CN105494257B - The method for promoting the method for giant salamander husking and extracting giant salamander genome - Google Patents
The method for promoting the method for giant salamander husking and extracting giant salamander genome Download PDFInfo
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- CN105494257B CN105494257B CN201511006587.8A CN201511006587A CN105494257B CN 105494257 B CN105494257 B CN 105494257B CN 201511006587 A CN201511006587 A CN 201511006587A CN 105494257 B CN105494257 B CN 105494257B
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- 238000000034 method Methods 0.000 title claims abstract description 25
- 230000001737 promoting effect Effects 0.000 title claims abstract description 6
- 241000157862 Dicamptodontidae Species 0.000 title claims abstract 17
- 238000000605 extraction Methods 0.000 claims abstract description 10
- 239000000463 material Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 11
- 241001465754 Metazoa Species 0.000 abstract description 3
- 238000013459 approach Methods 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 238000012252 genetic analysis Methods 0.000 abstract description 2
- 230000007613 environmental effect Effects 0.000 abstract 1
- 241001415306 Cryptobranchidae Species 0.000 description 54
- 238000001179 sorption measurement Methods 0.000 description 11
- 241001253208 Andrias davidianus Species 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 238000005119 centrifugation Methods 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 241000269328 Amphibia Species 0.000 description 2
- 241001253206 Andrias Species 0.000 description 2
- 241000269333 Caudata Species 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 238000005336 cracking Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241000395633 Andrias japonicus Species 0.000 description 1
- 241000086550 Dinosauria Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 235000009932 Zanthoxylum simulans Nutrition 0.000 description 1
- 244000089698 Zanthoxylum simulans Species 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 125000005909 ethyl alcohol group Chemical group 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 210000003097 mucus Anatomy 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 238000001507 sample dispersion Methods 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/02—Breeding vertebrates
-
- 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
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Biomedical Technology (AREA)
- Environmental Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Analytical Chemistry (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Biodiversity & Conservation Biology (AREA)
- Microbiology (AREA)
- Animal Husbandry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods for promoting the method for giant salamander husking and extract giant salamander genome.The present invention a set of makes it quickly generate the method for husking by being found that by environmental stimulus giant salamander; giant salamander can be fast and effectively collected into using this method to cast off a skin; material is provided for extraction giant salamander genome; extraction giant salamander genome is added to make the approach of genetic analysis; and effectively avoid damage giant salamander; achieve the purpose that protect giant salamander, also comply with the thought of animal protection.The present invention is simple and practicable, and of low cost, using effect is good.
Description
Technical field
The present invention relates to bioengineering art field, especially a kind of method for promoting giant salamander husking and extraction giant salamander genome
Method.
Background technology
Giant salamander（Andrias dauidianus）, " giant salamander " is commonly called as, is also cried " big Chinese pepper fish ", belongs to Amphibia
（Amphibia）Caudata（Caudata）Cryptobranchidae（Cryptobranchidae）Giant salamander category（Andrias）, it is that one kind is in
The aquatic excessive species between terrestrial, it is very original in evolution, research think giant salamander be with the coeval species of dinosaur, for me
The distinctive rare wild animal of state, has both high medical value and scientific research value, is mainly distributed in Middle And Upper Reaches of The Yangtze River, the Zhujiang River
In the deep mountain valleys streams of upstream and river Han upstream.Since giant salamander is very big to the degree of dependence of water environment, diffusivity is poor, mesh
Oneself drastically declines the distribution and resource of preceding Chinese giant salamander, Andrias davidianus, and habitat shows serious fragmentation, along with giant salamander gives birth to
Dis environment is worsening, such as artificially catches and kills, environmental pollution, the serious failure of giant salamander natural resources.
Existing wild stocks how is protected, the breeding of science is taken to match measure to avoid inbreeding depression and genetic diversity
Property lose, it has also become the key issue that faces in China Giant Salamander Protection and cultivation.Although in recent years, giant salamander artificial propagation
And propagation in scale technology makes a breakthrough, population quantity also has certain recovery, but due to being lacked to giant salamander genetic background
Weary understanding, therefore cannot fundamentally solve the problems, such as the in imminent danger of it.Therefore, the research of Giant Salamander Protection science of heredity is carried out, to giant salamander
Artificial fecundation and Germplasm resources management have important meaning.
So far, domestic and foreign scholars have been carried out the research in terms of a large amount of giant salamander science of heredity.Data shows, Andrias japonicus
Its gene structure is analyzed, the sample of use is tissue samples：Toe, tail tissue, skin histology and liver；Chinese giant salamander, Andrias davidianus line grain
The extraction of body genome is mostly either to be secreted by gathering the tail muscles of giant salamander, blood, liver after it is centainly stimulated
Mucus method.These methods currently taken can more or less cause giant salamander centainly to injure, and frightened latter section of giant salamander
It can influence to ingest in time, this is that raiser is reluctant to see.And in the angle for protecting wild giant salamander for be also can not
It takes.Husking is the product that giant salamander grows naturally, need not stimulate or shear any position of giant salamander body.But giant salamander exists
In the case of nature, 10 to 15 talentes are casted off a skin once, and in the wild or under conditions of simulating natural environment cultivation, it is also more difficult
To collect.
The content of the invention
The purpose of the present invention is：A kind of method for promoting the method for giant salamander husking and extract giant salamander genome, its energy are provided
Giant salamander husking is fast and effectively obtained, using the material as extraction giant salamander genome, avoids the damage caused by giant salamander.
What the present invention was realized in：Promote the method for giant salamander husking, giant salamander is placed under water environment, temperature 18-
23 DEG C, humidity 40-80%, when the time is 3-5 small；Then giant salamander is positioned in the water that water temperature is 18-23 DEG C again, when 1-2 is small
Afterwards, you can obtain the fresh husking of giant salamander.
The method for extracting giant salamander genome collects material of the fresh husking of giant salamander as extraction giant salamander genome.
By adopting the above-described technical solution, compared with prior art, the present invention a set of is pierced by being found that by environment
Sharp giant salamander makes it quickly generate the method for husking, and can fast and effectively be collected into giant salamander using this method casts off a skin, to extract giant salamander
Genome provides material, adds extraction giant salamander genome to make the approach of genetic analysis, and effectively avoids damage
Giant salamander achievees the purpose that protect giant salamander, also complies with the thought of animal protection.Simple and practicable, of low cost, using effect of the invention
It is good.
Specific embodiment
The embodiment of the present invention 1：Promote the method for giant salamander husking, giant salamander is placed under water environment, temperature 18-23
DEG C, humidity 40-80%, time 3-5；Then giant salamander is positioned in the water that water temperature is 18-23 DEG C again, after the 1-2 times, i.e.,
The fresh husking of giant salamander can be obtained.
The embodiment of the present invention 2：The method for extracting giant salamander genome
1）The fresh husking for the giant salamander being collected by embodiment 1 is used as to the material of extraction giant salamander genome.Acquisition
Giant salamander casts off a skin regardless of whether fresh, can extract genome.But after being influenced in order to prevent because of the degradation of giant salamander genome
Continuous experiment also for subsequent experimental energy accurate marker, preferably gathers husking fresh with giant salamander.
2）Extract genome：
It 1. taking the husking of 1g or so, is put into after cutting into fritter in the centrifuge tube of 1.5ml, adds in 180 μ l Buffer
GTL marks different articles for use.
2. adding in 20 μ l Proteinase Ks, whirlpool shakes thorough mixing.56 DEG C of water-baths, until tissue cracking completely, cracking process
In at regular intervals overturn or shake centrifuge tube make sample dispersion.
3. adding in 200 μ l Buffer GL, whirlpool shakes abundant mixing, 70 DEG C of water-baths 10 minutes.
4. adding in 200 μ l absolute ethyl alcohols after of short duration centrifugation, whirlpool shakes abundant mixing.
4. 5. of short duration centrifugation acquired solution will be transferred to collecting pipe（Collection Tube）Adsorption column (Spin
Column DM) in, it endless can add in once plus several times.8000rpm centrifuges 1min, outwells the waste liquid in collecting pipe, will adsorb
Column is placed back in collecting pipe.
6. the Buffer GW1 (using preceding addition absolute ethyl alcohol) of 500 μ l, 8000rpm centrifugations are added in into adsorption column
1min outwells the waste liquid received in adsorption column, adsorption column is placed back in collecting pipe.
7. the Buffer GW2 (using preceding addition absolute ethyl alcohol) of 500 μ l, 13000rpm centrifugations are added in into adsorption column
1min outwells the waste liquid received in adsorption column, adsorption column is placed back in collecting pipe.
8. 13000rpm centrifuges 2min, the waste liquid received in adsorption column is outwelled, adsorption column is placed in room temperature several minutes, with thorough
It dries（Remove remaining ethyl alcohol）.
9. adsorption column is placed in genome collecting pipe, 50-200 μ l Buffer are vacantly added in the centre of adsorption column
GE is placed at room temperature for 2-5 minutes, and 13000rpm is centrifuged 1 minute, collects DNA solution ,-20 DEG C of preservations.
3）Detect the genome obtained：It is detected using 1.5% agarose gel electrophoresis, DM2000 Maker, point sample amount
For 5 μ l, electrophoretic voltage 110V, 30 ~ 35min of time.
4）PCR amplification：It is designed according to known technology, the primer filtered out, PCR is reacted in the type PCR instruments of PTC -200
（TECHNE companies）Upper progress, reaction system are 40 μ l：Each 2 μ l of upstream and downstream primer；4 20 μ l of μ l, MIX of template DNA, add sterile
Deionized water is to 40 μ l；Response procedures are：95 DEG C of 3 min of pre-degeneration；94 DEG C of 45 S of denaturation, 56 DEG C of 45 S of renaturation, 72 DEG C extend 1
Min, and carry out 35 Xun Huans；72 DEG C of 10 min of extension.
5）Testing goal segment：PCR products are detected with 1.5% agarose gel electrophoresis, in the type disk electrophoresises of DYY-III 32
Slot（Liuyi Instruments Plant, Beijing）Upper progress.By the use of the reaction solution of template DNA is not added as blank control, to check whether there is pollution
In the presence of.Point sample amount is 5 μ l, voltage 100V, 35 ~ 40 min of time.
Claims (2)
- A kind of 1. method for promoting giant salamander husking, it is characterised in that：Giant salamander is placed under water environment, temperature is 18-23 DEG C, wet Spend for 40-80%, the time for 3-5 it is small when；Then giant salamander is positioned over water temperature again is in 18-23 DEG C of water, when 1-2 is small after, you can Obtain the fresh husking of giant salamander.
- A kind of 2. method of the extraction giant salamander genome based on the method as described in claim 1, it is characterised in that：Collect giant salamander Fresh husking as extraction giant salamander genome material.
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| CN201511006587.8A CN105494257B (en) | 2015-12-29 | 2015-12-29 | The method for promoting the method for giant salamander husking and extracting giant salamander genome |
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| CN201511006587.8A CN105494257B (en) | 2015-12-29 | 2015-12-29 | The method for promoting the method for giant salamander husking and extracting giant salamander genome |
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| CN105494257B true CN105494257B (en) | 2018-05-22 |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031307A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Kit and method for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN104223121A (en) * | 2014-09-16 | 2014-12-24 | 重庆馗旭生物科技股份有限公司 | Extracting device for giant salamander skin mucus and extracting method |
| CN104357522A (en) * | 2014-11-11 | 2015-02-18 | 四川龙王洞生态农业开发有限公司 | Method for extracting collagen by using ecdysis of giant salamander |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0701062D0 (en) * | 2007-01-19 | 2007-02-28 | Evocell Ltd | Biological materials and uses thereof |
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- 2015-12-29 CN CN201511006587.8A patent/CN105494257B/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102031307A (en) * | 2010-11-19 | 2011-04-27 | 西北农林科技大学 | Kit and method for detecting virus infecting situation of Chinese giant salamander colony iridovirus by use of ecdysis |
| CN103184215A (en) * | 2013-04-10 | 2013-07-03 | 中国水产科学研究院长江水产研究所 | Method for extracting genomic DNA from giant salamander skin mucus |
| CN104223121A (en) * | 2014-09-16 | 2014-12-24 | 重庆馗旭生物科技股份有限公司 | Extracting device for giant salamander skin mucus and extracting method |
| CN104357522A (en) * | 2014-11-11 | 2015-02-18 | 四川龙王洞生态农业开发有限公司 | Method for extracting collagen by using ecdysis of giant salamander |
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| Title |
|---|
| 成年大鲵实验室饲养及蜕皮和体表粘液收集方法;许永杰等;《遵义医学院学报》;20140228;第37卷(第1期);第115-118页 * |
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