CN108300664A - A kind of extracting method of Chlamydomonas reinhardtii chloroplaset - Google Patents

A kind of extracting method of Chlamydomonas reinhardtii chloroplaset Download PDF

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CN108300664A
CN108300664A CN201710019354.4A CN201710019354A CN108300664A CN 108300664 A CN108300664 A CN 108300664A CN 201710019354 A CN201710019354 A CN 201710019354A CN 108300664 A CN108300664 A CN 108300664A
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chloroplaset
frustule
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nitrogen
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薛松
杨淼
曹旭鹏
姜君鹏
褚亚东
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Dalian Institute of Chemical Physics of CAS
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Abstract

The invention discloses a kind of extracting methods of Chlamydomonas reinhardtii chloroplaset, by the way that pressure difference Mechanical Crushing, density gradient centrifugation are combined with aliphatic acid enrichment analysis method, can effective DNA purity and the high chloroplaset of integrity degree, with chlorophyll meter, chloroplaset extraction efficiency is 0.5~30%.This method is particularly suitable for the Chlamydomonas reinhardtii chloroplaset extraction that conventional method is difficult under the conditions of the coercing cultivation extracted, and effective biomaterial is provided for the research of microalgae subcellsular level.

Description

A kind of extracting method of Chlamydomonas reinhardtii chloroplaset
Technical field
The invention belongs to biotechnologies, are related to microalgae cell device separating and purifying technology field, and in particular to Yi Zhonglai The extracting method of mattress Chlamydomonas reinhardtii chloroplast.
Background technology
As photosynthetic reaction centre, chloroplaset is the synthesizing site of amino acid in plant and microalgae, aliphatic acid and chlorophyll, A variety of physiological metabolism processes are participated in, therefore the acquisition of chloroplaset can provide effectively for the various biochemical research of subcellsular level Biomaterial.Model organism Chlamydomonas reinhardtii has researcher and is detached from Chlamydomonas reinhardtii containing only there are one cup-shaped chloroplasets The chloroplaset under eutrophy growth conditions is gone out, these chloroplasets are obtained under particular growth condition and separation condition mostly , there is certain limitation, the chloroplaset quality evaluating method extracted in addition to be all based on traditional identification method, such as pass through The specific subcellular localization of enzyme determines chloroplast envelope to determine the purity of chloroplaset, put oxygen method of testing by the potassium ferricyanide Integrity degree or the purity and integrity degree of chloroplaset are determined by electron microscope direct observational method, these methods not only need Dependent on the physiological activity of inoculationin vitro, it is unfavorable for realizing the repetition extraction of chloroplaset, and take time and effort, especially electronics The preparation and observation of microscope example cannot determine the quality state for obtaining chloroplaset, so that chloroplaset is excellent in time Change extraction research stagnation.In addition, microalgae chloroplaset can accumulate amylum body or oil droplet under stress conditions, these metabolites Formation the membrane structure of chloroplaset can be made to be distorted, general extraction methods be no longer desirable for stress chloroplaset extraction.This Invention expands the scope of application of microalgae chloroplaset extraction, establishes a kind of mild smudge cells and efficient quick quality evaluation Method.
Invention content
The object of the present invention is to provide a kind of extracting methods of Chlamydomonas reinhardtii chloroplaset.
To achieve the goals above, the technical solution adopted in the present invention content is：
(1) chlamydonomas reinhardtii cells for taking certain quantity of fresh include complete excision by the acquisition of mild Mechanical Crushing Cell fragment concentrate；
(2) by density gradient centrifugation, chloroplaset is collected.
The chloroplaset of chlamydonomas reinhardtii cells needs to keep complete under optics or electron microscope in the step (1), especially It is that should keep integrality including nitrogen-free or without phosphorus frustule chloroplast membranes structure under nutrition condition of salt stress；
The Chlamydomonas reinhardtii either cultivate obtained biomass under the conditions of the eutrophy of Minimal culture mediums, The biomass obtained under the conditions of can also be the Nutrient Stress in nitrogen-free or without phosphorus Minimal culture mediums.
Frustule fragmentation procedure in the step (1) is：By the frustule of the chlorophyll containing 6~10mg with 30~50mM 7.5~8.0 eccentric cleanings of Hepes-KOH pH 1~2 time, centrifugal condition be 2000~3000g, 5~10min, 4~10 DEG C, abandon Supernatant；
Cell is resuspended with 30~50mM Hepes-KOH pH 7.5~8.0, again such as above-mentioned pelleted by centrifugation, repetitive operation 1 ~2 times, the frustule concentrate of acquisition can pass through Yeda press, Bioneb, French press or with equivalent broken The stainless steel syringe needle destroyer of effect is crushed by mechanical shear stress, wherein acting on the inert atmosphere gas of frustule concentrate The cracking pressure of body (such as nitrogen and/or helium) is 0.35~0.75MPa.
The operation of the step (2) is：By 10~15ml ml containing 0.3~0.5mg in step (1)-1The cell of chlorophyll is broken After broken liquid centrifuges 2~5min at 750~5000g and 4~10 DEG C, 2~5ml Chloroplast isolation buffer solutions are added, are uniformly mixed Gone to afterwards containing percent by volume be respectively 20~25%, 45~50%, 65~70% Percoll density gradient liquid in (it Volume ratio be 1~2: 1~3: 1~2)), at 4200~5000g and 4~10 DEG C centrifuge 15~30min；By 45~ Chloroplaset band between 65%Percoll gradient liquid be added be equivalent to the Chloroplast isolation buffer solution of its 10~15 times of volumes into Row dilution, centrifuges 1~5min at 670~5000g and 4~10 DEG C after mixing, abandons after supernatant and is added that be equivalent to bottom dense The chloroplaset of 1~5 times of volume of contracting object preserves liquid and is diluted, and chloroplaset, which preserves liquid group, becomes 30~50mM HEPES-KOH pH 8.0 and final concentration 0.3~0.5M sorbierites, as chloroplaset extracting solution after mixing.
The extracting method of the Chlamydomonas reinhardtii chloroplaset, Minimal culture medium groups become 7.48mM NH4Cl、0.41mM MgSO4·7H2O、0.35mM CaCl2·2H2O、5.37mM K2HPO4、2.67mM KH2PO4And the Hunters 1ml/L trace element (Hunter ' s trace elements), surplus is water；
The Hunter trace element composition is as follows：134.3μM Na2-EDTA·2H2O、76.5μM ZnSO4·7H2O、 184.4μM H3BO3、25.6μM MnCl2·4H2O、17.9μM FeSO4·7H2O、6.8μM CoCl2·6H2O、6.3μM CuSO4·5H2O and 0.9 μM of (NH4)6Mo7O24·4H2O, surplus are water.
Nitrogen-free Minimal culture mediums are that above-mentioned Minimal culture mediums remove NH4Cl；
Without phosphorus Minimal culture mediums are that above-mentioned Minimal culture mediums remove K2HPO4And KH2PO4。
The extracting method of the Chlamydomonas reinhardtii chloroplaset, stainless steel syringe needle destroyer include the tube body of upper and lower two end opening, Tube body upper open end is equipped with valve as charge door at charge door, tube body lower open end is connected with syringe needle, tube body lower open end with Syringe needle airtight connection, tube body middle and upper part are equipped with gas interface, gas interface by pipeline and inert atmosphere gases source (nitrogen and/ Or helium) be connected, tube body total length be 52cm, internal diameter of tube body 2cm, lower part syringe needle internal diameter be 2mm, the long 5cm of syringe needle, wherein The nitrogen and/or helium pressure for being passed through frustule concentrate are 0.35~0.75MPa, algae is thin after nitrogen and/or helium release of pressure The disposable crushing failure at high speed of born of the same parents passes through destroyer；Make frustule concentrate with 0.1~0.5ml s by controlling needle-valve-1Speed stream Go out destroyer, the centrifuge tube after being pre-chilled with ice under the conditions of 0~4 DEG C collects frustule and is crushed liquid.
The extracting method of the Chlamydomonas reinhardtii chloroplaset, Chloroplast isolation buffer solution group become 0.3M sorbierites, 50mM Hepes-KOH、2mM Na2-EDTA·2H2O、1mM MgCl2·6H2O and mass concentration 1%BSA, surplus are water.
The extracting method of the Chlamydomonas reinhardtii chloroplaset utilizes the chloroplaset institute fatty acids of equivalent frustule biomass 18：3n6、18：4n3、16：4n3 and 18：3n3 (carbon atom numbers：Number of double bonds-n- position of double bond, position of double bond is from methyl end Start several) relative abundance in equivalent frustule, evaluate the purity and integrity degree of chloroplaset.
The content of fatty acid of the extracting method of the Chlamydomonas reinhardtii chloroplaset, frustule or chloroplaset passes through direct transesterification Change and gas chromatography is combined to measure, aliphatic acid 18 in the normal chloroplaset of every gram of equivalent frustule biomass：3n6、18：4n3、 16：4n3 and 18：3n3 (carbon atom numbers：Number of double bonds-n- position of double bond, position of double bond is several since the methyl end) content accounts for The distribution proportion of corresponding content of fatty acid is respectively in equivalent frustule biomass：38~59%, 56~80%, 85~99% and 83~96%；
Aliphatic acid 18 in the stress chloroplaset of every gram of equivalent frustule biomass：3n6、18：4n3、16:4n3 and 18:3n3 The distribution proportion that content accounts for corresponding content of fatty acid in equivalent frustule biomass is respectively：30~48%, 38~52%, 87~ 99% and 89~96%；
18 in its Chloroplast:3n6 and 18:4n3 abundance is lower, and the chloroplaset purity of extraction is higher；In chloroplaset 16:4n3 and 18:3n3 abundance is higher, and the chloroplaset integrity degree of extraction is higher.
The extracting method of the Chlamydomonas reinhardtii chloroplaset, using the transesterification combination gas chromatography of a step acid catalysis, step Suddenly it is, it will be under the conditions of Minimal culture mediums richness nitrogen in claim 2 or nitrogen-free Minimal culture mediums or without phosphorus Minimal are cultivated 5~10ml 2%H2SO4-methanol (v/ is added in the fresh frustule or chloroplaset sample that are obtained under base nutrition condition of salt stress V), 1~2h of magnetic agitation is mixed at 70~75 DEG C, and after being cooled to room temperature, 2ml n-hexanes and 0.75ml deionizations is added Water goes to the n-hexane that fatty acid methyl ester is contained on upper layer in 2ml chromatogram bottles after mixing 30~60s, carries out gas-chromatography point Analysis.
The reagent preserves at 4 DEG C, and described to operate on ice or carried out at 4 DEG C, the centrifugally operated is in horizontal rotor It is carried out in centrifuge.
The extracting method of Chlamydomonas reinhardtii Chloroplast provided by the present invention has the following advantages that：
The present invention is mildly crushed frustule by pressure difference mechanical shear stress can be from eutrophy in conjunction with density-gradient centrifugation method And purity and the high chloroplaset of integrity degree are extracted in the microalgae cell grown under the conditions of Nutrient Stress, this method is easy to keep leaf The integrality of green body, and can guarantee the adequate, stability and reliability of the subcellular biomaterial.
The present invention establish it is a kind of the purity and integrity degree of chloroplaset are evaluated based on the method for quality control of aliphatic acid, compared with Western blot and transmission electron microscope method are simpler, efficient and applicable.
Description of the drawings
Fig. 1 is the schematic diagram of Chlamydomonas reinhardtii Chloroplast extraction process.
Fig. 2 is that chlamydonomas reinhardtii cells chlorophyll and the changes of contents schematic diagram of nitrogen, wherein Chl after N stress 4h are Chlorophyll content, N is nitrogen element content, with 106A frustule meter.
Fig. 3 is transmission electron microscope figure of chlamydonomas reinhardtii cells under the conditions of eutrophy, and scale represents 500nm.
Fig. 4 is transmission electron microscope figure of the chlamydonomas reinhardtii cells after N stress 4h, and scale represents 500nm.
Fig. 5 is the Percoll density gradient centrifugation result schematic diagrams of Chlamydomonas reinhardtii chloroplaset, and arrow instruction is chloroplaset Layer.
Fig. 6 is purity detecting result schematic diagram of the Chlamydomonas reinhardtii chloroplaset based on immunoblotting, and wherein BIP represents endoplasmic reticulum Protein marker-binding domain-immunoglobulin, cell represents frustule, and cp represents chloroplaset.
Fig. 7 is the transmission electron microscope phenogram of Chlamydomonas reinhardtii chloroplaset, and wherein E represents chloroplast envelope, and T represents class Utricule film, P represent pyrenoids, and S represents amylum body, and scale represents 500nm.
Specific implementation mode
The method and result of the present invention are illustrated below by specific embodiment.
Table 1 is chloroplaset extraction conditions and its yield, and wherein N+1 to N+6 is the chloroplaset that extracts under the conditions of eutrophy algae, N-1 to N-6 is the chloroplaset that extracts under the conditions of N stress.
Table 2 is the content of fatty acid of frustule and chloroplaset under each chloroplaset extraction conditions.
Minimal culture mediums, Chloroplast isolation buffer solution and Percoll density gradient liquid in the present invention refer to document：
Mason C B,Bricker T M,Moroney J V.A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii[J].Nature protocols, 2006,1(5):2227-2230.
1 algae culture of embodiment
Chlamydomonas reinhardtii can also be in Nutrient Stress condition either cultivate obtained biomass under the conditions of eutrophy Lower obtained biomass.
Frustule is grown on solidification agar Minimal culture mediums first, and monoclonal algae spot is then gone to liquid Photoautotrophy is carried out in Minimal culture mediums, it is dark (synchronization) that the photoperiod is set as 12h illumination/12h.Algae solution is synchronized Be forwarded in bioreactor (PBR) after culture 4d and continue to cultivate (Fig. 1), preceding 48h carries out continuous illumination culture, rear 52h into The synchronization photoperiod of row 12h illumination/12h dark cultivates, and intensity of illumination is 50 μm of ol m-2s-1, wait for that algae cell density increases to 1 ×107A ml-1When, part frustule, centrifugal condition 3000g, 5min, 4 DEG C are harvested, these frustules are eutrophy culture Biomass.A part of frustule under the conditions of above-mentioned eutrophy is continued into N stress culture, wait for 3000g, 5min, 4 DEG C from Frustule of cleaning is resuspended with nitrogen-free Minimal culture mediums after the heart, nitrogen-free is used after being centrifuged under the conditions of 3000g, 5min, 4 DEG C Minimal culture mediums adjust frustule OD value (750nm) to 0.752, are then forwarded to progress N stress culture in PBR 4h, intensity of illumination increase to 100 μm of ol m by 50-2s-1, frustule OD value harvests all frustules up to 0.95, centrifuges item Part be 3000g, 5min, 4 DEG C, these frustules be N stress culture biomass.
These above-mentioned frustules may be respectively used for extracting normal and stress-inducing chloroplaset.
Frustule after N stress 4h coerces characterization parameter
Show the stress level of its tolerance by the variation of the chlorophyll and nitrogen element content of frustule.
The method for measuring chlorophyll content of wherein frustule is：2ml algae solutions are taken, it is anhydrous that 2ml is added after supernatant is removed in centrifugation Ethyl alcohol, ultrasound in ice bath, centrifuges 2min after pigment is fully extracted under 12,000g, measures supernatant in 649 and 665nm Under absorbance value, calculate chlorophyll concentration：Chlorophyll concentration (μ g L-1)=18.08OD649+6.63OD665.Pass through hemocytometer Number plate calculates algae cell density, and the chlorophyll content of frustule is calculated in conjunction with chlorophyll concentration.
The nitrogen element content of frustule is measured by elemental analyser (Elementar, Germany), and when measurement need to use automatic point Analysis balance (Mettler Toledo XP6) accurately weighs the freeze-drying algae powder of 2~4mg.
Step (1-3) the result shows that, in N stress 4h, the chlorophyll and nitrogen element content of frustule decline respectively 19% and 32%, as shown in Fig. 2, showing that frustule economic stress increases.
The chloroplaset of chlamydonomas reinhardtii cells needs to keep complete (Fig. 3 and Fig. 4) under the microscope, especially under stress conditions Frustule chloroplast membranes structure should keep integrality.It is in frustule it is observed that equally distributed wherein under the conditions of eutrophy Chloroplast membranes structure, especially thylakoid membrane basal granule heap arrangement it is compact, while can see chloroplaset expoeridium film profile and The central outer pyrenoids by a circle amylome；Under the conditions of N stress, frustule Chloroplast membrane structure will appear decomposition, simultaneously Along with amylum body accumulation and be dispersed in thylakoid membrane so that albumen Assessment of Nuclear Volume is smaller and smaller, but remains to see that arrangement is dredged The film profile of the outer quilt of thylakoid membrane basal granule and chloroplaset of pine.It these results suggest that eutrophy condition and N stress in the embodiment Under the conditions of frustule chloroplast membranes structure keep complete.
The extraction of 2 chloroplaset of embodiment
With 50ml 50mM Hepes-KOH pH 7.5 by the frustule of the chlorophyll containing 10mg 3000g, 5min, 4 DEG C It is cleaned 2 times under centrifugal condition, 4ml 50mM Hepes-KOH pH 7.5 is added after abandoning supernatant.
20ml Chloroplast isolation buffer solutions are added, cell is resuspended, the frustule concentrate chlorophyll concentration of acquisition is 0.5mg ml-1, above-mentioned frustule concentrate is passed through into the stainless steel syringe needle destroyer (stainless steel with equivalent crushing effect as shown in Figure 1 Syringe needle destroyer) it is crushed, which includes the tube body of upper and lower two end opening, and tube body upper open end is as charge door, charging Valve is equipped at mouthful, tube body lower open end is connected with syringe needle, tube body lower open end and syringe needle airtight connection, and tube body middle and upper part is equipped with Gas interface, gas interface are connected by pipeline with inert atmosphere gases source, tube body total length be 52cm, internal diameter of tube body 2cm, Lower part syringe needle internal diameter be 2mm, the long 5cm of syringe needle, wherein be passed through frustule concentrate nitrogen cracking pressure be 0.35,0.55 or Frustule disposable crushing failure at high speed is passed through destroyer after nitrogen release of pressure by 0.75MPa.Make frustule concentrate by controlling needle-valve With 0.1ml s-1Speed flow out destroyer, ice-cold centrifuge tube collects frustule and is crushed liquid.
After the broken liquid of frustule in 10ml steps (2) is centrifuged 2min at 750 or 5000g and 4 DEG C, 2ml leaves are added Green body dissociating buffer is uniformly mixed.Sequentially add 3ml 65%, 3ml 45% and 3ml from bottom to top in round bottom test tube 20% Percoll gradient liquid is eventually adding the above-mentioned frustules of 3ml and is crushed liquid mixed liquor, centrifuged at 4200g and 4 DEG C 15min, the Chloroplast isolation that the chloroplaset band between 45~65%Percoll gradient liquid is added to 10 times of dilution volumes buffer Liquid centrifuges 1min at 670 or 5000g and 4 DEG C after mixing, and 5 times of 50mM HEPES-for diluting volume are added after abandoning supernatant KOH pH 8.0 and 0.3M sorbierites, after mixing as chloroplaset extracting solution.
The Chlamydomonas reinhardtii of chloroplaset extraction conditions in step (1-3) totally 6, eutrophy and Nutrient Stress CMC model is thin Totally 2 kinds of born of the same parents, as shown in table 1.
1 chloroplaset extraction conditions of table and its yield
The quality evaluation of 3 chloroplaset of embodiment
The productivity parameters of chloroplaset
The yield of chloroplaset=chloroplaset total chlorophyll content/frustule total chlorophyll content × 100%, for extracting leaf The frustule of green body is subject to the frustule of the chlorophyll containing 10mg.
The yield of chloroplaset under regular culture conditions
Chloroplaset yield under the normal condition obtained based on cracking pressure different in embodiment 2 and centrifugal force is 13~ 30%, as shown in table 1.
The yield of chloroplaset under the conditions of Nutrient Stress
The N stress chloroplaset yield obtained based on cracking pressure different in embodiment 2 and centrifugal force is 0.5~3%, As shown in table 1, the increasing for being remarkably decreased (as shown in Figure 5) and showing stress chloroplaset extraction difficulty of yield.
The purity and integrity degree of chloroplaset detect
The purity and integrity degree of chloroplaset are detected based on aliphatic acid abundance
The normal or stress chloroplaset and frustule that are extracted in embodiment 1 and 2 are passed through into the transesterification method knot of a step acid catalysis The composition that gas chromatography measures its aliphatic acid is closed, 5ml 2%H2SO4-first is added in fresh frustule or chloroplaset sample Alcohol (v/v) mixes magnetic agitation 1h at 70 DEG C, and after being cooled to room temperature, 2ml n-hexanes and 0.75ml deionized waters is added, After mixing 30s, the n-hexane that fatty acid methyl ester is contained on upper layer is gone in 2ml chromatogram bottles, carries out gas chromatographic analysis.Such as table 2 It is shown, aliphatic acid 18 in the normal chloroplaset of every gram of equivalent frustule biomass：3n6、18:4n3、16：4n3 and 18：3n3 contents Respectively 1.47~2.28,0.83~1.25,7.68~9.70 and 12.07~14.97mg account for phase in equivalent frustule biomass The distribution proportion of content of fatty acid is answered to be respectively：38~59%, 56~80%, 85~99% and 83~96%, wherein N+6 conditions The purity and integrity degree highest of lower richness nitrogen chloroplaset；Aliphatic acid 18 in the stress chloroplaset of every gram of equivalent frustule biomass： 3n6、18：4n3、16：4n3 and 18:3n3 contents are respectively 1.43~2.31,0.67~0.92,9.59~10.83 and 14.99~ 16.30mg, the distribution proportion for accounting for corresponding content of fatty acid in equivalent frustule biomass are respectively：30~48%, 38~52%, The purity and integrity degree highest of N stress chloroplaset under the conditions of 87~99% and 89~96%, wherein N-5.The above results show leaf 18 in green body:3n6 and 18:4n3 abundance is lower, and the chloroplaset purity of extraction is higher；16 in chloroplaset:4n3 and 18:3n3 Abundance is higher, and the chloroplaset integrity degree of extraction is higher.18 in chloroplaset:3n6 and 18:The abundance of 4n3 is not higher than 38%, and 16: 4n3 and 18:When the abundance of 3n3 is not less than 88%, purity and integrity degree are highest, and chloroplaset quality at this time is also best 's.
The content of fatty acid of frustule and chloroplaset under 2 chloroplaset extraction conditions of table
mg/g 18:3n6 18:4n3 16:4n3 18:3n3
N+1 1.94 0.93 8.31 12.83
N+2 1.57 0.83 8.43 12.97
N+3 2.28 1.14 9.70 14.97
N+4 1.56 0.97 8.91 13.61
N+5 1.76 1.17 8.71 13.35
N+6 1.47 0.83 9.01 13.74
N+ frustules 3.90 1.46 9.79 15.54
N-1 2.31 0.71 10.83 16.30
N-3 1.96 0.89 10.31 16.08
N-4 1.43 0.92 9.59 14.99
N-5 1.64 0.67 10.21 15.88
N- frustules 4.82 1.76 10.98 16.90
Chloroplaset purity is detected based on Western blot
The normal of the best in quality extracted in embodiment 1 and 2 or stress chloroplaset and frustule are passed through into Western blot The degree of chloroplaset pollution endoplasmic reticulum protein marker BIP is detected, as shown in fig. 6, the normal or stress chloroplaset of best in quality In equal light contamination endoplasmic reticulum, illustrate that the chloroplaset purity for using the method for the present invention to extract is higher, while confirming step (1) In based on aliphatic acid abundance detect chloroplaset purity reliability.
Chloroplaset purity and integrity degree are detected based on transmission electron microscope method
The normal of the best in quality extracted in embodiment 1 and 2 or stress chloroplaset and frustule are passed through into transmission electron microscope method The purity and integrity degree for detecting chloroplaset, as shown in fig. 7, under transmission electron microscope, these chloroplasets be morphologically it is complete, The complete envelope of visible chloroplaset expoeridium.Normal chloroplaset center has pyrenoids, and is surrounded by a circle amylome.Stress Chloroplast membranes structure shows a degree of recombination, and the basal granule heap compactness being embodied on thylakoid membrane reduces, stress Amylum body significantly accumulates in chloroplaset, and is all scattered between thylakoid membrane, leads to chloroplast membranes structural distortion and obscures.Lay Mattress reinhardtii cell remains to successfully be extracted a certain amount of chloroplaset after N stress, the quality of these stress chloroplasets, packet Purity and integrity degree are included, compared with normal chloroplaset, is not also traded off.As it can be seen that under the above transmission electron microscope chloroplaset table Sign observation further demonstrates in step (1) and detects chloroplaset purity and the reliability of integrity degree based on aliphatic acid abundance.

Claims (10)

1. a kind of extracting method of Chlamydomonas reinhardtii chloroplaset, includes the following steps：
(1) chlamydonomas reinhardtii cells for taking certain quantity of fresh include the cell of complete excision by the acquisition of mild Mechanical Crushing Fragment concentrate；
(2) by density gradient centrifugation, chloroplaset is collected.
2. the extracting method of Chlamydomonas reinhardtii chloroplaset according to claim 1, it is characterised in that：Lay in the step (1) The chloroplaset of mattress reinhardtii cell needs to keep complete under optics or electron microscope, includes nothing especially under nutrition condition of salt stress Nitrogen or without phosphorus frustule chloroplast membranes structure should keep integrality；
The Chlamydomonas reinhardtii also may be used either cultivate obtained biomass under the conditions of the eutrophy of Minimal culture mediums With the biomass obtained under the conditions of being the Nutrient Stress in nitrogen-free or without phosphorus Minimal culture mediums.
3. the extracting method of Chlamydomonas reinhardtii chloroplaset according to claim 1 or 2, it is characterised in that：In the step (1) Frustule fragmentation procedure be：By the frustule of the chlorophyll containing 6~10mg 30~50mM Hepes-KOH pH 7.5~8.0 Eccentric cleaning 1~2 time, centrifugal condition be 2000~3000g, 5~10min, 4~10 DEG C, abandon supernatant；
Cell is resuspended with 30~50mM Hepes-KOH pH 7.5~8.0, again such as above-mentioned pelleted by centrifugation, repetitive operation 1~2 Secondary, the frustule concentrate of acquisition by Yeda press, Bioneb, French press or can have equivalent crushing effect Stainless steel syringe needle destroyer be crushed by mechanical shear stress, wherein acting on the inert atmosphere gases of frustule concentrate The cracking pressure of (such as nitrogen and/or helium) is 0.35~0.75MPa.
4. the extracting method of Chlamydomonas reinhardtii chloroplaset according to claim 1, it is characterised in that：The behaviour of the step (2) As：By 10~15ml ml containing 0.3~0.5mg in step (1)-1The clasmatosis liquid of chlorophyll is in 750~5000g and 4~10 After centrifuging 2~5min at DEG C, 2~5ml Chloroplast isolation buffer solutions are added, go to after mixing by Percoll, Ficoll or In density gradient liquid prepared by sucrose, this density gradient liquid be equivalent to containing percent by volume be respectively 20~25%, 45~ 50%, 65~70% Percoll density gradients liquid (their volume ratio be 1~2: 1~3: 1~2), 4200~ 15~30min is centrifuged at 5000g and 4~10 DEG C；The chloroplaset band that will be equivalent between 45~65%Percoll gradient liquid adds The Chloroplast isolation buffer solution for entering to be equivalent to its 10~15 times of volumes is diluted, after mixing 670~5000g and 4~ 1~5min is centrifuged at 10 DEG C, abandon after supernatant be added the chloroplaset for being equivalent to 1~5 times of volume of bottom concentrate preserve liquid carry out it is dilute It releases, chloroplaset, which preserves liquid group, becomes 30~50mM HEPES-KOH pH 8.0 and final concentration 0.3~0.5M sorbierites, and mixing is equal It is chloroplaset extracting solution after even.
5. extracting method according to claim 2, it is characterised in that：
Minimal culture medium groups become 7.48mM NH4Cl、0.41mM MgSO4·7H2O、0.35mM CaCl2·2H2O、 5.37mM K2HPO4、2.67mM KH2PO4And the Hunters 1ml/L are micro- (Hunter ' s trace elements), surplus is Water；
The Hunter trace element composition is as follows：134.3μM Na2-EDTA·2H2O、76.5μM ZnSO4·7H2O、184.4μM H3BO3、25.6μM MnCl2·4H2O、17.9μM FeSO4·7H2O、6.8μM CoCl2·6H2O、6.3μM CuSO4·5H2O And 0.9 μM of (NH4)6Mo7O24·4H2O, surplus are water；
Nitrogen-free Minimal culture mediums are that above-mentioned Minimal culture mediums remove NH4Cl；
Without phosphorus Minimal culture mediums are that above-mentioned Minimal culture mediums remove K2HPO4And KH2PO4。
6. extracting method according to claim 3, it is characterised in that：
Stainless steel syringe needle destroyer includes the tube body of upper and lower two end opening, and tube body upper open end is set at charge door as charge door There are valve, tube body lower open end to be connected with syringe needle, tube body lower open end and syringe needle airtight connection, tube body middle and upper part connects equipped with gas Mouthful, gas interface is connected by pipeline with inert atmosphere gases source (nitrogen and/or helium), and tube body total length is 52cm, tube body Internal diameter is 2cm, and lower part syringe needle internal diameter is 2mm, the long 5cm of syringe needle, wherein being passed through the nitrogen and/or helium pressure of frustule concentrate Power is 0.35~0.75MPa, and the disposable crushing failure at high speed of frustule is passed through destroyer after nitrogen and/or helium release of pressure；Pass through control Needle-valve processed makes frustule concentrate with 0.1~0.5ml s-1Speed flow out destroyer, after being pre-chilled with ice be in 0~4 DEG C of condition Under centrifuge tube collect frustule be crushed liquid.
7. extracting method according to claim 4, it is characterised in that：Chloroplast isolation buffer solution group becomes 0.3M sorbs Alcohol, 50mM Hepes-KOH, 2mM Na2-EDTA·2H2O、1mM MgCl2·6H2O and mass concentration 1%BSA, surplus are water.
8. the extracting method of Chlamydomonas reinhardtii chloroplaset according to claim 1, it is characterised in that：It is given birth to using equivalent frustule The chloroplaset institute fatty acids 18 of substance:3n6、18:4n3、16:4n3 and 18：Relative abundances of the 3n3 in equivalent frustule, is commented The purity and integrity degree of valence chloroplaset.
9. the extracting method of Chlamydomonas reinhardtii chloroplaset according to claim 8, it is characterised in that：Frustule or chloroplaset Content of fatty acid is measured by directly transesterification combination gas chromatography, in the normal chloroplaset of every gram of equivalent frustule biomass Aliphatic acid 18：3n6、18：4n3、16：4n3 and 18：3n3 contents account for point of corresponding content of fatty acid in equivalent frustule biomass Cloth ratio is respectively：38~59%, 56~80%, 85~99% and 83~96%；
Aliphatic acid 18 in the stress chloroplaset of every gram of equivalent frustule biomass:3n6、18:4n3、16:4n3 and 18:3n3 contents The distribution proportion for accounting for corresponding content of fatty acid in equivalent frustule biomass is respectively：30~48%, 38~52%, 87~99% And 89~96%；
18 in its Chloroplast:3n6 and 18:4n3 abundance is lower, and the chloroplaset purity of extraction is higher；16 in chloroplaset: 4n3 and 18:3n3 abundance is higher, and the chloroplaset integrity degree of extraction is higher.
10. extracting method according to claim 9, it is characterised in that：Using the transesterification combination gas phase color of a step acid catalysis Spectrometry, step be, will be under the conditions of Minimal culture mediums richness nitrogen in claim 2 or nitrogen-free Minimal culture mediums or without phosphorus 5~10ml 2% is added in the fresh frustule or chloroplaset sample that are obtained under Minimal culture medium nutrition condition of salt stress H2SO4-methanol (v/v), at 70~75 DEG C mix 1~2h of magnetic agitation, after being cooled to room temperature, be added 2ml n-hexanes and 0.75ml deionized waters go to the n-hexane that fatty acid methyl ester is contained on upper layer in 2ml chromatogram bottles after mixing 30~60s, carry out Gas chromatographic analysis.
CN201710019354.4A 2017-01-11 2017-01-11 A kind of extracting method of Chlamydomonas reinhardtii chloroplaset Pending CN108300664A (en)

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