment of the present invention, in step S3, it specifically includes: lift the upper leaf chamber of the semi-open leaf chamber assembly, place the leaf to be sampled in the leaf chamber, then lower the upper leaf chamber, and adjust the tightness of the clamping of the semi-open leaf chamber assembly.

[0046] According to an embodiment of the present invention, in step S4, it specifically includes: set the environmental parameters of the semi-open leaf chamber assembly on the interface of the control system, and adjust the humidity control valve to adjust the humidity of the air flow.

[0047] According to an embodiment of the present invention, in step S7, it specifically includes: the control system controls and drives the freezing hammer to rapidly punch downward, cutting and smashing the leaf segment together with the transparent film into the freezing box;

[0048] After several seconds, the freezing hammer will automatically reset and the sampling is completed.

[0049] The positive and progressive effects of the present invention are as follows:

[0050] The rapid cryogenic sampling device, sampling system and sampling method for the original state of plant leaves according to the present invention highly automate the isotope labeling of plant leaves and the sampling process, greatly reduce the sampling time, improve the sampling accuracy and freezing reliability, and have a compact device structure design and strong scalability (multiple units can be connected as required), and multiple machines can be connected in parallel or in series according to experimental needs, so as to achieve high-throughput labeled sampling. BRIEF DESCRIPTION OF THE DRAWINGS

[0051] The above and other features, properties and advantages of the present invention will become more obvious through the following description in conjunction with the drawings and embodiments. In the drawings, the same reference numerals always represent the same features, where:

[0052] Figure 1 It is a schematic structural diagram of the device main body in the rapid cryogenic sampling device for the original state of plant leaves according to the present invention.

[0053] Figure 2 It is a schematic structural diagram of the semi-open leaf chamber in the rapid cryogenic sampling device for the original state of plant leaves according to the present invention.

[0054] Figure 3 It is a side view of the semi-open leaf chamber in the rapid cryogenic sampling device for the original state of plant leaves according to the present invention.

[0055] Figure 4 It is a schematic diagram of the peripheral gas path structure in the rapid cryogenic sampling device for the original state of plant leaves according to the present invention.

[0056] Figure 5 It is a schematic diagram of the four-unit series operation of the rapid cryogenic sampling device for the original state of plant leaves according to the present invention.

[0057]

REFERENCE NUMERALS

[0058] Device main body 10

[0059] Device peripheral gas path 20

[0060] Device bracket 11

[0061] Semi-open leaf chamber assembly 12

[0062] Freezing hammer 13

[0063] Freezing box 14

[0064] Upper leaf chamber 121

[0065] Lower leaf chamber 122

[0066] Sealing ring 123

[0067] Transparent film 15

[0068] Air inlet hole a

[0069] First air outlet b

[0070] Second air outlet c

[0071] Mounting table 111

[0072] High-speed cylinder 16

[0073] Connecting rod 17

[0074] Precision guide post 18

[0075] LED light source 19

[0076] Carrier gas source 21

[0077] First mass flow controller 22

[0078] Humidifier 23

[0079] Normal gas source 24

[0080] First gas path switching valve 25

[0081] Second mass flow controller 26

[0082] Gas mixer 27

[0083] Second gas path switching valve 28

[0084] Second mass flow meter 30

[0085] First check valve 31

[0086] Temperature and humidity sensor 32

[0087] Carbon dioxide sensor 33

[0088] Throttle valve 34

[0089] First mass flow meter 35

[0090] Bypass branch A

[0091] Control solenoid valve B

[0092] Second check valve 36

[0093] Isotope gas source 40

[0094] Third mass flow meter 41

[0095] Control host 50

[0096] Plant leaf 100 Specific implementation method

[0097] To make the above objects, features, and advantages of the present invention more apparent and understandable, the following provides a detailed description of the specific embodiments of the present invention in conjunction with the accompanying drawings.

[0098] Embodiments of the present invention will now be described in detail with reference to the accompanying drawings. Preferred embodiments of the present invention will now be described in detail, which are illustrated in the accompanying drawings. Whenever possible, the same reference numerals will be used throughout the drawings to represent the same or similar parts.

[0099] In addition, although the terms used in the present invention are selected from well-known and commonly used terms, some of the terms mentioned in the specification of the present invention may be selected by the applicant according to his or her judgment, and their detailed meanings are described in the relevant parts of the description herein.

[0100] In addition, it is required to understand the present invention not only through the actual terms used, but also through the meaning implied by each term.

[0101] Figure 1 It is a schematic structural diagram of the device main body in the rapid freezing sampling device for the original state of plant leaves of the present invention. Figure 2 It is a schematic structural diagram of the semi-open leaf chamber in the rapid freezing sampling device for the original state of plant leaves of the present invention. Figure 3 It is a side view of the semi-open leaf chamber in the rapid freezing sampling device for the original state of plant leaves of the present invention. Figure 4 It is a schematic structural diagram of the peripheral gas path in the rapid freezing sampling device for the original state of plant leaves of the present invention. Figure 5 It is a schematic diagram of the four-unit series operation of the rapid freezing sampling device for the original state of plant leaves of the present invention.

[0102] As Figures 1 to 5 shown, the present invention discloses a rapid freezing sampling device for the original state of plant leaves, which includes at least one device main body 10, a control system, and a device peripheral gas path 20. The device main bodies 10 are connected in series within the device peripheral gas path 20, and the device peripheral gas path 20 is connected to the control system. Each device main body 10 includes a device bracket 11, and a semi-open leaf chamber assembly 12, a freezing hammer 13, a freezing box 14, and a pneumatic driving structure installed on the device bracket 11. The freezing hammer 13 is located directly above the semi-open leaf chamber assembly 12, the freezing box 14 is installed below the semi-open leaf chamber assembly 12, and the pneumatic driving structure is connected to the freezing hammer 13 to drive the freezing hammer 13 to sample into the semi-open leaf chamber assembly 12 to obtain a sample, and the sample falls downward into the freezing box 14 for rapid freezing. A control solenoid valve B is provided on the device bracket 11, connecting the control system and the pneumatic driving structure.

[0103] Preferably, the semi-open leaf chamber assembly 12 includes an upper leaf chamber 121 with a through hole in the middle, a lower leaf chamber 122 with a through hole in the middle, and a sealing ring 123. The upper leaf chamber 121 and the lower leaf chamber 122 are stacked up and down, the sealing ring 123 is clamped between the upper leaf chamber 121 and the lower leaf chamber 122, and the plant leaf 100 is clamped in the through hole. A transparent film 15 is covered on both the upper surface of the upper leaf chamber 121 and the lower surface of the lower leaf chamber 122. Air inlet holes a are provided on the side parts of the same side of the upper leaf chamber 121 and the lower leaf chamber 122, air flow enters the leaf chamber from the air inlet holes a, a first air outlet hole b is opened on the side part of the other side of the lower leaf chamber 122, and a second air outlet hole c is opened on the side part of the other side of the upper leaf chamber 121. The first air outlet hole b and the second air outlet hole c are communicated with each other.

[0104] Furthermore, an installation table 111 is provided on the device support 11, and the semi-open leaf chamber assembly 12 is fixed on the installation table 111. The pneumatic driving structure includes a high-speed cylinder 16 and a connecting rod 17. The high-speed cylinder 16 is installed at the lower part of the device support 11. The semi-open leaf chamber assembly 12 and the freezing hammer 13 are connected by a precision guide post 18. The connecting rod 17 passes through the semi-open leaf chamber assembly 12, is connected to the freezing hammer 13 at one end, and is connected to the high-speed cylinder 16 at the other end. The freezing hammer 13 is driven by the high-speed cylinder 16 to move up and down.

[0105] Even further, the freezing hammer 13 is in a hollow cylindrical shape. The outer edge of the lower part of the freezing hammer 13 is the same size as the through hole, and the outer edge of the lower part of the freezing hammer 13 is in a cutting edge shape. The designed stroke of the high-speed cylinder 16 is 10 cm, and the time consumption of a one-way stroke is less than or equal to 0.05 seconds.

[0106] Before sampling, dry ice is placed in the hollow part of the freezing hammer 13 to achieve the pre-cooling effect of the freezing hammer 13. When reaching the preset time point of the program, the freezing hammer 13 is driven by the high-speed cylinder 16 to quickly accelerate and fall. The lower cutting edge will quickly cut the leaf chamber film and the leaf, and quickly push the cut film and leaf into the freezing box 14 located below the leaf chamber.

[0107] Preferably, the plant leaf original state rapid freezing sampling device further includes an LED light source 19. The LED light source 19 is arranged on both sides symmetrically above the semi-open leaf chamber assembly 12. The light transmittance of the transparent film 15 is greater than 90%, and the elastic strength is less than 1 kg·mm -2 . Here, the light intensity of the LED light source 19 can be controlled by the upper computer program to change. After passing through the transparent film, the maximum light intensity (PAR) needs to exceed 1200 mmol·m -2 s -1 , and there is an active cooling by a fan.

[0108] Here, the sealing ring 123 is preferably a foam sealing ring. After the semi-open leaf chamber assembly 12 clamps and fixes the leaf, the leaf chamber is relatively sealed. The air flow in the leaf chamber is controlled by the peripheral gas path, and the light in the leaf chamber is provided by the LED light source 19, so that the leaf in the leaf chamber is in an artificially set controllable and stable environment of light, humidity, and carbon dioxide.

[0109] Preferably, the freezing box 14 can be taken out for precooling during non-sampling periods. Before sampling, there is a removable foam heat insulation layer to prevent the low temperature from affecting the leaves in the leaf chamber. After sampling is completed, it can be taken out of the device, and the frozen leaves can be separately packed.

[0110] Furthermore, the peripheral gas path 20 of the device includes a carrier gas source 21, a mass flow controller 22, and a humidifier 23 connected in sequence, and a normal gas source 24, a first gas path switching valve 25, a second mass flow controller 26, and a second mass flow controller 26 are respectively connected to a gas mixer 27. The gas mixer 27 is connected to a second gas path switching valve 28, the second gas path switching valve 28 is connected to the device main body 10, and then comes out from the device main body 10 and is connected to a second mass flow measuring device 30, and the waste gas is discharged. A first check valve 31 is provided between the device main body 10 and the second mass flow measuring device 30.

[0111] Preferably, a temperature and humidity sensor 32 and a carbon dioxide sensor 33 are also provided between the humidifier 23 and the gas mixer 27. The humidifier 23 is connected in parallel with a throttle valve 34. A first mass flow measuring device 35 is also provided between the gas mixer 28 and the second gas path switching valve 28. A bypass branch A is also provided between the second gas path switching valve 28 and the second mass flow measuring device 30, and a second check valve 36 is provided in the bypass branch A.

[0112] In addition, the peripheral gas path 20 of the device also includes an isotope gas source 40 and a third mass flow measuring device 41, and the isotope gas source 40 and the third mass flow measuring device 41 are connected to the first gas path switching valve 25 in sequence.

[0113] The control system is directly controlled by a programmable logic controller, and the programmable logic controller works according to the instructions issued by the host computer.

[0114] According to the above description, the peripheral gas path of the device can achieve the following functions:

[0115] I. Control and detection of the input air flow of the semi-open leaf chamber: 2 - 40 L / min (accuracy < 1%);

[0116] II. Online ratio and detection of carbon dioxide (or other gases): concentration 0 - 2500 ppm (accuracy < 1%);

[0117] III. Humidity control and detection of the input air flow of the semi-open leaf chamber: 0 - 85% R.H. (no condensation, accuracy < 5%);

[0118] IV. Flow rate detection of the isotope gas source;

[0119] V. Flow rate detection of the output air flow of the semi-open leaf chamber.

[0120] The peripheral gas circuit of the device can use a variety of stable isotope-labeled gases according to experimental needs. Such as 13 CO2, 18 O2 and 15 NH3.

[0121] Such as Figure 5 As shown, the present invention also provides a rapid cryogenic sampling system for plant leaf in-situ, which includes at least one rapid cryogenic sampling device for plant leaf in-situ as described above. This structure can achieve very strong scalability (multiple units can be connected as required)

[0122] Specifically, the rapid cryogenic sampling device for plant leaf in-situ of the present invention can be arbitrarily connected in series and in parallel according to experimental needs to form an isotope labeling and cryogenic sampling array for plant leaves. The array shares the same set of peripheral gas circuit 20 and is uniformly coordinated and controlled by the same set of control host 50 and upper computer.

[0123] The present invention also provides a method for rapid cryogenic sampling of plant leaf in-situ, characterized in that the method for rapid cryogenic sampling of plant leaf in-situ uses the rapid cryogenic sampling device for plant leaf in-situ as described above, and includes the following steps:

[0124] S1. Assemble the transparent film onto the semi-open leaf chamber assembly.

[0125] The upper leaf chamber and the lower leaf chamber of the semi-open leaf chamber assembly are each covered by a layer of transparent film, which are disposable consumable parts and need to be assembled and fixed below and above the leaf chamber before each sampling. After assembly, the leaf chamber should ensure airtightness.

[0126] S2. Configure the gas source for the rapid cryogenic sampling device for plant leaf in-situ.

[0127] The gas environment in the leaf chamber of the semi-open leaf chamber assembly is completely artificially set, so it is necessary to connect the leaf chamber gas source in advance. Taking the experimental situation where it is necessary to control the carbon dioxide concentration in the leaf chamber as an example, it is necessary to connect the carrier gas source (a mixture of 71% nitrogen and the remaining oxygen) and the high-purity carbon dioxide gas source. If it is a metabolic flux experiment, that is, an isotope labeling experiment of 13 CO2 is required, it is also necessary to connect 13 the CO2 gas source at the same time. In addition, it is also necessary to connect the high-pressure gas source that drives the high-speed cylinder to work.

[0128] S3. Place the leaf in the semi-open leaf chamber assembly.

[0129] Preferably, step S3 specifically includes: lifting the upper leaf chamber of the semi-open leaf chamber assembly, placing the leaf to be sampled in the leaf chamber, then lowering the upper leaf chamber, and adjusting the tightness of the clamping of the semi-open leaf chamber assembly through a screw knob to make it as loose as possible on the premise of ensuring airtightness of the leaf chamber so as not to affect the leaf state.

[0130] S4. Set the parameters of each instrument.

[0131] First, it is necessary to set the environmental parameters of the leaf chamber on the control interface, including light intensity, gas flow rate, carbon dioxide concentration, etc. Secondly, it is also necessary to adjust the humidity control valve to adjust the humidity of the air flow. For isotope labeling experiments, it is also necessary to set experimental parameters such as labeling time.

[0132] S5. After the environmental parameters in the semi-open leaf chamber assembly are stable, allow a period of adaptation time to keep the metabolism of the leaf in a steady state.

[0133] This adaptation time varies according to different experimental materials and is generally 20 - 30 minutes.

[0134] S6. Pre-cool the freezing box and freezing hammer.

[0135] The freezing box can preferably be a liquid nitrogen box. Before sampling, it is necessary to pre-cool the liquid nitrogen box and the freezing hammer first. The liquid nitrogen box can be taken out for separate pre-cooling. The freezing hammer is not detachable, and dry ice (or other freezing media) needs to be placed into the hollow groove of the freezing hammer for pre-cooling.

[0136] When approaching sampling, after filling the pre-cooled liquid nitrogen box with sufficient liquid nitrogen, place it back inside the device and wait for sampling.

[0137] For isotope labeling experiments, after the adaptation is over, isotope labeling can be started through the control interface. The labeling time is based on the previously set value, and the instrument will automatically sample after reaching the set labeling time point.

[0138] S7. After the freezing box is placed in position, perform freezing sampling.

[0139] Under the control of the control host 50, the device drives the freezing hammer to rush down at high speed, cutting and smashing the leaf segment together with the leaf chamber film into the freezing box. After several seconds, the freezing hammer will automatically reset and the sampling is completed.

[0140] S8. Take out the freezing box and extract the sample.

[0141] Take out the freezing box, and tools such as forceps and filters can be used to take out the frozen leaves from the freezing box for subsequent metabolite extraction, instrument operation and other experimental operations.

[0142] S9. Sampling is completed.

[0143] Close and disconnect all gas sources. Clean the residual leaves and film fragments in the freezing box and the leaf chamber. Disconnect the power supply of the device and confirm that the freezing hammer is correctly reset.

[0144] The whole sampling process can be divided into three stages: sampling preparation stage, sampling progress stage and sampling ending stage. Among them, steps S1 to S4 are the sampling preparation stage, steps S5 to S7 are the sampling progress stage, and steps S8 and S9 are the sampling ending stage.

[0145] In summary, for the plant leaf original state rapid freezing sampling device and sampling method of the present invention, while highly automating the isotope labeling and sampling process of plant leaves, it also greatly reduces the sampling time, improves the sampling accuracy and freezing reliability, and the device has a compact structure design and strong scalability (multiple units can be connected as required), and multiple machines can be connected in parallel or in series according to experimental needs, so as to achieve high-throughput labeling sampling.

[0146] Although the specific implementation manners of the present invention have been described above, those skilled in the art should understand that these are only examples, and the protection scope of the present invention is defined by the appended claims. Without departing from the principle and essence of the present invention, those skilled in the art can make various changes or modifications to these implementation manners, but these changes and modifications all fall within the protection scope of the present invention.

Claims

1. A device for rapid cryogenic sampling of plant leaf in its original state, characterized in that, The rapid cryogenic sampling device for the original state of plant leaves includes at least one device main body, a control system, and a peripheral gas circuit of the device. The device main bodies are connected in series within the peripheral gas circuit of the device, and the peripheral gas circuit of the device is connected to the control system; Each device main body includes a device bracket, and a semi-open leaf chamber assembly, a freezing hammer, a freezing box, and a pneumatic drive structure mounted on the device bracket. The freezing hammer is located directly above the semi-open leaf chamber assembly. The freezing box is installed below the semi-open leaf chamber assembly. The pneumatic drive structure is connected to the freezing hammer to drive the freezing hammer to sample into the semi-open leaf chamber assembly to obtain a sample, and the sample falls downward into the freezing box for rapid freezing; The semi-open leaf chamber assembly includes an upper leaf chamber with a through hole in the middle, a lower leaf chamber with a through hole in the middle, and a sealing ring. The upper leaf chamber and the lower leaf chamber are stacked up and down, and the sealing ring is clamped between the upper leaf chamber and the lower leaf chamber. The plant leaf is clamped in the through hole, and a transparent film is covered on the upper surface of the upper leaf chamber and the lower surface of the lower leaf chamber.

2. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 1, characterized in that, Air inlet holes are provided on the side of the same side of the upper leaf chamber and the lower leaf chamber. Airflow enters the leaf chamber through the air inlet holes. A first air outlet hole is opened on the side of the other side of the lower leaf chamber, and a second air outlet hole is opened on the side of the other side of the upper leaf chamber. The first air outlet hole and the second air outlet hole are communicated with each other.

3. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 2, characterized in that, An installation table is provided on the device bracket, and the semi-open leaf chamber assembly is fixed on the installation table. The pneumatic drive structure includes a high-speed cylinder and a connecting rod. The high-speed cylinder is installed at the lower part of the device bracket, and the semi-open leaf chamber assembly and the freezing hammer are connected by a precision guide post; The connecting rod passes through the semi-open leaf chamber assembly, is connected to the freezing hammer at one end, and is connected to the high-speed cylinder at the other end. The high-speed cylinder is used to drive the freezing hammer to move up and down.

4. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 3, characterized in that, The designed stroke of the high-speed cylinder is 10 cm, and the time consumption of a single-way stroke is less than or equal to 0.05 seconds.

5. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 2, characterized in that, The freezing hammer is in the shape of a hollow cylinder. The outer edge of the lower part of the freezing hammer is the same size as the through hole, and the outer edge of the lower part of the freezing hammer is in the shape of a cutting edge.

6. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 1, characterized in that, The rapid cryogenic sampling device for the original state of plant leaves further includes an LED light source, and the LED light source is arranged on the symmetric two sides obliquely above the semi-open leaf chamber assembly.

7. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 1, characterized in that, The light transmittance of the transparent film is greater than 90%, and the elastic strength is less than 1 kg·mm -2 .

8. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 2, characterized in that, The peripheral gas circuit of the device includes a carrier gas source, a mass flow controller, and a humidifier connected in sequence, and a normal gas source, a first gas path switching valve, and a second mass flow controller connected in sequence. A temperature and humidity sensor and a carbon dioxide sensor are also arranged between the humidifier and a gas mixer. The carbon dioxide sensor and the second mass flow controller are respectively connected to the gas mixer; The gas mixer is connected to a second gas path switching valve. The second gas path switching valve is connected to the device main body, and then comes out from the device main body and is connected to a second mass flow meter, and the waste gas is discharged. A first check valve is arranged between the device main body and the second mass flow meter.

9. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 8, characterized in that, The humidifier is connected in parallel with a throttle valve.

10. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 8, characterized in that, A first mass flow meter is further provided between the gas mixer and the second gas path switching valve.

11. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 8, characterized in that, A bypass branch is further provided between the second gas path switching valve and the second mass flow meter, and a second check valve is provided in the bypass branch.

12. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 8, characterized in that, The peripheral gas path of the device further includes an isotope gas source and a third mass flow meter, and the isotope gas source and the third mass flow meter are sequentially connected to the first gas path switching valve.

13. The device for rapid cryogenic sampling of plant leaf in its original state according to claim 1, characterized in that, The control system is directly controlled by a programmable logic controller, and the programmable logic controller operates according to instructions issued by a host computer.

14. A system for rapid cryogenic sampling of plant leaf in its original state, characterized in that, The plant leaf original state rapid freezing sampling system includes at least one plant leaf original state rapid freezing sampling device as described in any one of claims 1-13.

15. A method for rapid cryogenic sampling of plant leaf in its original state, characterized in that, The plant leaf original state rapid freezing sampling method uses a plant leaf original state rapid freezing sampling device as described in any one of claims 1-13, and includes the following steps: S1. Assemble a transparent film onto the semi-open leaf chamber assembly; S2. Configure the gas source for the plant leaf original state rapid freezing sampling device; S3. Place a leaf in the semi-open leaf chamber assembly; S4. Set the parameters of each instrument; S5. After the environmental parameters in the semi-open leaf chamber assembly are stable, allow a period of adaptation time to keep the metabolism of the leaf in a steady state; S6. Pre-cool the freezing box and the freezing hammer; S7. After the freezing box is placed in place, perform freezing sampling; S8. Take out the freezing box and extract the sample; S9. End of sampling.

16. The method for rapid cryogenic sampling of plant leaf in its original state according to claim 15, wherein, Specifically, step S3 includes: lift the upper leaf chamber of the semi-open leaf chamber assembly, place the leaf to be sampled in the leaf chamber, then lower the upper leaf chamber, and adjust the clamping tightness of the semi-open leaf chamber assembly.

17. The method for rapid cryogenic sampling of plant leaf in its original state according to claim 16, wherein, Specifically, step S4 includes: set the environmental parameters of the semi-open leaf chamber assembly on the interface of the control system and adjust the air flow humidity.

18. The method for rapid cryogenic sampling of plant leaf in its original state according to claim 15, wherein, Specifically, step S7 includes: the control system controls and drives the freezing hammer to rapidly punch downward, cutting and smashing the leaf segment together with the transparent film into the freezing box; After several seconds, the freezing hammer will automatically reset and the sampling is completed.

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