CN107513534A - A kind of huge furuncle bat moth DNA bar code standard gene sequence and its application - Google Patents
A kind of huge furuncle bat moth DNA bar code standard gene sequence and its application Download PDFInfo
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Abstract
The present invention disclose a kind of huge furuncle bat moth [Endoclita davidi(Poujade, 1886), different namePhassus giganodus(Chu and Wang, 1985)] DNA bar code standard gene sequence, it is characterised in that the bar code standards detection gene is CO I genes, has gene order SEQ ID NO.1.Sequencing result carries out Blast similarity searchings by manual check and correction, sequence assembly in NCBI, it is ensured that gained sequence is target sequence.The Identification of Species of huge furuncle bat moth is realized by Morphological Identification, so as to ensure that the reliability of result.Huge furuncle bat moth DNA bar code standard gene sequence provided by the invention, is advantageously implemented to different worm states（Including adult, ovum, larva, pupa）With imperfect sample（Include the portion of tissue of muscle, foot, wing and body wall）And quick, the precise Identification of Cordyceps Militaris host, shorten qualification time.
Description
Technical field
The present invention relates to a kind of huge furuncle bat moth DNA bar code standard gene sequence, the identification for the species.
Background technology
Huge furuncle bat moth Endoclita davidi (Poujade, 1886) different name Phassus giganodus (Chu and
Wang, 1985) it is under the jurisdiction of Hepialidae (Hepialoidea) bat moth category (Endoclita), is a kind of moth dryness insect, 2 years
A generation, larva mainly eat into food Verenaceae (Verbenaceae) medicinal plant smalt (Clerodendrum cyrtophyllum
) etc. Turcz. stem and root.Meanwhile huge furuncle bat moth larvae can be by a kind of important nematode grass Cordycepps fungi snowy peak Cordyceps Militaris
(Ophiocordyceps xuefengensis) is parasitic and forms entomogenous fungi combination-snowy peak cordyceps sinensis.Snowy peak cordyceps sinensis is as precious jade medicine
Usage history is long, is mainly used in treating PUD D, after compatibility to hypertension, virus hepatitis, rheumatoid arthritis and
The diseases such as gout also have the effect of good.
Identification and identification to huge furuncle bat moth, are the bases that the worm Resources of Entomogenous Fungi is develop and useedd, and bat moth
The core procedure of section's Molecular Phylogeny of Some Species research.At present, the identification to huge furuncle bat moth relies primarily on veteran insect
Systematist realizes that the identification of other worm states needs to raise to adult, and breeding cycle is long, hardly possible according to the morphological feature of adult
Degree is big；Especially the host of bombys batryticatus state after the incomplete sample of form and Cordyceps Militaris parasitism is just more difficult to identify.Therefore, it is badly in need of
Seek a kind of new quick accurate method, to make up the deficiency of traditional form sorting technique.
DNA bar code technology (DNA Barcoding) be using standard, have enough variations, easily amplification and relatively
Short DNA fragmentation (DNA barcode) creates a kind of new from the specificity in species kind and the diversity of inter-species
Biological status identification system, it can carry out quickly automatic identification to species.In recent years, bar codes technique had proved to be
One effective bioassay means, it can not only make strong supplement to conventional identification method, and because it is more objective
See, be accurate, breakthrough was depended on unduly to experience in the past, can help to identify species, discovery novel species and hidden kind, reconstruction species and advanced
Evolvement of rank member etc..
It as the standard target gene of DNA bar code, on the one hand must enough guard, be easy to carry out greatly using universal primer
The amplification of scope；On the other hand enough variations distinguish different plant species.Therefore, identify that different species monoids needs to select
Select effective target gene.Cytochrome oxidase subunit I (Cytochrome Oxidase in mitochondrial genomes
Subunit I, CO I) gene is because having a variety of advantages：1. in animal life all there is obvious CO I bases in most stages
Because of sequence；2. there is the mitochondria of more than up to a hundred in most cells, but an only group chromosome, therefore in the sample of equivalent
The DNA of mitochondria is easier to be exaggerated and used；3. compared with the DNA of nucleus, the mutating speed of mitochondrial DNA is core DNA
10 times, make species separation it is more accurate；4. the mode of inheritance of mitochondria belongs to matrocliny：CO I genes are located at cell mitochondrial
In, can only be hereditary from parent, therefore the incidence of genetic recombination is low, and the gene of most cells core is from parent and male parent
What coinheritance got off, the incidence of genetic recombination is higher；5.CO I genes possess the feature common to protein coding gene, i.e.,
The bit base of codon the 3rd is not influenceed by natural selection pressure, can be with free variation；6.CO I genes can ensure to become enough
It is easy to be expanded by universal primer again while different, and its DNA sequence dna seldom has insertion and missing in itself, makes sequence alignment
Easily operation；7.Hebert etc. (2003) is to the animal kingdom, including vertebrate and invertebrate totally 11 13320 species
The comparative analysis of CO I gene sequences is drawn：In addition to coelenterate Cnidaria, the difference of 98% species genetic distance is being planted
It is inside 0%~2%, inter-species averagely can reach 11.3%.A fragment in the generally selected CO I genes of old friends is as DNA bars
Shape encoding gene.So far, existing many research has shown that CO I genes in birds, fish, lepidopterous insects, mosquito class, ant class
With it is effective in the taxonomic identification of the monoid such as Collembola.But the report of the related gene sequence of huge furuncle bat moth is there is no, this is not to
With the identification of worm state, the incomplete huge furuncle bat moth sample of form and Cordyceps Militaris host, and the molecular system of Hepialidae insect
It is an obstacle to develop research.
The content of the invention
It is an object of the invention to provide a kind of huge furuncle bat moth DNA bar code standard gene sequence, huge furuncle bat moth is realized
Quick and precisely identify.
A kind of huge furuncle bat moth DNA bar code standard detection gene, it is characterised in that the bar code standards detect gene and are
CO I genes, there is gene order SEQ ID NO.1 as described below：
The PCR primer of described huge furuncle bat moth DNA bar code standard detection gene, it is characterised in that PCR primer sequence is：
- the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 '
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of reverse primer 5 '.
The method for identifying molecules of the huge furuncle bat moth of the present invention, including：
1) the separation and Extraction DNA from huge furuncle bat moth tissue to be measured；
2) using the DNA as template pair of primers ,-the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 ', reversely draw
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of thing 5 ', huge furuncle bat moth CO I genes are gone out by PCR amplification,
PCR condition is：94 DEG C of pre-degeneration 10min；94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 40s, 32 circulations；72 DEG C of extension 10min；
PCR system is：25ul：2 × Mastermix12.5ul, upstream and downstream primer each 1ul, ddH2O7.5ul, template 3ul；
3) the CO I genes for taking the PCR amplification of appropriate step 2) to go out are separated with agarose electrophoresis, and uviol lamp is seen
Examine, according to the size result of determination of amplified production, specific amplification goes out 658bp or so band, send biotech firm to be sequenced；
4) according to sequencing result, the homology with the gene order SEQ ID NO.1 is more than 98%, you can described in judging
Sample, tissue and Cordyceps Militaris host to be measured are huge furuncle bat moth.
According to DNA bar code technical principle, using insect trace DNA templet preparation method, bar is reacted by improved PCR
Part, the CO I genes of huge furuncle bat moth are expanded by the primer synthesized, PCR primer sequence is sent to be determined by professional biotech firm.Sequencing knot
Fruit carries out Blast similarity searchings by manual check and correction, sequence assembly in NCBI, it is ensured that gained sequence is target sequence.
The Identification of Species of huge furuncle bat moth is realized by Morphological Identification, so as to ensure that the reliability of result.
The primer is described according to document and improved ,-the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 ', reversely
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of primer 5 '.
The present invention can use agarose gel electrophoresis detection method detection amplification.
Compared with traditional Morphological Identification method, the gene order that the present invention obtains can be used for the molecule mirror of huge furuncle bat moth
It is fixed, it can accurately and effectively shorten the qualification time of huge furuncle bat moth.
Brief description of the drawings
Fig. 1 is the techniqueflow chart of the present invention.
Fig. 2 is 16 sample CO I gene PCR amplification electroresis appraisal figures of the huge furuncle bat moth of the present invention.Number explanation
It is as follows：Swimming lane 1~16 is the huge furuncle bat moth difference worm state of field acquisition and CO I genes (totally 15 cephalont, wherein numbering of tissue
For 11,12 samples for being same polypide different tissues, 1) specifying information of each sample is shown in Table, the band that detection size is 658bp.
M is 2000plus DNA marker.
The sample information table of table 1
| Sample number | Test worm state | Test organization | Acquisition time (years months) | Gather worm state |
| 1 | Ovum | All | 2015/11 | Larva |
| 2 | Larva | Pereiopoda | 2015/11 | Larva |
| 3 | Larva | Pereiopoda | 2015/05 | Larva |
| 4 | Larva | Pereiopoda | 2015/05 | Larva |
| 5 | Larva | Pereiopoda | 2015/09 | Larva |
| 6 | Larva | Pereiopoda | 2015/05 | Larva |
| 7 | Larva | Pereiopoda | 2015/09 | Larva |
| 8 | Pupa | Body wall | 2015/05 | Larva |
| 9 | Pupa | Body wall | 2015/07 | Larva |
| 10 | Pupa | Body wall | 2015/09 | Larva |
| 11 | Adult | Pereiopoda | 2015/07 | Larva |
| 12 | Adult | Chest muscle | 2015/07 | Larva |
| 13 | Adult | Wing base portion | 2015/10 | Larva |
| 14 | Adult | Pereiopoda | 2015/05 | Larva |
| 15 | Cordyceps sinensis bombys batryticatus | Body wall | 2015/11 | Cordyceps sinensis bombys batryticatus |
| 16 | Cordyceps sinensis bombys batryticatus | Pereiopoda | 2016/03 | Cordyceps sinensis bombys batryticatus |
Fig. 3 is the snowy peak cordyceps sinensis bombys batryticatus CO I gene PCR amplification electroresis appraisal figures of the present invention.Numbering is described as follows：Swimming lane 1
~3 be the CO I genes of three wild snowy peak cordyceps sinensis bombys batryticatus of field acquisition, detects the band that size is 658bp or so.M is
2000plus DNA marker.
Embodiment
Embodiment 1
1st, the collection and preservation of huge furuncle bat moth sample
Larva and the equal field acquisition of cordyceps sinensis bombys batryticatus sample are obtained, and larva is stored in 75% alcohol, and cordyceps sinensis bombys batryticatus sample is naturally dry
It is put into after dry in the hermetic bag equipped with silica gel, is placed in Riker mount and preserves；Pupa and adult obtain for the larva raising of field harvest,
Pupa is stored in 75% alcohol, and adult is put in centrifuge tube in 20 DEG C of freezen protectives of ﹣；Ovum is produced by the adult of raising, is stored in 8 DEG C
Growth cabinet.
2nd, the pre-treatment of test specimen
15 are taken out from the huge furuncle bat moth sample of the different worm states of above-mentioned preservation, carefully a small amount of respective organization is removed with tweezers, moves
Enter in EP pipes, often pipe one, and carry out unique number (numbering 1 is simple grain worm's ovum, and 2~7 be respectively the pereiopoda of six larvas, 8~
10 be three pupa body wall tissues, and 11,12 be respectively pereiopoda and the chest muscle part of same head adult, and 13 be the wing base of an adult
Portion, 14 be the pereiopoda of an adult, and 15,16 be respectively body wall and the pereiopoda part of two end cordyceps sinensis bombys batryticatus) (details are shown in Table 1), use
Extracted in DNA.Remainder continues to preserve to carry out Identification of Species.
3rd, prepared by DNA profiling
Sample is extracted using blood tissues cellular genome extracts kit (DP304, Hunan inscriptions on bones or tortoise shells biological medicine Co., Ltd)
Product genomic DNA saves backup for 20 DEG C in ﹣.
4th, primer synthesizes
The present embodiment the primer is as follows：
- the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 '
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of reverse primer 5 '
5th, PCR is expanded
The PCR reaction systems of embodiment 1 are as shown in table 2.
The huge furuncle bat moth CO I genes PCR reaction systems of table 2 (25ul systems)
| Composition | Volume (ul) |
| Template | 3 |
| Primer 1 | 1 |
| Primer 2 | 1 |
| 2×Mastermix | 12.5 |
| ddH2O | 7.5 |
The response procedures of the present embodiment are as shown in table 3.
The huge furuncle bat moth CO I genes PCR response procedures of table 3
PCR primer saves backup in 4 DEG C.
6th, pcr amplification product detects
5ul pcr amplification products are taken, with 1% agargel electrophoresis (130V voltages, electrophoresis 10min).Gel imaging system detects,
The electrophoresis pattern of the amplified production of 1~No. 16 sample is referring to accompanying drawing 2.It can be seen that 16 samples of embodiment 1 can specificity
Amplify 658bp or so product.
7th, gene sequencing
The PCR products for expanding to obtain are sent and are sequenced after purification by biotech firm (sequencing primer is identical with PCR the primers), are obtained
It is objective gene sequence after the calibrated splicing of gene order.Sequencing result shows 16 samples and the gene order of the present invention
SEQ ID NO.1 homology can confirm that 16 samples of the different worm states belong to huge furuncle 98.7%~100%
Bat moth.
Embodiment 2
1st, the selection and preservation of sample
Field acquisition snowy peak cordyceps sinensis bombys batryticatus, it is put into after drying in the hermetic bag equipped with silica gel, is placed in Riker mount and preserves.
2nd, the pre-treatment of sample
The snowy peak cordyceps sinensis bombys batryticatus sample three preserved is taken out, the cordyceps sinensis bombys batryticatus body wall of sesame seed size is carefully removed with tweezers, is moved
Enter in EP pipes, and carry out unique number (numbering 1~3 is respectively the body wall of three snowy peak cordyceps sinensis bombys batryticatus of field harvest), be used for
DNA is extracted.Remainder continues to preserve stand-by.
3rd, prepared by DNA profiling
Sample is extracted using blood tissues cellular genome extracts kit (DP304, Hunan inscriptions on bones or tortoise shells biological medicine Co., Ltd)
Product genomic DNA saves backup for 20 DEG C in ﹣.
4th, primer synthesizes
The present embodiment the primer is as follows：
- the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 '
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of reverse primer 5 '
5th, PCR is expanded
The PCR reaction systems of the present embodiment are as shown in table 4.
The snowy peak cordyceps sinensis bombys batryticatus CO I gene PCR reaction systems (25ul systems) of table 4
The response procedures of the present embodiment are as shown in table 5.
The snowy peak cordyceps sinensis bombys batryticatus CO I gene PCR response procedures of table 5
PCR primer saves backup in 4 DEG C.
6th, pcr amplification product detects
5ul pcr amplification products are taken, with 1% agargel electrophoresis (130V voltages, electrophoresis 10min).Gel imaging system detects,
Electrophoresis pattern is respectively that the body wall of three snowy peak cordyceps sinensis bombys batryticatus extracts DNA amplified production referring to accompanying drawing 3,1~3.It can see
Go out three snowy peak cordyceps sinensis bombys batryticatus of embodiment 2 also can specific amplification go out 658bp or so product.
7th, gene sequencing
The PCR products for expanding to obtain are sent and are sequenced after purification by biotech firm (sequencing primer is identical with PCR the primers), are obtained
It is objective gene sequence after the calibrated splicing of gene order.Sequencing result shows three samples and the gene order of the present invention
SEQ ID NO.1 homology can confirm that the worm of three snowy peaks cordyceps sinensis bombys batryticatus sample 98.9%~99.7%
Careless bacterium host is huge furuncle bat moth.
Sequence table
<110>Hunan Normal University
<120>A kind of huge furuncle bat moth DNA bar code standard gene sequence and its application
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 658
<212> DNA
<213>Huge furuncle bat moth DNA
<400> 1
aactttatat tttatttttg gtatttgagc aggaataatt ggtacttcat taagattatt 60
aattcgaaca gaattaggaa acccaggatc tttaattgga gatgatcaaa tttataatgt 120
aattgtaaca gcacatgctt ttattataat tttttttata gttataccaa ttataattgg 180
tggatttggt aattgattag tgccattaat attaggagca ccagatatag cattcccacg 240
attaaataat ataagatttt gattattacc cccatcatta atattattaa tctctagaag 300
aattgttgaa aatggggcag gaacaggttg aactgtctat cccccattat ctgcaaatat 360
tgcacatgca ggaagatctg tagatttagc aattttttct ttacatttag caggtatttc 420
ttctatttta ggtgcagtta attttattac aactgtaatt aacatacgat cagaaggaat 480
atcttttgat cgcatacctt tatttgtttg aagagttgca attactgctt tattactttt 540
attatcctta cctgtattag caggagctat tactatatta ttaacagatc gaaatttaaa 600
tacctcattt tttgaccctg caggaggggg agatcctatt ttatatcaac atttattt 658
<210> 2
<211> 25
<212> DNA
<213>Forward primer
<400> 2
attcaaccaa tcataaagat attgg 25
<210> 3
<211> 26
<212> DNA
<213>Reverse primer
<400> 3
taaacttctg gatgtccaaa aaatca 26
Claims (3)
1. a kind of huge furuncle bat moth [Endoclita davidi(Poujade, 1886), different namePhassus giganodus
(Chu and Wang, 1985)] DNA bar code standard detection gene, it is characterised in that the bar code standards detect gene
For CO I genes, there is gene order SEQ ID NO.1 as described below：
aactttatat tttatttttg gtatttgagc aggaataatt ggtacttcat taagattatt 60
aattcgaaca gaattaggaa acccaggatc tttaattgga gatgatcaaa tttataatgt 120
aattgtaaca gcacatgctt ttattataat tttttttata gttataccaa ttataattgg 180
tggatttggt aattgattag tgccattaat attaggagca ccagatatag cattcccacg 240
attaaataat ataagatttt gattattacc cccatcatta atattattaa tctctagaag 300
aattgttgaa aatggggcag gaacaggttg aactgtctat cccccattat ctgcaaatat 360
tgcacatgca ggaagatctg tagatttagc aattttttct ttacatttag caggtatttc 420
ttctatttta ggtgcagtta attttattac aactgtaatt aacatacgat cagaaggaat 480
atcttttgat cgcatacctt tatttgtttg aagagttgca attactgctt tattactttt 540
attatcctta cctgtattag caggagctat tactatatta ttaacagatc gaaatttaaa 600
tacctcattt tttgaccctg caggaggggg agatcctatt ttatatcaac atttattt 658。
2. the PCR primer of the huge furuncle bat moth DNA bar code standard detection gene described in claim 1, it is characterised in that PCR primer
Sequence is：
- the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 '
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of reverse primer 5 '.
3. a kind of method for identifying molecules of huge furuncle bat moth, including：
1）The separation and Extraction DNA from huge furuncle bat moth tissue to be measured；
2）Using the DNA as template pair of primers ,-the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 ', reversely draw
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of thing 5 ', huge furuncle bat moth CO I genes are gone out by PCR amplification；
3）Take appropriate step 2）The CO I genes that go out of PCR amplification separated with agarose electrophoresis, uviol lamp is seen
Examine, according to the size result of determination of amplified production, specific amplification goes out 658bp or so band, send biotech firm to be sequenced；
4）According to sequencing result, if with the homology of the gene order SEQ ID NO.1 more than 98%, you can judge institute
Sample to be measured, tissue or the Cordyceps Militaris host stated is huge furuncle bat moth.
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