CN104152451B - A kind of primer and method of the identification of neosalanx taihuensis species molecule - Google Patents
A kind of primer and method of the identification of neosalanx taihuensis species molecule Download PDFInfo
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- CN104152451B CN104152451B CN201410409580.XA CN201410409580A CN104152451B CN 104152451 B CN104152451 B CN 104152451B CN 201410409580 A CN201410409580 A CN 201410409580A CN 104152451 B CN104152451 B CN 104152451B
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Abstract
The invention discloses the primers and method of a kind of identification of neosalanx taihuensis species molecule, belong to molecular biology DNA marker field.Primer sequence of the present invention：ND5L (5 ' CTTGGTGCAAATCCAAGCAGGAAC 3 '), ND5R (5 ' GCTTACTCGTGGCCTGAGCCGA 3 ')；Identification method：(1) PCR amplification is carried out with above-mentioned primer pair neosalanx taihuensis experimental specimen total DNA；(2) PCR product purifying, duplication clone, sequencing obtain ND5 gene complete sequences；(3) by the ND5 gene global alignments of experimental specimen and related nearly edge species, normal stream biosystem tree is built；(4) method as above obtains the ND5 gene complete sequences of fish sample to be measured, which is added step (3), rebuilds detection stream biosystem tree；(5) it analyzes and compares.The present invention is used for non-morphologic accurately species identification and classification, and for the artificial breeding of the species, hybridization, further genetics research provide scientific verification foundation.
Description
Technical field
The invention belongs to molecular biology DNA marker technical fields, more specifically to a kind of neosalanx taihuensis species
The primer and method of Molecular Identification.
Background technology
Silverfish (icefishes) is a kind of important economic fish in China.There are 15 kinds in 17 kinds of world silverfish in China,
In 6 kinds be Chinese unique wheat.Neosalanx taihuensis (Neosalanx taihuensi) is kind of a more typical silverfish, peculiar for China
Kind, belong to Salangidae (Salangidae) Neosalanx (Neosalanx).
Silverfish has higher nutritive value and economic value：Contain 8.2 grams of protein, fat per the fresh silverfish edible part of hectogram
0.3 gram of fat, 1.4 grams of carbohydrate, 258 milligrams of calcium etc..Full fish can be used as medicine, and meat nature and flavor are sweet, flat, have and nourish kidney tonifying, stomach invigorating
The effect of qi-restoratives, beneficial lung, sharp water；It can be used for treating splenasthenic diarrhea, nutritional deficiency, indigestion, the diseases such as children's sore product.Silverfish is
Important one of the outlet aquatic products of China, are exported to Europe, Asia, various countries of U.S., are known as in the international market " soft gold ".
The germplasm identification necessity of neosalanx taihuensis：China silverfish natural resources because reclaiming land from a lake, overfishing,
The influences of many factors such as environmental pollution and Habitat Fragmentation and continuous downturn, the natural resources of various silverfishes are all to some extent
Decline, species distribution range is reduced significantly, and individual species are gradually endangered, and the yield of neosalanx taihuensis also declines year by year.Therefore Taihu Lake is new
Germplasm identification, breeding, hybridization and the genetic diversity conservation of silverfish become particularly significant.On the other hand, silverfish flying fish
There are still many disputes, the affiliation according to morphology and each Salangidae species of waters classification is also very mixed for the classification of class
Disorderly.Especially economic fish neosalanx taihuensis species and Big Salangid similar in morphology, Neosalanx pseudotaihuensis and few tooth silverfish
Taxonomic identification is very difficult, this cultivates extraordinary silverfish, species hybridization, the production such as artificially breeding and genetic diversity conservation and section
It learns research and brings inconvenience.Therefore, it is identified using Molecular tools and Salangidae species of classifying is very necessary, identification of means also ten
Divide succinct and science.
Inventor once participates in the paper that research is delivered《The measurement of 2 kinds of silverfish mitochondria CO II and flank tRNA genes is analyzed
And its affiliation research》, in May, 2008, the 3rd phase of volume 38, was published in《Chinese Marine University's journal》, inquired into 4 kinds of silverfishes
And sweetfish mtDNA-COⅡ gene sequence variations and their affiliation, it measures and analysis silverfish mtDNA-COⅡ gene total order
Row and flank tRNA gene orders, and and sequence alignment, genealogical tree reconstruction is carried out to it with NJ methods and MP methods, deduces species
Between affiliation, seek molecule foundation for the genealogical classification of silverfish, used primer is in paper
YaL(5’-TCAGCCACATAACCGCTCTG-3’)；
YaR (5 '-CTTCTAGGAGGCGGAATTG-3 ') and
YbL(5’-CTGTAATTCTTATCCTCAT-3’)；
YbR(5’-GCTGGGTTGAGTTGAGGCATG-3’)。
But inventor's later stage study in find using above-mentioned primer detection silverfish mtDNA-COⅡ gene to build life
During object genealogical tree, primer amplification is not to stablize very much, and PCR product result is simultaneously insufficient to accurately, to accurately identify
Silverfish type needs to redesign a kind of primer that can stablize amplification.
Invention content
1. technical problems to be solved by the inivention
The present invention is to improve existing silverfish classification and identification technology, provides a kind of neosalanx taihuensis species molecule
The primer and method of identification.Technical solution using the present invention, by measuring and analyzing the mitochondrial ND5 gene sequence of silverfish,
The affiliation between species is deduced, and then obtains the taxonomic identification foundation of its science.
2. technical solution
In order to achieve the above objectives, technical solution provided by the invention is：
The primer and method of a kind of neosalanx taihuensis species molecule identification of the present invention, inventor choose neosalanx taihuensis line
Plastochondria ND5 gene design primers, primer sequence are as follows：
ND5L：5’-CTTGGTGCAAATCCAAGCAGGAAC-3’(SEQ ID NO.1)
ND5R：5’-GCTTACTCGTGGCCTGAGCCGA-3’(SEQ ID NO.2).
The method identified using the primer pair neosalanx taihuensis species molecule of above-mentioned mitochondrial ND5 gene, such as following step：
Step 1: structure normal stream biosystem tree
(1) total DNA of primer pair neosalanx taihuensis experimental specimen described in claim 1 carries out PCR amplification, obtains PCR
Product；
(2) PCR product is purified, is transferred to plasmid vector and replicates clone, bacterium solution is sequenced, obtains ND5 gene total orders
Row；
(3) neosalanx taihuensis experimental specimen is compared with the ND5 gene complete sequences of related nearly edge species, is utilized
MEGA6.0 software building NJ method normal stream biosystem trees；
Step 2: structure detection stream biosystem tree
(4) according to step 1 method, the mitochondrial ND5 gene complete sequence of fish sample to be measured is obtained, which is added
Step (3) rebuilds detection stream biosystem tree；
(5) analysis and standard of comparison stream biosystem tree and detection stream biosystem tree, identify whether sample to be tested is too
The new silverfish in lake.
Preferably, pcr amplification reaction volume is 25 μ l, and 1 μ l of template DNA (contain 10~50ng)；PCR cycle annealing temperature：
55℃。
The mitochondrial ND5 gene primer that the present invention designs obtains as follows：
A obtains the mitochondria complete sequence 3 of the nearly edge species of neosalanx taihuensis from NCBI；It is done using ClustalXv1.83
The comparison of two terminal sequences of ND5 finds out the conservative section of both ends 200bp or so, as primer candidate regions；
B analyzes candidate region sequence using OLIGO v6 softwares, primer sequence alternative condition：Length is selected to exist
20bp or so, G/C content 50% or so, the primer annealing temperature of software prediction does not form hair fastener between 50-55 degrees Celsius, primer
Structure, the primer efficiency that primer may expand the sequence of other positions in complete sequence are less than 200.After meeting these requirements, we
It is 1 pair selected, final primer of the upstream and downstream primer as PCR amplification, as ND5L, ND5R sequence of the invention.
The principle of identification method of the present invention is：
It chooses the mitochondrial ND5 gene design primer that evolutionary rate is moderate in neosalanx taihuensis and carries out PCR amplification, will obtain
PCR product purifying, be transferred to plasmid vector and replicate clone, and bacterium solution is sequenced, obtains ND5 gene complete sequences；Then root
The gene order of neosalanx taihuensis, related nearly edge species and sample to be measured is constructed into biosystem according to gene sequencing result
Tree, so that it is determined that whether sample to be measured is neosalanx taihuensis, with other silverfishes or the affiliation of neosalanx taihuensis.
3. advantageous effect
Using technical solution provided by the invention, compared with prior art, there is following remarkable result：
(1) primer and method of a kind of neosalanx taihuensis species molecule of the invention identification, compared with traditional form, point
Sub- identification method science, accurate, easily operated, carrying out identification to biological sample has a generality, scientific and repeatability, soon
Speed measures the affiliation of fish sample and neosalanx taihuensis to be measured, and relationship data judges between the species of neosalanx taihuensis
Foundation is provided with resource identification, breeding, hybridization and genetic diversity conservation.
(2) primer and method of a kind of neosalanx taihuensis species molecule of the invention identification, chooses the line of neosalanx taihuensis
Plastochondria ND5 genes, compared with the mtDNA-COⅡ gene chosen in inventor's paper before, the result of acquisition is more stable, if
The primer PCR of meter, can stablize amplification, PCR long-PCR, and PCR product is accurately reliable.
(3) primer and method of a kind of neosalanx taihuensis species molecule of the invention identification, by building normal stream biology
The method that genealogical tree and increase sample to be tested gene rebuild biosystem tree, is not only identifying and is inferring that species to be measured are
No is neosalanx taihuensis, has more accurate advantage with the affiliation of other silverfishes or neosalanx taihuensis aspect, moreover it is possible to
It is enough that the reproduction of affiliation can be carried out for the silverfish of the unknown kind of any type, and it is further used for their classification,
The scope of application is wide.
Description of the drawings
Fig. 1 is the flow diagram of neosalanx taihuensis species molecule identification method in the present invention；
Fig. 2 is the general flow chart of neosalanx taihuensis species molecule identification method in the present invention；
Fig. 3 is the biosystem tree structure figures of embodiment 1 in the present invention.
Specific implementation mode
To further appreciate that present disclosure, in conjunction with accompanying drawings and embodiments, the present invention is described in detail.
Embodiment 1
As shown in Figure 1, a kind of method and step that neosalanx taihuensis species molecule is identified of the present invention is：
Step 1: structure normal stream biosystem tree
It obtains that neosalanx taihuensis is complete, fresh individual specimen, reflects with sample comparison in Huainan Normal University's zoological specimens room
It is fixed, be tentatively confirmed as neosalanx taihuensis sample, then according to neosalanx taihuensis morphological feature (tongue and tooth numbers and position,
The position of fin and fin ray number), Morphological Identification is carried out using anatomical lens and microscope, confirms species, and new as Taihu Lake
Silverfish standard sample, the musculature that laboratory takes sample fresh, using SDS/ protease crackings, phenol chloroform extraction total DNA, fine jade
Sepharose electrophoresis detection and spectrophotometric determination total DNA content.
(1) nearly edge species correlated series are referred to, neosalanx taihuensis mitochondrial ND5 gene primer is designed, sequence is as follows：
ND5L：5’-CTTGGTGCAAATCCAAGCAGGAAC-3’(SEQ ID NO.1)
ND5R：5’-GCTTACTCGTGGCCTGAGCCGA-3’(SEQ ID NO.2).
PCR amplification is carried out with the total DNA of above-mentioned primer pair neosalanx taihuensis experimental specimen, obtains PCR product；PCR amplification
Reaction volume is 25 μ l, and 1 μ l of template DNA (contain 10~50ng)；PCR cycle annealing temperature：55℃.
(2) PCR product is purified by biotinylation kit, is transferred to plasmid vector and replicates clone, bacterium solution is sequenced, has
Body gives Shanghai biotech firm in the present embodiment, to the examining order of bacterium solution gene, obtains ND5 gene complete sequences.
The specific nucleotide of ND5 gene complete sequences of neosalanx taihuensis is as follows：1824bp
//atgtaccccctcaccaccattttaaactcctcgctagtactgatcttcgctcttctcctcttcccct
tgttcaccacggc
taacaaacactgggccctaacgcacgtcaacaccgcaatcaaagccgcattcctcgtcagccttatccccctctctg
ttt
tcctcgactctggggttgaaacagttgtctgtgcctgacaatgaatctgcacccgcacctttgacattagcatcagc
ttt
aaatttgacctttactccctcatcttcacccccgtcgctctttatgtaacatgagcaatcttagaattcgcccgctg
ata
tatacatgcagaccccaacatgaaccgattcttcaaatgccttctcctcttcctagtggcaatgattattatggtaa
cag
ccaacaacatgttccaactctttattggctgggaaggcattggtatcatgtcttttctactcatcgactgatgagac
ggg
cgagcagatgccggcgccgctgcccttcaggccgtcctatacaaccgagttggagacatcggacttgttcttagcat
agc
ttgattcgctgtaaatctaaactcttgagacatagagcaaatatctgtgtcttcccaagaccttgacctcaccctcc
ccc
tcataggcctaatcctggcagccactgcaaaatccgcgcaattcggcctccacccctgactccccgcggctatagaa
ggc
cctactcctgtctccgccctgcttcactccagcacaatggtggtcgcaggagtattccttctagtccggactagccc
tat
aatagaaaacaaccccatagcccttactacctgcttatgcctgggggccctcaccactctctttgccgccacctgcg
ctc
tgacccaaaacgacattaaaaaaatcgtcgccttctccacctcaagccagctcggactaatgatagtcgctgtcggt
ctt
aaccaaccacagctcgctttcctccatatctgcacacacgcatttttcaaagccatactgttcttgtgctcaggatc
aat
tatccacagcctaaatgatgaacaggacattcggaagatgggaggcctaagcaacctcgcccccttcacctcctctt
gcc
tcgctatcggcagcctcgccctcacagggacccccttcctggcaggcttcttctcaaaagacgccatcatggaagct
tta
aacacatcttacctgaacgcctgagccctggccctcaccctcctagccacctccttcaccgcaatctatagccttcg
tgt
ggcctactttgtagccatgggccacccccgatccccagccctctcccccatcaacgaaaataacccctgcgttatta
acc
ccattaaacgcctcgccttaggaagcatcattgccggcctcctaatcacctccaacctcctcccctccaaaacccct
gtc
ttaactatgccccccctcctcaaacttagcgcccttgtagtaactgtcatcggccttctcacagccctagaactcgc
ctc
cttgacttccaaacaatttaagacctccccctctctcaccccccacaacttctctaacatgctaggatttttccccg
caa
tcatccatcgatcaatcccctactgaagcctcttcctcggacaaacagttgcgagccaaatgcttgacaaaacatga
ttc
gagaaagtcggccccaaggcaatatccactatcaacctgcctgcagcttccaccactgccgaccttcaccagggcat
aat
caaaacctatctgtccctattccttctgacagtcgtcttggctattatcctgcccctcatctaa//
(SEQ ID NO.3)
(3) it utilizes biometric database NCBI to obtain the corresponding sequence of related nearly edge species, utilizes biosoftware clusterW
Neosalanx taihuensis experimental specimen is compared with the ND5 gene complete sequences of related nearly edge species, is based on mitochondrial ND5 gene core
Nucleotide sequence utilizes MEGA6.0 software building NJ method normal stream biosystem trees；Determine the classification position of neosalanx taihuensis.
Step 2: structure detection stream biosystem tree
(4) muscle sample for obtaining 2 unknown definite species X and Y of silverfish extracts them respectively according to step 1 method
DNA 2 mitochondria ND5 bases are obtained after sequencing using above-mentioned their mitochondrial ND5 gene of ND5L and ND5R primer amplifications
Because of complete sequence.Step (3) is added in the sequence, rebuilds detection stream biosystem tree, (as shown in Figure 3).
(5) analysis and standard of comparison stream biosystem tree and the tree-like difference of detection stream biosystem tree, identify sample to be tested
Whether be neosalanx taihuensis, muscle sample X forms a monosystem group with known neosalanx taihuensis, can determine whether for Taihu Lake it is newly silver-colored
Fish, muscle sample Y cannot form a monosystem group with known neosalanx taihuensis, not be neosalanx taihuensis, but itself and big silver
Fish forms a monosystem group, is Big Salangid.
The mitochondria of a kind of neosalanx taihuensis species molecule identification method of the present invention, neosalanx taihuensis and other species
ND5 genes exist in species and inter-species variation checks the frequency of mutation, can build biosystem tree by sequence alignment, reflect
Earnest kind.And it is more reliable according to the primer ND5L and ND5R of its design, it can be in neosalanx taihuensis and related nearly edge species fish
Stablize amplification in the group of class, the PCR product of PCR long-PCR, acquisition are stablized so that the species of neosalanx taihuensis differentiate more
Accurately, and by the structure of biosystem tree determine that the affiliation of species is more accurate apparent.
Schematically the present invention and embodiments thereof are described above, description is not limiting, institute in attached drawing
What is shown is also one of embodiments of the present invention, and practical situation is not limited thereto.So if the common skill of this field
Art personnel are enlightened by it, without departing from the spirit of the invention, are not inventively designed and the technical solution
Similar frame mode and embodiment, are within the scope of protection of the invention.
Claims (2)
1. a kind of primer of neosalanx taihuensis species molecule identification, feature exist：The primer sequence is as follows：
ND5L：5’-CTTGGTGCAAATCCAAGCAGGAAC-3’
ND5R：5’-GCTTACTCGTGGCCTGAGCCGA-3’.
2. a kind of method of neosalanx taihuensis species molecule identification, it is characterised in that：Include the following steps：
Step 1: structure normal stream biosystem tree
(1) the primer pair neosalanx taihuensis experimental specimen total DNA described in claim 1 carries out PCR amplification, obtains PCR product,
Pcr amplification reaction volume is 25 μ l, and 1 μ l of template DNA, template DNA is containing 10~50ng；PCR cycle annealing temperature：55℃；
(2) PCR product is purified, is transferred to plasmid vector and replicates clone, bacterium solution is sequenced, obtains ND5 gene complete sequences；
(3) neosalanx taihuensis experimental specimen is compared with the ND5 gene complete sequences of related nearly edge species, utilizes MEGA6.0
Software building NJ method normal stream biosystem trees；
Step 2: structure detection stream biosystem tree
(4) according to step 1 method, the mitochondrial ND5 gene complete sequence of fish sample to be measured is obtained, which is added step
(3), detection stream biosystem tree is rebuild；
(5) analysis and standard of comparison stream biosystem tree and detection stream biosystem tree, identify whether sample to be tested is that Taihu Lake is new
Silverfish.
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| CN106399500B (en) * | 2016-09-18 | 2019-06-28 | 江苏省淡水水产研究所 | A kind of molecular identification method of Big Salangid and neosalanx taihuensis |
| CN107885977B (en) * | 2017-11-30 | 2019-10-18 | 淮南师范学院 | A method of it is reset for detecting animal monoid mitochondrial genomes |
| CN108841941B (en) * | 2018-05-22 | 2021-11-02 | 广西壮族自治区水产引育种中心 | Method for accurately identifying Cyprinus carpioides by utilizing mitochondrial NADH5 gene |
| CN111944910A (en) * | 2020-08-28 | 2020-11-17 | 海南热带海洋学院 | Primer group, kit and method for detecting Pink paranieri population |
| CN113913534B (en) * | 2021-11-19 | 2022-05-13 | 中国水产科学研究院黑龙江水产研究所 | Primer for identifying big whitebait and Taihu new whitebait and identification method thereof |
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| CN104004752A (en) * | 2014-04-23 | 2014-08-27 | 江汉大学 | Neosalanxtaihuensis microsatellite DNA molecule labelled primers and preparation method thereof |
| CN104911182A (en) * | 2015-06-01 | 2015-09-16 | 中国水产科学研究院 | Fish mitochondria genome-wide DNA amplification |
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| CN104004752A (en) * | 2014-04-23 | 2014-08-27 | 江汉大学 | Neosalanxtaihuensis microsatellite DNA molecule labelled primers and preparation method thereof |
| CN104911182A (en) * | 2015-06-01 | 2015-09-16 | 中国水产科学研究院 | Fish mitochondria genome-wide DNA amplification |
Non-Patent Citations (3)
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| Genbank Accession：JX524196.1;www.ncbi.nlm.nih.gov/genbank;《www.ncbi.nlm.nih.gov/genbank》;20121017;全文 * |
| 太湖新银鱼mtDNA COII和Cytb基因的克隆与序列分析;张际峰 等;《激光生物学报》;20080415;第17卷(第2期);229-234 * |
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