CN110218694A - A kind of kiwifruit fruit amyloplaste extracting method - Google Patents
A kind of kiwifruit fruit amyloplaste extracting method Download PDFInfo
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- CN110218694A CN110218694A CN201910461128.0A CN201910461128A CN110218694A CN 110218694 A CN110218694 A CN 110218694A CN 201910461128 A CN201910461128 A CN 201910461128A CN 110218694 A CN110218694 A CN 110218694A
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- C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
- C12N5/04—Plant cells or tissues
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2509/00—Methods for the dissociation of cells, e.g. specific use of enzymes
Abstract
The present invention relates to a kind of kiwifruit fruit amyloplaste extracting methods, are mainly made into gradient separations liquid with Percoll stoste and carry out the amyloplaste that density gradient centrifugation is purified twice with this.The present invention originally isolates the amyloplaste of high-purity and high integrity degree from kiwifruit fruit for the first time, and in the present invention can from 30 grams of kiwifruit fruits isolated about 2g amyloplaste, it is higher to efficiently separate rate.
Description
Technical field
The present invention relates to photobiochemistry technical fields, and in particular to a kind of kiwifruit fruit amyloplaste extracting method.
Background technique
Kiwi berry is a kind of emerging fruit, and fruits nutrition is abundant, is rich in vitamin C and minerals, wherein Vitamin C content
It is several times to tens times more contour than citrus, apple, it is one of most comprehensive fruit of nutrition in fruit, and have and promote appetite, health care
The effect of beauty is a kind of fruit being loved by consumers, there is the laudatory title of " king of fruit ".
Plastid is the main cell device existed only in plant and alga cells, is played in biosynthesis and metabolic function
Important function.Plastid includes proplastid, chloroplaset, leucoplast, chromoplast, amyloplaste etc..Plastid is not only involved in photosynthesis, nitrogen
It absorbs, and the synthesis and storage of wide participation amino acid, fat, starch and secondary metabolites, is that regulation plant cell is basic
Crucial subcellular organelle needed for framework and function.
Amyloplaste is a kind of plastid of heterotroph, and major function is to synthesize and store a large amount of starch.Starch is storage
The main carbohydrate of official, there is a large amount of reports in the crops such as cereals, root crop, beans, and in garden crop
The report of starch research is less.Starch is the main dry matter of Kiwi berry, and Zhan Guoshi specific gravity is up to 50% or more.Kiwi berry and other
Many fruits garden crops are the same, mainly accumulate starch early period in development, and degrade rapidly in the maturity period.Kiwifruit fruit is adopted
Starch degradation and fruit softening are maintained close ties with afterwards, and directly related with the saccharic acid flavor of fruit.Therefore, separating-purifying completely forms sediment
Powder is of great significance to the biological function and starch industry of research starch.
Summary of the invention
In view of the above-mentioned problems, now providing the extraction side of a kind of purity and all excellent kiwifruit fruit amyloplaste of integrity degree
Method.
Specific technical solution is as follows:
A kind of kiwifruit fruit amyloplaste extracting method, specific implementation are the gradient separations liquid being made into Percoll stoste,
Density gradient centrifugation, the amyloplaste purified, step include: twice for progress
1) kiwifruit fruit, is taken, exocarp is pruned and removes and pulp is cut into bulk behind center pillar part, take 30 grams of samples,
It is blended after 90mL buffer solution A is added thereto to homogenate shape, obtains homogenate；
2) homogenate, is drawn into filter-cloth filtering by two layers of gauze and four layers of rice, obtains filtrate, filtrate is dispensed into 50mL centrifugation
Guan Zhong takes lower sediment with 800g centrifugation 8 minutes under the conditions of 4 DEG C；
3) 2mL buffer solution A, is added into precipitating, is gently resuspended, obtains stand-by sample I；
4) Percoll stoste, is diluted to the gradient separations liquid that volume fraction is 50% with buffer solution B, takes 5mL gradient point
Chaotropic is placed in the centrifuge tube of 10mL volume, and centrifuge tube is placed in ice chest and is pre-chilled 10 minutes；
5) sample I, is diluted to 5mL with buffer solution A, is then gently added in centrifuge tube on the liquid level of gradient separations liquid
Supernatant is gently outwelled with 500g centrifugation 8 minutes under the conditions of 4 DEG C by side, and is resuspended and is precipitated with the buffer solution A of 2mL, then to
The buffer solution A of 2 times of volumes is added and under the conditions of 4 DEG C wherein with 800g centrifugation 5 minutes, abandons supernatant, obtain sample II；
6), sample II is gently resuspended with 5mL buffer solution A, step 5) is repeated, obtains the Kiwi berry amyloplaste of purifying；
Wherein, buffer solution A, B formula are as follows:
Buffer solution A
0.2M hydroxyethyl piperazineethanesulfonic acid；
0.8M sorbierite；
1mM ethylenediamine tetra-acetic acid；
1mM potassium chloride；
2mM magnesium chloride；
2mM dithiothreitol；
Buffer solution B
0.4M hydroxyethyl piperazineethanesulfonic acid；
1.6M sorbierite；
2mM ethylenediamine tetra-acetic acid；
2mM potassium chloride；
4mM magnesium chloride；
4mM dithiothreitol.
Dithiothreitol need to use preceding addition in buffer in buffer solution A of the present invention, and buffer needs to use before use
Sodium hydroxide adjusts pH value to 7.5.
Dithiothreitol need to use preceding addition in buffer in buffer solution B of the present invention, and buffer needs to use before use
Sodium hydroxide adjusts pH value to 7.5.
Associated extraction equipment used should be in be pre-chilled 30 minutes or more in ice face when carrying out purification in the present invention, and purifying
Associated funnel, gauze, filter cloth etc. need to be soaked with 2mL buffer solution A when extraction.Purification needs on ice or 4 in the present invention
It is carried out in DEG C environment.
The beneficial effect of above scheme is:
1), the present invention originally isolates the amyloplaste of high-purity and high integrity degree from kiwifruit fruit for the first time；
2), the present invention using Percoll separating liquid can from 30 grams of kiwifruit fruits isolated about 10g amyloplaste, have
It is higher to imitate separation rate.
Detailed description of the invention
Figure 1A is the Western blot hybridization figure of the amyloplaste provided in the embodiment of the present invention, and Figure 1B is pulp and shallow lake
Powder albumen polyacrylamide gel electrophoresis figure；
Fig. 2 is the structure observation schematic diagram of the amyloplaste provided in the embodiment of the present invention 1；
Fig. 3 is the structure observation schematic diagram of the amyloplaste provided in the embodiment of the present invention 2.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, those of ordinary skill in the art without creative labor it is obtained it is all its
His embodiment, shall fall within the protection scope of the present invention.
It should be noted that in the absence of conflict, the feature in embodiment and embodiment in the present invention can phase
Mutually combination.
The present invention will be further explained below with reference to the attached drawings and specific examples, but not as the limitation of the invention.
A kind of kiwifruit fruit amyloplaste extracting method, includes the following steps:
1) rice of 100mL graduated cylinder, 500mL beaker, the funnel of 15 centimetres of outside diameters, 0.22 micron pore size, is drawn into filter cloth, doctor
It is placed in ice face and is pre-chilled 30 minutes or more with absorbent gauze, 50mL centrifuge tube and 10mL centrifuge tube, and will with 2mL buffer solution A
Funnel and gauze, filter cloth soak；
2) kiwifruit fruit, is taken, removes center pillar part after pruning the exocarp of 1 mm of thickness, pulp is cut into 0.5 centimetre
90mL buffer solution A is added in × 0.5 centimetre of bulk, 30 grams of samples of weighing, and is stirred sample with stirring mode with nine positive wall-breaking machines
Broken, 3 times repeatedly, each 3-5 seconds is in homogenate shape to it, obtains homogenate；
3), by the 2) homogenate that step obtains filter-cloth filterings are drawn by two layers of gauze and four layers of rice, filtrate is obtained, by filtrate
It is dispensed into 50mL centrifuge tube, with 800g centrifugation 8 minutes under the conditions of 4 DEG C, takes lower sediment；
4) 2mL buffer solution A gently, is added in the precipitating obtained into step 3), gently (specific method is centrifuge tube for resuspension
It is placed in ice face and loops, the precipitating not being resuspended for a long time can gently be inhaled with dropper beats), obtain stand-by sample I；
5) 100% Percoll stoste, is diluted to the gradient separations liquid that volume fraction is 50% with buffer solution B, is taken
5mL is placed in the centrifuge tube of 10mL volume, is pre-chilled 10 minutes in ice chest；
6) sample I, is diluted to 5mL with buffer solution A, is then gently added to gradient separations liquid in step 5) centrifuge tube
Ullage gently outwells supernatant with 500g centrifugation 8 minutes under the conditions of 4 DEG C, then precipitating is resuspended with the buffer solution A of 2mL
(specific method is that centrifuge tube is placed in ice face and loops, and the precipitating not being resuspended for a long time can gently be inhaled with dropper beats), adds 2
The buffer solution A of times volume obtains sample II with 800g centrifugation 5 minutes under the conditions of 4 DEG C；
7) (method is same as above) gently, is resuspended in sample II in step 6) with 5mL buffer solution A, step 6) is repeated, is purified
Kiwifruit fruit amyloplaste；
Wherein, buffer solution A, B formula are as follows:
Buffer solution A
0.2M hydroxyethyl piperazineethanesulfonic acid；
0.8M sorbierite；
1mM ethylenediamine tetra-acetic acid；
1mM potassium chloride；
2mM magnesium chloride；
2mM dithiothreitol；
Buffer solution B
0.4M hydroxyethyl piperazineethanesulfonic acid；
1.6M sorbierite；
2mM ethylenediamine tetra-acetic acid；
2mM potassium chloride；
4mM magnesium chloride；
4mM dithiothreitol；
Dithiothreitol need to use preceding addition in buffer in buffer solution A of the present invention, and buffer needs to use before use
Sodium hydroxide adjusts pH value to 7.5；
Dithiothreitol need to use preceding addition in buffer in buffer solution B of the present invention, and buffer needs to use before use
Sodium hydroxide adjusts pH value to 7.5；
Purification needs carry out on ice or in 4 DEG C of environment in the present invention.
Embodiment 1
Hayward kiwifruit fruit sample is picked in Hubei Province, Hayward Kiwi berry base, Chibi City in August, 2018, and
It is extracted according to method as above.
Embodiment 2
In the red kiwi fruit fruit sample that in August, 2018 is picked in Hubei Province, flowers and fruits mountain, Xianning Kiwi berry base, and
It is extracted according to method as above.
Pass through the purity of the amyloplaste of Western blot hybridization check judgement purification, cardinal principle in the present invention are as follows:
The plastid of purification tends to the pollution by mitochondria, cytoplasm and vacuole, is done using the labelled antibody of different subcellular organelles
Western blot hybridization can check whether the pollution there are other subcellular organelles.The present invention uses the verifying purification of following antibody
The purity of amyloplaste: (1) UGPase cytoplasmic marker antibody: cytoplasmic protein 51.6kDa, dilution ratio 1:2000；(2) VDAC1 line
Plastochondria outer membrane tag antibody: mitochondrial membrance protein 29kDa, dilution ratio 1:5000；(3) V-ATPase vacuole tag antibody: liquid
Steep protein 26-31kDa, dilution ratio 1:2000；(4) PGL35 (plastoglobulus labelled antibody): fibrillin 35, thinner ratio
Example: 1:2000.
Pass through the integrity degree of the amyloplaste of cytological observation verifying purification, cardinal principle are as follows: with phase in the present invention
Under the optical microscopy of potential difference function, typical membrane structure can be observed in complete amyloplaste；Meanwhile by scanning electron microscope and thoroughly
It penetrates electron microscope then and the fine structure on amyloplaste surface and inside can be observed.
Western blot hybridization check shows the purity of the amyloplaste purified in the embodiment of the present invention 1,2 up to 90%
More than, and it is not affected by the pollution (embodiment 1 (Figure 1A), embodiment 2 (Figure 1B)) of cytoplasm, mitochondria and vacuole.
Cytological structure observes (optical microscopy (Fig. 2A, D), transmission electron microscope (Fig. 2 B, E), scanning electron microscope (Fig. 2 C, F))
Observation shows the amyloplaste purified in the embodiment of the present invention 1 in unsetting, semi-crystal shape, and diameter is 2-10 μm, and is formed sediment
It is clear that amyloplast structure feature in powder, shows that its integrity degree is higher.
Cytological structure observes (optical microscopy (Fig. 3 A, D), transmission electron microscope (Fig. 3 B, E), scanning electron microscope (Fig. 3 C, F))
Observation shows the amyloplaste purified in the embodiment of the present invention 2 in unsetting, semi-crystal shape, and diameter is 2-10 μm, and is formed sediment
It is clear that amyloplast structure feature in powder, shows that its integrity degree is higher.
The foregoing is merely preferred embodiments of the present invention, are not intended to limit embodiments of the present invention and protection model
It encloses, to those skilled in the art, should can appreciate that all with made by description of the invention and diagramatic content
Equivalent replacement and obviously change obtained scheme, should all be included within the scope of the present invention.
Claims (1)
1. a kind of kiwifruit fruit amyloplaste extracting method, which is characterized in that the gradient separations liquid being made into Percoll stoste,
Density gradient centrifugation, the amyloplaste purified, step include: twice for progress
1) kiwifruit fruit, is taken, exocarp is pruned and removes and pulp is cut into bulk behind center pillar part, take 30 grams of samples, Xiang Qi
It is blended after middle addition 90mL buffer solution A to homogenate shape, obtains homogenate；
2) homogenate, is drawn into filter-cloth filtering by two layers of gauze and four layers of rice, filtrate is obtained, filtrate is dispensed into 50mL centrifuge tube
In, with 800g centrifugation 8 minutes under the conditions of 4 DEG C, take lower sediment；
3) 2mL buffer solution A, is added into precipitating, is gently resuspended, obtains stand-by sample I；
4) Percoll stoste, is diluted to the gradient separations liquid that volume fraction is 50% with buffer solution B, takes 5mL gradient separations liquid
It is placed in the centrifuge tube of 10mL volume, centrifuge tube is placed in ice chest and is pre-chilled 10 minutes；
5) sample I, is diluted to 5mL with buffer solution A, is then gently added to the ullage of gradient separations liquid in centrifuge tube,
With 500g centrifugation 8 minutes under the conditions of 4 DEG C, supernatant is gently outwelled, and is resuspended after precipitating with the buffer solution A of 2mL again to it
The middle buffer solution A that 2 times of volumes are added abandons supernatant, obtains sample II with 800g centrifugation 5 minutes under the conditions of 4 DEG C；
6), sample II is gently resuspended with 5mL buffer solution A, step 5) is repeated, obtains the Kiwi berry amyloplaste of purifying；
Wherein, buffer solution A, B formula are as follows:
Buffer solution A
Buffer solution B
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