CN113061567A - Method for extracting chloroplast of kochia scoparia - Google Patents
Method for extracting chloroplast of kochia scoparia Download PDFInfo
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- CN113061567A CN113061567A CN202110367518.9A CN202110367518A CN113061567A CN 113061567 A CN113061567 A CN 113061567A CN 202110367518 A CN202110367518 A CN 202110367518A CN 113061567 A CN113061567 A CN 113061567A
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Abstract
The invention discloses a method for extracting chloroplast from a hevea brasiliensis leaf, which comprises the steps of collecting the complete leaf of the hevea brasiliensis plant; crushing the leaf tissue, and then carrying out primary extraction to obtain a rough extract of the hevea brasiliensis chloroplast; adding the crude extract of Hevea brasiliensis chloroplast into the upper layer of cell separation liquid, and centrifuging at 4 deg.C for 10-15 min; and after the centrifugation is finished, sucking the precipitate of the cell separation liquid, washing the precipitate by chloroplast extracting solution, and then carrying out second ultra-high-speed centrifugation for 12min to obtain the complete melissa chloroplast. The invention can be applied to the separation of the chloroplast of the kochia kochiana, can also extract the chloroplast of the plants of the Compositae or the same genus, is suitable for being collected from general plant materials, and can also be applied to the separation of the chloroplast of recalcitrant plant leaves.
Description
Technical Field
The invention belongs to the technical field of plant tissue identification, and particularly relates to a method for extracting hevea brasiliensis chloroplast.
Background
Kokuba, wherein mevalonic acid (MVA) is used in the pathway for regulation of natural rubber biosynthesis, is diploid (2n ═ 16), has a relatively simple genome, is 1.29Gb in length, and comprises 46,731 predicted protein-encoding genes, and kokuba roots contain high molecular weight rubber molecules, with properties comparable to those of the rubber latex of hevea brasiliensis, and can be used as an alternative rubber crop, with high rubber content of kokuba roots, representing 3% to 28% of their total dry weight, and they are also a new source of inulin (linear β - (2-1) -linked fructan). In addition, the kokstroemia indica can be widely grown in cold and temperate regions in a relatively short life cycle, and is easy to harvest and genetically transform, and thus can be used as an ideal model plant for the gene function of wheat and a biosynthetic material of natural rubber.
Chloroplasts are the basis for the architecture of organelles, cortical chloroplasts, that function in photosynthesis. The acquisition of the complete chloroplast is a precondition for developing chloroplast energy conversion, electron transfer, carbon fixation and downstream omics research, but the extraction of the hevea brasiliensis chloroplast is blank, and the physiological and molecular mechanism research of cortical photosynthesis is severely restricted, so that the hevea brasiliensis is used as a material to establish an extraction and purification system of the hevea brasiliensis chloroplast, and the research of the photosynthesis mechanism of the hevea brasiliensis is promoted at the level of organelles, subcellular organelles and molecules. Therefore, a method for extracting the chloroplast of the kochia scoparia is needed.
Disclosure of Invention
The invention provides a method for extracting and purifying hevea brasiliensis chloroplast.
The invention comprises the following steps:
a, collecting complete leaves of a rubber grass plant;
b, crushing the leaf tissue, and carrying out primary extraction to obtain a rough extract of the hevea brasiliensis chloroplast;
c, adding the rough extract of the rubberella chloroplast into the upper layer of the cell separation liquid, and then carrying out first ultra-high-speed centrifugation for 10-15min at 4 ℃;
and D, after the centrifugation is finished, sucking the precipitate of the cell separation solution, washing the precipitate by using chloroplast extracting solution, and then carrying out second ultra-high-speed centrifugation for 12min to obtain the complete melissa chloroplast.
The method for the initial extraction comprises the following steps of: 1 adding PVPP powder, fully grinding, adding 5mL of chloroplast extracting solution into the ground sample of the leaf tissue according to each gram of sample, and filtering the grinding solution to add the chloroplast extracting solution.
Further, the preparation method of the extract comprises adding 0.3mM/L of sorbusAlcohol, 5mM/L EDTA, 1mM/LMgCL2，10mM/LNaHCO30.5mM/L dithiothreitol DTT in a volume ratio of 1:1:1:1:2, mixing with ddH2O is added to 850mL to be completely dissolved; then adding 100mL of buffer solution, adjusting the pH value to 7.5, mixing the solution and the solution evenly, and then using ddH2And (4) performing constant volume to 1L to obtain chloroplast extract.
Furthermore, 1mL of the 70% cell separation solution, 2mL of the 50% cell separation solution, 3mL of the 30% cell separation solution, and 4mL of the 10% cell separation solution were applied to the cell separation solution in this order from bottom to top, and 1mL of the crude chloroplast extract was added to the uppermost layer.
Further, the ultracentrifugation is at 8000 RCF.
The invention has the beneficial effects that:
the invention can be applied to the separation of the chloroplast of the kochia kochiana, can also extract the chloroplast of the plants of the Compositae or the same genus, is suitable for being collected from general plant materials, and can also be applied to the separation of the chloroplast of recalcitrant plant leaves.
Drawings
FIG. 1 is a schematic flow diagram of the present invention;
FIG. 2 is a comparison graph of 3 biological repeated verifications of the extract of the chloroplast of the rubber grass in this example;
Detailed Description
The principles and features of this invention are described below in conjunction with examples which are set forth to illustrate, but are not to be construed to limit the scope of the invention.
As shown in fig. 1, the present invention comprises the steps of:
a, collecting complete leaves of a rubber grass plant;
preparing a chloroplast extracting solution:
sorbitol (0.3mM/L), EDTA (5mM/L), LMgCL2(1mM/L)，NaHCO3(10mM/L), dithiothreitol DTT (0.5mM/L), 4-hydroxyethylpiperazine ethanesulfonic acid buffer (50mM/L), crospovidone PVPP2 g; a plurality of absorbent cotton gauzes;
mixing the above reagents according to the volume ratio of 1:1:1:1:2, and then usingddH2The volume of O is determined to 850mL, and a magnetic stirrer is used for uniformly mixing until the solid is completely dissolved; adding 100mL of buffer solution, adjusting the pH value to 7.5 by KOH, mixing the solution completely, and then adding ddH2And O, fixing the volume to 1L, and finally storing the chloroplast extract in a refrigerator at 4 ℃.
Chloroplast chromatography liquid preparation (100mL system, using Percoll multipurpose density gradient cell separation liquid manufactured by Sigma-Aldrich Co.): percoll 70% solution Percoll 50% solution, Percoll 30% solution, Percoll 10% solution, and the obtained chloroplast extract is stored at 4 ℃.
B, crushing the leaf tissue, and carrying out primary extraction to obtain a rough extract of the hevea brasiliensis chloroplast;
in this example, chloroplast extraction was performed by keeping the temperature of the chloroplast on (low temperature) ice at 4 ℃, 10g of fresh rubber grass plant leaves were cut into pieces and placed in a mortar, 0.1g of PVPP powder in grams of sample was added, 5mL of chloroplast extract was added per gram of sample, and the mixture was ground thoroughly; filtering the grinding fluid with middle 8-10 layers of gauze, centrifuging the filtrate at 4 deg.C under 800RCF for 3 min; transferring the supernatant into a new tube, centrifuging at 4 deg.C for 5min at 5000RCF ultra high speed, and collecting precipitate;
adding 5-6mL of chloroplast extract into the precipitate, and fully suspending and precipitating to prepare rough extract of the melissa officinalis chloroplast;
c, adding the rough extract of the rubberella chloroplast into the upper layer of the cell separation liquid, and then carrying out first ultra-high-speed centrifugation for 10-15min at 4 ℃;
taking a 15mL centrifuge tube, and sequentially spreading 70% Percoll1mL → from bottom to top
50% Percoll2mL → 30% Percoll3mL → 10% Percoll4mL, spreading 1mL chloroplast crude extract on the top, and centrifuging at 4 deg.C and 8000RCF for 12 min;
c, after the centrifugation is finished, sucking the precipitate of the cell separation liquid, washing the precipitate by chloroplast extracting solution, and then carrying out second ultra-high-speed centrifugation for 12min to obtain complete melissa chloroplast;
sucking dark green precipitate between Percoll 50-30% with a rubber head dropper, placing into a new centrifuge tube, cleaning the precipitate with chloroplast extract for 2 times, centrifuging at 8000RCF ultra high speed for 12min, precipitating to obtain Hevea brasiliensis chloroplast, and storing at-80 deg.C.
And (4) after chloroplast sediment is obtained, taking chloroplasts, and placing the chloroplasts under an optical microscope for microscopic examination to determine whether the integrity of the chloroplasts is qualified. The chloroplasts were then stored at-80 ℃ until use.
As shown in figure 2, the microscopic observation of the individual bodies of the hevea brasiliensis chloroplast is clear, the distribution of the chloroplasts is relatively dispersed, and the tissue residues are less, which proves that the extraction efficiency of the method is higher. By combining the experimental results, the method has higher yield of the extracted hevea brasiliensis leaves, and can be suitable for the related research of the extraction of the hevea brasiliensis chloroplast genome and chloroplast. Particularly, the invention has certain theoretical basis in the aspects of plant germplasm resource screening, SSR positioning, agricultural core germplasm resource gene positioning and the like, can be applied to separation of the chloroplast of the kochia kochiana, can also extract the chloroplast of the plant of the Compositae or the congeneric family, is not only suitable for collecting common plant materials, but also can be used for separating the chloroplast of recalcitrant plant leaves.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (5)
1. A method for extracting turfgrass chloroplast is characterized by comprising the following steps:
a, collecting complete leaves of a rubber grass plant;
b, crushing the leaf tissue, and carrying out primary extraction to obtain a rough extract of the hevea brasiliensis chloroplast;
c, adding the rough extract of the rubberella chloroplast into the upper layer of the cell separation liquid, and then carrying out first ultra-high-speed centrifugation for 10-15min at 4 ℃;
and D, after the centrifugation is finished, sucking the precipitate of the cell separation solution, washing the precipitate by using chloroplast extracting solution, and then carrying out second ultra-high-speed centrifugation for 12min to obtain the complete melissa chloroplast.
2. The method for extracting the chloroplast of the rubber grass as claimed in claim 1, wherein the initial extracting method comprises the following steps of: 1 adding PVPP powder, fully grinding, adding 5mL of chloroplast extracting solution into the ground sample of the leaf tissue according to each gram of sample, and filtering the grinding solution to add the chloroplast extracting solution.
3. The method for extracting chloroplast from Hevea brasiliensis according to claim 1, wherein the chloroplast extract is prepared by mixing 0.3mM/L sorbitol, 5mM/L EDTA, 1mM/LMgCL2，10mM/LNaHCO30.5mM/L dithiothreitol DTT, mixed at a volume ratio of 1:1:1:1:2, and then treated with ddH2O is added to 850mL to be completely dissolved; then adding 100mL of buffer solution, adjusting the pH value to 7.5, mixing the solution and the solution evenly, and then using ddH2And (4) performing constant volume to 1L to obtain the chloroplast extract.
4. The method for extracting the chloroplast of the rubber grass as claimed in claim 1, wherein 1mL of the cell separating medium with a concentration of 70%, 2mL of the cell separating medium with a concentration of 50%, 3mL of the cell separating medium with a concentration of 30% and 4mL of the cell separating medium with a concentration of 10% are spread in the cell separating medium from bottom to top in sequence, and 1mL of the crude chloroplast extract is added to the uppermost layer.
5. The method for extracting the chloroplast of rubber grass as claimed in claim 1, wherein the ultracentrifugation is carried out at 8000 RCF.
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Citations (11)
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| WO2006027865A2 (en) * | 2004-09-09 | 2006-03-16 | Research Institute Of Innovative Technology For The Earth | Method of transforming chloroplast of composite plant |
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| CN106967714A (en) * | 2017-06-07 | 2017-07-21 | 唐山师范学院 | The bar shaped leaf plant chloroplast DNA high-quality extracting method of high-purity |
| CN107365765A (en) * | 2017-07-26 | 2017-11-21 | 南京农业大学 | The highly effective extraction method of strawberry chloroplast genomic dna |
| CN108179131A (en) * | 2016-12-08 | 2018-06-19 | 中国林业科学研究院林业研究所 | A kind of preparation method of dry land willow blade complete excision |
| CN108300664A (en) * | 2017-01-11 | 2018-07-20 | 中国科学院大连化学物理研究所 | A kind of extracting method of Chlamydomonas reinhardtii chloroplaset |
| US20190352346A1 (en) * | 2016-12-05 | 2019-11-21 | Total Raffinage Chimie | Targeting Nuclear-Encoded Recombinant Proteins to the Chloroplast in Microalgae |
| CN111117943A (en) * | 2020-01-10 | 2020-05-08 | 北京林业大学 | Chloroplast membranaceous substance separation method |
| CN111440759A (en) * | 2020-03-06 | 2020-07-24 | 河南农业大学 | Method for simultaneously separating and extracting corn nucleus and chloroplast |
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2021
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| CN106676054A (en) * | 2016-11-17 | 2017-05-17 | 华南农业大学 | Rapid and simple method for extracting complete arabidopis thaliana chloroplast |
| US20190352346A1 (en) * | 2016-12-05 | 2019-11-21 | Total Raffinage Chimie | Targeting Nuclear-Encoded Recombinant Proteins to the Chloroplast in Microalgae |
| CN108179131A (en) * | 2016-12-08 | 2018-06-19 | 中国林业科学研究院林业研究所 | A kind of preparation method of dry land willow blade complete excision |
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| CN106967714A (en) * | 2017-06-07 | 2017-07-21 | 唐山师范学院 | The bar shaped leaf plant chloroplast DNA high-quality extracting method of high-purity |
| CN107365765A (en) * | 2017-07-26 | 2017-11-21 | 南京农业大学 | The highly effective extraction method of strawberry chloroplast genomic dna |
| CN111117943A (en) * | 2020-01-10 | 2020-05-08 | 北京林业大学 | Chloroplast membranaceous substance separation method |
| CN111440759A (en) * | 2020-03-06 | 2020-07-24 | 河南农业大学 | Method for simultaneously separating and extracting corn nucleus and chloroplast |
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