CN105255872A - Amplification primer group of whole enome sequence of manis javanica mitochondrion as well as amplification method and application of amplification primer group - Google Patents
Amplification primer group of whole enome sequence of manis javanica mitochondrion as well as amplification method and application of amplification primer group Download PDFInfo
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- CN105255872A CN105255872A CN201510752411.0A CN201510752411A CN105255872A CN 105255872 A CN105255872 A CN 105255872A CN 201510752411 A CN201510752411 A CN 201510752411A CN 105255872 A CN105255872 A CN 105255872A
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Abstract
The invention discloses an amplification primer group of a whole enome sequence of manis javanica mitochondrion as well as an amplification method and application of the amplification primer group. The amplification primer group comprises the following five pairs of amplification primers: an Lsc primer pair: Lsc F: 5'-AGCGGTCAAATCCAGACTAA C-3', and Lsc R: 5'-AGAATGGGGTCACCTCCTCC-3'; an Lcc primer pair: Lcc F: 5'-CGTCAATCCTTGGGGCTATC-3', and Lcc R: 5'-GCCTGGGTTATGTGGTTTCG-3'; an Lcn primer pair: Lcn F: 5'-G GCATAATCCTGTTCATTGTCT-3', and Lcn R: 5'-AACGGGTGTCTGTCATCCTCT-3'; an Lnc primer pair: LncF: 5'-ACACAAACTATGAGCGAACCC-3', and Lnc R: 5'-AGGATGAAGTGAAGAGCGAAG-3'; and an Lcs primer pair: Lcs F: 5'-GGATTCTCGGTAGACAAAGC-3', and Lcs R: 5'-TCAATTAACGGATTATCTGGAC-3'.
Description
Technical field
The invention belongs to biology field, be specifically related to a kind of amplimer group of Manis javanica plastosome whole genome sequence and amplification method and application thereof.
Background technology
Manis javanica (Manisjavanica) is under the jurisdiction of Pholidota, Squama Manis section, Squama Manis genus, is mainly distributed in the ground such as Thailand, Malaysia, Burma, Indonesia, Singapore, lives in low altitude area forest and secondary forest.The scale of Manis javanica has very high pharmaceutical use, and in recent years, transition is caught and caused Manis javanica wild resource day by day to reduce, and is classified as pole danger animal by IUCN.In order to protect wild resource, recovery Manis javanica wild stocks quantity, studies its Genetic Constitution of Population and wild population heritable variation level is very necessary.
Molecule marker is the direct reflection to genetic diversity on DNA level, has quantity many, and polymorphism is good, the advantages such as inheritance stability, is the Perfected process of research heritable variation.Wherein, Mitochondrial Genome Overview is a very effective genetic marker, compared with Matrix attachment region, and the hereditary property of the uniquenesses such as Mitochondrial Genome Overview has matrilinear inheritance, structure is simple, evolutionary rate is fast; Compared with single chondriogen, mitochondrial genome complete sequence can provide the information of more horn of plenty.
Therefore, plastosome full-length genome is the effective tool carrying out Manis javanica group structure and Research on Genetic Variation, sets up the mitochondrial effective ways of a kind of amplification Manis javanica significant to wild resource protection and development.
Summary of the invention:
The object of this invention is to provide a kind of amplimer group of Manis javanica plastosome whole genome sequence; utilize this amplimer group specificly can amplify Manis javanica plastosome whole genome sequence, the Molecular Identification for the conservative genetics of Manis javanica, evolutionary genetics and species thereof provides basis.
The amplimer group of Manis javanica plastosome whole genome sequence of the present invention, is characterized in that, comprises following 5 pairs of amplimers:
Lsc primer pair:
LscF:5 '-AGCGGTCAAATCCAGACTAAC-3 ' (its nucleotide sequence is as shown in SEQIDNO.1)
LscR:5 '-AGAATGGGGTCACCTCCTCC-3 '; (its nucleotide sequence is as shown in SEQIDNO.2)
Lcc primer pair:
LccF:5 '-CGTCAATCCTTGGGGCTATC-3 ' (its nucleotide sequence is as shown in SEQIDNO.3)
LccR:5 '-GCCTGGGTTATGTGGTTTCG-3 '; (its nucleotide sequence is as shown in SEQIDNO.4)
Lcn primer pair:
LcnF:5 '-GGCATAATCCTGTTCATTGTCT-3 ' (its nucleotide sequence is as shown in SEQIDNO.5)
LcnR:5 '-AACGGGTGTCTGTCATCCTCT-3 '; (its nucleotide sequence is as shown in SEQIDNO.6)
Lnc primer pair:
LncF:5 '-ACACAAACTATGAGCGAACCC-3 ' (its nucleotide sequence is as shown in SEQIDNO.7)
LncR:5 '-AGGATGAAGTGAAGAGCGAAG-3 '; (its nucleotide sequence is as shown in SEQIDNO.8)
Lcs primer pair:
LcsF:5 '-GGATTCTCGGTAGACAAAGC-3 ' (its nucleotide sequence is as shown in SEQIDNO.9)
LcsR:5 '-TCAATTAACGGATTATCTGGAC-3 ' (its nucleotide sequence is as shown in SEQIDNO.10).
Second object of the present invention is to provide a kind of method of the Manis javanica plastosome whole genome sequence that increases, it is characterized in that, using Manis javanica complete genome DNA as template, using above-mentioned 5 pairs of amplimers as primer pair, carry out pcr amplification, then, after the PCR primer order-checking obtained increasing, splicing obtains Manis javanica plastosome whole genome sequence (its nucleotide sequence is as shown in SEQIDNO.11).
3rd object of the present invention is to provide the amplimer group of above-mentioned Manis javanica plastosome whole genome sequence in the application obtained in Manis javanica plastosome whole genome sequence of increasing.
The present invention sets up the effective ways of amplification Manis javanica Mitochondrial Genome Overview; utilize this amplimer group specificly can amplify Manis javanica plastosome whole genome sequence, the Molecular Identification for the conservative genetics of Manis javanica, evolutionary genetics and species thereof provides basis.
Embodiment:
Following examples further illustrate of the present invention, instead of limitation of the present invention.
Embodiment 1:
The amplimer of Manis javanica plastosome whole genome sequence of the present invention is 5 right, primer sequence and amplification scope as shown in table 1:
Table 1: Manis javanica Mitochondrial Genome Overview amplified fragments and primer
| Amplified fragments | Upstream primer | Downstream primer |
| Lsc(2280-6014) | AGCGGTCAAATCCAGACTAAC | AGAATGGGGTCACCTCCTCC |
| Lcc(5792-9091) | CGTCAATCCTTGGGGCTATC | GCCTGGGTTATGTGGTTTCG |
| Lcn(8850-11696) | GGCATAATCCTGTTCATTGTCT | AACGGGTGTCTGTCATCCTCT |
| Lnc(11155-14695) | ACACAAACTATGAGCGAACCC | AGGATGAAGTGAAGAGCGAAG |
| Lcs(14640-2324) | GGATTCTCGGTAGACAAAGC | TCAATTAACGGATTATCTGGAC |
(1) extraction of Manis javanica genomic dna
Tissue, in 2mL centrifuge tube, shreds by clip Manis javanica muscle tissue, and add 800 μ L and digest damping fluid, 10 μ L Proteinase Ks (20mg/mL), spend the night in 55 DEG C of digestion after mixing.After digestion clearly thoroughly, add the saturated phenol of 800 μ L, put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant.Add the saturated phenol of 400 μ L, 400 μ L chloroform/primary isoamyl alcohol (24:1), put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant.Add 800 μ L chloroform/primary isoamyl alcohol (24:1), put upside down the centrifugal 10min of mixing 20min, 10000g gently, extracting supernatant.Add the dehydrated alcohol of 1mL precooling, in-20 DEG C of centrifugal 15min of freezing 2h, 12000g, abandon supernatant.Add 70% ethanol of precooling, careful washing DNA precipitates the centrifugal 5min of twice, 12000g, abandons supernatant.Dry ethanol, add 50 μ L ultrapure water dissolution precipitations, be placed in-20 DEG C and save backup.Obtain Manis javanica genomic dna
(2) pcr amplification 5 long segment
The long round pcr of Mitochondrial Genome Overview DNA fragmentation increases, LA-PCR amplification system is 25 μ L, comprise the Manis javanica genomic dna that about 10ng step (1) obtains, 2.5 μ L10 × LAPCRbufferII, 0.4mMdNTP, (5 pairs of primers in table 1 increase upstream and downstream primer respectively, namely increase respectively with Lsc primer pair, Lcc primer pair, Lcn primer pair, Lnc primer pair and Lcs primer pair) each 0.4 μM, 1.25ULATaq polysaccharase (Takara).Pcr amplification condition is: first 94 DEG C of denaturation 4min, and then carry out 30 circulations, each circulation comprises 94 DEG C of sex change 30sec, 55 DEG C of annealing 30sec, and 72 DEG C extend 2-4min, and last 72 DEG C extend 10min.Directly carry out two-way order-checking after PCR primer purifying, then sequencing primer in the middle of design, walks the method for moving by primer and progressively surveys logical whole fragment.Sequencing result sequencher4.2 (GeneCodes, AnnArbor, MI, USA) software splicing, first by complete for each long segment splicing, five long segment be stitched together, obtain complete Manis javanica mitochondrial genome complete sequence (its nucleotide sequence is as shown in SEQIDNO.11), its structure is as shown in table 2 again.
The 2280-6014bp of Lsc primer pair amplifies Manis javanica plastosome whole genome sequence, the 14640-2324bp of 11155-14695bp and the Lcs primer pair amplifies Manis javanica plastosome whole genome sequence of 8850-11696bp, Lnc primer pair amplifies Manis javanica plastosome whole genome sequence of 5792-9091bp, Lcn primer pair amplifies Manis javanica plastosome whole genome sequence of Lcc primer pair amplifies Manis javanica plastosome whole genome sequence.
Table 2 Manis javanica Mitochondrial Genome Overview structure
Claims (3)
1. an amplimer group for Manis javanica plastosome whole genome sequence, is characterized in that, comprises following 5 pairs of amplimers:
Lsc primer pair:
LscF：5’-AGCGGTCAAATCCAGACTAAC-3’
LscR：5’-AGAATGGGGTCACCTCCTCC-3’；
Lcc primer pair:
LccF：5’-CGTCAATCCTTGGGGCTATC-3’
LccR：5’-GCCTGGGTTATGTGGTTTCG-3’；
Lcn primer pair:
LcnF：5’-GGCATAATCCTGTTCATTGTCT-3’
LcnR：5’-AACGGGTGTCTGTCATCCTCT-3’；
Lnc primer pair:
LncF：5’-ACACAAACTATGAGCGAACCC-3’
LncR：5’-AGGATGAAGTGAAGAGCGAAG-3’；
Lcs primer pair:
LcsF：5’-GGATTCTCGGTAGACAAAGC-3’
LcsR：5’-TCAATTAACGGATTATCTGGAC-3’。
2. the method for Manis javanica plastosome whole genome sequence that increases, it is characterized in that, using Manis javanica complete genome DNA as template, using 5 pairs of amplimers according to claim 1 as primer pair, carry out pcr amplification, then, after the PCR primer order-checking obtained increasing, splicing obtains Manis javanica plastosome whole genome sequence.
3. the amplimer group of Manis javanica plastosome whole genome sequence according to claim 1 is in the application obtained in Manis javanica plastosome whole genome sequence of increasing.
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| CN106434632A (en) * | 2016-10-10 | 2017-02-22 | 南方医科大学 | DNA extraction method of squama manitis |
| CN109430564A (en) * | 2018-12-29 | 2019-03-08 | 广西壮族自治区林业科学研究院 | A kind of Manis javanica special feed and preparation method thereof |
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| CN109430564A (en) * | 2018-12-29 | 2019-03-08 | 广西壮族自治区林业科学研究院 | A kind of Manis javanica special feed and preparation method thereof |
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Application publication date: 20160120 |