CN113069790A - Extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP47 - Google Patents
Extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP47 Download PDFInfo
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- CN113069790A CN113069790A CN202110340709.6A CN202110340709A CN113069790A CN 113069790 A CN113069790 A CN 113069790A CN 202110340709 A CN202110340709 A CN 202110340709A CN 113069790 A CN113069790 A CN 113069790A
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Abstract
The invention provides an extraction preparation device and method for optical system II internal peripheral antennas CP43 and CP47, the device comprises a centrifuge main body, a blade processing device, a liquid adding device and a liquid transferring device, wherein the centrifuge main body comprises a centrifugal groove, a centrifugal column, a centrifugal motor, a test tube groove and an automatic press seal device, the blade processing device comprises a fixed frame, a homogenizer, a homogenizing container and a filter, the liquid adding device comprises a rotating frame, a low-permeability buffer liquid tank and a solubilizing liquid tank, and the liquid transferring device comprises a liquid transferring frame, a negative pressure liquid taking machine and a telescopic liquid taking pipe; the method comprises the following steps: leaf processing, chloroplast separation, thylakoid membrane preparation, PSII particle preparation, PSII oxygen release core complex preparation and extraction preparation of CP43 and CP 47. In a word, the invention has the advantages of novel structure, convenient operation, high extraction efficiency and the like.
Description
Technical Field
The invention belongs to the technical field of plant research, and particularly relates to an extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP 47.
Background
Solar energy is the prime mover for life activities on the earth. Radiant energy from the sun can only be converted into the various forms of chemical energy necessary for life activities through photosynthesis by photosynthetic organisms. The most potential and efficient way for improving the yield of grains is to improve the light energy conversion and utilization efficiency of crops, and by disclosing the mechanism of efficient energy absorption, energy transmission and energy conversion of photosynthesis, the new way for developing solar energy utilization is an important way for solving the energy shortage in the next century. Many redox components and electron transfer molecular mechanisms in photosynthetic organs all show obvious bistable characteristics, so that the molecular logic device which is operated by single electron can be made into an ideal functional material of a future computer. Furthermore, the absorption of carbon dioxide by photosynthesis to release oxygen plays a crucial role in regulating the increasingly deteriorating atmospheric environment. Therefore, the research on photosynthesis can generate great promotion effect on the survival and the continuous development of the human society.
Strong light inhibits and destroys photosynthesis of higher plants, and when the amount of light energy received by the plant photosynthesis mechanism exceeds the amount that can be utilized by photosynthesis, the photosynthesis function is reduced, which is expressed as a reduction in quantum efficiency and light energy conversion efficiency, which is called photoinhibition of photosynthesis. The action mechanism of photoinhibition is very complex, and the phenomenon of photodamage can be observed in leaves, chloroplasts, thylakoid membranes, photosystem II granules or oxygen release granules under the stress of light. In the middle of the 80's of the 20 th century, there was consensus on PSII for the primary and major sites of light suppression. Plants are subject to intense light stress in the natural environment, which causes photoinhibition and further affects various physiological activities including photosynthesis.
In PSII, light energy is first absorbed by the outer perimeter antenna and then transmitted by the inner perimeter antennas (CP43 and CP47) to the reaction center where the photochemical reaction occurs. The research on CP43 and CP47 is one of the hot spots in the research on photosynthesis nowadays, and the structural and functional elucidation of the PSII plays an important role in analyzing the spatial structure of the PSII, revealing the mechanism of absorption, distribution and transmission of excitation energy in the PSII and the mechanism of oxygen release of the PSII. The invention designs an extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP 47.
Disclosure of Invention
In view of the above problems, the present invention provides an extracting and preparing apparatus and method for optical system II internal peripheral antennas CP43 and CP 47.
The technical scheme of the invention is as follows: the extraction and preparation device for the light system II inner peripheral antennas CP43 and CP47 mainly comprises a centrifuge body, a blade processing device, a liquid adding device and a liquid transferring device,
the centrifuge main body is fixed on a horizontal table board, a cylindrical hollow groove is arranged in the middle of the centrifuge main body, a centrifugal column is arranged in the centrifugal groove, a centrifugal motor connected with the bottom end of the centrifugal column is arranged at the center of the bottom of the centrifuge main body, four test tube grooves for placing test tubes are arranged at the edge of the inner part of the centrifugal column in a cross manner, an automatic press sealer for automatically pressing and sealing the test tubes in the test tube grooves is arranged at the center of the centrifugal column,
the blade processing device comprises a fixed frame fixedly arranged at the left end of the centrifuge main body, a homogenizer arranged at the upper end of the fixed frame, a homogenizing container arranged at the middle part of the fixed frame and a filter arranged at the middle lower part of the fixed frame,
the homogenate container comprises a loop bar movably sleeved on the fixed frame and a homogenate cup movably arranged at the far end of the loop bar, the lower end of the homogenate cup is provided with a discharge funnel, the junction of the discharge funnel and the homogenate cup is provided with a liquid discharge valve,
the filter comprises a movable loop bar sleeved on the fixed frame and a liquid collecting cup movably arranged at the far end of the movable loop bar, the upper end of the liquid collecting cup is provided with a plurality of layers of disposable gauze, the lower part of the side wall of the liquid collecting cup is provided with a liquid outlet valve,
the liquid adding device comprises a rotating frame fixedly arranged at the rear side end of the centrifuge main body, and a hypotonic buffer liquid tank and a solubilization liquid tank which are respectively arranged at two sides of the rotating frame, wherein a first liquid adding valve is arranged at the outer side of the lower end of the side wall of the hypotonic buffer liquid tank, a second liquid adding valve is arranged at the outer side of the lower end of the side wall of the solubilization liquid tank, the first liquid adding valve and the second liquid adding valve can both rotate to a position right above the test tube groove,
the liquid transfer device comprises a liquid transfer frame fixedly arranged at the right side end of the centrifuge body, a negative-pressure liquid taking machine fixedly arranged at the bottom of the liquid transfer frame and a telescopic liquid taking pipe arranged at the top end of the liquid transfer frame.
Furthermore, a control panel is arranged on the front side end face of the centrifuge body and used for setting operation parameters of the extraction and preparation device.
Further, be equipped with in the test tube groove and be used for fixing the fixation clamp of test tube avoids the test tube to break away from the test tube groove when centrifugation or unseal the test tube.
Further, automatic press seal ware is including fixed setting at the inside miniature hydraulic pump of centrifugation post, connection the hydraulic lifting rod of miniature hydraulic pump upside output, install the automatically controlled rotating head on hydraulic lifting rod top, be cross fixed mounting four connecting rods of automatically controlled rotating head side with set up respectively four the test tube closing cap of connecting rod distal end, automatic press seal device can realize the automatic closing cap of test tube before the centrifugation, need not artificial operation, use manpower sparingly and can avoid operating personnel to forget the closing cap test tube.
Further, still be equipped with negative pressure generator in the centrifugal column, negative pressure generator passes through inner tube and four test tube closing cap intercommunication for make the in vitro negative pressure environment that forms, the bursting of chloroplast can be accelerated to the negative pressure environment at the chloroplast bursting in-process, and is efficient.
Further, homogenate cup upper end activity is equipped with the splashproof lid, the aircraft nose of refiner bottom can run through the splashproof lid is arranged in inside the homogenate cup, avoid splashing at homogenate in-process homogenate liquid.
Further, the edge of the upper port of the liquid collecting cup is provided with a magnetic ring, a magnetic pressing ring is magnetically adsorbed on the magnetic ring, and the multilayer disposable gauze clamp is arranged between the magnetic pressing ring and the magnetic ring, so that the disposable gauze is convenient to fix and replace.
Furthermore, the hypotonic buffer solution tank and the solubilization solution tank are both temperature-insulated storage tanks, the hypotonic buffer solution and the solubilization solution pipe both need to be stored at low temperature, and the temperature-insulated storage tanks can prolong the storage time.
Furthermore, the lower end of the telescopic liquid taking pipe is movably sleeved with a replaceable disposable liquid taking pipe, so that the problem that the prepared solution is mixed due to the fact that one liquid taking pipe is used for multiple times is avoided.
The method for extracting and preparing the light system II inner peripheral antennas CP43 and CP47 by using the extraction and preparation device mainly comprises the following steps:
s1: blade treatment
Cutting the blades, putting the cut blades into a homogenizing cup, adding a buffer solution into the homogenizing cup, covering a splash-proof cover, moving a homogenizing container upwards, extending a machine head of a homogenizer into the homogenizing cup, starting the homogenizer to homogenize the blades in the homogenizing cup, opening a drain valve after the treatment is finished, discharging the homogenized solution into a filter, filtering the homogenized solution by a plurality of layers of disposable gauze to remove residual blades, and collecting the homogenized solution in a liquid collecting cup;
s2: chloroplast isolation
Placing a test tube in a test tube tank, rotating the test tube to the position below a liquid outlet valve, opening the liquid outlet valve to add filtered homogenate into the test tube, sealing the test tube by using an automatic pressure sealing device, then starting a centrifugal motor for centrifugation, and taking out chloroplast positioned in the middle of the test tube by using a liquid transfer device after the centrifugation is finished;
s3: preparation of thylakoid membranes
Rotating a blank test tube to the lower part of a telescopic liquid taking tube of a liquid transferring device, transferring the chloroplast obtained in the step S2 into the blank test tube, then rotating a low-permeability buffer liquid tank of the liquid adding device to the upper part of the test tube, adding a low-permeability buffer liquid into the test tube, sealing the test tube by using an automatic pressure sealing device, then starting a negative pressure generator to form a negative pressure condition in the test tube, accelerating the bursting of the chloroplast, then starting a centrifugal motor to carry out centrifugation, replacing the disposable liquid taking tube sleeved on the telescopic liquid transferring tube after the centrifugation is finished, and then extracting the supernatant containing the thylakoid membrane in the test tube by using the liquid transferring device;
s4: PSII particle preparation
Rotating a blank test tube to the lower part of a telescopic liquid taking tube of a liquid transferring device, transferring the supernatant obtained in the step S3 into the blank test tube, then rotating a solubilizing liquid tank of the liquid adding device to the upper part of the test tube, adding a solubilizing liquid into the test tube, sealing the test tube by using an automatic pressure sealing device, then starting a centrifugal motor for centrifugation, and after the centrifugation is finished, extracting the supernatant containing an optical system II in the test tube by using the liquid transferring device;
s5: preparation of PSII oxygen-releasing core Complex
Subjecting the supernatant obtained in the step S4 to electrophoresis treatment, and eluting with an ion exchange column to obtain a PSII oxygen-releasing core compound;
s6: extraction and preparation of CP43 and CP47
The PSII oxygen-evolving core complex prepared in step S5 was eluted separately using ion exchange columns filled with different buffers to give CP43 and CP 47.
The invention has the beneficial effects that: the extraction and preparation device and the method for the light system II inner peripheral antennas CP43 and CP47 are particularly suitable for laboratory research, the blade processing device is integrated on the centrifuge body, the homogenate, filtration and collection of the blades can be completed only by putting the cut blades into the blade processing device, and the homogenate can be conveniently added into the test tube for further processing through the liquid outlet valve, an automatic press sealer which can automatically seal the test tube is arranged in the centrifuge body, the test tube can be automatically sealed before centrifugation, and the test tube closing cap is communicated with a negative pressure generator, so that a negative pressure environment can be formed inside the test tube, the bursting of chloroplasts is accelerated, a liquid adding device is further arranged, hypotonic buffer liquid and solubilization liquid can be accurately added, the liquid adding device capable of replacing the disposable liquid taking tube is arranged, and the liquid amount can be accurately moved. In a word, the invention has the advantages of novel structure, convenient operation, high extraction efficiency and the like.
Drawings
FIG. 1 is a schematic view of the overall structure of the present invention;
FIG. 2 is a schematic structural view of the main body of the centrifuge of the present invention;
FIG. 3 is a schematic view of a spin column according to the present invention;
FIG. 4 is a top view of a test tube well according to the present invention;
FIG. 5 is a schematic diagram of the construction of the homogenization vessel of the invention;
fig. 6 is a schematic view of the filter construction of the present invention.
Wherein, 1-centrifuge body, 11-centrifuge groove, 12-centrifuge column, 13-centrifuge motor, 14-test tube groove, 141-fixing clamp, 15-automatic press seal device, 151-micro hydraulic pump, 152-hydraulic lifting rod, 153-electric control rotating head, 154-connecting rod, 155-test tube sealing cover, 16-control panel, 17-negative pressure generator, 2-blade processing device, 21-fixing frame, 22-homogenizer, 23-homogenizing container, 231-loop bar, 232-homogenizing cup, 233-splash-proof cover, 234-discharging funnel, 235-discharging valve, 24-filter, 241-movable loop bar, 242-collecting cup, 243-magnetic ring, 244-magnetic press ring, 245-discharging valve, 3-liquid adding device, 31-rotating frame, 32-hypotonic buffer liquid tank, 321-liquid adding valve I, 33-solubilizing liquid tank, 331-liquid adding valve II, 4-liquid transferring device, 41-liquid transferring frame, 42-negative pressure liquid taking machine, 43-telescopic liquid taking pipe and 44-disposable liquid taking pipe.
Detailed Description
For the convenience of understanding the technical scheme of the present invention, the present invention is further explained with reference to the accompanying fig. 1 to 6 and the specific embodiments, which are not to be construed as limiting the scope of the present invention.
Example (b): as shown in FIG. 1, the extraction and preparation device for optical system II inner peripheral antennas CP43 and CP47 mainly comprises a centrifuge body 1, a blade processing device 2, a liquid adding device 3 and a liquid transferring device 4,
the centrifuge body 1 is fixed on a horizontal table, as shown in fig. 2, a cylindrical hollow groove 11 is arranged in the middle of the centrifuge body 1, a centrifugal column 12 is arranged in the centrifugal groove 11, a centrifugal motor 13 connected with the bottom end of the centrifugal column 12 is arranged at the center of the bottom of the centrifuge body 1, as shown in fig. 3, four test tube slots 14 for placing test tubes are arranged at the edge of the inside of the centrifugal column 12 in a cross shape, as shown in fig. 4, a fixing clamp 141 for fixing the test tubes is arranged in the test tube slots 14, an automatic press sealer 15 for automatically press-sealing the test tubes in the test tube slots 14 is arranged at the center of the centrifugal column 12,
the automatic press-sealing device 15 comprises a micro hydraulic pump 151 fixedly arranged inside the centrifugal column 12, a hydraulic lifting rod 152 connected to the upper output end of the micro hydraulic pump 151, an electrically controlled rotating head 153 arranged at the top end of the hydraulic lifting rod 151, four connecting rods 154 fixedly arranged on the side edges of the electrically controlled rotating head 153 in a cross shape, and test tube sealing caps 155 respectively arranged at the distal ends of the four connecting rods 154,
the centrifugal column 12 is also provided with a negative pressure generator 17, the negative pressure generator 17 is communicated with four test tube sealing covers 155 through an internal pipeline and is used for forming a negative pressure environment in the test tube, the front side end face of the centrifuge body 1 is provided with a control panel 16,
the blade processing device 2 comprises a fixed frame 21 fixedly arranged at the left end of the centrifuge main body 1, a homogenizer 22 arranged at the upper end of the fixed frame 21, a homogenizing container 23 arranged at the middle part of the fixed frame 21 and a filter 24 arranged at the middle lower part of the fixed frame 21,
as shown in fig. 5, the homogenizing container 23 comprises a sleeve rod 231 movably sleeved on the fixing frame 21 and a homogenizing cup 232 movably installed at the far end of the sleeve rod 231, a splash-proof cover 233 is movably arranged at the upper end of the homogenizing cup 232, a head at the bottom end of the homogenizing machine 22 can penetrate through the splash-proof cover 233 and is arranged inside the homogenizing cup 232, a discharge funnel 234 is arranged at the lower end of the homogenizing cup 232, a drain valve 235 is arranged at the connection part of the discharge funnel 234 and the homogenizing cup 232,
as shown in fig. 6, the filter 24 comprises a movable loop bar 241 sleeved on the fixed frame 21 and a liquid collecting cup 242 movably mounted at the distal end of the movable loop bar 241, the upper end of the liquid collecting cup 242 is provided with a plurality of layers of disposable gauze, the edge of the upper port of the liquid collecting cup 242 is provided with a magnetic ring 243, the magnetic ring 243 is magnetically adsorbed with a magnetic pressing ring 244, the plurality of layers of disposable gauze are clamped between the magnetic pressing ring 244 and the magnetic ring 243, the lower part of the side wall of the liquid collecting cup 242 is provided with a liquid outlet valve 245,
the liquid adding device 3 comprises a rotating frame 31 fixedly arranged at the rear side end of the centrifuge body 1, a hypotonic buffer liquid tank 32 and a solubilization liquid tank 33 respectively arranged at two sides of the rotating frame 31, the hypotonic buffer liquid tank 32 and the solubilization liquid tank 33 are both heat insulation storage tanks, a liquid adding valve I321 is arranged at the outer side of the lower end of the side wall of the hypotonic buffer liquid tank 32, a liquid adding valve II 331 is arranged at the outer side of the lower end of the side wall of the solubilization liquid tank 33, the liquid adding valve I321 and the liquid adding valve II 331 can both rotate to the position right above the test,
the liquid transferring device 4 comprises a liquid transferring frame 41 fixedly arranged at the right side end of the centrifuge body 1, a negative pressure liquid taking machine 42 fixedly arranged at the bottom of the liquid transferring frame 41 and a telescopic liquid taking pipe 43 arranged at the top end of the liquid transferring frame 41, wherein the lower end of the telescopic liquid taking pipe 43 is movably sleeved with a replaceable disposable liquid taking pipe 44,
the electronic components are not specially designated, and common products sold in the market can be selected as long as the use requirements of the electronic components can be met.
The method for extracting and preparing the light system II inner peripheral antennas CP43 and CP47 by using the extraction and preparation device in the embodiment mainly comprises the following steps:
s1: blade treatment
Cutting the blades, putting the cut blades into a homogenizing cup 232, adding a buffer solution into the homogenizing cup 232, covering a splash-proof cover 233, moving a homogenizing container 23 upwards, extending a machine head of a homogenizing machine 22 into the homogenizing cup 232, starting the homogenizing machine 22 to homogenize the blades in the homogenizing cup 232, opening a drain valve 235 after the homogenization is finished, discharging the homogenate into a filter 24, filtering the homogenate by multiple layers of disposable gauze to remove residual blades, and collecting the homogenate in a liquid collecting cup 242;
s2: chloroplast isolation
Placing a test tube in a test tube groove 14, rotating the test tube to the position below a liquid outlet valve 245, opening the liquid outlet valve 245 to add the filtered homogenate into the test tube, sealing the test tube by using an automatic pressure sealing device 15, then starting a centrifugal motor 13 for centrifugation, and taking out the chloroplast positioned in the middle of the test tube by using a liquid transfer device 4 after the centrifugation is finished;
s3: preparation of thylakoid membranes
Rotating a blank test tube to the lower part of a telescopic liquid taking tube 43 of a liquid transferring device 4, transferring the chloroplast obtained in the step S2 into the blank test tube, then rotating a low-permeability buffer liquid tank 32 of the liquid adding device 3 to the upper part of the test tube, adding a low-permeability buffer liquid into the test tube, sealing the test tube by using an automatic pressure sealing device 15, then starting a negative pressure generator 17 to form a negative pressure condition in the test tube, accelerating the bursting of the chloroplast, then starting a centrifugal motor 13 to carry out centrifugation, replacing a disposable liquid taking tube 44 sleeved on the telescopic liquid transferring tube 43 after the centrifugation is finished, and then extracting the supernatant containing the thylakoid membrane in the test tube by using the liquid transferring device 4;
s4: PSII particle preparation
Rotating a blank test tube to the lower part of the telescopic liquid taking tube 43 of the liquid transferring device 4, transferring the supernatant obtained in the step S3 into the blank test tube, then rotating the solubilizing liquid tank 33 of the liquid adding device 3 to the upper part of the test tube, adding the solubilizing liquid into the test tube, sealing the test tube by using the automatic pressure sealing device 15, then starting the centrifugal motor 13 for centrifugation, and after the centrifugation is finished, extracting the supernatant containing the light system II in the test tube by using the liquid transferring device 4;
s5: preparation of PSII oxygen-releasing core Complex
Subjecting the supernatant obtained in the step S4 to electrophoresis treatment, and eluting with an ion exchange column to obtain a PSII oxygen-releasing core compound;
s6: extraction and preparation of CP43 and CP47
The PSII oxygen-evolving core complex prepared in step S5 was eluted separately using ion exchange columns filled with different buffers to give CP43 and CP 47.
Claims (10)
1. The extraction and preparation device for the light system II internal peripheral antennas CP43 and CP47 is characterized by mainly comprising a centrifuge body (1), a blade processing device (2), a liquid adding device (3) and a liquid transferring device (4),
the centrifuge main body (1) is fixed on a horizontal table board, a cylindrical hollow groove type centrifugal groove (11) is arranged in the middle of the centrifuge main body (1), a centrifugal column (12) is arranged in the centrifugal groove (11), a centrifugal motor (13) connected with the bottom end of the centrifugal column (12) is arranged at the center of the bottom of the centrifuge main body (1), four test tube grooves (14) for placing test tubes are arranged at the edge of the inner part of the centrifugal column (12) in a cross manner, an automatic press sealer (15) for automatically pressing and sealing the test tubes in the test tube grooves (14) is arranged at the center of the centrifugal column (12),
the blade processing device (2) comprises a fixed frame (21) fixedly arranged at the left end of the centrifuge main body (1), a refiner (22) arranged at the upper end of the fixed frame (21), a homogenizing container (23) arranged in the middle of the fixed frame (21) and a filter (24) arranged at the middle lower part of the fixed frame (21),
the homogenate container (23) comprises a loop bar (231) movably sleeved on the fixed frame (21) and a homogenate cup (232) movably arranged at the far end of the loop bar (231), the lower end of the homogenate cup (232) is provided with a discharge funnel (234), the connection part of the discharge funnel (234) and the homogenate cup (232) is provided with a liquid discharge valve (235),
the filter (24) comprises a movable loop bar (241) sleeved on the fixed frame (21) and a liquid collecting cup (242) movably arranged at the far end of the movable loop bar (241), the upper end of the liquid collecting cup (242) is provided with a plurality of layers of disposable gauze, the lower part of the side wall of the liquid collecting cup (242) is provided with a liquid outlet valve (245),
the liquid adding device (3) comprises a rotating frame (31) fixedly arranged at the rear side end of the centrifuge main body (1), and a hypotonic buffer liquid tank (32) and a solubilization liquid tank (33) which are respectively arranged at two sides of the rotating frame (31), wherein a first liquid adding valve (321) is arranged at the outer side of the lower end of the side wall of the hypotonic buffer liquid tank (32), a second liquid adding valve (331) is arranged at the outer side of the lower end of the side wall of the solubilization liquid tank (33), and the first liquid adding valve (321) and the second liquid adding valve (331) can both rotate to the position right above the test tube groove (14),
the liquid transfer device (4) comprises a liquid transfer frame (41) fixedly arranged at the right side end of the centrifuge body (1), a negative pressure liquid taking machine (42) fixedly arranged at the bottom of the liquid transfer frame (41) and a telescopic liquid taking pipe (43) arranged at the top end of the liquid transfer frame (41).
2. Extraction preparation device for light system II internal perimeter antennas CP43 and CP47 according to claim 1, characterized in that a control panel (16) is provided on the front side end face of the centrifuge body (1).
3. The extraction preparation apparatus for optical system II internal peripheral antennas CP43 and CP47 according to claim 1, wherein a fixing clip (141) for fixing the cuvette is provided in the cuvette slot (14).
4. The extraction and preparation device for optical system II internal peripheral antennas CP43 and CP47 according to claim 1, wherein the automatic press-sealing device (15) comprises a micro hydraulic pump (151) fixedly disposed inside the centrifugal column (12), a hydraulic lifting rod (152) connected to an upper output end of the micro hydraulic pump (151), an electrically controlled rotating head (153) installed at a top end of the hydraulic lifting rod (151), four connecting rods (154) fixedly installed at sides of the electrically controlled rotating head (153) in a cross shape, and test tube sealing caps (155) respectively disposed at distal ends of the four connecting rods (154).
5. Extraction preparation device for photosystem II internal perimeter antennas CP43 and CP47 as claimed in claim 4, characterized in that a negative pressure generator (17) is further provided in the spin column (12), said negative pressure generator (17) is connected to the four cuvette lids (155) through internal piping for creating a negative pressure environment in the cuvette.
6. The device for extracting and preparing light system II internal peripheral antennas CP43 and CP47 of claim 1, wherein a splash cover (233) is movably provided at the upper end of the homogenizing cup (232), and a head at the bottom end of the homogenizing machine (22) can be placed inside the homogenizing cup (232) through the splash cover (233).
7. Extraction preparation device for photosystem II internal perimeter antennas CP43 and CP47 as claimed in claim 6, characterized in that a head at the bottom end of the refiner (22) is placeable inside the refiner cup (232) through the splash cover (233).
8. Extraction preparation device for photosystem II internal perimeter antennas CP43 and CP47 as claimed in claim 1, characterized in that the hypotonic buffer liquid tank (32) and the solubilization liquid tank (33) are both insulated holding tanks.
9. The extraction and preparation device for optical system II internal peripheral antennas CP43 and CP47 as claimed in claim 1, wherein the lower end of said telescopic liquid-taking tube (43) is movably sleeved with a replaceable disposable liquid-taking tube (44).
10. The method for extracting and preparing light system II internal peripheral antennas CP43 and CP47 by using the extraction and preparation apparatus as claimed in any one of claims 1 to 9, mainly comprising the steps of:
s1: blade treatment
The method comprises the steps of cutting blades, putting the cut blades into a homogenizing cup (232), adding a buffer solution into the homogenizing cup (232), covering a splash-proof cover (233), moving a homogenizing container (23) upwards to enable a head of a homogenizing machine (22) to extend into the homogenizing cup (232), starting the homogenizing machine (22) to homogenize the blades in the homogenizing cup (232), opening a drain valve (235) after treatment is finished to discharge the homogenized liquid into a filter (24), filtering the homogenized liquid through multiple layers of disposable gauze to remove residual blades, and collecting the homogenized liquid in a liquid collecting cup (242);
s2: chloroplast isolation
Placing a test tube in a test tube groove (14), rotating the test tube to the position below a liquid outlet valve (245), opening the liquid outlet valve (245) to add filtered homogenate into the test tube, sealing the test tube by using an automatic pressure sealing device (15), then starting a centrifugal motor (13) for centrifugation, and taking out chloroplast positioned in the middle of the test tube by using a liquid transfer device (4) after the centrifugation is finished;
s3: preparation of thylakoid membranes
Rotating a blank test tube to the lower part of a telescopic liquid taking tube (43) of a liquid transferring device (4), transferring the chloroplasts obtained in the step S2 into a blank test tube, then rotating a low-permeability buffer liquid tank (32) of the liquid adding device (3) to the upper part of the test tube, adding a low-permeability buffer liquid into the test tube, sealing the test tube by using an automatic pressure sealing device (15), then starting a negative pressure generator (17) to form a negative pressure condition in the test tube, accelerating the bursting of the chloroplasts, then starting a centrifugal motor (13) to carry out centrifugation, replacing a disposable liquid taking tube (44) sleeved on the telescopic liquid transferring tube (43) after the centrifugation is finished, and then extracting the supernatant containing thylakoid membranes in the test tube by using the liquid transferring device (4);
s4: PSII particle preparation
Rotating a blank test tube to the lower part of a telescopic liquid taking tube (43) of a liquid transferring device (4), transferring the supernatant obtained in the step S3 into the blank test tube, then rotating a solubilizing liquid tank (33) of the liquid adding device (3) to the upper part of the test tube, adding a solubilizing liquid into the test tube, sealing the test tube by using an automatic pressure sealing device (15), then starting a centrifugal motor (13) for centrifugation, and after the centrifugation is finished, extracting the supernatant containing a photosystem II in the test tube by using the liquid transferring device (4);
s5: preparation of PSII oxygen-releasing core Complex
Subjecting the supernatant obtained in the step S4 to electrophoresis treatment, and eluting with an ion exchange column to obtain a PSII oxygen-releasing core compound;
s6: extraction and preparation of CP43 and CP47
The PSII oxygen-evolving core complex prepared in step S5 was eluted separately using ion exchange columns filled with different buffers to give CP43 and CP 47.
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| Application Number | Priority Date | Filing Date | Title |
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| CN202110340709.6A CN113069790B (en) | 2021-03-30 | 2021-03-30 | Extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP47 |
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| CN202110340709.6A CN113069790B (en) | 2021-03-30 | 2021-03-30 | Extraction and preparation device and method for light system II inner peripheral antennas CP43 and CP47 |
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| CN113069790A true CN113069790A (en) | 2021-07-06 |
| CN113069790B CN113069790B (en) | 2022-08-30 |
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| CN115251387A (en) * | 2022-07-29 | 2022-11-01 | 上海交通大学 | Method for preparing nanoscale chlorophyll protein complex and application thereof |
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| CN113069790B (en) | 2022-08-30 |
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