Symbiotic algae chloroplast extraction method

Abstract

The invention relates to the technical field of biology, in particular to a symbiotic algae chloroplast extraction method, the symbiotic algae chloroplast extraction method has a remarkable effect on symbiotic algae chloroplast purification, the limitation of an existing Percoll density gradient centrifugation technology is effectively broken through, the traditional technology needs two times of centrifugation and sample transfer, the process is tedious, and sample loss is easily caused. And in the aspects of purity and recovery rate, through reasonable setting of sucrose gradient and precise centrifugation, complete chloroplast is precisely layered, impurities are effectively separated, the purity and recovery rate are greatly improved, the accuracy of subsequent experiments is guaranteed, in the aspect of cost, expensive Percoll is replaced with cheap sucrose, the economic pressure is reduced, and meanwhile, the cost is reduced. The method is simple and convenient to operate and easy to master by experimenters, reduces errors and time consumption, improves efficiency, and provides a better purification scheme for symbiotic algae chloroplast research.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, and in particular to a method for extracting symbiotic algal chloroplasts. BACKGROUND

[0002] Symbiotic algae belong to dinoflagellate, which is a kind of single-cell algae that forms symbiotic relationship with marine invertebrates such as corals. The chloroplast type of symbiotic algae is peridininplastid type, which is commonly found in dinoflagellate. The chloroplast genome of symbiotic algae is significantly different from other algae, mainly in that the genome is divided into multiple small rings from a large ring, and each small ring contains only one to several genes. Although some peridininplastid chloroplast genes of dinoflagellate have been identified, the traditional method of extracting total DNA, separating circular DNA and performing enzyme digestion and sequencing often leads to the loss of chloroplast genes with low copy number.

[0003] With the advancement of technology, new methods for obtaining chloroplast DNA mainly rely on the separation of intact chloroplasts, and the extraction of chloroplast DNA to obtain a more comprehensive chloroplast genome. In the process of separating symbiotic algal chloroplasts, differential centrifugation combined with density gradient centrifugation is mainly used to obtain high-purity symbiotic algal chloroplasts. In the differential centrifugation process, first, centrifugation is performed at 1,800 rmp to remove unbroken cells, nuclei and other large organelles, and then centrifugation is performed at 5,000 rmp to remove mitochondria and other small organelles. Then, Percoll density gradient centrifugation is used, which is divided into two steps: the first step is to resuspend the precipitate obtained in the previous step and add it to 50% Percoll density gradient separation liquid, centrifuge at 3,000 rmp for 20 min, and collect the supernatant; the second step is to add the supernatant to 15% Percoll density gradient separation liquid, also centrifuge at 3,000 rmp for 20 min, and collect the precipitate. Finally, the obtained precipitate is washed and collected, and its morphology and purity are observed by fluorescence microscope to ensure that the purified chloroplasts are obtained.

[0004] When the existing method uses Percoll density gradient centrifugation technology, two centrifugation steps need to be performed, and sample transfer during the process increases sample loss, and the operation process is more complicated. At the same time, the purity of the separated chloroplasts is low, which affects the subsequent experimental results, and the cost of Percoll is high. SUMMARY

[0005] The present application aims to provide a symbiotic algal chloroplast extraction method, which aims to solve the technical problems in the prior art that the Percoll density gradient centrifugation technology needs to implement two centrifugation steps, the sample transfer during which causes an increase in sample loss, and the operation process is more complicated; meanwhile, the purity of the separated chloroplasts is low, which further affects the subsequent experimental results, and the cost of Percoll is high.

[0006] To achieve the above-mentioned purpose, the present application adopts a symbiotic algal chloroplast extraction method, which comprises the following steps:

[0007] First, the algal liquid is pretreated, then centrifuged at 4℃, 5,000rmp for 10min, the precipitate is collected, and the separation buffer A is used for suspension and repeated centrifugation steps;

[0008] 300μL supernatant and precipitate are reserved, and glass beads with a diameter of 0.5mm and 0.1mm are added in an equal volume to the precipitate, then broken, and the separation buffer A is added to suspend the sample, centrifuged at 500rmp for 30s, the supernatant is reserved, and the precipitate glass beads are discarded, to prepare a crude extract;

[0009] The crude extract is placed in a centrifuge, centrifuged at 4℃, 1,800rmp for 10min, the precipitate is removed, and then the supernatant is centrifuged at 4℃, 5,000rmp for 10min, the supernatant is removed, and the precipitate is collected, which is the crude extract of chloroplasts;

[0010] A special centrifuge tube is constructed: 5mL ice-precooled 55% sucrose buffer is laid on the lowermost layer of the HITACHI CP100 centrifuge tube, and 5mL ice-precooled 40% sucrose buffer is laid on the 55% sucrose buffer, forming a gradient;

[0011] After the crude chloroplast extract is suspended with the separation buffer B, the resuspended buffer suspension is uniformly laid on the upper layer of the 13.2mL HITACHI CP100 special centrifuge tube;

[0012] Centrifuged at 4℃, 40,000g for 3h, the complete chloroplasts are concentrated between the 40% and 55% sucrose gradient, and this layer is sucked into a 2mL centrifuge tube;

[0013] Dilute 5 times with ice-precooled separation buffer B, and centrifuged at 6,000rmp for 30min to remove excess sucrose, and repeat this step once;

[0014] Collect the precipitate to obtain the crude chloroplast product, and the obtained precipitate is the purified chloroplast.

[0015] In the step of "firstly, the algal liquid is pretreated, then centrifuged at 4℃ and 5,000 rpm for 10 min, the precipitate is collected, and the separation buffer A is used to suspend and repeat the centrifugation step", the number of repeated centrifugation steps is not less than two times.

[0016] In the step of "300 μL supernatant and precipitate are reserved, an equal volume of (0.5 mm:0.1 mm=3:1) glass beads is added to the precipitate, and then crushing is performed", the specific way of crushing is as follows:

[0017] The FastPrep-24 homogenizer is used, the frequency is 6 m / s, crushing is performed for 60 s, the suspension is placed on ice for 60 s after each crushing, and the process is repeated for three times.

[0018] In the step of "the resuspended buffer suspension is uniformly laid on the upper layer of a 13.2 mL HITACHI CP100 special centrifuge tube", the gap above the suspension is reserved to be 1-2 μm.

[0019] In the construction of the special centrifuge tube, the composition of the 40% sucrose buffer is 40% (w / v) sucrose, 50 mM HEPES, 300 mM Sorbitol.

[0020] The composition of the 55% sucrose buffer is 55% (w / v) sucrose, 50 mM HEPES, 300 mM Sorbitol.

[0021] In the step of "the resuspended buffer suspension is uniformly laid on the upper layer of a 13.2 mL HITACHI CP100 special centrifuge tube", the gap above the suspension is reserved to be 1-2 μm.

[0022] The composition of the separation buffer B is 50 mM HEPES, 300 mM Sorbitol, 5 mM MgCl2, 50 mM EDTA, 5 mM C6H7O6Na, and the pH is 7.5-7.8, which is stored at 4℃.

[0023] In the step of "the resuspended buffer suspension is uniformly laid on the upper layer of a 13.2 mL HITACHI CP100 special centrifuge tube", the gap above the suspension is reserved to be 1-2 μm.

[0024] In the step of "an equal volume of glass beads with a diameter of 0.5 mm and 0.1 mm is added to the precipitate", the ratio of the 0.5 mm glass beads to the 0.1 mm glass beads is 3:1.

[0025] The present invention discloses a method for extracting chloroplasts from symbiotic algae. In practical application, the algal solution is first pretreated, then centrifuged at 4°C and 5,000 rpm for 10 min. The precipitate is collected, resuspended in separation buffer A, and the centrifugation process is repeated. 300 μL of supernatant and precipitate are retained, and glass beads with diameters of 0.5 mm and 0.1 mm, in equal volumes to the precipitate, are added and crushed. The sample is then resuspended in separation buffer A, centrifuged at 500 rpm for 30 s, and the supernatant is retained while the precipitate glass beads are discarded to obtain a crude extract. The crude extract is then centrifuged at 4°C and 1,000 rpm for 10 min. After centrifugation at 800 rpm for 10 min, remove the precipitate. Then, centrifuge the supernatant at 5,000 rpm for 10 min at 4°C, remove the supernatant, and collect the precipitate. This is the crude chloroplast extract. Construct a dedicated centrifuge tube: Place 5 mL of ice-cold 55% sucrose buffer at the bottom of a HITACHICP 100 centrifuge tube, and then place 5 mL of ice-cold 40% sucrose buffer on top of this 55% sucrose buffer to form a gradient. After suspending the crude chloroplast extract in separation buffer B, evenly spread the resuspended buffer suspension on 13.2 mL of HITACHICP 100 centrifuge tube. The upper layer of the CP100 centrifuge tube was centrifuged at 40,000g for 3 hours at 4°C. Intact chloroplasts were concentrated between the 40% and 55% sucrose gradients. This layer was transferred to a 2mL centrifuge tube. The chloroplast was diluted 5-fold with ice-cold separation buffer B and centrifuged at 6,000rpm for 30 minutes to remove excess sucrose. This step was repeated once. The precipitate was collected to obtain the initial chloroplast sample, which is the purified chloroplast. This method solves the problems of existing technologies using Percoll density gradient centrifugation: requiring two centrifugation steps, increasing sample loss due to sample transfer, and making the operation more cumbersome; the resulting chloroplasts have lower purity, affecting subsequent experimental results; and Percoll is also costly. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0027] Figure 1 This is a schematic diagram of the symbiotic algal chloroplast extraction method of the present invention.

[0028] Figure 2 This is a comparison image of the chloroplasts extracted by the symbiotic algal chloroplast extraction method of the present invention under a fluorescence microscope with chloroplasts separated by the symbiotic algal cells before disruption and by Percoll density gradient centrifugation. Detailed Implementation

[0029] The embodiments of the present application are described in detail below, examples of which are shown in the drawings, the embodiments described below by referring to the drawings are exemplary, and are intended to explain the present application, and cannot be understood as a limitation of the present application.

[0030] Please refer to Figure 1 and Figure 2 , Figure 1 is a schematic diagram of the symbiotic algae chloroplast extraction method of the present application. Figure 2 is a comparison diagram of the extracted chloroplast of the symbiotic algae chloroplast extraction method of the present application under a fluorescence microscope with the symbiotic algae cells before crushing and the chloroplast separated by the Percoll density gradient centrifugation method.

[0031] The present application provides a symbiotic algae chloroplast extraction method, comprising the following steps:

[0032] S1, first pretreat the algae liquid, then centrifuge at 4℃, 5,000rmp for 10min, collect the precipitate, and suspend with separation buffer A and repeat the centrifugation step;

[0033] For this specific embodiment, the pretreatment method of the algae liquid is:

[0034] 500mL of algae liquid reaching the late exponential growth phase after 10 days of culture is placed at 4℃ for dark treatment for 24h.

[0035] The number of repeated centrifugation steps is not less than two times.

[0036] S2, reserve 300μL of supernatant and precipitate, add glass beads with a diameter of 0.5mm and 0.1mm in an equal volume to the precipitate, then crush, add separation buffer A to suspend the sample, centrifuge at 500rmp for 30s, reserve the supernatant, discard the precipitate glass beads, and prepare a crude extract;

[0037] The specific method of crushing is:

[0038] Use FastPrep-24 homogenizer, frequency is 6m / s, crush for 60s, rest on ice for 60s after each crushing, repeat for 3 times.

[0039] The composition of separation buffer A is 50mM HEPES, 300mM Sorbitol, 5mM MgCl2, 50mM EDTA, 5mMC6H7O6Na, 1%β-Mercaptoethanol, pH is 7.5-7.8, and is stored at 4℃.

[0040] For this specific embodiment, the ratio of 0.5mm glass beads and 0.1mm glass beads is 3:1.

[0041] S3, the crude liquid is placed in a centrifuge, centrifuged at 4°C, 1,800 rpm for 10 min, the precipitate is removed, and then the supernatant is centrifuged at 4°C, 5,000 rpm for 10 min, the supernatant is removed, and the precipitate is collected, which is the crude chloroplast extract;

[0042] S4, a special centrifuge tube is constructed: 5 mL of ice-precooled 55% sucrose buffer is laid at the bottom of the HITACHI CP100 centrifuge tube, and 5 mL of ice-precooled 40% sucrose buffer is laid on the 55% sucrose buffer, forming a gradient;

[0043] For this specific embodiment, the composition of the 40% sucrose buffer is 40% (w / v) sucrose, 50 mM HEPES, and 300 mM Sorbitol when the special centrifuge tube is constructed.

[0044] The composition of the 55% sucrose buffer is 55% (w / v) sucrose, 50 mM HEPES, and 300 mM Sorbitol.

[0045] S5, after the chloroplast crude extract is suspended with separation buffer B, the resuspended buffer suspension is uniformly laid on the upper layer of the 13.2 mL HITACHI CP100 special centrifuge tube;

[0046] For this specific embodiment, the composition of the separation buffer B is 50 mM HEPES, 300 mM Sorbitol, 5 mM MgCl2, 50 mM EDTA, 5 mM C6H7O6Na, and the pH is 7.5-7.8, which is stored at 4°C.

[0047] In the step of "laying the resuspended buffer suspension uniformly on the upper layer of the 13.2 mL HITACHI CP100 special centrifuge tube", the gap above the suspension is reserved for 1-2 μm.

[0048] S6, centrifugation at 4°C, 40,000 g for 3 h, the intact chloroplasts are concentrated between the 40% and 55% sucrose gradient, and this layer is absorbed into a 2 mL centrifuge tube;

[0049] For this specific embodiment,

[0050] S7, dilute the ice-precooled separation buffer B by 5 times, and centrifuge at 6,000 rpm for 30 min to remove excess sucrose, and repeat this step once;

[0051] S8, collect the precipitate to obtain the chloroplast primary product, and the obtained precipitate is the purified chloroplast.

[0052] For this specific embodiment, after obtaining the purified chloroplasts, the morphology and purity were observed under a fluorescence microscope. Please refer to Figure 2 Wherein A is the symbiotic algae cell before being broken under fluorescence irradiation, B is the chloroplast separated by Percoll density gradient centrifugation under fluorescence irradiation, and C is the chloroplast separated by sucrose density gradient centrifugation under fluorescence irradiation. The red fluorescence is the chloroplast excitation fluorescence. As can be seen from C, most of the samples collected by the present application are symbiotic algae chloroplasts.

[0053] The content of chlorophyll in the chloroplasts was determined by a spectrophotometer to evaluate the quality and activity of the chloroplasts, and based on the detection results, the reasons were analyzed and the parameters of the foregoing steps were adjusted to improve the purification effect.

[0054] In specific use, the symbiotic algae chloroplast extraction method of the present application is used to first pretreat the algae liquid, then centrifuge at 4°C and 5,000 rmp for 10 min, collect the precipitate, suspend it with separation buffer A and repeat the centrifugation step; retain 300 μL supernatant and precipitate, add glass beads with a diameter of 0.5 mm and 0.1 mm in an amount equal to that of the precipitate, then break, add separation buffer A to suspend the sample, centrifuge at 500 rmp for 30 s, retain the supernatant, discard the precipitate glass beads, and prepare a crude extract; centrifuge the crude extract at 4°C and 1,800 rmp for 10 min, remove the precipitate, centrifuge the supernatant at 4°C and 5,000 rmp for 10 min, remove the supernatant, and collect the precipitate, which is the chloroplast crude extract; construct a special centrifuge tube: lay 5 mL ice-precooled 55% sucrose buffer at the bottom of a HITACHI CP100 centrifuge tube, and lay 5 mL ice-precooled 40% sucrose buffer on the 55% sucrose buffer, forming a gradient; suspend the chloroplast crude extract with separation buffer B, and evenly lay the resuspended buffer suspension on the upper layer of the 13.2 mL HITACHI CP100 special centrifuge tube; centrifuge at 4°C and 40,000 g for 3 h, and the intact chloroplasts are concentrated between the 40% and 55% sucrose gradients, and this layer is absorbed into a 2 mL centrifuge tube; dilute the ice-precooled separation buffer B by 5 times, and centrifuge at 6,000 rmp for 30 min to remove excess sucrose, and repeat this step once; collect the precipitate to obtain the chloroplast primary product, and the obtained precipitate is the purified chloroplast. In this way, the technical problems of the prior art, such as the need to implement two centrifugation steps, the increase in sample loss caused by sample transfer during the process, and the more complicated operation process, are solved. At the same time, the purity of the separated chloroplasts is relatively low, which further affects the subsequent experimental results, and the cost of Percoll is relatively high.

[0055] The above disclosed is only a preferred embodiment of the present application, of course, cannot be limited by this, the person skilled in the art can understand that the all or part of the processes of the above-mentioned embodiment are realized, and the equivalent changes made according to the claims of the present application still belong to the scope covered by the present application.

Claims

1. A method for extracting chloroplasts from symbiotic algae, comprising the following steps: firstly, pretreating the algae liquid, then centrifuging at 5,000 rpm for 10 min at 4°C, collecting the precipitate, and suspending the precipitate with separation buffer A and repeating the centrifugation step; retaining 300 μL of supernatant and precipitate, adding glass beads with a diameter of 0.5 mm and 0.1 mm in an equal volume to the precipitate, then crushing, suspending the sample with separation buffer A, centrifuging at 500 rpm for 30 s, retaining the supernatant, and discarding the precipitate and glass beads to obtain a crude extract; centrifuging the crude extract at 1,800 rpm for 10 min at 4°C to remove the precipitate, then centrifuging the supernatant at 5,000 rpm for 10 min at 4°C to remove the supernatant and collect the precipitate, which is the crude chloroplast extract; constructing a special centrifuge tube: placing 5 mL of ice-precooled 55% sucrose buffer at the bottom of a HITACHI CP100 centrifuge tube, and placing 5 mL of ice-precooled 40% sucrose buffer on the 55% sucrose buffer to form a gradient; suspending the crude chloroplast extract with separation buffer B, and uniformly placing the resuspended buffer suspension on the upper layer of a 13.2 mL HITACHI CP100 special centrifuge tube; centrifuging at 40,000 g for 3 h at 4°C, and collecting the intact chloroplasts between the 40% and 55% sucrose gradient, and then sucking the layer into a 2 mL centrifuge tube; diluting the ice-precooled separation buffer B by 5 times, and centrifuging at 6,000 rpm for 30 min to remove the excess sucrose, and repeating the step once; collecting the precipitate to obtain the crude chloroplast product, and the obtained precipitate is the purified chloroplast.

2. The method for extracting chloroplasts from symbiotic algae according to claim 1, wherein the number of repeated centrifugation steps in the step of firstly pretreating the algae liquid, then centrifuging at 5,000 rpm for 10 min at 4°C, collecting the precipitate, and suspending the precipitate with separation buffer A and repeating the centrifugation step is not less than two times.

3. The method for extracting chloroplasts from symbiotic algae according to claim 2, wherein the specific method for crushing in the step of retaining 300 μL of supernatant and precipitate, adding glass beads with a diameter of 0.5 mm and 0.1 mm in an equal volume to the precipitate, then crushing is as follows: using a FastPrep-24 homogenizer, crushing at a frequency of 6 m / s for 60 s, resting on ice for 60 s after each crushing, and repeating three times.

4. The method for extracting chloroplasts from symbiotic algae according to claim 3, wherein the gap above the suspension is retained to be 1-2 μm when uniformly placing the resuspended buffer suspension on the upper layer of a 13.2 mL HITACHI CP100 special centrifuge tube in the step.

5. The method for extracting chloroplasts from symbiotic algae according to claim 4, wherein the composition of the 40% sucrose buffer is 40% (w / v) sucrose, 50 mM HEPES, and 300 mM sorbitol when constructing the special centrifuge tube. ​ ​ ​ ​ ​ 55% sucrose buffer solution is composed of 55% (w / v) sucrose, 50 mM HEPES, 300 mM Sorbitol.

6. The method of claim 5, wherein the purified chloroplasts are obtained by the steps of: Separation buffer A is composed of 50 mM HEPES, 300 mM Sorbitol, 5 mM MgCl2, 50 mM EDTA, 5 mM C6H7O6Na, 1% β-Mercaptoethanol, pH 7.5-7.8, stored at 4°C.

7. The method of claim 6, wherein the purified chloroplasts are obtained by the steps of: Separation buffer B is composed of 50 mM HEPES, 300 mM Sorbitol, 5 mM MgCl2, 50 mM EDTA, 5 mM C6H7O6Na, pH 7.5-7.8, stored at 4°C.

8. The method of claim 7, wherein the purified chloroplasts are obtained by the steps of: After obtaining the purified chloroplasts, the morphology and purity of the chloroplasts are observed under a fluorescence microscope.

9. The method of claim 8, wherein the purified chloroplasts are obtained by the steps of: In the step of "adding glass beads with a diameter of 0.5 mm and 0.1 mm to the same volume as the precipitation", the ratio of 0.5 mm glass beads to 0.1 mm glass beads is 3:1.

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