CN104531846A - Amplification primer of mitochondrial genome control region of spider and reaction system of amplification primer - Google Patents
Amplification primer of mitochondrial genome control region of spider and reaction system of amplification primer Download PDFInfo
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- CN104531846A CN104531846A CN201410753795.3A CN201410753795A CN104531846A CN 104531846 A CN104531846 A CN 104531846A CN 201410753795 A CN201410753795 A CN 201410753795A CN 104531846 A CN104531846 A CN 104531846A
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- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
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Abstract
The invention discloses an amplification primer of a mitochondrial genome control region of a spider and a reaction system of the amplification primer. The amplification primer consists of two single strain oligonucleotides, wherein a forward primer Ctrl-F is shown in SEQ ID NO: 1 and a reverse primer Ctrl-R is shown in SEQ ID NO: 2. The amplification primer disclosed by the invention can be used for quickly, efficiently and specifically amplifying the mitochondrial genome control regions of various spiders, has important meaning in the aspects of rapid identification of the types of the spiders, analysis of population diversity, protection and utilization of genetic resources and the like and further provides a powerful tool for analyzing and researching evolution of different taxonomic category systems of the spiders.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of amplimer and reaction system thereof of spider Mitochondrial Genome Overview control region.
Background technology
Spider is the predacious arthropods of a class, main episite, they insect biological control, safeguard the ecosystem balance in play very important effect.Note Protect natural enemies kind storehouse, promote that Natural Enemies is rebuild, can effectively control many Agricultural pests, such as the occurrence and harm of plant hopper, leafhopper, snout moth's larva, cotten aphid etc.Spider is various, to its species identification and to carry out fauna sort research very important fast and accurately, to the available protecting of spider germ plasm resource with make full use of aspect and have great importance.
Arthropods Mitochondrial Genome Overview DNA is a kind of in virus covalently closed circular, has self-replicating, transcribes the gene with translation ability outside nucleus.The advantages such as compared with core DNA, Mitochondrial Genome Overview DNA has matrilinear inheritance, and copy number is many, and recombination fraction is low, and rate of evolution is very fast.Become arthropodan population genetics, a tool of the research field such as phylogenetics and conservation biology.It is generally by 13 protein genes, 22 tRNA genes, 2 rRNA genes (12S rRNA and 16S rRNA) totally 37 encoding genes and one section of non-coding region (control region) composition.Wherein control region is the region that in Mitochondrial Genome Overview, evolutionary rate is the fastest, its variation rate is about 5 to 10 times of other regional DNAs of plastosome, thus be applicable to very much the comparative studies between the very near colony of sibship, can be used as the important genetic marker of similar kind and the research aspect such as different groups qualification and Investigation on Diversity.In addition, also there is tandem sequence repeats variable region in various degree in mitochondria control region, for the evolutionary mechanism and mitochondrial DNA Replication transcriptional control mechanism studying control region provides good research material.With belong to compared with arthropodan insect, to the research significantly backwardness of the plastosome full-length genome of spider.Utilizing mitochondria control region sequence to carry out spider genetics and evolutionary biology research is at present mostly special primer for a certain specific species, does not also have a kind of universal primer of most of spider Mitochondrial Genome Overview control region of can effectively increasing.Therefore, the universal primer of design amplification spider plastosome Mitochondrial Genome Overview control region, on the one hand for the analysis of spider Investigation on Diversity provides powerful, also for the efficient spider plastosome whole genome sequence that obtains lays the foundation.
Summary of the invention
For above-mentioned the deficiencies in the prior art, the object of the present invention is to provide a kind of amplimer and reaction system thereof of spider Mitochondrial Genome Overview control region.
For realizing object of the present invention, contriver provides following technical scheme:
The universal amplification primer of spider Mitochondrial Genome Overview control region designs according to the spider plastosome whole genome sequence announced in GenBank database, forward primer Ctrl-F is as shown in SEQ ID NO:1, and reverse primer Ctrl-R is as shown in SEQ ID NO:2:
SEQ ID NO:1 : 5′- AAGGGTGACGGGCGATAT-3′;
SEQ ID NO:2 : 5′- ATAGCTCTATTAGCTGACT-3′。
Utilize the universal amplification primer of above-mentioned a pair spider Mitochondrial Genome Overview control region, after pcr amplification order-checking, all can obtain the 10 kinds of spider Mitochondrial Genome Overview control region base sequences gathered in farmland, Zhejiang Province.Specifically comprise the following steps:
(1) DNA extraction agent box is adopted to extract the total genomic dna of spider.For avoiding polluting, casting out a belly, only extracting DNA from leg.
(2) utilize above-mentioned pair of primers, with the good spider genome of extracting for template, carry out PCR amplification and order-checking (the results are shown in Figure 1).PCR reaction system is 25 μ 1, wherein: 2 μ l template DNAs (about 20ng), and 2.5 μ l 10 × PCR buffer, 2 μ l 2. 5mM MgCl
2, the forward and reverse primer of 1 μ l 10 mM dNTPs, each 1 μ l 10 mM, 0.3 μ l 5U/ μ l Taq DNA polymerase, adds deionized water and is adjusted to final volume 25 μ 1; PCR reaction conditions is: 95 ° of C denaturations 3 minutes, 94 ° of C sex change 30 seconds, and 50 ° of C anneal 40 seconds, and 72 ° of C extend 3 minutes; After 35 circulations, 72 ° of C extend 10 minutes again.
(3) pcr amplification product carries out gel electrophoresis, all obtains the fragment of 1000-3500 bp, and the homologous sequence in order-checking and comparison GenBank, determines that institute's amplified fragments is mitochondria control region sequence.
As preferably, in authentication method of the present invention, the spider wherein described in step (1) includes but not limited to following: intend water tarantula (
pirata subpiraticus), Misumenops tricuspidatus (
ebrechtella tricuspidata), golden spider (
argiope bruennichi), cone abdomen Tetragnathidae (
tetragnatha maxillosa), magnificent Tetragnathidae (
tetragnatha nitens), dark brown new round spider (
neoscona theisi), arteries and veins Wa spider (
wadicosa venatrix), black spider that feeds on flies (
plexippus paykulli), Araneus ventricosus (
araneus ventricosus), cat jump spider (
carrhotus xanthogramma).
Advantage of the present invention is:
1. spider plastosome Mitochondrial Genome Overview control region provided by the invention amplimer, can the Mitochondrial Genome Overview control region complete sequence of the multiple spider of efficient specific amplification, instead of the Partial Fragment sequence that only increases, the different taxonomic category phyletic evolution of spider and sort research can be applied to;
2. universal primer of the present invention and method of design thereof can increase as universal primer PCR the specific examples of principle and key parameter research, promote the development of the field that the invention relates to, thus possess important invention value and theory significance;
Accompanying drawing explanation
Fig. 1 be in the present invention spider Mitochondrial Genome Overview control region amplimer in order to the electrophoretogram of the different sorts spider plastosome ND4 gene complete sequence that increases.Wherein, M:DNA molecular weight standard (Mark IV); 1-10 swimming lane is followed successively by intends water tarantula, Misumenops tricuspidatus, golden spider, cone abdomen Tetragnathidae, magnificent Tetragnathidae, and dark brown new round spider, arteries and veins Wa spider, black spider that feeds on flies, Araneus ventricosus, cat jumps spider.
Embodiment
Below in conjunction with specific embodiment, set forth the present invention further
embodiment 1:
(1)the collection of spider sample, qualification and preservation
In embodiment, spider used all picks up from Yuyao City farmland, Zhejiang Province with trapping method.Sample of adopting is taken back laboratory and is carried out information registering, all with stereoscopic microscope qualification, to determine kind, for the spider sample of molecular biology experiment with 100% alcohol-pickled and to be stored in 4 ° of C refrigerators for subsequent use.The spider gathered comprises: intend water tarantula, three spend prominent spider, golden spider, cone abdomen Tetragnathidae, magnificent Tetragnathidae, dark brown new round spider, arteries and veins Wa spider, black spider that feeds on flies, and Araneus ventricosus, cat jump spider;
(2)the extraction of spider genome DNA
DNA extraction agent box is adopted to extract the total genomic dna of different sorts spider.For avoiding polluting, casting out a belly, only extracting DNA from leg.DNA extraction directly utilizes DNA extraction agent box (Tian Gen biochemical technology company limited) to extract.The Zong Ji extracted saves backup Yin Zu – 20 ° of C;
(3)the amplimer of design and synthesis spider Mitochondrial Genome Overview control region
The GenBank database logged in NCBI website receives the spider plastosome whole genome sequence that rope has been announced, through the comparison of ClustalX software homology, find out conservative sequence in control region upstream and downstream, utilize Premier primer 5.0 software design go out spider Mitochondrial Genome Overview control region amplimer and send Nanjing Genscript Biotechnology Co., Ltd. to synthesize.Forward primer Ctrl-F is as shown in SEQ ID NO:1, and reverse primer Ctrl-R is as shown in SEQ ID NO:2:
SEQ ID NO:1 : 5′- AAGGGTGACGGGCGATAT-3′;
SEQ ID NO:2 : 5′- ATAGCTCTATTAGCTGACT-3′；
(4)pcr amplification Mitochondrial Genome Overview control region complete sequence and sequencing
Utilize above-mentioned pair of primers, with the good spider genome of extracting for template, carry out PCR amplification.Wherein, PCR reaction system is 25 μ 1, wherein: 2 μ l template DNAs (about 20ng), and 2.5 μ l 10 × PCR buffer, 2 μ l 2. 5mM MgCl
2, the forward and reverse primer of 1 μ l 10 mM dNTPs, each 1 μ l 10 mM, 0.3 μ l 5U/ μ l Taq DNA polymerase, adds deionized water and is adjusted to final volume 25 μ 1; PCR reaction conditions is: 95 ° of C denaturations 3 minutes, 94 ° of C sex change 30 seconds, and 50 ° of C anneal 40 seconds, and 72 ° of C extend 3 minutes; After 35 circulations, 72 ° of C extend 10 minutes again.
The PCR primer sepharose of 1.5% carries out electrophoresis detection (the results are shown in Figure 1), and amplified fragments gel reclaims test kit (Tian Gen biochemical technology company limited) rubber tapping and reclaims.Reclaiming product send Nanjing Genscript Biotechnology Co., Ltd. to check order.Sequencing result is compared with homologous sequence known in GenBank, determines that it is Mitochondrial Genome Overview Control region sequence.
SEQUENCE LISTING
The <110> China Measures Institute
The amplimer of a <120> spider Mitochondrial Genome Overview control region and reaction system thereof
<130> 123456
<160> 2
<170> PatentIn version 3.3
<210> 1
<211> 18
<212> DNA
<213> artificial sequence
<400> 1
aagggtgacg ggcgatat 18
<210> 2
<211> 19
<212> DNA
<213> artificial sequence
<400> 2
atagctctat tagctgact 19
Claims (4)
1. an amplimer for spider Mitochondrial Genome Overview control region, it is characterized in that being made up of two single strain oligonucleotides, wherein forward primer Ctrl-F is as shown in SEQ ID NO:1, and reverse primer Ctrl-R is as shown in SEQ ID NO:2:
SEQ ID NO:1: 5′- AAGGGTGACGGGCGATAT-3′;
SEQ ID NO:2: 5′- ATAGCTCTATTAGCTGACT-3′。
2. the amplimer of spider Mitochondrial Genome Overview control region as claimed in claim 1, is characterized in that described spider comprises: intend water tarantula, Misumenops tricuspidatus, golden spider, cone abdomen Tetragnathidae, magnificent Tetragnathidae, dark brown new round spider, arteries and veins Wa spider, black spider that feeds on flies, Araneus ventricosus, cat jumps spider.
3. the amplimer of spider Mitochondrial Genome Overview control region according to claim 1, it is characterized in that the method for design of primer is: log in the spider plastosome whole genome sequence that the GenBank database search in NCBI website has been announced, sequence homology comparison is carried out through ClustalX software, find out conservative sequence, recycling Premier primer 5.0 software design goes out the amplimer of spider Mitochondrial Genome Overview control region.
4. adopt a reaction system for primer PCR amplification spider mitochondria control region described in claim 1, it is characterized in that reaction system is 25 μ 1, wherein: 2 μ l template DNAs, 2.5 μ l 10 × PCR buffer, 2 μ l 2.5 mM MgCl
2, the forward and reverse primer of 1 μ l 10 mM dNTPs, each 1 μ l 10 mM, 0.3 μ l 5U/ μ l Taq DNA polymerase, adds deionized water and is adjusted to final volume 25 μ l; PCR reaction conditions is: 95 ° of C denaturations 5 minutes, 94 ° of C sex change 30 seconds, and 50 ° of C anneal 40 seconds, and 72 ° of C extend 3 minutes; After 35 circulations, 72 ° of C extend 10 minutes again.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048457A (en) * | 2017-12-13 | 2018-05-18 | 安徽师范大学 | A kind of Bai Dingxi mynas mitochondria control region amplification nested primers and amplification, clone and sequencing approach |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086014A (en) * | 2007-05-25 | 2007-12-12 | 中山大学 | Fish mitochondrion DNA control area amplification primer and its design method and uses |
| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101086014A (en) * | 2007-05-25 | 2007-12-12 | 中山大学 | Fish mitochondrion DNA control area amplification primer and its design method and uses |
| CN102409051A (en) * | 2011-10-18 | 2012-04-11 | 浙江海洋学院 | Marine fish mitochondrial genome control area amplification primer, as well as design and amplification methods |
Non-Patent Citations (1)
| Title |
|---|
| ZHENG LIANG WANG ET AL: "The complete mitochondrial genome of wolf spider Pirata subpiraticus Boes.et str.（Araneae：Lycosidae）", 《MITOCHONDRIAL DNA》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108048457A (en) * | 2017-12-13 | 2018-05-18 | 安徽师范大学 | A kind of Bai Dingxi mynas mitochondria control region amplification nested primers and amplification, clone and sequencing approach |
| CN108048457B (en) * | 2017-12-13 | 2021-04-13 | 安徽师范大学 | Nested amplification primer for mitochondrial control region of Baijixi myna and amplification, cloning and sequencing methods |
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