CN102758023B - CH4 molecular marker denaturing gel gradient electrophoresis (DGGE) identifying method for aquaculture of catfish hybrids - Google Patents
CH4 molecular marker denaturing gel gradient electrophoresis (DGGE) identifying method for aquaculture of catfish hybrids Download PDFInfo
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- CN102758023B CN102758023B CN2012102784298A CN201210278429A CN102758023B CN 102758023 B CN102758023 B CN 102758023B CN 2012102784298 A CN2012102784298 A CN 2012102784298A CN 201210278429 A CN201210278429 A CN 201210278429A CN 102758023 B CN102758023 B CN 102758023B
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Abstract
The invention relates to a CH4 molecular marker DGGE identifying method for aquaculture of catfish hybrids. Southern catfishes, catfishes and hybrids of the southern catfishes and the catfishes serve as objects of study, CH4 gene sections in catfishes are selected to serve as molecular markers, the sequence difference the CH4 gene sections between the southern catfishes and the catfishes, denaturing gel gradient electrophoresis detection is performed and the southern catfishes, the catfishes and hybrids of the southern catfishes and the catfishes are identified successfully. The method has the advantages of being fast in experiment, convenient to operate, visual in identification and free from sequencing.
Description
Technical field
The present invention relates to the molecular marker identification method of fish hybrid, be specifically related to the CH of aquaculture catfish hybrid
4molecule marker DGGE authentication method.
Background technology
The SILURIFORMES fish belong to carnivorous demersal fish, are one of Teleosteis the most flourishing during fish are evolved.Silurus meridionalis Chen (crying again large mouthful catfish) (
silurus meridionalischen) and catfish (
silurus asotuslinnaeus) all belonging to SILURIFORMES, catfish section, catfish and belong to, both delicious flavours, be of high nutritive value, is the high added value economic fish that China various places mainly cultivate.Silurus meridionalis Chen is distributed in the water systems such as China's Zhujiang River, the Min River, Xiang River, the Changjiang river, is a kind of predacious fish of large-scale ferociousness, has the characteristics such as growth is fast, individual greatly, disease resistance is strong, happiness inhabits opens wide water body, and gravel section, Tuo, river bed, gulf, fish brood amount is large, and the spawning time is long.Catfish is distributed in each water system of the whole nation except Qinghai-Tibet Platean and Xinjiang, and body colour is different and change to some extent with habitat, mainly inhabits the middle lower floor of rivers master stream or larger water bodys such as tributary, large-size lake and reservoir, its strong adaptability, inhabit hydrostatic or unhurried current waters, individuality is less, and growth is fast.Silurus meridionalis Chen and catfish body profile similarity, the Silurus meridionalis Chen of little individuality and catfish adult confuse lives together, large individual and catfish isolation life.It is reported, in the Silurus meridionalis Chen colony of artificial propagation, male ratio is very low.Investigation shows, when male Silurus meridionalis Chen lacks, may carry out the hybridization that female Silurus meridionalis Chen and other male catfish belong to kind, thereby the cross-breeding between species occurred in catfish produces.
In order to meet the needs of social production, obtain the seed that has above 2 kinds of fish good characters concurrently, the bright grade of imperial court (cross experiment of large mouthful catfish and catfish, fresh water fishery, 2004,34(6): 41 ~ 43; The RAPD of three kinds of catfish genetic diversities analyzes, fresh water fishery, and 2005,35(4): 14 ~ 17) carried out the cross-breeding test of Silurus meridionalis Chen and catfish, and done certain hybrid identification work, filtered out 22 random primers for Silurus meridionalis Chen, catfish and hybridization F thereof
1generation three population diversity analyses, found that hybridization F
1in generation, inherited 52 characteristic strips of large mouthful catfish and 32 characteristic strips of catfish.Yet the RAPD mark that its evaluation work adopts, be subject to the restriction of method itself, detection efficiency is low and repeated not high, has had a strong impact on promoting the use of of this method.In addition, only have Longhua etc. (catfish, large mouthful catfish and hybridization catfish blood physiology biochemical indicator thereof relatively reach the Transferrins,iron complexes alleles analysis, Changjiang University's journal (from section's version), 2006,3(2): 161 ~ 164; The blood type analysis of catfish, large mouthful of catfish and hybridization catfish thereof, water conservancy related fisheries, 2007,27(3): 19 ~ 20) to the research of the aspects such as blood physiology, blood type analysis and isozyme of Silurus meridionalis Chen, catfish and hybridization catfish thereof.Visible, be necessary to carry out molecule marker and the authenticate technology research of Silurus meridionalis Chen, catfish and hybrid generation thereof, for the hybrid identification in Silurus meridionalis Chen cultivation and breeding practice provides reliable method.
At present, RAPD, AFLP and micro-satellite all are applicable to the Idioplasm identification of population level, and still, the RAPD detection efficiency is low, and usually attempting tens ofly just has ten pairs of left and right to use to random primer, and repeatability is not good; The AFLP circulation ratio is higher, can produce the polymorphic bands of a large amount of (common more than 100), but not easy to operate, when amplifying difference between species, has more aggravated intraspecies variation, has a large amount of false positive results, has limited the hybrid identification accuracy; Although micro-satellite has polymorphism and the susceptibility of species specificity, codominance and height, in the analyses such as hybrid identification and Introgression In Hatchery Stocks, be widely used, but need the tool species specificity for the site of differentiating hybrid, to judge the verity of amplified production in analytic process, especially when differentiating sibling species hybridization, allelic size and the number particularly important that seems in correct judgement sibling species, this has increased the use difficulty of micro-satellite to a great extent.
Denaturing gradient gel electrophoresis (DGGE) technology is widely used in the evaluation of the grand genome kind of microorganism, also never is introduced into the hybrid identification field.Denaturing gradient gel electrophoresis is a kind of method of finding point mutation or SNP site in nucleotide sequence, the difference of the Denaturing that this electrophoretic technique direct basis DNA sequence dna difference produces and carrying out is identical according to fragment length but DNA molecular mobilities in the conformation glue that contains linear denaturing agent gradient that based composition is different are different and can be separated from each other.By DGGE can separation length at 500 bp with the interior DNA fragmentation with 50 % sequence differences, and after an end of giving DNA fragmentation adds the clamping plate of high GC content, the DNA fragmentation of 1 bp even can separation sequence difference be only arranged.In the hybrid identification field, make in this way its allelotrope to be separated finding when there are differences the site nuclear gene, and then judge intuitively on glue figure whether species exist hybridization, thereby improved the agility of hybrid identification, and overcome to a certain extent in sepharose or common polyacrylamide gel electrophoresis and can not carry out allelotrope and separate the defect of differentiating.
Summary of the invention
Purpose of the present invention is for identifying hybrid, the hybrid the molecular biology aspect, form is not easily distinguishable and purebred resolution of Silurus meridionalis Chen and catfish.The present invention be take Silurus meridionalis Chen, catfish and hybrid generation thereof as research object, at the catfish CH that belonged in fish screening design
4gene fragment, as molecule marker, is applied CH
4the denaturing gradient gel electrophoresis of gene fragment (DGGE) detects, and has successfully identified Silurus meridionalis Chen, catfish and hybrid generation thereof.
CH
4gene fragment is the part of heavy chain immunoglobulin encoding gene exon, high conservative between sibling species.The present invention finds CH
4gene fragment is sequence difference between existence between Silurus meridionalis Chen and catfish is planted, by CH
4the denaturing gradient gel electrophoresis of gene fragment (DGGE) detects the evaluation for Silurus meridionalis Chen, catfish and hybrid generation thereof, has experiment fast, and evaluation is directly perceived, without the advantage of order-checking.
For achieving the above object, the present invention adopts following technical scheme:
The CH of aquaculture catfish hybrid
4molecule marker DGGE authentication method is characterized in that: the CH of the Silurus meridionalis Chen after the application pcr amplification
4the CH of gene fragment and catfish
4sequence difference between the kind existed between gene fragment, identify Silurus meridionalis Chen, catfish and hybrid generation thereof.
The CH of described aquaculture catfish hybrid
4molecule marker DGGE authentication method comprises the following steps:
A gets respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts DNA;
It is template that B be take respectively total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof, uses specific primer to carry out pcr amplification;
C DGGE analyzing and testing difference between species, detect through DGGE, occurs two gene bands of Silurus meridionalis Chen and catfish on a swimming lane simultaneously, is the cross-fertilize seed of Silurus meridionalis Chen and catfish.
Specific primer in described step B is amplification CH
4the specific primer of gene fragment, its base sequence is:
Forward primer (SEQID NO:3) 5 '-TCCCCAAGGTTTACTTGCTCGCTCC-3 ';
Reverse primer (SEQID NO:4) 5 '-CGATGGATCTGGATATGTGGCGCAC-3 ';
And the 5 ' end at forward primer has added GC clamping plate (SEQID NO:5) 5 '-CGGGGGCCGGGCCGGGCGGGCGTGGCGGGCGCTGCGGGCG-3 '.
Described CH
4the specific primer of gene fragment, the nucleotide sequence of the gene fragment of the Silurus meridionalis Chen amplified (SEQID NO:1) is:
CCAGAGACTT CAGAATCAGT GACCCTGACT TGCTATGTAA AAGACTTCTA CCCTAAGGAG GTGTATGTGT CTTGGCTTGC TGATGATAAA CCAGTGGAGG GTCTGAGCTA CTATAAGGAG AACACCACCG TTCCCCAGAA TGACAAACAA TTTTCAATGT ATAGTCAGC TGATTGTTGA TTCCCAATCT TGGAAGAAA GGCGTTGTGT ACACCTGCAG GGTTTATCAC GAGTCCATCG AGGACCCA；
Described CH
4the specific primer of gene fragment, the nucleotides sequence of the gene fragment of the catfish amplified is classified as:
CCAGAGACTT CAGAATCAGT GACCCTGACT TGCTATGTAA AAGACTTCTA CCCTAAGGAG GTGTATGTGT CTTGGCTTGC TGATGATAAA CCAGTGGAGG GTCTGAGCTA CTATAAGGAG AACACCACCA TTCCCCAGAA TGAGAAACAA TTTTCAATGT ATAGTCAGCT GATTGTTGAT TCCCAATCTT GGAAGAAAGG CGTTGTGTAC ACCTGCAGGG TTTATCACGA GTCCATCGAG GACCCA。
DGGE in described step C, the denaturing gradient gel component of using refers to the mixed solution of the coagulant liquid that denaturing agent concentration is 30% and 60%.Adopt the denaturing gradient gel component of this combination, DGGE is ideal as a result, shows as band electrophoresis in gel abundant, and in the middle of when electrophoresis finishes, pillar location approaches, the resolution of band is good etc.
DGGE in described step C carries out in 1 * TAE electrophoretic buffer of 60 ° of C of constant temperature, at first under 80V voltage, moves 20min, then under 150V voltage, moves 2.5h.Its advantage is, shortens electrophoresis time under the prerequisite that does not affect result as far as possible, raises the efficiency, because present method is separate allelotrope and do not need the long-time electrophoresis of low voltage, therefore more traditional DGGE electrophoresis duration (20-40h) has shortened a lot.
Beneficial effect of the present invention shows:
(1) the present invention is used for the hybrid identification field by PCR-DGGE first, has advantages of quicklook, and is particularly useful for the species that those lack associated sequence information.This method does not rely on sequencing result to a certain extent, as long as selected suitable molecule marker, the DGGE electrophorogram just can intuitively be showed qualification result, do not need that the PCR product is carried out to the allelotrope order-checking and analyze to contribute, or find suitable restriction enzyme site according to the sequence difference between sibling species and carry out the PCR-RFLP analysis.
(2) mixed solution of the coagulant liquid that the denaturing gradient gel component that DGGE method provided by the invention is used is 30% and 60%.Adopt the denaturing gradient gel component of this combination, the band of DGGE electrophoresis in gel is abundant, and in the middle of when electrophoresis finishes, pillar location approaches, the resolution of band is good.
(3) DGGE method provided by the invention is carried out in 1 * TAE electrophoretic buffer of 60 ° of C of constant temperature, at first under 80V voltage, moves 20min, then under 150V voltage, moves 2.5h.Its advantage is, shortens electrophoresis time under the prerequisite that does not affect experimental result as far as possible, raises the efficiency, because present method is separate allelotrope and do not need the long-time electrophoresis of low voltage, therefore more traditional DGGE electrophoresis duration (20-40h) has shortened a lot.
The accompanying drawing explanation
Fig. 1 is the CH that DGGE detects parent's Silurus meridionalis Chen and catfish
4the gene histogram.
Fig. 2 is for detecting the CH of Silurus meridionalis Chen, catfish and hybrid generation thereof
4the gene histogram.
In figure, be labeled as: D1～D3 is the Silurus meridionalis Chen sample, and A1～A3 is the catfish sample; Z1～Z3 is the quadrature filial generation, and F1～F3 is the reciprocal cross filial generation.
Embodiment
Below in conjunction with embodiment, essentiality content of the present invention is described in further detail.
The CH of aquaculture catfish hybrid
4molecule marker DGGE authentication method, with Silurus meridionalis Chen, catfish and hybridization F thereof
1in generation,, as research object, comprise the following steps:
The first step, get respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts genomic dna.
Because hybridization fry individuality is very little, so extract test kit (Tiangen company) with the ocean tissue DNA, extract the fry sample genomic dna.
With traditional phenol-chloroform extraction process (Sambrook & Russell, 2001) extract the genomic dna of parent's Silurus meridionalis Chen and catfish fin ray sample:
A draws materials: get soaked in absolute ethyl alcohol the fin ray sample a little, the scissors disinfected in alcohol shreds, and puts into 1.5 ml EP pipes.
B digestion: add 500 μ L lysis buffer (10 mmol/L Tris-Cl; 0.1 mol/L EDTA; 0.5 % SDS; PH 8.0), 5 μ L Proteinase Ks (20 mg/ml), mix, and puts into the shaking table of 55 ℃, and 60-70 rpm digestion is spent the night.
C extracting 1: take out the EP pipe, treat that solution is cooled to room temperature, add the saturated phenol of isopyknic Tris (approximately 500 μ L), slowly mix 10 min, centrifugal 10 min of 12000 rpm, slowly move to supernatant liquor in another EP pipe with 1 ml rifle head.
D extracting 2: repeat the step of extracting 1, with saturated phenol again extracting once, supernatant liquor is proceeded in another EP pipe.
E extracting 3: add the phenol-chloroform (1:1) of 400 μ L, slowly mix 5 min, centrifugal 10 min of 12000 rpm, proceed to supernatant liquor in another EP pipe gently.
F extracting 4: add the chloroform of 300 μ L, slowly mix 5 min, centrifugal 10 min of 12000 rpm, supernatant liquor is transferred in another EP pipe.
G precipitation: add the freezing dehydrated alcohol of pipe internal volume two volumes, in-20 ℃ of precipitation 20 min.Centrifugal 1 min of 8000 rpm.
H rinsing 1: add the freezing alcohol of 150 μ L 75 %, after rinsing, centrifugal 1 min of 8000 rpm.
I rinsing 2: with rinsing 1 step.
J rinsing 3: with the freezing dehydrated alcohol post rinse of 150 μ L once, after centrifugal 1 min of 8000 rpm, discard supernatant, natural air drying.
K dissolves: add 50 μ L MilliQ ultrapure waters, the DNA of dissolution precipitation.
Second step, the total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof of take respectively is template, at CH
4carry out pcr amplification under the guiding of gene fragment specific primer;
(1) pcr amplification Silurus meridionalis Chen, catfish and hybridization F thereof
1the heavy chain conserved regions DNA exon sequence CH of the immunoglobulin M in generation
4gene fragment.CH
4primer adopts CH4-F(5 '-TCCCCAAGGTTTACTTGCTCGCTCC-3 ') and CH4-R (5 '-CGATGGATCTGGATATGTGGCGCAC-3 ').For obtaining DGGE separating effect preferably, added GC clamping plate (5 '-CGGGGGCCGGGCCGGGCGGGCGTGGCGGGCGCTGCGGGCG-3 ') at the 5 ' end of primer CH4-F, above primer is synthetic by Invitrogen company.
CH4-F and CH4-R are amplification CH
4the primer pair of purpose fragment, F wherein represents forward primer (or upstream primer), R represents reverse primer (or downstream primer).
(2) pcr amplification
Each component is mixed to (reaction system is 50 μ L) in the following order in the PCR of 0.2 ml pipe:
1. ultrapure water 36 μ L
2. Taq enzyme 10 * buffer 5 μ L
③MgCl
2（1.5 mM） 4 μL
④dNTPs（2.5 mM） 1.5 μL
5. primer GC-CH4-F(25 pM) 1 μ L
6. primer CH4-R(25 pM) 1 μ L
7. template DNA (approximately 100 ng) 1 μ L
8. Taq archaeal dna polymerase (2.5 U μ L
-1) 0.5 μ L
Through the PCR reaction amplification step of optimizing, be:
1. 94 ° of C denaturation 4 min
2. 94 ° of C sex change 40 s
3. 60 ° of C, 30 s that anneal
4. 72 ° of C extend 30 s
5. repeating step is 2.-4. 32 times
6. 72 ° of C fully extend 10 min
(3) PCR product purification and order-checking
Separate PCR product fragment with the agarose gel electrophoresis of 1 %, and put into the EP pipe of 1.5 mL after cutting purpose band under ultraviolet lamp, reclaim and adopt DNA Agarose Extraction kit (Omega company) to reclaim test kit.Reclaim product after electrophoresis detection, send biotech firm's order-checking.
Its sequence information of sequence verification, find CH
4sequence difference between gene fragment exists and plants, for carrying out the DGGE method hybrid identification in later stage.
The 3rd step, DGGE analyzing and testing difference between species.
DGGE is at the Dcode of Bio-Rad company
tMin general sudden change detection system, carry out, the polyacrylamide concentration of selecting is 8 %, and wherein denaturing agent (urea and deionized formamide) gradient is through being optimized for 30%～60%.Compound method is: at first according to table 1, prepare respectively low denaturing agent concentration (30%) coagulant liquid 15mL and high sex change agent concentration (60%) coagulant liquid 15 mL, and respectively add 150 μ L APS and 8 μ L TEMED before encapsulating; Then use the gradients setup device that 2 kinds of coagulant liquids are injected simultaneously in the sandwich glass plywood be vertically placed in horizontal table top gel groove, wherein high sex change agent concentration coagulant liquid injection speed is very fast, and encapsulating is plugged the point sample comb after finishing; Finally, due to action of gravity, form the polyacrylamide gel that the denaturing agent gradient is 30%～60% from top to bottom.
In gel, by the 1 * TAE electrophoretic buffer preheating in the DGGE instrument, when temperature approaches 60 ° of C, by accompanying the sandwich clamping plate that solidify gel, put into electrophoresis chamber, during loading, each swimming lane adds approximately 25 μ L of PCR product.DGGE carries out in 1 * TAE electrophoretic buffer of 60 ° of C of constant temperature, at first under 80V voltage, moves 20min, then under 150V voltage, moves 2.5h.After DGGE finishes, by polyacrylamide gel lucifuge dyeing 40min in the 1 * TAE electrophoretic buffer that adds 25 Gold View, then in the UV gel imaging system, detect and electrophoresis result is analyzed.The mixed solution that DGGE denaturing gradient gel component is the denatured gradient denaturing agent that is 30% and 60%, each component sees the following form:
Table 1 DGGE denaturing gradient gel component
| Denaturing agent concentration | The acrylamide mother liquor | 50×TAE | Urea | Deionized formamide | MilliQ |
| 30% | 3 mL | 0.3 mL | 1.89 g | 1.8 mL | Benefit to 15 mL |
| 60% | 3 mL | 0.3 mL | 3.78 g | 3.6 mL | Benefit to 15 mL |
Agar gel preparation method of the present invention is as follows:
The preparation method of the sepharose of 1 %
Weigh in the balance and get 0.3 g agarose, pour in 1 * TAE damping fluid of 30 ml, shake up to be placed in microwave oven and be heated to dissolve fully, take out after slightly cool and (prevent that high temperature from making the EB volatilization), add EB 2 μ L.Glue plate (Bio-Rad company) is positioned over to level attitude, then inserts the sample comb.Pour the sepharose of preparation before into glue plate inside groove.Take out comb cooling half an hour after gel solidifies, gel is put into to electrophoresis chamber (Bio-Rad company) and added 1 * TAE electrophoretic buffer to make its submergence gel.The point sample damping fluid (tetrabromophenol sulfonphthalein) of 1/6 volume on selecting on point template.
1.5 the preparation method of the sepharose of %
Weigh in the balance and get 0.3 g agarose, pour in 1 * TAE damping fluid of 20 ml, shake up to be placed in microwave oven and be heated to dissolve fully, take out after slightly cool and (prevent that high temperature from making the EB volatilization), add EB 2 μ L.Glue plate (Bio-Rad company) is positioned over to level attitude, then inserts the sample comb.Pour the sepharose of preparation before into glue plate inside groove.Take out comb cooling half an hour after gel solidifies, gel is put into to electrophoresis chamber (Bio-Rad company) and added 1 * TAE electrophoretic buffer to make its submergence gel.The point sample damping fluid (tetrabromophenol sulfonphthalein) of 1/6 volume on selecting on point template.
The key instrument that the present invention uses is as follows:
PCR instrument: Bio-Rad iCycler and MJ100
Whizzer: Eppendorf Centrifuge 5415D
Voltage stabilization and current stabilization electrophoresis apparatus: BIO-RAD power PAC 300
Gel imaging system: Alphalmage Multimage Light Cabinet
Sudden change detection system: Bio-Rad Dcode
tMgeneral sudden change detection system
Electronic balance, incubator, steam sterilizer, shaking table and the experimental installation that some are commonly used.
Above-described embodiment result shows: the CH of Silurus meridionalis Chen and catfish
4gene fragment all has no report in GenBank, and as shown in the gene histogram of Fig. 1 and Fig. 2, two gene bands of Silurus meridionalis Chen and catfish appear in the hybrid generation catfish simultaneously, consistent with 2 kinds of parental arrays respectively through sequence verification.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
sequence table
<110 > Tongwei Co., Ltd.
<120>CH of aquaculture catfish hybrid
4molecule marker DGGE authentication method
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 246
<212> DNA
<213 > Silurus meridionalis Chen (Silurus meridionalis)
<400> 1
ccagagactt cagaatcagt gaccctgact tgctatgtaa aagacttcta ccctaaggag 60
gtgtatgtgt cttggcttgc tgatgataaa ccagtggagg gtctgagcta ctataaggag 120
aacaccaccg ttccccagaa tgacaaacaa ttttcaatgt atagtcagct gattgttgat 180
tcccaatctt ggaagaaagg cgttgtgtac acctgcaggg tttatcacga gtccatcgag 240
gaccca 246
<210> 2
<211> 246
<212> DNA
<213 > catfish (Silurus asotus)
<400> 2
ccagagactt cagaatcagt gaccctgact tgctatgtaa aagacttcta ccctaaggag 60
gtgtatgtgt cttggcttgc tgatgataaa ccagtggagg gtctgagcta ctataaggag 120
aacaccacca ttccccagaa tgagaaacaa ttttcaatgt atagtcagct gattgttgat 180
tcccaatctt ggaagaaagg cgttgtgtac acctgcaggg tttatcacga gtccatcgag 240
gaccca 246
<210> 3
<211> 25
<212> DNA
<213 > artificial sequence
<400> 3
tccccaaggt ttacttgctc gctcc 25
<210> 4
<211> 25
<212> DNA
<213 > artificial sequence
<400> 4
cgatggatct ggatatgtgg cgcac 25
<210> 5
<211> 40
<212> DNA
<213 > artificial sequence
<400> 5
cgggggccgg gccgggcggg cgtggcgggc gctgcgggcg 40
Claims (6)
1. the CH of aquaculture catfish hybrid
4molecule marker DGGE authentication method is characterized in that: the CH of the Silurus meridionalis Chen after the application pcr amplification
4the CH of gene fragment and catfish
4between the kind existed between gene fragment, sequence difference is identified Silurus meridionalis Chen, catfish and hybrid generation thereof;
Described CH
4the specific primer of gene fragment, base sequence is:
Forward primer: 5 '-TCCCCAAGGTTTACTTGCTCGCTCC-3 ';
Reverse primer: 5 '-CGATGGATCTGGATATGTGGCGCAC-3 ';
And the 5 ' end at forward primer has added the GC clamping plate:
5’- CGGGGGCCGGGCCGGGCGGGCGTGGCGGGCGCTGCGGGCG-3’。
2. the CH of aquaculture catfish hybrid according to claim 1
4molecule marker DGGE authentication method is characterized in that: comprise the following steps:
A gets respectively Silurus meridionalis Chen, catfish and hybridization sample thereof, extracts genomic dna;
It is template that B be take respectively total DNA of Silurus meridionalis Chen, catfish and hybridization sample thereof, uses specific primer to carry out pcr amplification;
C DGGE analyzing and testing difference between species, detect through DGGE, occurs two gene bands of Silurus meridionalis Chen and catfish on a swimming lane simultaneously, is the cross-fertilize seed of Silurus meridionalis Chen and catfish.
3. according to the CH of claim 1 or 2 described aquaculture catfish hybrids
4molecule marker DGGE authentication method is characterized in that: described CH
4the specific primer of gene fragment, the nucleotides sequence of the gene fragment of the Silurus meridionalis Chen amplified is classified as:
CCAGAGACTT CAGAATCAGT GACCCTGACT TGCTATGTAA AAGACTTCTA CCCTAAGGAG GTGTATGTGT CTTGGCTTGC TGATGATAAA CCAGTGGAGG GTCTGAGCTA CTATAAGGAG AACACCACCG TTCCCCAGAA TGACAAACAA TTTTCAATGT ATAGTCAGC TGATTGTTGA TTCCCAATCT TGGAAGAAA GGCGTTGTGT ACACCTGCAG GGTTTATCAC GAGTCCATCG AGGACCCA。
4. according to the CH of claim 1 or 2 described aquaculture catfish hybrids
4molecule marker DGGE authentication method is characterized in that: described CH
4the specific primer of gene fragment, the nucleotides sequence of the gene fragment of the catfish amplified is classified as:
CCAGAGACTT CAGAATCAGT GACCCTGACT TGCTATGTAA AAGACTTCTA CCCTAAGGAG GTGTATGTGT CTTGGCTTGC TGATGATAAA CCAGTGGAGG GTCTGAGCTA CTATAAGGAG AACACCACCA TTCCCCAGAA TGAGAAACAA TTTTCAATGT ATAGTCAGCT GATTGTTGAT TCCCAATCTT GGAAGAAAGG CGTTGTGTAC ACCTGCAGGG TTTATCACGA GTCCATCGAG GACCCA。
5. the CH of aquaculture catfish hybrid according to claim 2
4molecule marker DGGE authentication method is characterized in that: the DGGE in described step C, the denaturing gradient gel component of using refers to the mixed solution of the denaturing agent that denatured gradient is 30% and 60%.
6. the CH of aquaculture catfish hybrid according to claim 2
4molecule marker DGGE authentication method is characterized in that: the DGGE in described step C, in 1 * TAE electrophoretic buffer of 60 ° of C of constant temperature, carry out, and at first under 80V voltage, move 20min, then under 150V voltage, move 2.5h.
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|---|---|---|---|---|
| TWI290585B (en) * | 2004-03-18 | 2007-12-01 | Jau-Der Chen | RAPD markers |
| CN102080124A (en) * | 2009-11-30 | 2011-06-01 | 青岛生物能源与过程研究所 | Method for preparing denaturing gradient gel electrophoresis (DGGE) Marker |
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| Publication number | Priority date | Publication date | Assignee | Title |
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