CN104531688A - Spider mitochondria genome complete sequence amplification primer and amplification method - Google Patents
Spider mitochondria genome complete sequence amplification primer and amplification method Download PDFInfo
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Abstract
The invention discloses a spider mitochondria genome complete sequence amplification primer and an amplification method. The method comprises the steps of extracting a general DNA of the spider genome, designing a mitochondria genome complete sequence universal amplification primer, and amplifying and sequencing the long PCR (L-PCR) of the DNA of the mitochondria genome. The spider mitochondria genome complete sequence amplification primer consists of 6 pairs of single strain oligonucleotides. Multiple spider mitochondria genome complete sequences can be quickly and efficiently amplified by adopting the universal amplification primer, and a material basis is established to spider species identification, population diversity analysis, genetic resource protection and utilization, systematic inheritance evolution and other researches.
Description
Technical field
The invention belongs to field of molecular biotechnology, be specifically related to a kind of spider mitochondrial genome complete sequence amplimer and amplification method.
Background technology
Plastosome is an important organoid in cell, and its genomic dna is virus covalently closed circular, has self-replicating, transcribes and translation ability.Plastosome full-length genome is generally by 13 protein genes, and 22 tRNA genes, totally 37 encoding genes and one section of non-coding region (control region) form 2 rRNA genes (12S rRNA and 16S rRNA).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, as the population genetics of animal, a kind of effective tool of the research field such as phylogenetics and conservation biology.As some genes on plastosome (as COI, CytB, 16S rRNA etc.) can be used as important genetic molecule tag application in species taxonomy identification research.Mitochondrial genome complete sequence information (genome structure composition and sequence in the gene mode etc.) provides best evidence for animal molecular system.Therefore, very important and necessary to the research of animal mitochondria genome structure.
Spider is the predacious arthropods of a class, main episite, they agricultural pests 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.With belong to compared with arthropodan insect, to the research significantly backwardness of the plastosome full-length genome of spider.The plastosome full genome of current 13 kinds of spiders of only having checked order, and spider is various, also has the kind chondrioid genome sequencing work of extreme portions not complete.Therefore, universal primer and the amplification method of the amplification of design spider plastosome whole genome sequence are particularly important, and at the Sustainable use of spider, evolutionary analysis and genetic marker selection aspect play an important role.
Summary of the invention
The object of the invention is to overcome the deficiencies in the prior art, a kind of spider mitochondrial genome complete sequence amplimer and amplification method are provided.
Spider mitochondrial genome complete sequence amplimer of the present invention, is made up of 6 pairs of single strain oligonucleotides, and title and the sequence of 6 pairs of universal primers are as follows:
Pair of primers:
Forward primer 1 is as shown in SEQ ID NO:1: 5 '-AGCTAATAAAGCTAATGAGTTCATA-3 ';
Reverse primer 1 is as shown in SEQ ID NO:2: 5 '-TAATTAATTTAAGAGACTAACACCT-3 ';
Second pair of primer:
Forward primer 2 is as shown in SEQ ID NO:3: 5 '-AATGTTCTGAAATTTGTGGTG-3 ';
Reverse primer 2 is as shown in SEQ ID NO:4: 5 '-CATACTACATCTACAAAATGTCA-3 ';
3rd pair of primer:
Forward primer 3 is as shown in SEQ ID NO:5: 5 '-TGACATTTTGTAGATGTAGTATG-3 ';
Reverse primer 3 is as shown in SEQ ID NO:6: 5 '-AAGCTCATGTAGAAGCTCC-3 ';
4th pair of primer:
Forward primer 4 is as shown in SEQ ID NO:7: 5 '-GGAGCTTCTACATGAGCTT-3 ';
Reverse primer 4 is as shown in SEQ ID NO:8: 5 '-GAATTAGTATCAGGATTTAATGT-3 ';
5th pair of primer:
Forward primer 5 is as shown in SEQ ID NO:9: 5 '-ACATTAAATCCTGATACTAATTC-3 ';
Reverse primer 5 is as shown in SEQ ID NO:10: 5 '-TGCTAAGGTAGCATAATCATT-3 ';
6th pair of primer:
Forward primer 6 is as shown in SEQ ID NO:11: 5 '-AATGATTATGCTACCTTAGCA-3 ';
Reverse primer 6 is as shown in SEQ ID NO:12: 5 '-TATGAACCCATTAGCTT-3 '.
Utilize above-mentioned 6 pairs of spider mitochondrial genome complete sequence amplimers, through pcr amplification, order-checking and splicing can obtain spider mitochondrial genome complete sequence information, and concrete technical scheme comprises the following steps:
(1) DNA extraction agent box extracts the total genomic dna of farmland spider.For avoiding polluting, casting out a belly, only extracting DNA from leg.
(2) six pairs of above-mentioned primers are utilized, with the good spider genome of extracting for template, carrying out PCR amplification (the results are shown in Figure 1) PCR reaction system is 25 μ 1, wherein: 2 μ l template DNAs (about 20ng), 2.5 μ l 10 × LA PCR buffer, 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 LATaq enzyme, adds deionized water and is adjusted to final volume 25 μ 1; PCR reaction conditions is as follows: 95 ° of C denaturations 5 minutes; 92 ° of C sex change 10 seconds, 48 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations; 92 ° of C sex change 10 seconds, 50 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations, and per cycling time increases 20s; 72 ° of C extend 10 minutes again.
(3) pcr amplification product gel electrophoresis, all obtains the fragment of size between 1.0-4.0 kb, and order-checking and the comparison with homologous sequence on GenBank are reclaimed in rubber tapping, turn out to be the specific amplified product of Mitochondrial Genome Overview sequence fragment.Each sequence through DNAMAN software splicing after mitochondrial genome complete sequence.
As preferably, in amplification method of the present invention, the spider wherein described in step (1) includes but not limited to following 10 kinds of spiders: 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 mitochondrial genome complete sequence amplimer provided by the invention, the mitochondrial genome complete sequence of multiple spider of can efficiently and specifically increasing, can be applied to the different taxonomic category phyletic evolution of spider and sort research;
2. spider mitochondrial genome complete sequence amplimer provided by the invention, primer pair quantity is few, time saving and energy saving, and saves reagent cost.
Accompanying drawing explanation
Fig. 1 is that spider mitochondrial genome complete sequence amplimer increases the agarose gel electrophoretogram of different spider plastosome complete sequence.
Wherein, A-J is respectively and intends 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, and Araneus ventricosus, cat jump spider; M and 1-6 swimming lane is followed successively by the amplification of DNA molecular amount standard (Mark IV) and primer pair 1-6.
Embodiment
embodiment 1:
(1)the collection of spider sample, qualification and preservation
In embodiment, spider used all picks up from Zhejiang Province's Yuyao City or Tongxiang County farmland with trapping method.Sample of adopting is taken back laboratory and is carried out information registering, all with stereoscopic microscope qualification, to determine kind.The good spider sample of Identification of Species is 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, and dark brown new round spider, arteries and veins Wa spider, black spider that feeds on flies, 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 design of the universal amplification primer of spider mitochondrial genome complete sequence and synthesis
The GenBank database logged in NCBI website receives the full Mitochondrial Genome Overview sequence of spider that rope has been announced, through the comparison of ClustalW software homology, find out relatively conservative region, utilize Premier primer 5.0 software design to go out 6 universal primers to amplification spider mitochondrial genome complete sequence, and send Nanjing Genscript Biotechnology Co., Ltd. to synthesize.6 pairs of amplimers are respectively:
Pair of primers:
Forward primer 1 is as shown in SEQ ID NO:1: 5 '-AGCTAATAAAGCTAATGAGTTCATA-3 ';
Reverse primer 1 is as shown in SEQ ID NO:2: 5 '-TAATTAATTTAAGAGACTAACACCT-3 ';
Second pair of primer:
Forward primer 2 is as shown in SEQ ID NO:3: 5 '-AATGTTCTGAAATTTGTGGTG-3 ';
Reverse primer 2 is as shown in SEQ ID NO:4: 5 '-CATACTACATCTACAAAATGTCA-3 ';
3rd pair of primer:
Forward primer 3 is as shown in SEQ ID NO:5: 5 '-TGACATTTTGTAGATGTAGTATG-3 ';
Reverse primer 3 is as shown in SEQ ID NO:6: 5 '-AAGCTCATGTAGAAGCTCC-3 ';
4th pair of primer:
Forward primer 4 is as shown in SEQ ID NO:7: 5 '-GGAGCTTCTACATGAGCTT-3 ';
Reverse primer 4 is as shown in SEQ ID NO:8: 5 '-GAATTAGTATCAGGATTTAATGT-3 ';
5th pair of primer:
Forward primer 5 is as shown in SEQ ID NO:9: 5 '-ACATTAAATCCTGATACTAATTC-3 ';
Reverse primer 5 is as shown in SEQ ID NO:10: 5 '-TGCTAAGGTAGCATAATCATT-3 ';
6th pair of primer:
Forward primer 6 is as shown in SEQ ID NO:11: 5 '-AATGATTATGCTACCTTAGCA-3 ';
Reverse primer 6 is as shown in SEQ ID NO:12: 5 '-TATGAACCCATTAGCTT-3 '.
(4)pcr amplification Mitochondrial Genome Overview DNA
Utilize six pairs of above-mentioned 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), 2.5 μ l 10 × LA PCR buffer, 1 μ l 10 mM dNTPs, the forward and reverse primer of each 1 μ l 10 mM, 0.3 μ l 5U/ μ l LATaq enzyme, adds deionized water and is adjusted to final volume 25 μ 1; PCR reaction conditions is as follows: 95 ° of C denaturations 5 minutes; 92 ° of C sex change 10 seconds, 48 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations; 92 ° of C sex change 10 seconds, 50 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations, and per cycling time increases 20s; 72 ° of C extend 10 minutes again.
(5)pcr amplification product sequencing and splicing
The PCR primer sepharose of 1.0% carries out electrophoresis detection (the results are shown in Figure 1), all obtains the fragment of size between 1.0-4.0 kb.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.Homologous sequence comparison on sequencing result and GenBank, verified is after the specific amplified product of Mitochondrial Genome Overview sequence, utilizes DNAMAN software that each sequence is spliced to obtain mitochondrial genome complete sequence successively.
SEQUENCE LISTING
The <110> China Measures Institute
<120> spider mitochondrial genome complete sequence amplimer and amplification method
<130> 123456
<160> 12
<170> PatentIn version 3.3
<210> 1
<211> 25
<212> DNA
<213> artificial sequence
<400> 1
agctaataaa gctaatgagt tcata 25
<210> 2
<211> 25
<212> DNA
<213> artificial sequence
<400> 2
taattaattt aagagactaa cacct 25
<210> 3
<211> 21
<212> DNA
<213> artificial sequence
<400> 3
aatgttctga aatttgtggt g 21
<210> 4
<211> 23
<212> DNA
<213> artificial sequence
<400> 4
catactacat ctacaaaatg tca 23
<210> 5
<211> 23
<212> DNA
<213> artificial sequence
<400> 5
tgacattttg tagatgtagt atg 23
<210> 6
<211> 19
<212> DNA
<213> artificial sequence
<400> 6
aagctcatgt agaagctcc 19
<210> 7
<211> 19
<212> DNA
<213> artificial sequence
<400> 7
ggagcttcta catgagctt 19
<210> 8
<211> 23
<212> DNA
<213> artificial sequence
<400> 8
gaattagtat caggatttaa tgt 23
<210> 9
<211> 23
<212> DNA
<213> artificial sequence
<400> 9
acattaaatc ctgatactaa ttc 23
<210> 10
<211> 21
<212> DNA
<213> artificial sequence
<400> 10
tgctaaggta gcataatcat t 21
<210> 11
<211> 21
<212> DNA
<213> artificial sequence
<400> 11
aatgattatg ctaccttagc a 21
<210> 12
<211> 17
<212> DNA
<213> artificial sequence
<400> 12
tatgaaccca ttagctt 17
Claims (6)
1. a spider mitochondrial genome complete sequence amplimer, is characterized in that described amplimer is 6 pairs of amplimers: wherein,
Pair of primers:
Forward primer 1 is as shown in SEQ ID NO:1: 5 '-AGCTAATAAAGCTAATGAGTTCATA-3 ';
Reverse primer 1 is as shown in SEQ ID NO:2: 5 '-TAATTAATTTAAGAGACTAACACCT-3 ';
Second pair of primer:
Forward primer 2 is as shown in SEQ ID NO:3: 5 '-AATGTTCTGAAATTTGTGGTG-3 ';
Reverse primer 2 is as shown in SEQ ID NO:4: 5 '-CATACTACATCTACAAAATGTCA-3 ';
3rd pair of primer:
Forward primer 3 is as shown in SEQ ID NO:5: 5 '-TGACATTTTGTAGATGTAGTATG-3 ';
Reverse primer 3 is as shown in SEQ ID NO:6: 5 '-AAGCTCATGTAGAAGCTCC-3 ';
4th pair of primer:
Forward primer 4 is as shown in SEQ ID NO:7: 5 '-GGAGCTTCTACATGAGCTT-3 ';
Reverse primer 4 is as shown in SEQ ID NO:8: 5 '-GAATTAGTATCAGGATTTAATGT-3 ';
5th pair of primer:
Forward primer 5 is as shown in SEQ ID NO:9: 5 '-ACATTAAATCCTGATACTAATTC-3 ';
Reverse primer 5 is as shown in SEQ ID NO:10: 5 '-TGCTAAGGTAGCATAATCATT-3 ';
6th pair of primer:
Forward primer 6 is as shown in SEQ ID NO:11: 5 '-AATGATTATGCTACCTTAGCA-3 ';
Reverse primer 6 is as shown in SEQ ID NO:12: 5 '-TATGAACCCATTAGCTT-3 '.
2. based on a spider mitochondrial genome complete sequence amplification method for amplimer as claimed in claim 1, it is characterized in that the method is undertaken by with following step: (1) DNA extraction agent box method extracts the STb gene of spider; (2) the universal amplification primer of spider mitochondrial genome complete sequence is designed; (3) with spider total genomic dna for template, carry out long pcr amplification; (4) amplified production 1.0% agarose gel electrophoresis, amplified fragments is reclaimed in RNA isolation kit rubber tapping, order-checking and sequence assembly.
3. method according to claim 2, is characterized in that, for avoiding the pollution of spider intestinal contents in described step (1), casts out a belly, only extracts DNA from its leg.
4. method according to claim 2, is characterized in that, the universal amplification primer of described step (2) is 6 pairs of amplimers according to claim 1.
5. method according to claim 2, it is characterized in that, the PCR reaction system of described step (3) is 25 μ 1, wherein: 2 μ l template DNAs, 2.5 μ l 10 × LA PCR buffer, 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 LATaq enzyme, adds deionized water and is adjusted to final volume 25 μ 1; PCR reaction conditions is as follows: 95 ° of C denaturations 5 minutes; 92 ° of C sex change 10 seconds, 48 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations; 92 ° of C sex change 10 seconds, 50 ° of C anneal 10 seconds, and 68 ° of C extend 3 minutes, 20 circulations, and per cycling time increases 20s; 72 ° of C extend 10 minutes again.
6. method according to claim 2, is characterized in that, in described step (4), gel electrophoresis adopts 1.0% agarose gel electrophoresis; After order-checking is reclaimed in rubber tapping, each sequence splices to obtain mitochondrial genome complete sequence successively from beginning to end.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255872A (en) * | 2015-11-04 | 2016-01-20 | 广东省昆虫研究所 | Amplification primer group of whole enome sequence of manis javanica mitochondrion as well as amplification method and application of amplification primer group |
| 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 |
| CN109868272A (en) * | 2019-03-18 | 2019-06-11 | 湖南农业大学 | The amplimer and amplification method of dark fund reddish tone mouse mitochondria whole genome sequence |
-
2014
- 2014-12-11 CN CN201410753794.9A patent/CN104531688B/en active Active
Non-Patent Citations (4)
| Title |
|---|
| SHIN-JU PARK ET AL.: "The complete mitochondrial genome of the sea spider Acheliabituberculata (Pycnogonida, Ammotheidae): arthropod ground pattern of gene arrangement", 《BMC GENOMICS》 * |
| SUSAN E. MASTA AND JEFFREY L. BOORE: "The Complete Mitochondrial Genome Sequence of the Spider Habronattus oregonensis Reveals Rearranged and Extremely Truncated tRNAs", 《MOL. BIOL. EVOL.》 * |
| SUSAN E. MASTA: "Mitochondrial Sequence Evolution in Spiders: Intraspecific Variation in tRNAs Lacking the TCC Arm", 《MOL. BIOL. EVOL.》 * |
| ZHENG-LIANG WANG ET AL.: "The Complete Mitochondrial Genome of two Tetragnatha Spiders (Araneae: Tetragnathidae): Severe Truncation of tRNAs and Novel Gene Rearrangements in Araneae", 《INT. J. BIOL. SCI.》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105255872A (en) * | 2015-11-04 | 2016-01-20 | 广东省昆虫研究所 | Amplification primer group of whole enome sequence of manis javanica mitochondrion as well as amplification method and application of amplification primer group |
| 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 |
| CN109868272A (en) * | 2019-03-18 | 2019-06-11 | 湖南农业大学 | The amplimer and amplification method of dark fund reddish tone mouse mitochondria whole genome sequence |
| CN109868272B (en) * | 2019-03-18 | 2020-07-24 | 湖南农业大学 | Amplification primer and amplification method of blackhead red-headed mouse mitochondrion whole genome sequence |
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