CN106577416A - Cultivation method of fluorescent protein-based luminous pearl - Google Patents
Cultivation method of fluorescent protein-based luminous pearl Download PDFInfo
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- CN106577416A CN106577416A CN201611123324.XA CN201611123324A CN106577416A CN 106577416 A CN106577416 A CN 106577416A CN 201611123324 A CN201611123324 A CN 201611123324A CN 106577416 A CN106577416 A CN 106577416A
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- pearl
- fluorescin
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- breeding method
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/80—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in fisheries management
- Y02A40/81—Aquaculture, e.g. of fish
Abstract
The invention relates to the field of a biotechnology, in particular to a cultivation method of a fluorescent protein-based luminous pearl. After plasmid containing fluorescent protein genes is transferred into a pearl-cultivating shell tissue, the pearl-cultivating shell which is transfected with the plasmid is cultivated, subjected to sheet production, sheet insertion or core insertion and then cultivated to obtain the luminous pearl.
Description
Technical field
The present invention relates to biological technical field, in particular to a kind of legendary luminous pearl breeding method based on fluorescin.
Background technology
Legendary luminous pearl is the rare jewellery of nature, is mankind's treasure, with very high investment and reserve value.One
Weigh 2800 grams, Jing illumination 15 minutes can in 72 hours continuous luminous light grey fluorite "night luminescent bead", be confirmed to be at that time
Maximum in the world, priceless rare treasure.It is said that sold with 40,000,000 yuans first, later again with 18,000,000 dollars
High price sell give position France collector.From the mineral with phosphorescent glow and rock, more is that " night is bright for fluorite to legendary luminous pearl
Pearl ".Fluorite hardness is 4, because being often presented light blue, emerald green, blackish green containing various rare elements.Fluorite is put into ultraviolet fluorescent
Shine under lamp, it can send the fluorescence of beauty.
Nature has many materials, is excited by outside energy (such as heat energy, luminous energy, electron beam), just sends naked eyes
Visible light, in specialty " fluorescence (Fluorescence) " is referred to as；Luminous referred to as " phosphorus can also be continued after stopping such as excitation energy
Light (Phosphorescence) "；Title long afterglow material (the Brightness Matter for of luminous energy last longer
Long more then Time).Have now been found that the mineral such as fluorite, diamond, calcite, spodumene in extraneous energy
Visible phosphorescence can be sent under exciting.
1973, Mr. Zhang Yuanpei of Hunan Province's Aquatic product institute was prepared into radiosiotope permanent light powder etc.
Pearl core, is then transplanted to the pearl core with fluorescent characteristic and mantle piece in pearl culturing clam outer embrane connective tissue, to produce " night
Jewel " (fluorescence Margarita).From the point of view of practice, using radioelement as the legendary luminous pearl of luminous body, breeding environment is on the one hand polluted,
Bio life is poisoned；On the other hand, although it is said that radioactivity is very low, but also has an impact the healthy worry of user, therefore
It is not applied and promotes.If modern high technology can be used, produce nontoxic, the treasure of bioluminescence can be produced again
Pearl is " legendary luminous pearl ", will produce huge commercial value.
In view of this, it is special to propose the present invention.
The content of the invention
It is an object of the invention to provide a kind of legendary luminous pearl breeding method based on fluorescin, what described method was provided
Legendary luminous pearl luminosity is stablized, and without radioactivity, to no toxic biological, with very high ornamental value.
Before compound, compositionss, protein, peptide in the description present invention etc. and method, it will be appreciated that these enforcements
Mode is not limited to described ad hoc approach, scheme and reagent, this is because they can change.It is also understood that herein
The term for being used is only used for describing particular implementation purpose, and is not intended to limit the model of present embodiment or claim
Enclose.
In order to realize the above-mentioned purpose of the present invention, spy employs the following technical solutions：
A kind of legendary luminous pearl breeding method based on fluorescin, by the plasmid containing fluorescence protein gene pearl shellfish of growing cultured pearls is proceeded to
After tissue, to transfected plasmids after pearl shellfish of growing cultured pearls cultivated and carried out film-making, inserted sheet or slotting core, cultivation obtains legendary luminous pearl.
The legendary luminous pearl that the present invention is produced, green fluorescent protein is nested in nacreous layer, and albumen is in what is be isolated from the outside
State, not degradable, fluorescence will not extract and go out.
Preferably, the fluorescin is selected from green fluorescent protein, blue fluorescent protein, yellow fluorescence protein, orange glimmering
Photoprotein, red fluorescent protein and optics highlight one or more in fluorescin；
It is furthermore preferred that the fluorescin is green fluorescent protein, the plasmid is pEGFP-C1.
Blue fluorescent protein can be selected from EBFP, Azuritc, TagBFP etc.；Yellow fluorescence protein can selected from EYFP,
Ypct, PhiYFP etc.；Orange fluorescent protein can be selected from mKO, mOrange, mBanana etc.；Red fluorescent protein can be selected from
TagRFP、mRuby、mCherry、mKatc。
Green fluorescent protein (greenfluorescent protein, GFP) is a kind of very common fluorescence in this area
Albumen, is made up of, long 2.6kb 3 exons；The monomeric protein being made up of 238 aminoacid, relative molecular mass is
27.0kD(26888D)；Its property of protein is sufficiently stable, is resistant to 60 DEG C of process.With 92 DEG C of 6mol/L guanidine hydrochlorides, 2min or acid
Process (pH2), after 5min degeneration, GFP remains to renaturation and recovers fluorescence, and its half renaturation time is less than 5min, again multiple in pH7.0
Property GFP maximum excitations peak be 398nm, emission peak is 508nm, as natural GFP, anti-trypsin hydrolysis process.
The luminous mechanism of GFP is to fold cyclisation after GFP expression, in the presence of oxygen, by the amino of 65～67
Sour residue (Ser-Tyr-Gly) cyclisation, forms chromophore (chromophore), so after GFP gene expressions, typically there is several little
When period of delay, just it is observed that green glow.Under ultraviolet excitation, GFP sends green fluorescence；Its maximum absorption band in 395nm,
Another little absworption peak is in 470nm；Maximum emission peak is 509nm.Send out during the conformation change that GFP is studied under different absolute temperature
Existing, there is the B states of high-energy and low-energy A states in GFP, Tyr66 is relevant with A states, and Ser65 is relevant with B states.
GFP genes of the present invention can be type commonly used in the art, the GFP genes of such as wild type；So far
The wild type gfp gene complete encoding sequences logged in GenBank have 5,4 (GenBank therein：AEVGFPA
M62653, AVGFP1X83959, AVGFP2X83960, AEVGFP L29345) it both is from the luminous of U.S.West Coast Its Adjacent Waters
Jellyfish Aequorea Victoria.This 4 sequences are carried out with the comparison of nucleotide and aminoacid, the homology of nucleotide is found
96% or so, with the presence of 8 site variations in 238 aminoacid.Other one is to be isolated from the big of Chinese Xiamen east marine site
The wild type Green Fluorescent protein gene gfpxm (GenBank of many siphonohores of type (Aequorea macrodactyla)：
AY013824).Can also adopt through improved GFP genes, such as enhancement mode GFP gene EGFP etc..
PEGFP-C1 carriers are the expression vectors produced by Clontech companies, and Clontech article No.s are 6084-1.
PEGFP-C1 carriers contain an EGFP fluorescent protein sequence, and EGFP fluorescence eggs can be expressed in mammalian cell
In vain.PEGFP-C1 is applied well in Fish etc. and Mollusca.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, the pearl shellfish tissue of growing cultured pearls is selected from educates
The embryo of pearl pearl shellfish, or one or more in shellfish gonad, outer embrane and its in vitro tissue.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, described that plasmid is proceeded to pearl shellfish of growing cultured pearls
The method of tissue is electroporation, micro- embryo's injection or direct injection.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, the plasmid concentration is 5～15%；
Import 50～200ul of dosage.
Preferably, the legendary luminous pearl breeding method of fluorescin is based on as above, and the condition of the electroporation is：
20～150V/cm of electric field intensity；Electroporation 1～8 time, every minor tick 1～2 minute, shocks by electricity 10～80 milliseconds every time.
It is furthermore preferred that the condition of the electroporation is：
60～120V/cm of electric field intensity；Electroporation 3～8 times, every minor tick 1～2 minute, shocks by electricity 30～50 milliseconds every time.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, will contain green fluorescence protein gene
Plasmid proceed to grow cultured pearls pearl shellfish tissue before, also include：
Clean freshwater mussel body is organized and is cooled to 6～15 DEG C.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, the pearl shellfish of growing cultured pearls is including Margarita freshwater mussel
Section, Unionidae, Ostreidae or Pteriidae.
Preferably, the legendary luminous pearl breeding method based on fluorescin as above, the pearl shellfish of growing cultured pearls is selected from jib
Freshwater mussel, pond dish freshwater mussel, cristaria plicata, anodon, beautiful freshwater mussel, Pinctadamaxima (Jameson), Pinctada margaritifera (Linnaeus), pteria martensii (Pinctada fucata), enterprise
Goose Margarita.
Legendary luminous pearl breeding method based on fluorescin as above cultivates the legendary luminous pearl for obtaining.
The legendary luminous pearl luminosity prepared by the legendary luminous pearl breeding method based on fluorescin that the present invention is provided is stablized, and
It is a kind of legendary luminous pearl producer highly promoted with very high ornamental value to no toxic biological without radioactivity
Method.
Specific embodiment
Unless otherwise defined, all technologies for using of the present invention and scientific terminology have with belonging to disclosed embodiment
The identical implication that the those of ordinary skill in field is generally understood that.Although similar or equivalent with method of the present invention and material
Method and material can be used in the practice or test of present embodiment, but hereafter still describe suitable method and material.
All publications, patent application, patent and other lists of references that the present invention is referred to are incorporated into this by quoting full content
Wen Zhong.In the case of a conflict, this specification (including definition) will play dominating role.Additionally, material, method and embodiment are only
It is merely illustrative, and is not intended to limit.Other feature and advantage of embodiment will be from following detailed description of book and right
Become in requirement obvious.
In order to promote to understand implementations described herein this purpose, with reference to some embodiments, and will will use
Language-specific is describing these embodiments.Term as used herein is only used for describing specific embodiment purpose, and not purport
Limiting the scope of the present disclosure.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the present invention.It is unreceipted concrete in embodiment
Condition person, the condition advised according to normal condition or manufacturer is carried out.Agents useful for same or the unreceipted production firm person of instrument, are
The conventional products that commercially available purchase is obtained can be passed through.
Embodiment 1
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
The outer embrane epithelial tissue for cutting hydriopsis cumingii (or cristaria plicata) is cut into small pieces and is cooled to 6～15 DEG C, immersion
In plasmid solution, plasmid concentration is 5%, imports dosage 200ul；
Hydriopsis cumingii (or cristaria plicata) shell is opened into 0.5～1.0cm with gag, above-mentioned outer embrane epithelial tissue is little
Block is implanted into outer embrane or internal sac, and cultivation is carried out 3～5 years with conventional method, you can formed with fluorescence without nucleus pearl.
Embodiment 2
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
1), pEGFP-C1 plasmids are injected in the embryo of pearl shellfish of growing cultured pearls using microinjector, plasmid concentration is 13%,
Import dosage 1ul；
Preferably, any one of pearl Becquerel of growing cultured pearls in Pinctadamaxima (Jameson), Pinctada margaritifera (Linnaeus), pteria martensii, Pteria penguin.
By the embryo under the conditions of 18～20 DEG C, conventional method cultivates into operation shellfish.Using general Margarita culture side
Formula, behind operation Bei Chahe areas, one pearl nucleus of insertion, while transplant one piece of mantle piece, mantle piece around nucleosis into
Pearl capsule, the epithelial cell of capsule secretes nacre on core face, deposits nacreous layer and forms nucleated pearl.Under the conditions of 18～20 DEG C
Cultivation 4 months, its nacreous layer can present week fluorescent, when the nacreous layer of nucleated pearl grows into more than 0.5mm by adopt
Pluck, the Margarita for obtaining can be considered night bright Margarita.
Embodiment 3
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
1), clean pteria martensii and be cooled to 6～15 DEG C, shell is opened into 0.5～1.0cm with gag；User
Ripple electroporation apparatuss carry out electricity and turn, and electrode is individually positioned in outer embrane and internal sac position, pEGFP-C1 plasmid electricity are rotated into outer
Mantle.Plasmid concentration is 10%, imports dosage 150ul；
The condition of electroporation is：
Electric field intensity 100V/cm；Electroporation 6 times, every minor tick 2 minutes, shocks by electricity 50 milliseconds every time.
2), performed the operation using general slotting kernel mode, after inserting a pearl nucleus in pteria martensii body, while with
Step 1) mantle tissue that obtains inserts as the mantle piece of transplanting, mantle piece around nucleosis beading capsule, capsule it is upper
Nacre is secreted on chrotoplast core face, nacreous layer is deposited and is formed nucleated pearl.Cultivate 3 months under the conditions of 20～18 DEG C, its
Shell pearl layer can present week fluorescent, when the nacreous layer of nucleated pearl grows into more than 0.5mm can harvesting, obtain
Margarita can be considered night bright Margarita.
Embodiment 4
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
1) clean spinnaker freshwater mussel body and be cooled to 6～15 DEG C, concha Anodonta seu Cristaria is opened into 0.5～1.0cm with gag；Electrode
Outer embrane and internal sac position are individually positioned in, pEGFP-C1 plasmid electricity is rotated into into outer embrane.Plasmid concentration is 15%, imported agent
Amount 200ul；
The condition of electroporation is：
Electric field intensity 90V/cm；Electroporation 8 times, every minor tick 2 minutes, shocks by electricity 10 milliseconds every time.
2), performed the operation using general slotting kernel mode, in the internal sac or outer embrane of hydriopsis cumingii a Margarita is inserted
After core, while with step 1) mantle tissue that obtains inserts as the mantle piece of transplanting, mantle piece around nucleosis into
Pearl capsule, secretes nacre on the epithelial nucleus face of capsule, deposit nacreous layer and form nucleated pearl.Support under the conditions of 20～18 DEG C
Grow 3 months, its shell pearl layer can be presented week fluorescent, when the nacreous layer of nucleated pearl grows into more than 0.5mm
Pluck, the Margarita for obtaining can be considered night bright Margarita.
Embodiment 5
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
1), clean cristaria plicata freshwater mussel body and be cooled to 6～15 DEG C, concha Anodonta seu Cristaria is opened into 0.5～1.0cm with gag；Electricity
Pole is individually positioned in outer embrane and internal sac position, and pEGFP-C1 plasmid electricity is rotated into into outer embrane.Plasmid concentration is 8%, is imported
Dosage 90ul；
The condition of electroporation is：
Electric field intensity 90V/cm；Electroporation 8 times, every minor tick 2 minutes, shocks by electricity 10 milliseconds every time.
2), performed the operation using general slotting kernel mode, in the internal sac or outer embrane of cristaria plicata a Margarita is inserted
After core, while with step 1) mantle tissue that obtains inserts as the mantle piece of transplanting, mantle piece around nucleosis into
Pearl capsule, secretes nacre on the epithelial nucleus face of capsule, deposit nacreous layer and form nucleated pearl.Support under the conditions of 20～18 DEG C
Grow 3 months, its shell pearl layer can be presented week fluorescent, when the nacreous layer of nucleated pearl grows into more than 0.5mm
Pluck, the Margarita for obtaining can be considered night bright Margarita.
Embodiment 6
A kind of production method of bright Margarita of night is present embodiments provided, it is comprised the following steps：
1) spinnaker freshwater mussel freshwater mussel body, is cleaned, checks that gonad confirms male and female by conventional method, select high-quality female individuals 10
It is individual, male 5, and it is cooled to 6～15 DEG C, concha Anodonta seu Cristaria is opened into 0.5～1.0cm with gag；Electrode is individually positioned in
Outer embrane and internal sac position, by pEGFP-C1 plasmids, respectively electricity rotates into the internal sac gonad area of female, male freshwater mussel.Plasmid concentration is
10%, import dosage 120ul；
The condition of electroporation is：
Electric field intensity 90V/cm；Electroporation 8 times, every minor tick 2 minutes, shocks by electricity 10 milliseconds every time.
2), above-mentioned male and female freshwater mussel is carried out by combo breeding using conventional method, obtains the little freshwater mussel for carrying fluorescence protein gene.With
Used as operation freshwater mussel of growing cultured pearls, routinely the operation of inserted sheet method, by the cultivation of 3-5 years, can obtain with fluorescent characteristic this little freshwater mussel
Without nucleus pearl；Routinely nuclear insertion method inserts core and is implanted into above-mentioned mantle piece in internal sac or outer embrane, cultivates 1-2 years, you can
Obtain the nucleated pearl with fluorescent characteristic.
The night bright Margarita that the present invention is provided, without radioactivity, to no toxic biological, with very high ornamental value,
View and admire under daylight and possess the normal gloss of Margarita, and at night, faint corresponding fluorescence can be glowed, be highly suitable as head
Decorations, craft decoration carry out deep processing.
The main component of Margarita is the Calcium Carbonate of aragonite, calcite both structures, by way of monocrystalline or polycrystalline,
Complete structure is combined together to form with conchiolin.The nacreous layer that per layer of aragonite is combined to form with conchiolin.It is green
Color fluorescin is closed in the middle of nacreous layer, and property is more stable, the invasion without microorganism and the interference of external environment, egg
White structure will not occur degeneration.The legendary luminous pearl fluorescent lifetime is long, and luminosity is stable, can keep the luminous of several years even many decades
The stabilized illumination of time.
Finally it should be noted that：Various embodiments above only to illustrate technical scheme, rather than a limitation；To the greatest extent
Pipe has been described in detail with reference to foregoing embodiments to the present invention, but it will be understood by those within the art that：Its
Still the technical scheme described in foregoing embodiments can be modified, either to which part or all technical characteristic
Carry out equivalent；And these modifications or replacement, do not make the essence disengaging various embodiments of the present invention skill of appropriate technical solution
The scope of art scheme.
Claims (10)
1. a kind of legendary luminous pearl breeding method based on fluorescin, it is characterised in that turn the plasmid containing fluorescence protein gene
The pearl shellfish that enters to grow cultured pearls tissue after, to transfected plasmids after pearl shellfish of growing cultured pearls cultivated and carried out film-making, inserted sheet or slotting core, cultivation is obtained
Legendary luminous pearl.
2. the legendary luminous pearl breeding method based on fluorescin according to claim 1, it is characterised in that the fluorescin
Highlight in fluorescin selected from green fluorescent protein, yellow fluorescence protein, orange fluorescent protein, red fluorescent protein and optics
One or more；
Preferably, the fluorescin is green fluorescent protein, and the plasmid is pEGFP-C1.
3. the legendary luminous pearl breeding method based on fluorescin according to claim 1, it is characterised in that the pearl shellfish of growing cultured pearls
Tissue is selected from the embryo of pearl shellfish of growing cultured pearls, or one or more in shellfish gonad, outer embrane and its in vitro tissue.
4. the legendary luminous pearl breeding method based on fluorescin according to claim 3, it is characterised in that described to turn plasmid
The method of the pearl shellfish that enters to grow cultured pearls tissue is electroporation, micro- embryo's injection or direct injection.
5. the legendary luminous pearl breeding method based on fluorescin according to claim 4, it is characterised in that the plasmid concentration
For 5～15%；Import 50～200ul of dosage.
6. the legendary luminous pearl breeding method based on fluorescin according to claim 4, it is characterised in that the electroporation
Condition be：
20～150V/cm of electric field intensity；Electroporation 1～8 time, every minor tick 1～2 minute, shocks by electricity 10～80 milliseconds every time.
7. the legendary luminous pearl breeding method based on fluorescin according to claim 3, it is characterised in that will be containing fluorescence egg
The plasmid of white gene is proceeded to before pearl shellfish tissue of growing cultured pearls, and is also included：
Clean freshwater mussel body is organized and is cooled to 6～15 DEG C.
8. the legendary luminous pearl breeding method based on fluorescin according to claim 1, it is characterised in that the pearl shellfish of growing cultured pearls
Including Margaritanidae, Unionidae, Ostreidae or Pteriidae.
9. the legendary luminous pearl breeding method based on fluorescin according to claim 8, it is characterised in that the pearl shellfish of growing cultured pearls
Selected from hydriopsis cumingii, pond dish freshwater mussel, cristaria plicata, anodon, beautiful freshwater mussel, Pinctadamaxima (Jameson), Pinctada margaritifera (Linnaeus), pteria martensii, penguin Margarita
Shellfish.
10. the legendary luminous pearl breeding method based on fluorescin described in any one of claim 1～9 cultivates the legendary luminous pearl for obtaining.
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Application publication date: 20170426 |