Method for extracting chloroplasts from Chlamydomonas reinhardtii

An extraction method and chloroplast technology are applied in the field of Chlamydomonas reinhardtii chloroplast extraction, which can solve the problems of distorted chloroplast membrane structure, time-consuming and labor-intensive, unfavorable repeated chloroplast extraction, etc., and achieve the effect of maintaining integrity and ensuring sufficiency.

Inactive Publication Date: 2018-07-20
DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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Problems solved by technology

The model organism Chlamydomonas reinhardtii only contains one cup-shaped chloroplast. Some researchers have isolated chloroplasts from Chlamydomonas reinhardtii under eutrophic growth conditions. Most of these chloroplasts are obtained under specific growth conditions and isolation conditions, which have certain limitations. In addition, the extracted chloroplast quality evaluation methods are based on traditional identification methods, such as determining the purity of chloroplasts through specific subcellular localization of enzymes, determining the integrity of chloroplast capsules through potassium ferricyanide oxygen release test method or The purity and integrity of chloroplasts are determined by direct observation of electron microscopes. These methods not only need to rely on the physiological activity of isolated chloroplasts, but are not conducive to the repeated extraction of chloroplasts, and are time-consuming and labor-intensive, especially the preparation of electron microscope samples. Observation, the quality status of the obtained chloroplasts cannot be determined in time, which makes the research on the optimal extraction of chloroplasts stagnant
In addition, microalgal chloroplasts can accumulate starch grains or oil droplets under stress conditions, and the formation of these metabolites can distort the membrane structure of chloroplasts. Conventional extraction methods are no longer suitable for the extraction of stressed chloroplasts.

Method used

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  • Method for extracting chloroplasts from Chlamydomonas reinhardtii
  • Method for extracting chloroplasts from Chlamydomonas reinhardtii
  • Method for extracting chloroplasts from Chlamydomonas reinhardtii

Examples

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Embodiment 1

[0039] Embodiment 1 algae culture

[0040] Chlamydomonas reinhardtii can be either the biomass obtained under eutrophic conditions or the biomass obtained under nutrient stress conditions.

[0041] Algal cells were first grown on solidified agar Minimal medium, and then the monoclonal algae spots were transferred to liquid Minimal medium for photoautotrophy, and the photoperiod was set to 12h light / 12h dark (synchronization). The algal liquid was cultured synchronously for 4 days and then transferred to the photobioreactor (PBR) to continue the culture ( figure 1 ), cultured under continuous light for the first 48 h, and then cultured with a synchronous photoperiod of 12 h light / 12 h dark for the next 52 h, with a light intensity of 50 μmol m -2 the s -1 , when the algae cell density increased to 1×10 7 ml -1 When , some algal cells were harvested, and the centrifugation conditions were 3000g, 5min, 4°C. These algal cells were the biomass of eutrophic culture. A part of t...

Embodiment 2

[0049] The extraction of embodiment 2 chloroplasts

[0050] Wash the algae cells containing 10mg chlorophyll twice with 50ml 50mM Hepes-KOH pH 7.5 under centrifugation conditions of 3000g, 5min, 4°C, discard the supernatant and add 4ml 50mM Hepes-KOH pH 7.5.

[0051] Add 20ml of chloroplast separation buffer to resuspend the cells, and the chlorophyll concentration of the obtained algae cell concentrate is 0.5mgml -1 , pass the above-mentioned algae cell concentrate through a stainless steel needle disruptor (such as figure 1 The stainless steel needle crusher with equivalent crushing effect shown above) is used for crushing. The crusher includes a pipe body with upper and lower ends open. The upper opening end of the pipe body is used as a feeding port. A needle is connected, the lower opening end of the tube is airtightly connected with the needle, the upper part of the tube is provided with a gas interface, and the gas interface is connected with an inert atmosphere gas so...

Embodiment 3

[0057] The quality evaluation of embodiment 3 chloroplasts

[0058] Chloroplast Yield Parameters

[0059] Yield of chloroplast = total chlorophyll content of chloroplasts / total chlorophyll content of algal cells × 100%, and the algal cells used to extract chloroplasts shall be those containing 10 mg of chlorophyll.

[0060] Yield of chloroplasts under normal culture conditions

[0061] The chloroplast yield under normal conditions obtained based on different crushing pressures and centrifugal forces in Example 2 is 13-30%, as shown in Table 1.

[0062] Yield of chloroplasts under nutrient stress conditions

[0063] The nitrogen stress chloroplast productive rate obtained based on different crushing pressures and centrifugal force in embodiment 2 is 0.5~3%, as shown in table 1, the significant decline of its productive rate (as Figure 5 (shown) shows that the difficulty of extracting chloroplasts under stress increases.

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Abstract

The invention discloses a method for extracting chloroplasts from Chlamydomonas reinhardtii. The chloroplasts with a high purity and a high completeness can be effectively extracted by combining differential pressure mechanical crushing, density gradient centrifugation and fatty acid abundance analysis technologies, and the extraction yield of the chloroplasts by chlorophyll is 0.5-30%. The methodis especially suitable for extracting the chloroplasts from Chlamydomonas reinhardtii under stress culture conditions which are difficult to extract by conventional methods, and provides an effectivebiomaterial for the subcellular level research of microalgae.

Description

technical field [0001] The invention belongs to the field of biological technology, relates to the technical field of separation and purification of microalgae organelles, in particular to a method for extracting Chlamydomonas reinhardtii chloroplast. Background technique [0002] As the center of photosynthesis, chloroplasts are the synthesis sites of amino acids, fatty acids and chlorophyll in plants and microalgae, and participate in a variety of physiological metabolic processes. Therefore, the acquisition of chloroplasts can provide effective biological materials for various biochemical studies at the subcellular level. The model organism Chlamydomonas reinhardtii only contains one cup-shaped chloroplast. Researchers have isolated chloroplasts from Chlamydomonas reinhardtii under eutrophic growth conditions. Most of these chloroplasts are obtained under specific growth conditions and isolation conditions, which have certain limitations. In addition, the extracted chloro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/12C12N1/06
CPCC12N1/066C12N1/12
Inventor 薛松杨淼曹旭鹏姜君鹏褚亚东
Owner DALIAN INST OF CHEM PHYSICS CHINESE ACAD OF SCI
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