CN106591298A - Amplification primer and amplification method of complete mitochondrial genome sequence of nibea miichthioides chu - Google Patents

Amplification primer and amplification method of complete mitochondrial genome sequence of nibea miichthioides chu Download PDF

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CN106591298A
CN106591298A CN201611027807.XA CN201611027807A CN106591298A CN 106591298 A CN106591298 A CN 106591298A CN 201611027807 A CN201611027807 A CN 201611027807A CN 106591298 A CN106591298 A CN 106591298A
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primer
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nucleotide sequence
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胡则辉
柴学军
王跃斌
胡成硕
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Zhejiang Marine Fisheries Research Institute
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Abstract

The invention discloses an amplification primer and an amplification method of a complete mitochondrial genome sequence of nibea miichthioides chu. The amplification primer comprises 4 klenow fragment amplification primer pairs and 19 genome walking primers. According to the amplification primer and the amplification method provided by the invention, the complete mitochondrial genome sequence of nibea miichthioides chu, which is obtained by conducting PCR amplification on genome of the nibea miichthioides chu, can provide a material base for conservation genetics and evolutionary genetics of the nibea miichthioides chu and for marker-assisted selection of high-quality breeding varieties.

Description

A kind of amplimer of catfish shape Nibea albiflora mitochondrion whole genome sequence and its amplification Method
Technical field
The present invention relates to gene engineering technology field, more particularly to a kind of catfish shape Nibea albiflora mitochondrion whole genome sequence Amplimer and its amplification method.
Background technology
Catfish shape Nibea albiflora (Nibea miichthioides Chu, Lo＆Wu) also known as catfish perch, cunner, be under the jurisdiction of Perciformess, Sciaenidae, Nibea, are distributed mainly on China East Sea middle and south and the South Sea, are that lower floor is large-scale carnivorous in offshore water warm Fish.Because its premunition is strong, growth is fast, it has also become Fujian, Zhejiang, the coastal excellent cage culture kind in Guangdong.
Catfish shape Nibea albiflora is large edible Fish, and maximum individuality can reach 1.5 meters.Due to cruel fishing for a long time it is indiscriminate Catch and habitat destruction, cause catfish shape Nibea albiflora resource to fail increasingly, and the catfish shape Nibea albiflora of artificial cultivation there is also germplasm and move back Change, disease takes place frequently the problems such as declining with culture benefit.Therefore, be protect this excellent sea-farming kind, systematic research with Rational exploitation seems particularly important.It is with regard to the phylogeny of catfish shape Nibea albiflora, Zhu Yuanding etc. (1963) and safe into celebrating (1987) 4 kinds of Nibea albiflora, Nibea coibor, Nibea diacanthus and catfish shape Nibea albiflora are attributed to into Nibea.Hereafter Talwar (1995) think that catfish shape Nibea albiflora should be Xiamen silver body(Argyrosomus amoyensis).Due to for Sciaenidae point The morphological criteria of class is inconsistent using form, counting and anatomical features (such as fish glue and otolith), and the classification of catfish shape Nibea albiflora is returned Category problem yet suffers from larger dispute, especially catfish shape Nibea albiflora and Japanese croaker because profile it is extremely similar, not only easily by edge Extra large fisherman is considered as Fish of the same race, and some have speculated that both Fish are because the subspecies produced by geographical isolation.
Mitochondrial genome is biological important hereditary material, and wherein metazoa mitochondrial DNA is that typical ring-type is double Chain molecular structure, length encodes 37 genes mostly between 14-18kb, including 13 protein genes, 22 tRNA and 12S rRNA and 16S rRNA (Wolstenholme DR, 1992).Mitochondrial DNA is extranuclear inheritance material, with molecular weight The features such as little, simple structure, maternal inheritance, become important point in the research of bio-diversity, population genetics and phyletic evolution Sub- labelling (Miya M and Takeshima H, 2003；Lavou S and Miya M, 2007).At present, mitochondrial gene has been As Identification of Species and the important molecular markers of Phylogenetic Analysis, and mitochondrion ND2,16SrRNA and Cyt b gene, D- Loop sequences have been applied to (the Su Tian phoenix 2007 of shellfish in aquatic animal；The Wang Gui tinkling of pieces of jade 2009), shell-fish (Wang Chenghui, 2008), Fish (Li Lin, 2011；Jiang Zongliang, 2011) species genetic diversity and Phylogenetic Relationships are analyzed.It is also not right so far The mitochondrial genome sequence of the catfish shape Nibea albiflora report related to Genetic conservation.
The content of the invention
It is an object of the invention to provide a kind of amplimer of catfish shape Nibea albiflora mitochondrion whole genome sequence, by 4 Large fragment amplification primers pair and 19 genomic walking primer pair catfish shape Nibea albiflora genomes are entered by performing PCR amplification and obtain catfish Shape Nibea albiflora mitochondrion whole genome sequence, can be conservative genetics, evolutionary genetics and the excellent cultivation product of catfish shape Nibea albiflora The molecular marker assisted selection planted provides material base.
Present invention also offers the amplification method of catfish shape Nibea albiflora mitochondrion whole genome sequence.
The technical solution adopted for the present invention to solve the technical problems is：
A kind of amplimer of catfish shape Nibea albiflora mitochondrion whole genome sequence, including 4 pairs of large fragment amplification specificitys Primer pair, 4 pairs of large fragment amplification specific primers are to specific as follows：
The forward primer of the 1st pair of primer pair is：Nucleotide sequence (5'- shown in SEQ ID NO.1 AGCCCTAAACATCGACAACAAC-3'), downstream primer is：Nucleotide sequence (5'- shown in SEQ ID NO.2 TAGGCGCAGGTAAAAGTATAGGCTAA-3')；
The forward primer of the 2nd pair of primer pair is：Nucleotide sequence (5'- shown in SEQ ID NO.3 TGCATTAGCCACTTCCTGAACCAA-3'), downstream primer is：Nucleotide sequence (5'- shown in SEQ ID NO.4 ACCTATTCGGCTCACTCTAGGCCTC-3')；
The forward primer of the 3rd pair of primer pair is：Nucleotide sequence (5'- shown in SEQ ID NO.5 ATTCTGGCTCCCTCAAATGACC-3'), downstream primer is：Nucleotide sequence (5'- shown in SEQ ID NO.6 CCTTATTTTGCTGCTTTGGGCTTG-3')；
The forward primer of the 4th pair of primer pair is：Nucleotide sequence (5'- shown in SEQ ID NO.7 TACCCAAGAAGTCTCATATCACCCC-3'), downstream primer is：Nucleotide sequence (5'- shown in SEQ ID NO.8 ACGACTTGCCTCCCCTTTACTGTG-3')；
Preferably, the amplimer of catfish shape Nibea albiflora mitochondrion whole genome sequence also includes that 19 genomic walkings are used Specific primer, 19 genomic walkings are specific as follows with specific primer：
1st article primer is：Nucleotide sequence (5'-GCAAGGGAACGCTGAAAGAG-3') shown in SEQ ID NO.9；
2nd article primer is：Nucleotide sequence (5'-ACCCCCCACCGAACCTTAAC-3') shown in SEQ ID NO.10；
3rd article primer is：Nucleotide sequence (5'-CGATCAACGAACCGAGTTAC-3') shown in SEQ ID NO.11；
4th article primer is：Nucleotide sequence (5'-ACACTCTGAGCACCTATACC-3') shown in SEQ ID NO.12；
5th article primer is：Nucleotide sequence (5'-CCGATACGACCAACTTATGC-3') shown in SEQ ID NO.13；
6th primer is：Nucleotide sequence (5'-GCGCTAGCCTACTTCTTCAG-3') shown in SEQ ID NO.14；
7th article primer is：Nucleotide sequence (5'-CTTAATTACAGCGGTCCTCC-3') shown in SEQ ID NO.15；
8th article primer is：Nucleotide sequence (5'-CACTTCCACTATGTCCTCTC-3') shown in SEQ ID NO.16；
9th article primer is：Nucleotide sequence (5'-CCGCGTGTCTTGCACCATTA-3') shown in SEQ ID NO.17；
10th article primer is：Nucleotide sequence (5'-GGCCAACCATAGCTTCATAC- shown in SEQ ID NO.18 3')；
Sub_clause 11 primer is：Nucleotide sequence (5'-TCAACCTAGGCCTTGCAGTC- shown in SEQ ID NO.19 3')；
12nd article primer is：Nucleotide sequence (5'-TCCTGCTAAGTGACTCTGAC- shown in SEQ ID NO.20 3')；
13rd article primer is：Nucleotide sequence (5'-TAATTCTTGCTGCCGTCCTC- shown in SEQ ID NO.21 3')；
14th article primer is：Nucleotide sequence (5'-CGAGAACACCTCCTGGTAAC- shown in SEQ ID NO.22 3')；
15th article primer is：Nucleotide sequence (5'-TCCTTAACGAAGGCGCAGAG- shown in SEQ ID NO.23 3')；
16th article primer is：Nucleotide sequence (5'-AAACAGCCCTCACCCTCTGC- shown in SEQ ID NO.24 3')；
17th article primer is：Nucleotide sequence (5'-AACCTCCTATCCGCCGTACC- shown in SEQ ID NO.25 3')；
18th article primer is：Nucleotide sequence (5'-CAGAGCGTAGCACCGGTCTT- shown in SEQ ID NO.26 3')；
19th article primer is：Nucleotide sequence (5'-CCCATGCCGAGCGTTCTTTC- shown in SEQ ID NO.27 3')。
A kind of amplification method of catfish shape Nibea albiflora mitochondrion whole genome sequence, with the catfish shape Nibea albiflora genome for extracting DNA is template, first with 4 pairs of large fragment amplification primers to carrying out 4 large fragment product amplifications, then by genomic walking Method, respectively using expand 4 large fragments as template use 19 genomic walking primers enter performing PCR amplification, PCR produce The sequencing of thing Jing sepharose electrophoresis detection Hou Song biotech firms.
The invention has the beneficial effects as follows：
1st, the present invention devises 4 pairs of large fragment amplification specific primers for the catfish shape Nibea albiflora of Perciformess Sciaenidae Pair and 19 genomic walking specific primers can enter performing PCR amplification to catfish shape Nibea albiflora mitochondrial DNA, and obtain line grain Body full-length gene.
2nd, the present invention obtains first catfish shape Nibea albiflora mitochondrial genome full length sequence, and Jing analyses draw：Sequence 16490bp, including 13 protein coding genes, 22 tRNA genes, 2 rRNA genes.
3rd, 4 pairs of large fragment amplification specific primers pair of the invention and 19 genomic walking specific primers can Performing PCR amplification is entered with the catfish shape Nibea albiflora genomic DNA to different groups.
4th, the catfish shape Nibea albiflora mitochondrion whole genome sequence for obtaining, can be the conservative genetics of catfish shape Nibea albiflora, evolve The molecular marker assisted selection of hereditism and excellent breed variety provides material base.
Specific embodiment
Below by specific embodiment, technical scheme is described in further detail.
In the present invention, if not refering in particular to, the raw material for being adopted and equipment etc. are commercially available or commonly used in the art. Method in following embodiments, if no special instructions, is the conventional method of this area.
Embodiment：
With extract catfish shape Nibea albiflora genomic DNA as template, first with 4 pairs of large fragment amplification specific primers to entering 4 large fragment product amplifications of row, the then method of genomic walking uses 19 using 4 large fragments expanding as template respectively Bar genomic walking primer enters performing PCR amplification, and the sequencing of the sepharose electrophoresis of PCR primer Jing 1.0% detection Hou Song biotech firms is right Sequencing result is spliced using biosoftware to sequencing result, is comprised the following steps that：
1st, 5 × TBE Buffer are prepared
Weigh tromethane 54g, Na2EDTA·2H2O 3.72g, boric acid 27.5g is in 1L beakers；Add about 800ml Deionized water, stirs；With NaOH pH is adjusted to 8.3, plus deionized water is settled to after 1L, room temperature preservation is standby.
2nd, DNA extraction
Using high salt method catfish shape Nibea albiflora DNA, step is as follows：
(1) take catfish shape Nibea albiflora (sample is by the offer of Zhoushan Fo Du plants) muscle of back to organize 50mg to be put into and be equipped with 360 μ L high salt extraction buffer (0.4M NaCl, 10mM Tris-HCl pH8.0,2mM EDTA pH 8.0.Aljanabi SM,Martinez I(1997)Universal and rapid salt-extraction of high quality genomic DNA for PCR-based techniques.Nucleic Acids Res 25:4692-4693) centrifuge tube In, quickly shredded with the eye scissors of sterilizing, it is then respectively adding 40 μ L 20%SDS, 10 μ l Proteinase K (20mg/ Ml) solution, gentle reverse mixing, in 56 DEG C of placement 1h or overnight, until tissue is completely dissolved.
(2) 300 μ L 6M NaCl vortex 30s are added, in 4 DEG C of high speed centrifugation 30min of 12000rpm, supernatant is moved into new Centrifuge tube in.
(3) isopyknic phenol is added：Chloroform：Isoamyl alcohol (25:24:1), gentle reverse mixing 5min, under room temperature 12000rpm is centrifuged 10min, and supernatant is moved in new centrifuge tube.
(4) isopyknic chloroform is added：Isoamyl alcohol (24:1), gentle reverse mixing 5min, 12000rpm centrifugations under room temperature 10min, supernatant is moved in new centrifuge tube.
(5) isopropanol of 2/3 volume pre-cooling, gentle reverse mixing, white linear to appear is added to precipitate, 12000rpm, 4 DEG C of centrifugation 15min, abandons supernatant.
(6) 75% ethanol of 800 μ L pre-coolings is added in pipe, turn upside down 5min, in 12000rpm, 4 DEG C of centrifugations 15min, abandons supernatant.
(7) repetitive operation step 6.
(8) the DNA white precipitates of extraction are placed in ventilated chamber and are dried 20min, add 50～100 μ L aquesterilisa or Tris-EDTA buffer (TE, 10mM Tris, 1mM EDTA, pH8.0) dissolve, and -20 DEG C save backup.
3rd, agarose gel electrophoresiies detection
Weighing takes 5g agar Icing Sugar and adds the conical flask containing 1 × tbe buffer liquids of 50mL, and microwave oven is interior to heat 3 times extremely repeatedly Agar powder dissolving and bubble-free, are cooled to 70 DEG C or so and add 3 μ L ethidium bromides (EB) nucleic acid dyes, mix, and are cooled to non-scald on hand, Gently pour in offset plate.After 30min, comb is gently pulled out in agarose gel shaping.Jia 1 × tbe buffer liquid in electrophoresis tank, Sequentially add in loading wells the μ L and DNA/Hind III Standard molecular weight markers of catfish shape Nibea albiflora DNA extraction sample 5 (or DL2000marker) 5 μ l, electrophoretic voltage is set to 5V/cm, detects in gel imaging instrument after electrophoresis 30min.
4th, design of primers
With reference to Japanese croaker (Nibea japonica, GenBank:KT184692), Sciaenops ocellatus (Sciaenops Ocellatus, GenBank:JQ286004.1), Xiamen silver bodyArgyrosomus amoyensis, GenBank: KM257863) mitochondrial genome complete sequence puts in order, and with biosoftware DNAMAN above-mentioned reference Fish mitochondrion base is analyzed Because of conserved region, specific primer is designed in gene conserved region with biosoftware Oligo 6, primer is by Shanghai Sheng Gong biotech firms Synthesis.
5th, PCR amplifications
Using Dalian treasured biological engineering company limited PrimeHS DNA Polymerase (R010Q) expands mesh Gene, reaction system：2 μ L catfish shape Nibea albiflora DNA profilings, 2.5 μ L10 × Buffer (contain Mg2+),4μL dNTP(2.5mmol/ L), 1 μ L forward primers (10 μm of ol/L), 1 μ L reverse primers (10 μm of ol/L), 0.2 μ L HS Taq archaeal dna polymerases (5U/ μ L), Remaining uses sterilizing distilled water polishing.PCR amplification conditions are：94 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 55～58 DEG C of annealing 30s, 72 DEG C extend 5min (according to the length of amplified production), totally 35 circulations；Most 72 DEG C extension 10min.PCR primer adopts fine jade Sepharose electrophoresis detection (with the 3rd step) purpose band, 4 pairs of large fragment amplification specific primers after amplification to obtaining respectively The amplified fragments of 4.4kb, 5.2kb, 4.5kb and 3.5kb.
6th, genomic walking
Using Dalian treasured biological engineering company limited TaKaRa Ex(RR53A) amplifying target genes, respectively with expansion Used as template, wherein 4.4kb products are expanded 4 large fragments for increasing using the 1st～5 article of primer；5.2kb products use the 6th ～11 primers, 4.5kb products use the 12nd～16 article of primer, 3.5kb products to use the 17th～19 article of primer.Reaction system：2 μ L DNA profilings, 5.0 μ L10 × Buffer (contain Mg2+), 8 μ L dNTP (2.5mmol/L), 2 μ L forward primers (10 μm of ol/L), 2 μ L reverse primers (10 μm of ol/L), 0.25 μ L Ex Taq archaeal dna polymerases (5U/ μ L), remaining uses sterilizing distilled water polishing.PCR Amplification condition is：95 DEG C of denaturations 3min；94 DEG C of degeneration 30s, 55～58 DEG C of annealing 30s, 72 DEG C of extension 1.5min, totally 35 are followed Ring；Most 72 DEG C extension 10min.PCR primer is using agarose gel electrophoresiies detection (with the 3rd step) purpose band.
7th, pcr amplification product sequencing and sequence assembly and sequence information analysis
The PCR primer of purposeful band is directly served into the sequencing of Hai Jieli biotech firms.Censorship sequencing result is biological Software ContigExpress is spliced, and is as a result checked by ContigExpress softwares with series quality, is gone by hand Except mistake splicing.Sequence assembly result software DOGMA (http://dogma.ccbb.utexas.edu/)、tRNAscan- SE1.21 and online BLAST (http://www.ncbi.nlm.nih.gov/BLAST) matching identification protein coding gene, RRNA genes and tRNA genes.
8th, catfish shape Nibea albiflora mitochondrial gene homology analysis
Catfish shape Nibea albiflora nearly edge species biology (Japanese croaker KT184692, Xiamen are downloaded in Genebank data bases Silver-colored bodyKM257863) Mitochondrial gene sequence, Jing is compared, with Japanese croaker and Xiamen silver bodyThere is higher homology, But it is respectively present 43 and 46 variant sites.9. different groups catfish shape Nibea albiflora analysis of genetic diversity
With different groups catfish shape Nibea albiflora individuality (Fuding colony, Zhoushan Fo Du colonies, Xiamen colony, Zhangpu colony and tide Shan colony) as sample, different groups catfish shape Nibea albiflora Mitochondrial gene sequence is obtained with reference to 1～7 step, it is soft with MEGA5.0 The sequence that part carries out compares and makes a variation detection, definitive variation site and haplotype.Nucleotide diversity is calculated with DnaSP5.0 (Nucleotide diversity, π), haplotype diversity (haplotype diversity, h) with nucleotide difference degree (Pairwise divergence)。
Jing is analyzed, and the variant sites that 5 for being gathered colony is present are respectively 10,12,8,15 and 18, list Times type number is respectively 5,8,6,11 and 14.Nucleotide diversity (π) is respectively 0.00245,0.00279, 0.00252,0.00285 with 0.00298.Haplotype diversity (h) is respectively 0.645,0.790,0.687,0.824 and 0.841. Nucleotide difference degree is respectively 1.237,1.562,1.317,1.932 and 2.226.
Sequence information (the GenBank accession of catfish shape Nibea albiflora mitochondrion whole genome sequence:KU738606)
Embodiment described above is one kind preferably scheme of the present invention, not makees any pro forma to the present invention Limit, also have other variants and remodeling on the premise of without departing from the technical scheme described in claim.
SEQUENCE LISTING
<110>Zhejiang Prov. Inst. of Marine Products
<120>A kind of amplimer and its amplification method of catfish shape Nibea albiflora mitochondrion whole genome sequence
<130> 2016.11.2
<160> 27
<170> PatentIn version 3.3
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taggcgcagg taaaagtata ggctaa 26
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tgcattagcc acttcctgaa ccaa 24
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acctattcgg ctcactctag gcctc 25
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attctggctc cctcaaatga cc 22
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ccttattttg ctgctttggg cttg 24
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acgacttgcc tcccctttac tgtg 24
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gcaagggaac gctgaaagag 20
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accccccacc gaaccttaac 20
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cgatcaacga accgagttac 20
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acactctgag cacctatacc 20
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gcgctagcct acttcttcag 20
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cttaattaca gcggtcctcc 20
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ccgcgtgtct tgcaccatta 20
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ggccaaccat agcttcatac 20
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aaacagccct caccctctgc 20
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aacctcctat ccgccgtacc 20
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Claims (3)

1. a kind of amplimer of catfish shape Nibea albiflora mitochondrion whole genome sequence, it is characterised in that including 4 pairs of large fragments amplifications With specific primer pair, 4 pairs of large fragment amplification specific primers are to specific as follows：
The forward primer of the 1st pair of primer pair is：Nucleotide sequence shown in SEQ ID NO.1, downstream primer is：SEQ ID Nucleotide sequence shown in NO.2；
The forward primer of the 2nd pair of primer pair is：Nucleotide sequence shown in SEQ ID NO.3, downstream primer is：SEQ ID Nucleotide sequence shown in NO.4；
The forward primer of the 3rd pair of primer pair is：Nucleotide sequence shown in SEQ ID NO.5, downstream primer is：SEQ ID Nucleotide sequence shown in NO.6；
The forward primer of the 4th pair of primer pair is：Nucleotide sequence shown in SEQ ID NO.7, downstream primer is：SEQ ID Nucleotide sequence shown in NO.8.
2. amplimer according to claim 1, it is characterised in that the expansion of catfish shape Nibea albiflora mitochondrion whole genome sequence Increasing primer also includes 19 genomic walking specific primers, and 19 genomic walkings are specific as follows with specific primer：
1st article primer is：Nucleotide sequence shown in SEQ ID NO.9；
2nd article primer is：Nucleotide sequence shown in SEQ ID NO.10；
3rd article primer is：Nucleotide sequence shown in SEQ ID NO.11；
4th article primer is：Nucleotide sequence shown in SEQ ID NO.12；
5th article primer is：Nucleotide sequence shown in SEQ ID NO.13；
6th primer is：Nucleotide sequence shown in SEQ ID NO.14；
7th article primer is：Nucleotide sequence shown in SEQ ID NO.15；
8th article primer is：Nucleotide sequence shown in SEQ ID NO.16；
9th article primer is：Nucleotide sequence shown in SEQ ID NO.17；
10th article primer is：Nucleotide sequence shown in SEQ ID NO.18；
Sub_clause 11 primer is：Nucleotide sequence shown in SEQ ID NO.19；
12nd article primer is：Nucleotide sequence shown in SEQ ID NO.20；
13rd article primer is：Nucleotide sequence shown in SEQ ID NO.21；
14th article primer is：Nucleotide sequence shown in SEQ ID NO.22；
15th article primer is：Nucleotide sequence shown in SEQ ID NO.23；
16th article primer is：Nucleotide sequence shown in SEQ ID NO.24；
17th article primer is：Nucleotide sequence shown in SEQ ID NO.25；
18th article primer is：Nucleotide sequence shown in SEQ ID NO.26；
19th article primer is：Nucleotide sequence shown in SEQ ID NO.27.
3. a kind of amplification method of catfish shape Nibea albiflora mitochondrion whole genome sequence, it is characterised in that with the catfish shape Huang aunt for extracting Fish genomic DNA is template, first with 4 pairs of large fragment amplification primers to carrying out 4 large fragment product amplifications, then by base Because of the method that group step is moved, use 19 genomic walking primers to enter performing PCR as template using 4 large fragments of amplification respectively and expand Increase, the sequencing of PCR primer Jing sepharose electrophoresis detection Hou Song biotech firms.
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CN107904234A (en) * 2017-11-20 2018-04-13 浙江海洋大学 A kind of clupeoid chondrioid genom sequence universal primer and design and amplification method
CN108085366A (en) * 2017-12-12 2018-05-29 浙江海洋大学 A kind of lawver mitochondrial genome complete sequence amplimer and its design and amplification method

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CN107904234A (en) * 2017-11-20 2018-04-13 浙江海洋大学 A kind of clupeoid chondrioid genom sequence universal primer and design and amplification method
CN108085366A (en) * 2017-12-12 2018-05-29 浙江海洋大学 A kind of lawver mitochondrial genome complete sequence amplimer and its design and amplification method
CN108085366B (en) * 2017-12-12 2021-04-20 浙江海洋大学 Burbot mitochondrial genome complete sequence amplification primer and design and amplification method thereof

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