CN101805785B - Molecular biological method for fast identifying sex of bighead seedlings - Google Patents
Molecular biological method for fast identifying sex of bighead seedlings Download PDFInfo
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- CN101805785B CN101805785B CN2009102078029A CN200910207802A CN101805785B CN 101805785 B CN101805785 B CN 101805785B CN 2009102078029 A CN2009102078029 A CN 2009102078029A CN 200910207802 A CN200910207802 A CN 200910207802A CN 101805785 B CN101805785 B CN 101805785B
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Abstract
The invention provides a molecular biological method for fast identifying the sex of bighead seedlings, which relates to molecular biology. The invention has the goal of providing the molecular biological method capable of fast identifying the sex of the bighead seedlings. The invention has the technical scheme that the method comprises the following steps: carrying out RCP amplification through using bighead individual genome DNA as a template; carrying out gel electrophoresis separation by amplification products through 1.5 percent agarose; carrying out dyeing by ethidium bromide; observing and taking photos for recording under a gel imaging system; and using a 100 bpDNAladder as a molecular weight marker. Individuals with target belts with the size of 518 bp are female individuals, and individuals without belts are male individuals. The invention is used for identifying the sex of the bighead seedlings.
Description
Technical field:
The present invention relates to a kind of molecular biology, particularly a kind of molecular biology method of quick discriminating bighead seedling sex.
Background technology:
There is abundant fish stock in China, and wherein many fish exist gender difference on growth velocity, and therefore, control has very high theory directive significance for fish sex to carry out the research of these fish sexs decision genes involveds and sex specific mark.Flathead (Aristichthys mobilis) belongs to Cypriniformes, Cyprinidae, and the silver carp subfamily, flathead belongs to.Commonly known as: bighead carp, variegated carp, black carp, yellow carp, bonito, Yong fish, bullhead.The fish name, bighead has another name called " bighead ", " variegated carp ", " black silver carp ", and head is very big, lives in the fresh water.Flathead growth rapidly, 3 age fish can reach the 4-5 kilogram, maximum individuality can reach 40 kilograms, natural output is very high.Disease is few, is prone to raise, and be one of " four large Chinese carps " in China's freshwater aquaculture industry, be the important economic fish of China.Bighead is female to grow soon, and growth rapidly.At present artificial gynogenesis and the hormonal cultured population that combines to create gynoecy that reverses is the main direction of bighead breeding.But, can't judge its sex character through mode of appearance period, so how from seedling colony, simply identify the prerequisite that genetic sex becomes bighead success seed selection fast seedling because bighead sexual maturity for the first time needs 4～5 years at all.
The RAPD technology occurred in recent years, can seek a kind of effective ways of molecule marker fast.In the genome of some animal, seek the sex mark with it and have successfully report.As in the rainbow trout genome, having found two male special molecule markers, can be used as the probe of identification male and female.Kovacs etc. 2000 scan female, the male gene pool of African catfish (Clarias gariepinus) with RAPD, find the relevant RAPD mark of two male sexs.But still there is not the report that relevant molecule marker is differentiated bighead seedling sex method at present both at home and abroad.
Summary of the invention:
The purpose of this invention is to provide a kind of to the female specific primer sequence of bighead.
Another object of the present invention provides a kind of molecular biology method that can differentiate bighead seedling sex fast.Technical scheme of the present invention is that the female specific primer sequence of bighead is characterized in that: its specific primer sequence is the upper reaches 5 ' CGCATACTCAAACATCACATAC3 '; Downstream 5 ' TCACTCACTGTCTGCTCACTG 3 '.Utilizing the special primer sequence of design is that template is carried out pcr amplification with bighead genes of individuals group DNA, and reaction system is 25ul, and reaction consists of the TaqDNA polysaccharase of 1U; The dNTP of 2ul, 100～200ng genomic dna wherein contains each 0.1 μ mol/L of female special upstream and downstream primer; Carrying out pcr amplification by following loop parameter on the PCR appearance: behind 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec; 72 ℃ are extended 60sec totally 30 circulations, and last 72 ℃ are extended 10min, and amplified production separates with 1.5% agarose gel electrophoresis; Ethidium bromide staining is observed and Taking Pictures recording under gel imaging system, and 100bpDNAladder makes molecular weight marker; The appearance size is a female individuals for 518bp purpose band, does not have the male that is of band.
The present invention and prior art relatively have the remarkable advantage that can quick and precisely identify sex in the bighead seedling phase.
Description of drawings:
Fig. 1 utilizes the checking electrophoresis result figure of specific PCR method to the sex specific fragment.In male and female 10 individuals, the crt gene band is high-visible in all individualities, proves that the pcr amplification program is no problem.1,2,3,4,5 do not amplify any band except that house-keeping gene, be male.And 6,7,8,9,10 swimming lanes all occurred the size be the amplified band of 518bp, be female individuals; M is the dna molecular amount standard of 100bp gradient.Arrow 1 indication is that size is the male specific band of 518bp; Arrow 2 indications are the crt gene band.
Fig. 2 is for utilizing female special primer to bighead seedling sex identification electrophoresis result figure.24 bighead seedling of picked at random individuality carries out sex identification.Wherein 1,2,3,4,5,6,11,12,13,14,16,18,19,21,22,23, the band of a 518bp size is arranged on the electrophoresis band of 240 seven individuals, be female individuals; 7,8,9,10,15,17,20 7 individuals do not have band on the 518bp position, be female individuals.M is the dna molecular amount standard of 100bp gradient.Arrow 1 indication is that size is the male specific band of 518bp; Arrow 2 indications are the crt gene band.
Embodiment:
1. the checking of the acquisition of female special primer and sex-specific
After sexual gland is dissected and identified sex, extract male and female genes of individuals group DNA, utilize 220 random primers to carry out the RAPD-PCR amplification, when using random primer S2120, in female individuals, amplify stable bands of a spectrum, this band line does not exist in male.This band is the female specific band of bighead; Obtained the female specific fragment of one section about 518bp through cloning and checking order, called after Amff (its dna sequence dna such as table 1), this fragment belongs to non-coding sequence; This sequence is carried out sequence alignment on Genebank, do not find homologous sequence with it.Order-checking back a pair of female special primer Amff1 of design and Amff2; Through the GAPDH crt gene of NCBI search bighead, design a pair of can be as the primer (sequence is 5 ' GCCTCCTGCACCACCAACTG3 ' and 5 ' CGGAAGGCCATGCCGGTCAG3 ') of PCR internal contrast.Special primer that utilization designs and crt gene primer carry out the specific PCR amplification in the individuality of 10 known sexes; The PCR reaction conditions is: behind 95 ℃ of preparatory sex change 5min, and 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec; 72 ℃ are extended 60sec totally 30 circulations, and last 72 ℃ are extended 10min.4 ℃ of preservations of amplified production.The result is as shown in Figure 1, and in male and female 10 individuals, the crt gene band is high-visible in all individualities, proves that the pcr amplification program is no problem.1,2,3,4,5 are male, except that house-keeping gene, do not amplify any band.And 6,7,8,9,10 be female individuals, and swimming lane size all occurred and has been the amplified band of 518bp; This shows that designed primer of the present invention and method can be used in the evaluation of bighead sex.
2. the discriminating of bighead seedling sex
To test fish vein haemospasia a little (being no more than 0.5ml), and add the antithrombotics ACD of twice at least, the centrifugal 10min of 4000rpm removes supernatant; Add 5ml lysate and 20 μ g RNA enzymes, 37 incubation 3h; Add Proteinase K to final concentration 100 μ g/ μ l, 50 ℃ are spent the night.Through phenol → phenol chloroform isoamyl alcohol → chloroform isoamyl alcohol extracting successively, absolute ethyl alcohol deposition, 70% washing with alcohol.After the drying, the bighead genomic dna is dissolved in TE or the aseptic double-distilled water, respectively numbering.Utilize the female special primer of design that 24 unknown sex chub fry individualities are carried out pcr amplification, reaction system is 25 μ l, 1U Taq enzyme, 2 μ l dNTP, 100～200ng genomic dna, each 0.1 μ mol/L of female special upstream and downstream primer.After loop parameter is 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30sec, 60 ℃ of annealing 30sec, 72 ℃ are extended 60sec totally 30 circulations, and last 72 ℃ are extended 10min.Amplified production separates with 1.5% agarose gel electrophoresis, and ethidium bromide staining is observed and Taking Pictures recording under gel imaging system.As Fig. 2 find 17 occur size for 518bp purpose band be female individuals, other 7 males that are that do not have band.
Below all show the discriminating that special primer that the present invention designs and method can be used in chub fry male and female sex.
The female specific fragment Amff of table 1. bighead sequence
CGCATACTCAAACATCACATACAAAAGCAACAAAAAAAAGGCAATGCACCGAGA
AGCCACAAAACAGTGTAAAGAAATGAAAGCGTGATGCGTCATGTAGTATGAACA
GACTTTAAAGTCATATTCACAGCATATCCACATATTTGGGATGACATAAATACAT
GCATTAGGCTTTTTTTAAATGAAATAATAATAACTCTGAATTCACACTAAGCCTG
AAATGTCACTGCAATATTTTTATTAAAAATAAATAATTATATGTATTATTGACTAT
TTAAATTTAAATTATGCATTGGTTTTAGTGTGAATAATTTAAGTGTGAAAAATTA
CAGTGAAAATAAGACATTCTATAGTTTCAGATTTGGTGTGAACAGGCATTAACTG
AAGCAGAGCGAAGATGAAGCCATCTGTTACAGCAGATGAGTGTACAATTTGAAA
ACAATGACAGTTTATGGAAGATTTGCTTTTGGGAAATGACTGCATAAAGCGGCAC
AAAGCAGTGAGCAGACAGTGAGTGA
The female specific primer sequence of table 2. bighead
A kind of molecular biology method .ST25 of quick discriminating bighead seedling sex
SEQUENCE?LISTING
< 110>He'nan Normal University
< 120>a kind of molecular biology method of quick discriminating bighead seedling sex
<140>200910207802.9
<141>2009-04-23
<160>1
<170>PatentIn?version?3.3
<210>1
<211>518
<212>DNA
< 213>bighead
<400>1
cgcatactca?aacatcacat?acaaaagcaa?caaaaaaaag?gcaatgcacc?gagaagccac 60
aaaacagtgt?aaagaaatga?aagcgtgatg?cgtcatgtag?tatgaacaga?ctttaaagtc 120
atattcacag?catatccaca?tatttgggat?gacataaata?catgcattag?gcttttttta 180
aatgaaataa?taataactct?gaattcacac?taagcctgaa?atgtcactgc?aatattttta 240
ttaaaaataa?ataattatat?gtattattga?ctatttaaat?ttaaattatg?cattggtttt 300
agtgtgaata?atttaagtgt?gaaaaattac?agtgaaaata?agacattcta?tagtttcaga 360
tttggtgtga?acaggcatta?actgaagcag?agcgaagatg?aagccatctg?ttacagcaga 420
tgagtgtaca?atttgaaaac?aatgacagtt?tatggaagat?ttgcttttgg?gaaatgactg 480
cataaagcgg?cacaaagcag?tgagcagaca?gtgagtga 518
Claims (2)
1. the female specific primer sequence of bighead, it is characterized in that: its specific primer sequence does
The upper reaches: 5 ' CGCATACTCAAACATCACATAC3 ';
Downstream: 5 ' TCACTCACTGTCTGCTCACTG3 '.
2. a molecular biology method of differentiating bighead seedling sex fast is characterized in that: the special primer sequence A mff1:5 ' CGCATACTCAAACATCACATAC 3 ' that utilizes design; Amff2:5 ' TCACTCACTGTCTGCTCACTG 3 ' is that template is carried out pcr amplification with bighead genes of individuals group DNA, and reaction system is 25ul; Reaction consists of the TaqDNA polysaccharase of 1U, the dNTP of 2ul, 100～200ng genomic dna; Wherein contain special primer Amff1, each 0.1 μ mol/L of Amff2, carrying out pcr amplification by following loop parameter on the PCR appearance: behind 95 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30sec; 60 ℃ of annealing 30sec, 72 ℃ are extended 60sec totally 30 circulations, and last 72 ℃ are extended 10min; Amplified production separates with 1.5% agarose gel electrophoresis, and ethidium bromide staining is observed and Taking Pictures recording under gel imaging system; 100bpDNAladder makes molecular weight marker, and the appearance size is a female individuals for 518bp purpose band, does not have the male that is of band.
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| CN2009102078029A CN101805785B (en) | 2009-04-23 | 2009-10-30 | Molecular biological method for fast identifying sex of bighead seedlings |
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| CN112553347B (en) * | 2020-12-28 | 2022-04-15 | 中国科学院水生生物研究所 | Development method of bighead carp sex identification molecular marker by taking Gapdh gene as reference |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814793A (en) * | 2005-12-15 | 2006-08-09 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1814793A (en) * | 2005-12-15 | 2006-08-09 | 中国水产科学研究院黄海水产研究所 | Cynoglossus semilaevis gunther specific molecular label and genetic sex identifying method |
Non-Patent Citations (5)
| Title |
|---|
| 常重杰等.鱼类的性别决定和性染色体.《淡水渔业》.2002,第32卷(第2期), * |
| 张锡元等.白鲢和鳙鱼的随机扩增多态DNA分析.《生物化学与生物物理进展》.1999,第26卷(第5期), * |
| 文爱韵等.鱼类性别决定与分化相关基因研究进展.《海洋科学》.2008,第32卷(第1期), * |
| 李静等.鱼类性别相关基因及性别特异标记的研究进展.《海洋水产研究》.2006,第27卷(第4期), * |
| 王忠卫等.快速建立鲢、鳙纯系的初步研究.《自然科学进展》.2003,第13卷(第10期), * |
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