CN105494257A - Method for promoting ecdysis of giant salamander and method for extracting giant salamander genome - Google Patents
Method for promoting ecdysis of giant salamander and method for extracting giant salamander genome Download PDFInfo
- Publication number
- CN105494257A CN105494257A CN201511006587.8A CN201511006587A CN105494257A CN 105494257 A CN105494257 A CN 105494257A CN 201511006587 A CN201511006587 A CN 201511006587A CN 105494257 A CN105494257 A CN 105494257A
- Authority
- CN
- China
- Prior art keywords
- giant salamander
- genome
- extracting
- skin
- salamander
- 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
- 238000000034 method Methods 0.000 title claims abstract description 22
- 241000157862 Dicamptodontidae Species 0.000 title claims abstract 17
- 230000001737 promoting effect Effects 0.000 title abstract description 3
- 239000000463 material Substances 0.000 claims abstract description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 8
- 238000000605 extraction Methods 0.000 claims description 6
- 238000011331 genomic analysis Methods 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 abstract description 3
- 230000007613 environmental effect Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract 1
- 241001415306 Cryptobranchidae Species 0.000 description 54
- 238000001179 sorption measurement Methods 0.000 description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 241001253208 Andrias davidianus Species 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
- 238000005266 casting Methods 0.000 description 3
- 230000009514 concussion Effects 0.000 description 3
- 230000006378 damage Effects 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 241000269328 Amphibia 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
- 210000004185 liver Anatomy 0.000 description 2
- 241000894007 species Species 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 238000011144 upstream manufacturing Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 241001253206 Andrias 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
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 235000009932 Zanthoxylum simulans Nutrition 0.000 description 1
- 244000089698 Zanthoxylum simulans Species 0.000 description 1
- 238000013459 approach Methods 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
- 230000008859 change Effects 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 230000007423 decrease Effects 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
- 238000013461 design Methods 0.000 description 1
- 238000009826 distribution Methods 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
- 239000012634 fragment Substances 0.000 description 1
- 238000013467 fragmentation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000009399 inbreeding Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000002438 mitochondrial effect Effects 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
- 230000008569 process Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004153 renaturation Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000001507 sample dispersion Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
Classifications
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Genetics & Genomics (AREA)
- Zoology (AREA)
- Environmental Sciences (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Molecular Biology (AREA)
- Plant Pathology (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Crystallography & Structural Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Analytical Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for promoting ecdysis of a giant salamander and a method for extracting a giant salamander genome. The method pushes the giant salamander to generate exuviae quickly through environmental stimulus, and by means of the method, giant salamander exuviae can be collected quickly and effectively, materials are provided for extracting the giant salamander genome, a path of extracting the giant salamander genome for genetics analysis is added, the giant salamander is effectively prevented from being hurt, the purpose of protecting the giant salamander is achieved, and the method also conforms to the animal protection concept. The method is simple and easy to implement, low in cost and good in using effect.
Description
Technical field
The present invention relates to biotechnology art field, especially a kind of promote giant salamander to cast off a skin method and extract giant salamander genomic method.
Background technology
Giant salamander (
andriasdauidianus), be commonly called as " giant salamander ", also cry " large Chinese pepper fish ", genus Amphibia (
amphibia) Caudata (
caudata) Cryptobranchidae (
cryptobranchidae) giant salamander genus (
andrias), a kind ofly be in aquatic excessive species between Lu Sheng, very original in evolution, research thinks that giant salamander is coeval species with dinosaur, for the distinctive rare wild animal of China, have high medical value and scientific research value concurrently, be mainly distributed in the deep mountain valleys streams of Middle And Upper Reaches of The Yangtze River, Zhujiang River middle and upper reaches and river Han upstream.Because giant salamander is very large to the degree of dependence of water environment, diffusivity is poor, the distribution of current Chinese giant salamander, Andrias davidianus and resource oneself sharply decline, habitat presents serious fragmentation, add giant salamander living environment to go from bad to worse, as people for catching and killing, environmental pollution etc., the serious exhaustion of giant salamander natural resources.
How to protect existing wild stocks, take the breeding of science measure of matching to lose to avoid inbreeding depression and genetic diversity, become the key issue faced in China Giant Salamander Protection and cultivation.In the last few years, although giant salamander artificial propagation and propagation in scale technology make a breakthrough, population quantity also had certain recovery, owing to being short in understanding to giant salamander genetic background, therefore fundamentally can not solve its problem in imminent danger.Therefore, carry out the genetic research of Giant Salamander Protection, have important meaning to the artificial fecundation of giant salamander and Germplasm resources management.
So far, Chinese scholars has carried out the research of a large amount of giant salamander genetics aspect.Data shows, and Andrias japonicus analyzes its gene structure, and the sample of employing is tissue samples: toe, tail tissue, skin histology and liver; The extraction of Chinese giant salamander, Andrias davidianus mitochondrial genomes be mostly tail muscles by gathering giant salamander, blood, liver or its be subject to the method for the mucus of secretion after certain stimulation.Current these methods taked more or less can cause certain injury to giant salamander, and giant salamander can affect in frightened rear a period of time and ingests, and this is that raiser is reluctant to see.And protection wild giant salamander angle on be also worthless.Casting off a skin is the product of giant salamander self-sow, and not needing stimulates or shear any position of giant salamander health.But giant salamander is in natural situation, and 10 to 15 talentes are casted off a skin once, and in the wild or simulating natural environment cultivation condition under, also compare be difficult to collect.
Summary of the invention
The object of the invention is: provide a kind of promote giant salamander to cast off a skin method and extract giant salamander genomic method, it can obtain giant salamander fast and effectively and cast off a skin, and using as the genomic material of extraction giant salamander, avoids the damage caused giant salamander.
The present invention is achieved in that the method promoting that giant salamander casts off a skin, and under giant salamander is placed in dried up environment, temperature is 18-23 DEG C, and humidity is 40-80%, and the time is 3-5 hour; And then giant salamander is positioned over water temperature is in the water of 18-23 DEG C, after 1-2 hour, the fresh of giant salamander can be obtained and cast off a skin.
Extract the genomic method of giant salamander, collect the fresh of giant salamander and cast off a skin as the genomic material of extraction giant salamander.
Owing to have employed technique scheme; compared with prior art; the present invention is by finding a set ofly to make it produce the method for casting off a skin fast by environmental stimulus giant salamander; utilize the method can collect giant salamander fast and effectively to cast off a skin; thering is provided material for extracting giant salamander genome, adding and extracting giant salamander genome to make the approach of genetic analysis, and effectively avoid damage giant salamander; reach the object of protection giant salamander, also meet the thought of protection of animals.The present invention is simple, with low cost, and result of use is good.
Embodiment
Embodiments of the invention 1: promote the method that giant salamander casts off a skin, under giant salamander is placed in dried up environment, temperature is 18-23 DEG C, and humidity is 40-80%, and the time is 3-5; And then giant salamander is positioned over water temperature is in the water of 18-23 DEG C, after the 1-2 time, the fresh of giant salamander can be obtained and cast off a skin.
Embodiments of the invention 2: extract the genomic method of giant salamander
1) the fresh of the giant salamander of being collected by embodiment 1 is casted off a skin as the genomic material of extraction giant salamander.No matter fresh whether the giant salamander gathered cast off a skin, and can extract genome.But in order to prevent because the genomic degraded of giant salamander affects subsequent experimental, also in order to subsequent experimental energy accurate marker, preferably gather that giant salamander is fresh to cast off a skin with it.
2) genome is extracted:
1. get casting off a skin of about 1g, after cutting into fritter, put into the centrifuge tube of 1.5ml, add 180 μ lBufferGTL, different articles for use are carried out mark.
2. 20 μ l Proteinase Ks are added, whirlpool concussion thoroughly mixing.56 DEG C of water-baths, until organize complete cracking, put upside down at set intervals in cracking process or shake centrifuge tube and make sample dispersion.
3. 200 μ lBufferGL are added, whirlpool concussion fully mixing, 70 DEG C of water-baths 10 minutes.
4. of short duration centrifugal after add 200 μ l absolute ethyl alcohols, whirlpool concussion fully mixing.
5. of short duration centrifugal, will 4. proceed in the adsorption column (SpinColumnDM) of collecting pipe (CollectionTube) by gained solution, and once cannot not add completely and can add several times.The centrifugal 1min of 8000rpm, outwells the waste liquid in collecting pipe, is placed back in collecting pipe by adsorption column.
6. in adsorption column, add the BufferGW1 (adding absolute ethyl alcohol before use) of 500 μ l, the centrifugal 1min of 8000rpm, outwell the waste liquid received in adsorption column, adsorption column is placed back in collecting pipe.
7. in adsorption column, add the BufferGW2 (adding absolute ethyl alcohol before use) of 500 μ l, the centrifugal 1min of 13000rpm, outwell the waste liquid received in adsorption column, adsorption column is placed back in collecting pipe.
8. the centrifugal 2min of 13000rpm, outwells the waste liquid received in adsorption column, adsorption column is placed in room temperature number minute, thoroughly to dry (removing remaining ethanol).
9. adsorption column is placed in genome collecting pipe, the centre to adsorption column is unsettled adds 50-200 μ lBufferGE, and room temperature is placed 2-5 minutes, centrifugal 1 minute of 13000rpm, collects DNA solution ,-20 DEG C of preservations.
3) genome obtained is detected: adopt the agarose gel electrophoresis of 1.5% to detect, DM2000 is Maker, and point sample amount is 5 μ l, electrophoretic voltage 110V, time 30 ~ 35min.
4) pcr amplification: according to known technology design, the primer filtered out, PCR reaction is carried out in PTC-200 type PCR instrument (TECHNE company), and reaction system is 40 μ l: each 2 μ l of upstream and downstream primer; Template DNA 4 μ l, MIX20 μ l, add aseptic deionized water to 40 μ l; Response procedures is: 95 DEG C of denaturation 3min; 94 DEG C of sex change 45S, 56 DEG C of renaturation 45S, 72 DEG C extend 1min, and carry out 35 circulations; 72 DEG C extend 10min.
5) testing goal fragment: PCR primer 1.5% agarose gel electrophoresis detects, DYY-III 32 type disk electrophoresis groove (Liuyi Instruments Plant, Beijing) carries out.Utilize the reactant liquor not adding template DNA as blank, exist to have checked whether to pollute.Point sample amount is 5 μ l, and voltage is 100V, time 35 ~ 40min.
Claims (2)
1. promote to it is characterized in that the method that giant salamander casts off a skin: under giant salamander is placed in dried up environment, temperature is 18-23 DEG C, and humidity is 40-80%, and the time is 3-5 hour; And then giant salamander is positioned over water temperature is in the water of 18-23 DEG C, after 1-2 hour, the fresh of giant salamander can be obtained and cast off a skin.
2. based on the genomic method of extraction giant salamander of the method for claim 1, it is characterized in that: collect the fresh of giant salamander and cast off a skin as the genomic material of extraction giant salamander.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| 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 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105494257A true CN105494257A (en) | 2016-04-20 |
| CN105494257B CN105494257B (en) | 2018-05-22 |
Family
ID=55702875
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201511006587.8A Expired - Fee Related CN105494257B (en) | 2015-12-29 | 2015-12-29 | The method for promoting the method for giant salamander husking and extracting giant salamander genome |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105494257B (en) |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008087442A1 (en) * | 2007-01-19 | 2008-07-24 | Evocell Limited | Biological materials and uses thereof |
| 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 |
-
2015
- 2015-12-29 CN CN201511006587.8A patent/CN105494257B/en not_active Expired - Fee Related
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2008087442A1 (en) * | 2007-01-19 | 2008-07-24 | Evocell Limited | Biological materials and uses thereof |
| 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 |
Non-Patent Citations (2)
| Title |
|---|
| 许永杰等: "成年大鲵实验室饲养及蜕皮和体表粘液收集方法", 《遵义医学院学报》 * |
| 邬文华: "蟾蜍自然脱衣及加工法", 《农技服务》 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105494257B (en) | 2018-05-22 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Lahmar et al. | Transmission dynamics of the Echinococcus granulosus sheep–dog strain (G1 genotype) in camels in Tunisia | |
| Li et al. | A new species of the Asian toad genus Megophrys sensu lato (Amphibia: Anura: Megophryidae) from Guizhou Province, China | |
| CN102134593B (en) | Gender-specific microsatellite marker for Cynoglossus semilaevis and application of same in identification of superfemale Cynoglossus semilaevis | |
| CN105123621A (en) | Establishment method of goat resource population | |
| CN113755566B (en) | PCR primer set, site, method and kit for rapidly identifying sex of wattle-necked softshell turtle | |
| Boora et al. | Evaluation of genetic diversity in Jatropha curcas L. using RAPD markers | |
| CN109680076B (en) | Molecular marker and method for identifying genetic sex of Chinese softshell turtles | |
| CN102899321A (en) | Method for selecting specific SRAP marking tapes for coilia ectenes genders and gender determination method | |
| Aziz et al. | Molecular DNA identification of medicinal plants used by traditional healers in Malaysia | |
| CN103882116B (en) | Four planting fruit-trees heart-eating worm Molecular Identification primer and using method | |
| Lopez-Osorio et al. | Phylogenomic analysis of yellowjackets and hornets (Hymenoptera: Vespidae, Vespinae) | |
| CN103642797B (en) | A kind of Extraction method of total RNA of intermuscular bone of megalobrama amblycephala | |
| CN103789441A (en) | SSR (Simple Sequence Repeat) molecular marker method for identifying two hippocampus populations | |
| Pengyan et al. | Transgenic mouse model integrating siRNA targeting the foot and mouth disease virus | |
| Brasileiro et al. | A new species of Physalaemus from central Brazil (Anura: Leptodactylidae) | |
| CN105494257A (en) | Method for promoting ecdysis of giant salamander and method for extracting giant salamander genome | |
| CN101878756B (en) | Method for storing monochamus alternatus hope sample and method for quickly detecting bursaphelenchus xylophilus therein | |
| CN105586398A (en) | Method for identifying aloe vera species with ITS sequence | |
| CN103232997B (en) | Method for extracting total RNA from leucaena leucocephala leaf tissue | |
| Zhao et al. | A new species of the genus Rana from Henan, central China (Anura, Ranidae) | |
| CN104987376A (en) | Channel protein of green plant bug for sensing damage temperature, as well as coding gene and application thereof | |
| CN104650220A (en) | Green plant bug temperature and smell receptor protein as well as coding gene and application thereof | |
| Mo et al. | A new species of Kaloula (Amphibia: Anura: Microhylidae) from southern Guangxi, China | |
| Guo et al. | Skin secretion and shedding is a good source for non-destructive genetic sampling in the Chinese giant salamander (Andrias davidianus) | |
| Marikar et al. | Usefulness of short sequence repeat markers in goat genetic diversity studies on the Asian and African continents |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180522 Termination date: 20211229 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |