CN106893690A - The preparation method of Chilean fragrant plant mentioned in ancient texts protoplast - Google Patents
The preparation method of Chilean fragrant plant mentioned in ancient texts protoplast Download PDFInfo
- Publication number
- CN106893690A CN106893690A CN201611230340.9A CN201611230340A CN106893690A CN 106893690 A CN106893690 A CN 106893690A CN 201611230340 A CN201611230340 A CN 201611230340A CN 106893690 A CN106893690 A CN 106893690A
- Authority
- CN
- China
- Prior art keywords
- enzyme liquid
- tissue
- preparation
- protoplast
- sea cucumber
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
- 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
-
- 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 preparation method of Chilean fragrant plant mentioned in ancient texts protoplast, belong to alga cells engineering field.The preparation method includes the steps such as the preparation of tissue enzyme liquid, Chilean fragrant plant mentioned in ancient texts tissue enzymolysis, and the tissue enzyme liquid preparation process is：Used 1 10 times of seawater of sea cucumber intestine volume, 0 DEG C of homogenate broken；4 18h are extracted at 4 DEG C, then adjusts pH value to 7.0 8.0, refrigerated centrifuge 5min leaching liquor, taken supernatant liquor and obtain final product sea cucumber enzyme liquid；Tissue enzyme liquid will be obtained final product after sea cucumber enzyme liquid, cellulase and sea water mixing.
Description
Technical field
The present invention relates to a kind of preparation method of Chilean fragrant plant mentioned in ancient texts protoplast, belong to alga cells engineering field.
Background technology
Plant protoplast refers to remove cell membrane is surrounded by plasma membrane, the viable cell of tool, and its structure includes
Cell membrane, cytoplasm (including various organelles, cytoskeleton system and cytoplasmic matrix) and part etc. nucleus.Protoplast Technique
Refer to protoplast be separately cultured basis on a series of technical operations for carrying out, mainly including Plant Regeneration from Protoplast,
Protoplast fusion and cell hydridization, transgenosis etc. cultivate biotechnology of variation new type etc..Protoplast cuhnre and plant
Regeneration techniques has the advantages that using scope is wide, feasibility is strong, is the basis of plant biological engineering.
Prepared by plant protoplast include the selection of material and pretreatment, tissue breakdown and protoplast tentatively dissociate and
The purifying of protoplast and viability examination.The suitable material of selection is the basis for being successfully separated protoplast；At enzymolysis or machinery
Reason is preceding can be improved broken wall efficiency and reduce plasmic damage for the pretreatment of material；The selection of rational enzyme preparation and
The regulation of concentration, the selection of osmotic pressure regulator are all the keys for improving plasm yield and activity.Although in recent years for original
The culture technique of raw matter achieves rapidly development, but also there is the problems such as protoplast vigor is not high, and cultivation cycle is long.
Chilean fragrant plant mentioned in ancient texts (Gracilaria chilensis) has fast, the resistant to elevated temperatures characteristic of growth, is southern potential foster
Grow one of kind.At present, fragrant plant mentioned in ancient texts is mainly bred by way of vegetative reproduction.Seed breeds by temperature, seawater salinity
Influence etc. envirment factor is very big, great unstability.Protoplast has totipotency, can be a large amount of under the conditions of manually operated
Quick breeding, can alleviate seed breeds pressure.Additionally, containing thicker colloid in the cell membrane of fragrant plant mentioned in ancient texts, it is impossible to directly
The genetic manipulations such as cell fusion, cell hydridization are carried out, the plasm obtained using enzyme dissolving means conventional in the prior art
Body is often active relatively low, and yield is also undesirable.
Patent CN2016101618265 discloses a kind of preparation method of fragrant plant mentioned in ancient texts protoplast of hanging, and it passes through to disappear using Bao
Change gland enzyme liquid to digest fragrant plant mentioned in ancient texts tissue of hanging, so as to prepare protoplast.But present invention discover that the enzyme from Bao digestion tissue
Liquid is not good to the hydrolysis result that Chilean fragrant plant mentioned in ancient texts is organized.
The content of the invention
It is an object of the invention to provide the preparation method of Chilean fragrant plant mentioned in ancient texts protoplast.Meanwhile, according to marine alga somatocyte development
Totipotency, decompose Chilean fragrant plant mentioned in ancient texts using toolenzyme and organize, dissociate somatoplasm body, or genetic engineering research is carried
For preferable receptor system.
In one embodiment, the preparation method includes that prepared by tissue enzyme liquid, Chilean fragrant plant mentioned in ancient texts organizes the steps such as enzymolysis,
It is described tissue enzyme liquid preparation process be：It is broken with the 0 DEG C of homogenate of the 1-10 times of seawater of sea cucumber intestine volume；4- is extracted at 4 DEG C
18h, then adjusts pH value to 7.0-8.0, refrigerated centrifuge 5min leaching liquor, takes supernatant liquor and obtains final product sea cucumber enzyme liquid；By sea cucumber enzyme
Tissue enzyme liquid is obtained final product after liquid, cellulase and sea water mixing.
In a specific embodiment, the tissue enzyme liquid preparation process is：Used 2 times of seawater of sea cucumber intestine volume
0 DEG C of homogenate is broken；12h is extracted at 4 DEG C, then adjusts pH value to 7.6, refrigerated centrifuge 5min leaching liquor, take supernatant liquor i.e.
Obtain sea cucumber enzyme liquid；Tissue enzyme liquid will be obtained final product after sea cucumber enzyme liquid, cellulase and sea water mixing.
In scheme is further carried out, the tissue enzyme liquid specifically comprises 2.4ml cellulases, 0.5ml sea cucumbers
Enzyme liquid and 17.1ml sterilization seawater.
In another embodiment, the Chilean fragrant plant mentioned in ancient texts tissue enzymolysis step will to take Chilean fragrant plant mentioned in ancient texts tissue scalpel
Frond is shredded, and the tissue enzyme liquid that addition is prepared is digested, and is then filtered with the bolting silk of 200 mesh, is removed indigested
Cell, cell mass and fragment, go supernatant to be centrifuged, 800rpm centrifugation 10min, single-celled protozoal plastid is sunk, cell fragment
Stay in supernatant, abandoning supernatant, centrifugation 2-3 times is rinsed with the sterilization seawater of the glucose containing 1mol/L, collect
Precipitation rear overhang obtains final product protoplast floating to 2ml.
In one embodiment, the enzymatic hydrolysis condition is that addition organizes the constant temperature that the frond tissue of enzyme liquid is put into 25 DEG C to shake
Dark enzymolysis 5h is carried out on bed.
Brief description of the drawings
Fig. 1：(× 20 times) of the fluoroscopic examination figure of protoplast is left：Protoplast；It is right：Protoplast after fluorescent staining,
Display is red
Fig. 2：Microscope figure of the Protoplast cuhnre after 12 days
Specific embodiment
Also the present invention further can be understood by embodiment, wherein the embodiment illustrates that some are prepared or user
Method.It is to be appreciated, however, that these embodiments do not limit the present invention.Currently known or further exploitation change of the invention
Change is considered within the scope of the invention described herein and claimed below.
Embodiment 1
It is prepared by tissue enzyme liquid：Used 2 times of seawater of sea cucumber intestine volume, 0 DEG C of homogenate broken；12h is extracted at 4 DEG C, then
Adjust pH value to 7.6, refrigerated centrifuge 5min leaching liquor, take supernatant liquor and obtain final product sea cucumber enzyme liquid；By sea cucumber enzyme liquid, cellulase and
Tissue enzyme liquid is obtained final product after sea water mixing.It is described tissue enzyme liquid specifically comprise 2.4ml cellulases, 0.5ml sea cucumbers enzyme liquid and
17.1ml sterilization seawater.
The preparation of protoplast：2g Chile river hedge material is taken, it is with scalpel that frond chopping is (more broken after being blotted with blotting paper
It is better), the tissue enzyme liquid that addition is prepared, being put into carries out dark enzymolysis on 25 DEG C of constant-temperature table.Per 30min microscopies once,
Observe and take pictures.After 5h, filtered with the bolting silk of 200 mesh, removed indigested cell, cell mass and fragment, removed supernatant
Centrifugation, 800rpm centrifugation 10min, makes single-celled protozoal plastid sink, and cell fragment is stayed in supernatant, abandoning supernatant, is used
The sterilization seawater of the glucose containing 1mol/L is rinsed centrifugation 2-3 times, collects precipitation rear overhang floating to 2ml, obtains final product plasm
Body.
Embodiment 2
The detection of protoplast：
The measure of cell density：The measure of cell density is carried out with blood counting chamber
Concrete outcome is as follows：
Comparative example 1 is that it uses 2 times of seawater of sea cucumber intestine volume, 0 DEG C of homogenate broken for the preparation method of sea cucumber enzyme liquid in tissue enzyme liquid
It is broken；12h is extracted at 4 DEG C, then adjusts pH value to 7.0, refrigerated centrifuge 5min leaching liquor, taken supernatant liquor and obtain final product sea cucumber enzyme
Liquid；Other tissue enzyme liquids and method for preparing protoplast are with the embodiment of the present invention 1.
Comparative example 2 is that it uses 2 times of seawater of sea cucumber intestine volume, 0 DEG C of homogenate broken for the preparation method of sea cucumber enzyme liquid in tissue enzyme liquid
It is broken；12h is extracted at 4 DEG C, then adjusts pH value to 8.0, refrigerated centrifuge 5min leaching liquor, taken supernatant liquor and obtain final product sea cucumber enzyme
Liquid；Other tissue enzyme liquids and method for preparing protoplast are with the embodiment of the present invention 1.
Comparative example 3 is 2.4ml cellulases, 0.5ml abalones enzyme liquid and 17.1ml sterilization seawater for the composition of tissue enzyme liquid；Abalone
Referring to the embodiment 1 in patent CN2016101618265 specifications, other organize enzyme liquid and plasm systems to enzyme liquid preparation method
Preparation Method is with the embodiment of the present invention 1.
Comparative example 4 specifically comprises 2.0ml cellulases, 0.9ml sea cucumbers enzyme liquid and 17.1ml sterilization seawater for tissue enzyme liquid,
Other tissue enzyme liquids and method for preparing protoplast are with the embodiment of the present invention 1.
Comparative example 5 specifically comprises 2.7ml cellulases, 0.2ml sea cucumbers enzyme liquid and 17.1ml sterilization seawater for tissue enzyme liquid,
Other tissue enzyme liquids and method for preparing protoplast are with the embodiment of the present invention 1.
Survival rate is determined：Dyeed with 0.5%Evans Blue, protoplast living is in glassy yellow, dead cell is in navy blue.
5 visuals field are randomly choosed under the microscope, the survival ratio of protoplast living is calculated, and concrete outcome is as follows：
Embodiment 3
The protoplast for collecting the preparation of embodiment 1 carries out dim light culture in hypertonic seawater, and intensity of illumination is 100-
200lx, after protoplast regeneration wall, gradually reduces penetration of sea water and is depressed into ordinary sea water, and intensity of illumination is gradually increased to 3000lx
Cultivated.
As shown in Fig. 2 after cultivating 42 hours, protoplast has not yet been formed complete cell membrane, and after cultivating 4 days, Duo Shuoyuan
Give birth to plastid regenerative cell's wall.After culture 7 days, multiple cells are split into, cell arrangement is relatively regular, after cultivating 12 days,
Cell number is continuously increased, and is arranged in different shapes.
Present invention merely illustrates some claimed specific embodiments, one of them or more skill
Described technical characteristic can be combined with arbitrary one or more technical schemes in art scheme, these are combined and obtain
Technical scheme also in the application protection domain, technical scheme is disclosed in the present invention just as obtained from these are combined
It is specific in content to record the same.
Claims (5)
1. a kind of preparation method of Chilean fragrant plant mentioned in ancient texts protoplast, the preparation method include tissue enzyme liquid prepare, Chilean fragrant plant mentioned in ancient texts group
The steps such as enzymolysis are knitted, the tissue enzyme liquid preparation process is：It is broken with the 0 DEG C of homogenate of the 1-10 times of seawater of sea cucumber intestine volume；
4-18h is extracted at 4 DEG C, then adjusts pH value to 7.0-8.0, refrigerated centrifuge 5min leaching liquor, taken supernatant liquor and obtain final product sea cucumber
Enzyme liquid；Tissue enzyme liquid will be obtained final product after sea cucumber enzyme liquid, cellulase and sea water mixing.
2. preparation method according to claim 1, it is characterised in that the tissue enzyme liquid preparation process is：Used 2 times
0 DEG C of homogenate of seawater of sea cucumber intestine volume is broken；12h is extracted at 4 DEG C, leaching liquor is then adjusted into pH value to 7.6, refrigerated centrifuge
5min, takes supernatant liquor and obtains final product sea cucumber enzyme liquid；Tissue enzyme liquid will be obtained final product after sea cucumber enzyme liquid, cellulase and sea water mixing.
3. preparation method according to claim 1, it is characterised in that the tissue enzyme liquid to specifically comprise 2.4ml fine
The plain enzyme of dimension, 0.5ml sea cucumbers enzyme liquid and 17.1ml sterilization seawater.
4. preparation method according to claim 1, it is characterised in that the Chilean fragrant plant mentioned in ancient texts tissue enzymolysis step is to take Chile
Fragrant plant mentioned in ancient texts tissue scalpel shreds frond, and the tissue enzyme liquid that addition is prepared is digested, and is then carried out with the bolting silk of 200 mesh
Filtering, removes indigested cell, cell mass and fragment, goes supernatant to be centrifuged, 800rpm centrifugation 10min, makes single-celled protozoal
Plastid sinks, and cell fragment is stayed in supernatant, and abandoning supernatant is rushed with the sterilization seawater of the glucose containing 1mol/L
Centrifugation 2-3 times is washed, precipitation rear overhang is collected floating to 2ml, protoplast is obtained final product.
5. preparation method according to claim 4, it is characterised in that the enzymatic hydrolysis condition is the frond of addition tissue enzyme liquid
Tissue is put on 25 DEG C of constant-temperature table carries out dark enzymolysis 5h.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611230340.9A CN106893690B (en) | 2016-12-27 | 2016-12-27 | Preparation method of Gracilaria chilies protoplast |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201611230340.9A CN106893690B (en) | 2016-12-27 | 2016-12-27 | Preparation method of Gracilaria chilies protoplast |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106893690A true CN106893690A (en) | 2017-06-27 |
| CN106893690B CN106893690B (en) | 2020-04-07 |
Family
ID=59198555
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201611230340.9A Active CN106893690B (en) | 2016-12-27 | 2016-12-27 | Preparation method of Gracilaria chilies protoplast |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106893690B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107711488A (en) * | 2017-10-19 | 2018-02-23 | 汕头大学 | A kind of method for preparing different Zhijiang Li protoplasts |
| CN109511550A (en) * | 2018-12-07 | 2019-03-26 | 浙江海洋大学 | Seaweed protoplast is separately cultured the method with regeneration plant |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806526A (en) * | 2004-12-24 | 2006-07-26 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding breeding technology |
| CN101173261A (en) * | 2007-10-29 | 2008-05-07 | 大连工业大学 | Separation purification technique for lysozyme in holothurian intestines |
| CN105754927A (en) * | 2016-03-22 | 2016-07-13 | 中国海洋大学 | Preparation method of gracilaria salicornia protoplast |
-
2016
- 2016-12-27 CN CN201611230340.9A patent/CN106893690B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1806526A (en) * | 2004-12-24 | 2006-07-26 | 中国海洋大学 | Sargassum thunbergii offspring breeding method based on somatocyte seeding breeding technology |
| CN101173261A (en) * | 2007-10-29 | 2008-05-07 | 大连工业大学 | Separation purification technique for lysozyme in holothurian intestines |
| CN105754927A (en) * | 2016-03-22 | 2016-07-13 | 中国海洋大学 | Preparation method of gracilaria salicornia protoplast |
Non-Patent Citations (2)
| Title |
|---|
| 吴湛霞等: "国内江蓠提取琼胶加工工艺的研究进展", 《广西轻工业》 * |
| 张学成等: "江蓠属海藻龙须菜的基础研究与大规模栽培", 《中国海洋大学学报（自然科学版）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107711488A (en) * | 2017-10-19 | 2018-02-23 | 汕头大学 | A kind of method for preparing different Zhijiang Li protoplasts |
| CN107711488B (en) * | 2017-10-19 | 2020-09-04 | 汕头大学 | Method for preparing Gracilaria heterochorifolia protoplast |
| CN109511550A (en) * | 2018-12-07 | 2019-03-26 | 浙江海洋大学 | Seaweed protoplast is separately cultured the method with regeneration plant |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106893690B (en) | 2020-04-07 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105417877B (en) | A kind of waste water of livestock poultry treatment process | |
| CN107207282A (en) | symbiotic algae system with loop reactor | |
| CN101263778B (en) | Method for fast establishing DSE and plant symbiosis cultivation system and uses thereof | |
| Rameshprabu et al. | A newly isolated green alga, Pediastrum duplex Meyen, from Thailand with efficient hydrogen production | |
| CN106893690A (en) | The preparation method of Chilean fragrant plant mentioned in ancient texts protoplast | |
| CN106479940A (en) | A kind of separation of Qinghai-Tibet Platean uvioresistant cyanophyceae and cultural method | |
| CN105754927B (en) | It hangs the preparation method of fragrant plant mentioned in ancient texts protoplast | |
| CN105993909A (en) | Pyropia haitanensis pure line seedling cultivation method | |
| JP2014113082A (en) | Method for culturing microalgae on the liquid surface characterized by collecting seed algae from the microalgae on the liquid surface and culturing the seed algae in another culture vessel | |
| CN104357327A (en) | Method for culturing microalgae on large scale by bean product wastewater | |
| CN103667072B (en) | A kind of Huperzia serrata endogenetic epiphyte and the application at preparation 8 α, 15 α-epoxidation selagine thereof | |
| CN105132354B (en) | A kind of extracting method of eddo fragrant plant mentioned in ancient texts protoplast | |
| US20140242641A1 (en) | Method for obtaining an open phototrophic culture with improved storage compound production capacity | |
| CN103555584A (en) | Acquisition and application of grease-producing monoraphidium LB50 | |
| Jesus et al. | Autochthonous microalgae cultivation with anaerobic effluent: isolation of strains, survivorship, and characterization of the produced biomass | |
| Abedi et al. | Bioenergy production using Trichormus variabilis–a review | |
| JP2007111023A (en) | Method for obtaining microorganism by using agarase | |
| CN104232559B (en) | The method of cultivating microalgae and the method for producing grease | |
| Erkelens et al. | The influence of protozoa with a filtered and non-filtered seawater culture of Tetraselmis sp., and effects to the bacterial and algal communities over 10 days | |
| JP3837589B2 (en) | Seawater microalgae producing ethanol | |
| JP2009261287A (en) | Chlorella/hydrogen production method and chlorella/hydrogen production apparatus | |
| CN107299059A (en) | A kind of method for preparing scenedesmus obliquus cell protoplast | |
| KR101155435B1 (en) | Novel strain Scenedesmus obliquus YSW15 | |
| CN106701587A (en) | Method used for recycling microalgae residue and producing spirulina rich in polysaccharides | |
| CN110157622A (en) | A kind of screening technique of the chlorella algae strain for production of biodiesel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |