CN107523577A - A kind of specific DNA sequences for identifying furuncle bat moth - Google Patents
A kind of specific DNA sequences for identifying furuncle bat moth Download PDFInfo
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Abstract
The present invention, which disclose, a kind of identifies furuncle bat moth Endoclita nodus (Chu and Wang, 1985) (different name：Phassus nodus Chu et Wang, 1985) specific DNA sequences, it is characterised in that the specific 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 particular target DNA sequence dna.The Identification of Species of furuncle bat moth is realized by Morphological Identification, so as to ensure that the reliability of result.Furuncle bat moth specific DNA sequences provided by the invention, it is advantageously implemented quick, precise Identification to furuncle bat moth difference worm state and imperfect sample.
Description
Technical field
The present invention relates to a kind of furuncle bat moth DNA bar code standard gene sequence, the quick and precisely identification for the species.
Background technology
Furuncle bat mothEndoclita nodus(Chu and Wang, 1985) (different name： Phassus nodus ChuetWang, 1985）, Hepialidae（Hepialoidea）Bat moth category（Endoclita）, be southern region of China it is common one
Kind moth dryness insect, especially likes the seeds such as thermophilic smalt.Larva moth food bast and xylem, the lighter make killed plant strain growth not
Good, timber can not utilize, and easily folding is or withered for severe one plant.Its larva can be formed entomogenous fungi and be combined by Cordyceps Militaris parasitism
Body --- cordyceps sinensis.Cordyceps sinensis is rare medicinal herbs special simply, can strengthen the immunity of body, nourishing lung and kidney, to lung cancer, liver cancer
Etc. there is obvious inhibitory action.Clinically to chronic cough of deficiency lung, asthma, hemoptysis of pulmonary tuberculosis, night sweat, kidney deficiency soreness of waist and knee joint, impotence
Pass out semen, the red blood cell decreased after neurasthenia and chemotherapy, radiotherapy is all effective in cure.
Accurately identifying and identifying to furuncle bat moth, both contributed to forest department take targetedly controlling measurement its to forest
Harm, it is the basis of the worm grass resources utilization of the worm again, meanwhile, and Hepialidae Molecular Phylogeny of Some Species research
Core procedure.At present, it is special according to the morphology of adult to rely primarily on veteran classification of insect scholar for the identification to furuncle bat moth
Levy to realize, the identification of other worm states needs to raise to adult.Because the worm breeding cycle is long, difficulty is big, especially to form not
Complete sample, different worm states and larva are just more difficult to distinguish and identify, are badly in need of seeking by the bombys batryticatus state after Cordyceps Militaris parasitism
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.Briefly, it is exactly by using one or one
A little short DNA genetic fragments carry out technology that is quick, accurately identifying to species as bar code.In recent years, bar code skill
Art has proved to be an effective bioassay means, can not only make strong supplement to conventional identification method, and
And because it is more objective, accurate, breakthrough was depended on unduly to experience in the past, it can help to identify species, discovery novel species and hidden kind, again
Build Evolvement of species and higher level taxa etc..
Preferable DNA barcoding should meet following standard：(1) there is enough variability to distinguish different things
Kind, while there is relative conservative；(2) must be that the region of DNA of a segment standard differentiates different taxons as far as possible；(3)
Target dna area should include enough phyletic evolution information to position position of the species in categorizing system (section, category etc.)；(4)
Should be highly conserved design of primers area in order to the design of universal primer；(5) target dna area should be enough it is short in order to
There is the DNA of Partial digestion amplification.At present, wide variety of marker gene is ribosomes 12S and 16S in systematic growth research
Gene, but because it has substantial amounts of insertion and deficient phenomena, bar coding gene should not be used as.Present in mitochondrial genomes
Cytochrome oxidase subunit I in 13 protein coding genes（Cytochrome Oxidase Subunit I,CO I）Gene
Meet gene insertion and deficient phenomena is less, length easily designs universal primer in the two conditions of 900bp or so, therefore
People finally select a fragment in CO I genes as DNA bar coding genes.So far, existing many research has shown that
CO I genes are effective in the taxonomic identification of the monoids such as birds, fish, lepidopterous insects, mosquito class, ant class and Collembola, because
This, can use and carry out Molecular Identification to furuncle bat moth based on the DNA bar code technology of CO I genes.This method and traditional classification side
Method is proved each other, can not only solve the problems, such as the species identification of furuncle bat moth difference worm state and incomplete sample, and be advantageous to reality
The now Rapid identification of the worm.At present, in American National Biotechnology Information center（National Center for
Biotechnology Information, NCBI）In database, the relative recording of furuncle bat moth is there is no, also without any related report
Road.DNA bar code standard gene sequence provided by the invention, it is possible to achieve the Rapid identification of furuncle bat moth, shorten qualification time.
The content of the invention
It is an object of the invention to provide a kind of furuncle bat moth DNA bar code standard gene sequence, the quick of furuncle bat moth is realized
Precise Identification.
A kind of furuncle bat moth DNA bar code standard detection gene, it is characterised in that the bar code standards detection gene is CO
I gene, there is gene order SEQ ID NO.1 as described below：
ttttattttt ggtatttgag caggaataat tggaacttcg ttaagattat taattcgaac 60
agaattaggt aatcctggat ctttaattgg agatgatcaa atttataatg taattgtaac 120
agcacatgct tttattataa ttttttttat agttatacct attataattg gtggatttgg 180
taattgatta gtacctttaa tattaggagc tcctgatata gcttttccac gattaaataa 240
tataagattt tgattattac ccccttcatt aatactatta atttctagaa gaattgtaga 300
aaatggagct ggaacaggtt gaacagtcta tcccccatta tctgcaaata ttgcacatgc 360
tggtagatca gtggatttag caattttttc cttacattta gcaggtattt catctatttt 420
aggggcagtc aattttatta caactgtaat taatatacga tcggaaggaa tatcttttga 480
ccgaatacca ttatttgtat gaagagttgc aattactgct ttattacttt tattatcttt 540
acctgtatta gcaggagcta ttactatatt actaacagac cgaaatttaa atacttcatt 600
ttttgaccct gctgggggag gggatcctat tttataccaa catttatttt gatttttt 658。
The PCR primer of described 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 furuncle bat moth of the present invention, including：
1）The separation and Extraction DNA from furuncle bat moth tissue to be measured；
2）Using the DNA as template pair of primers：- the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 ', reversely draws
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of thing 5 ', 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 50s, 32 circulations；72 DEG C of extension 10min.
PCR system is：2 × Mastermix 12.5ul, upstream and downstream primer each 1ul, ddH2O 7.5ul, template 3ul, common 25ul；
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, the homology with the gene order SEQ ID NO.1 is more than 98%, you can described in judging
Sample to be measured is 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 furuncle bat moth are expanded by the primer synthesized, PCR primer sequence is sent to be determined by professional biotech firm.Sequencing result
By manual check and correction, sequence assembly, and Blast similarity searchings are carried out in NCBI, it is ensured that gained sequence is target sequence.Furuncle
The Identification of Species of 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 ', instead
To-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 furuncle bat moth species
It is fixed, it can accurately and effectively shorten qualification time.
Brief description of the drawings
Fig. 1 is the techniqueflow chart of the present invention.
Fig. 2 is 11 samples of the furuncle bat moth of the embodiment of the present invention 1（Numbering 1~11, including adult and pupa）CO I genes
PCR amplification electroresis appraisal figures.Numbering is described as follows：Swimming lane 1~10 is the CO I genes of ten furuncle bat moth adult extractions, is swum
Road 11 is the CO I genes of furuncle bat chrysalis extraction（The specifying information of each sample is shown in Table 1）, detect the band that size is 658bp.M is
2000 plus DNA marker.
Fig. 3 is 11 samples of the furuncle bat moth of the embodiment of the present invention 2（Numbering 12~22, including ovum, larva and pupa）CO I bases
Because of PCR amplification electroresis appraisal figures.Numbering is described as follows：Swimming lane 12 is the CO I genes of furuncle bat moth ovum extraction, and 13~19 are
The CO I genes of seven larva different tissues extractions of furuncle bat moth, 20~22 be the CO I bases of three pupa different tissues extractions of furuncle bat moth
Cause（The specifying information of each sample is shown in Table 1）, detect the band that size is 658bp.M is 2000plus DNA marker.
The sample information table of table 1
Embodiment
Embodiment 1
1st, the collection and preservation of furuncle bat moth sample
Field acquisition pupa, indoor feeding to adult, Morphological Identification are defined as furuncle bat moth.In 20 DEG C of ﹣ in adult sealed plastic pipe
Preserve, pupa is stored in 75% alcohol.
2nd, the pre-treatment of test specimen
11 are taken from the furuncle bat moth sample of above-mentioned preservation（Numbering 1 ~ 3 picks up from Wangcheng County of Hunan Province, that numbering 4 ~ 6 picks up from Hunan Province is peaceful
Hunan Province's Dongkou County is picked up from county of township, numbering 7 ~ 9, numbering 10 and 11 picks up from Hunan Province Miluo City）, carefully remove adult one with tweezers
Pereiopoda, pupa take its body wall, move into EP pipes, often pipe one, and carry out unique number, are extracted for DNA.Remainder continues to
Preserve in case carrying out further Identification of Species.
3rd, prepared by DNA profiling
Using blood tissues cellular genome extracts kit（DP304, Hunan inscriptions on bones or tortoise shells biological medicine Co., Ltd）Extract sample
Product genomic DNA saves backup for 20 DEG C in ﹣.
4th, primer synthesizes
The primer of the present embodiment 1 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 furuncle bat moth CO I gene PCR reaction systems of table 2（25ul systems）
The response procedures of the present embodiment 1 are as shown in table 3.
The furuncle bat moth CO I gene 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, and 1
The electrophoresis pattern of the amplified production of~No. 11 samples is referring to accompanying drawing 2（Swimming lane numbering is corresponding with sample number into spectrum）.It can be seen that implement
11 samples of example 1 energy specific amplification goes 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）, obtain
It is objective gene sequence after the calibrated splicing of gene order.The judgement of qualification result：Sequencing result shows 11 samples and this
The gene order SEQ ID NO.1 of invention homology can confirm that the COI genes can be used for more than 99.2%
The Identification of Species of furuncle bat moth.
Embodiment 2
1st, the collection and preservation of furuncle bat moth sample
Field harvest larva and pupa, indoor feeding to adult, larva and pupa are stored in 75% alcohol, and adult is stored in -20 DEG C of ice
Case；Ovum is produced by the furuncle bat moth adult of raising, is stored in 8 DEG C of growth cabinets.
2nd, the pre-treatment of sample
11 samples are taken out from the furuncle bat moth sample of the different worm states of above-mentioned preservation（Numbering is 12~22）, carefully removed with tweezers
A small amount of respective organization（Locality, the details for gathering worm state, test organization, test organization and acquisition time are shown in Table 1）, move into EP pipes
In, often pipe one, and carry out unique number（Numbering 12 is simple grain worm's ovum, and numbering 13~19 is respectively pereiopoda, the stern of seven larvas
Foot and body wall, 20~22 be the body wall or in-vivo tissue of three pupas,）, extracted for DNA.Remainder continue to preserve in case
Carry out Identification of Species.
3rd, prepared by DNA profiling
Using blood tissues cellular genome extracts kit（DP304, Hunan inscriptions on bones or tortoise shells biological medicine Co., Ltd）Extract sample
Product genomic DNA saves backup for 20 DEG C in ﹣.
4th, primer synthesizes
The primer of the present embodiment 2 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 2 are as shown in table 4.
The furuncle bat moth CO I gene PCR reaction systems of table 4（25ul systems）
The response procedures of the present embodiment 2 are as shown in table 5.
The furuncle bat moth 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,
For electrophoresis pattern referring to accompanying drawing 3, swimming lane 12~22 is respectively the amplified production that reference numeral sample extracts DNA.It is it can be seen that real
Apply the furuncle bat moths of the different worm states of example 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）, obtain
It is objective gene sequence after the calibrated splicing of gene order.Sequencing result shows 11 tested samples（Numbering 12~22）
Homology with the gene order SEQ ID NO.1 of the present invention can confirm that tested ovum, larva and pupa up to 98.4%
Sample is for furuncle bat moth.
Sequence table
<110>Hunan Normal University
<120>A kind of specific DNA sequences for identifying furuncle bat moth
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 658
<212> DNA
<213>Furuncle bat moth DNA
<400> 1
ttttattttt ggtatttgag caggaataat tggaacttcg ttaagattat taattcgaac 60
agaattaggt aatcctggat ctttaattgg agatgatcaa atttataatg taattgtaac 120
agcacatgct tttattataa ttttttttat agttatacct attataattg gtggatttgg 180
taattgatta gtacctttaa tattaggagc tcctgatata gcttttccac gattaaataa 240
tataagattt tgattattac ccccttcatt aatactatta atttctagaa gaattgtaga 300
aaatggagct ggaacaggtt gaacagtcta tcccccatta tctgcaaata ttgcacatgc 360
tggtagatca gtggatttag caattttttc cttacattta gcaggtattt catctatttt 420
aggggcagtc aattttatta caactgtaat taatatacga tcggaaggaa tatcttttga 480
ccgaatacca ttatttgtat gaagagttgc aattactgct ttattacttt tattatcttt 540
acctgtatta gcaggagcta ttactatatt actaacagac cgaaatttaa atacttcatt 600
ttttgaccct gctgggggag gggatcctat tttataccaa catttatttt gatttttt 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. one kind identification furuncle bat mothEndoclita nodusThe DNA standard detection gene orders of (Chu and Wang, 1985),
It is characterized in that the standard detection gene is CO I genes, there is gene order SEQ ID NO.1 as described below：
ttttattttt ggtatttgag caggaataat tggaacttcg ttaagattat taattcgaac 60
agaattaggt aatcctggat ctttaattgg agatgatcaa atttataatg taattgtaac 120
agcacatgct tttattataa ttttttttat agttatacct attataattg gtggatttgg 180
taattgatta gtacctttaa tattaggagc tcctgatata gcttttccac gattaaataa 240
tataagattt tgattattac ccccttcatt aatactatta atttctagaa gaattgtaga 300
aaatggagct ggaacaggtt gaacagtcta tcccccatta tctgcaaata ttgcacatgc 360
tggtagatca gtggatttag caattttttc cttacattta gcaggtattt catctatttt 420
aggggcagtc aattttatta caactgtaat taatatacga tcggaaggaa tatcttttga 480
ccgaatacca ttatttgtat gaagagttgc aattactgct ttattacttt tattatcttt 540
acctgtatta gcaggagcta ttactatatt actaacagac cgaaatttaa atacttcatt 600
ttttgaccct gctgggggag gggatcctat tttataccaa catttatttt gatttttt 658。
2. the PCR primer of the furuncle bat moth DNA standard detection genes 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 furuncle bat moth, including：
1）The separation and Extraction DNA from furuncle bat moth tissue to be measured；
2）Using the DNA as template pair of primers ,-the ATTCAACCAATCATAAAGATATTGG-3 ' of forward primer 5 ', reversely
- the TAAACTTCTGGATGTCCAAAAAATCA-3 ' of primer 5 ', 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, if energy specific amplification goes out 658bp or so band, send biotech firm to survey
Sequence；
4）According to sequencing result, if with the homology of the gene order SEQ ID NO.1 more than 98%, you can judge institute
Insect specimen to be measured, tissue and the Cordyceps Militaris host stated is furuncle bat moth.
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| CN109735554A (en) * | 2019-02-01 | 2019-05-10 | 中山大学 | It is a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata |
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| CN109735554A (en) * | 2019-02-01 | 2019-05-10 | 中山大学 | It is a kind of for identifying the COI gene and method for identifying molecules of triatoma rubrofasciata |
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