CN107513534B - DNA barcode standard gene sequence of giant furuncle moth and application thereof - Google Patents
DNA barcode standard gene sequence of giant furuncle moth and application thereof Download PDFInfo
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Abstract
The invention discloses a giant furuncle bat moth [ alpha ]Endoclita davidi(Poujade,1886), synonymsPhassus giganodus(Chu and Wang,1985)]The DNA barcode standard gene sequence of (1), which is characterized in that the barcode standard detection gene is a CO I gene and has a gene sequence SEQ ID NO. 1. The sequencing result is subjected to manual proofreading and sequence splicing, and Blast similarity search in NCBI (national center for Biotechnology information) is carried out to ensure that the obtained sequence is a target sequence. The species identification of the giant furuncle bat moths is realized by morphological identification, so that the reliability of results is ensured. The DNA barcode standard gene sequence of the giant furuncle moth provided by the invention is beneficial to realizing the rapid and accurate identification of different insect states (including imagoes, eggs, larvae and pupae), incomplete specimens (including muscles, feet, wings and partial tissues of body walls) and cordyceps hosts, and shortening the identification time.
Description
Technical Field
The invention relates to a DNA barcode standard gene sequence of a giant furuncle moth, which is used for identifying the species.
Background
Giant furuncle moth endogilica davidi (Poujade,1886) the synonym Phassus gigandus (Chu and wang,1985) belongs to the hepialidae (Hepialoidea) armyworm (endogilicia), is a stem and root of a stem-boring insect which takes two years, and the larva mainly eats the medicinal plant of Verbenaceae (Verbenaceae), namely the green (Clerodendrum cyrtophyllum Turcz.) and the like. Meanwhile, the giant furuncle moth larvae can be parasitized by an important nematodiaceae fungus, namely cordyceps xuefeng (ophiomorphyceps xuefengensis), to form an entomomycete combination, namely cordyceps xuefeng. The Xuefeng Chinese caterpillar fungus has a long use history as a Yao medicine, is mainly used for treating lung diseases, and has good curative effects on hypertension, viral hepatitis, rheumatoid arthritis, gout and other diseases after being combined.
The identification and recognition of the giant furuncle bat moth are the basis for the development and utilization of the insect and fungus resources and are also the core step of the research on the molecular phylogenetic relationship of the bat moth family. At present, the identification of the giant furuncle bat moth is mainly realized by an experienced insect classificator according to the morphological characteristics of adults, the identification of other insect states needs to be carried out to adults, the feeding period is long, and the difficulty is high; especially, the method is more difficult to identify specimens with incomplete forms and hosts in the state of the parasitic stiff worms of the cordyceps sinensis. Therefore, a new fast and accurate method is urgently needed to be found to make up for the defects of the traditional morphological classification method.
The DNA Barcoding technology (DNA Barcoding) is a new biological identification system created by using the specificity and interspecies diversity of standard, sufficiently mutated, easily amplified and relatively short DNA fragments (DNA barcode) per se, and can rapidly and automatically identify the species. In recent years, the bar code technology has proved to be an effective biological identification means, which not only can make a powerful supplement to the traditional identification method, but also can help identify species, discover new species and hidden species, reconstruct the evolution relationship of species and high-level order, etc. because it is more objective and accurate, breaks through the past over-dependence on experience.
As a standard target gene of a DNA bar code, on one hand, the target gene must be sufficiently conserved so as to be convenient for carrying out large-scale amplification by utilizing a universal primer; on the other hand, there should be enough variation to distinguish different species. Therefore, the identification of different species groups requires the selection of efficient genes of interest. Cytochrome oxidase subunit I (CO I) gene in the mitochondrial genome has various advantages: 1. the obvious CO I gene sequence exists in most stages in animal life; 2. most cells have more than a hundred mitochondria, but only one set of chromosomes, so that mitochondrial DNA is more easily amplified and used in an equal amount of sample; 3. compared with the DNA of a cell nucleus, the mutation speed of the mitochondrial DNA is 10 times that of the nuclear DNA, so that the species separation is more accurate; 4. the inheritance pattern of mitochondria belongs to maternal inheritance: the CO I gene is located in cell mitochondria and can only be inherited from a mother body, so the incidence rate of gene recombination is low, while most of the genes of cell nuclei are inherited from the mother body and a father body together, and the incidence rate of gene recombination is high; the CO I gene has the common characteristics of protein coding genes, namely, the 3 rd base of the codon is not influenced by natural selection pressure and can be freely mutated; the CO I gene can ensure enough variation and is easily amplified by a universal primer, and the DNA sequence of the CO I gene rarely has insertion and deletion, so that the sequence comparison is easy to operate; comparative analysis of the CO I gene sequences of a total of 11 phyla 13320 species of the kingdom Ames of animals, including vertebrates and invertebrates, by Hebert et al (2003) yields: with the exception of the coelenterate Cnidaria, the difference in genetic distance of 98% of species is 0% to 2% within species, with an average of 11.3% between species. It is common to select a fragment of the CO I gene as the DNA barcode. To date, many studies have demonstrated that the CO I gene is effective in identifying groups such as birds, fish, lepidopteran insects, mosquitoes, ants, and pinworms. However, no report on the related gene sequence of the giant furuncle swift moth exists, and the identification of giant furuncle swift moth specimens with different insect states and incomplete forms and hosts of cordyceps sinensis and the molecular phylogenetic research of the hepiales are obstacles.
Disclosure of Invention
The invention aims to provide a DNA barcode standard gene sequence of giant furuncle swift moth, and realize the rapid and accurate identification of the giant furuncle swift moth.
A DNA barcode standard detection gene of giant furuncle moth is characterized in that the barcode standard detection gene is a CO I gene and has a gene sequence SEQ ID NO. 1:
the PCR primer of the DNA barcode standard detection gene of the giant furuncle swift moth is characterized in that the sequence of the PCR primer is as follows:
forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3'
The reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3'.
The molecular identification method of the giant furuncle bat moth comprises the following steps:
1) separating and extracting DNA from the tissue of the giant furuncle swift moth to be detected;
2) the DNA is used as a template, a pair of primers, a forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3' and a reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3' are used for amplifying a CO I gene of the giant furuncle moth by polymerase chain reaction, and the PCR conditions are as follows: pre-denaturation at 94 ℃ for 10 min; 30s at 94 ℃ and 5532 cycles of 30s at 72 ℃ and 40 s; extending for 10min at 72 ℃; the PCR system is as follows: 25 ul: 2 XMastermix 12.5ul, 1ul each of the upstream and downstream primers, ddH2O7.5ul, template 3 ul;
3) taking a proper amount of the CO I gene amplified by the polymerase chain reaction in the step 2), carrying out agarose electrophoresis separation, observing by using an ultraviolet lamp, judging a result according to the size of an amplification product, specifically amplifying a band of about 658bp, and sending the band to a biological company for sequencing;
4) according to the sequencing result, the homology with the gene sequence SEQ ID NO.1 is more than 98 percent, and the specimen, the tissue and the cordyceps host to be detected can be judged to be the giant furuncle bat moth.
According to the technical principle of DNA bar codes, a preparation method of insect micro DNA templates is adopted, the CO I gene of the giant furuncle bat moth is amplified by a synthesized primer through improved PCR reaction conditions, and the sequence of a PCR product is determined by a professional biological company. The sequencing result is subjected to manual proofreading and sequence splicing, and Blast similarity search in NCBI (national center for Biotechnology information) is carried out to ensure that the obtained sequence is a target sequence. The species identification of the giant furuncle bat moths is realized by morphological identification, so that the reliability of results is ensured.
The primers were modified according to literature, forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3', reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3'.
The invention can detect the amplification result by an agarose gel electrophoresis detection method.
Compared with the traditional morphological identification method, the gene sequence obtained by the invention can be used for molecular identification of the giant furuncle swift moth, and can accurately and effectively shorten the identification time of the giant furuncle swift moth.
Drawings
FIG. 1 is a technical flow chart of the present invention.
FIG. 2 is the electrophoresis identification chart of the 16 samples of the giant furuncle moth of the present invention for the CO I gene PCR amplification result. The numbering is as follows: lane 1-16 are CO I genes of different insect states and tissues of the giant furuncle bat moth collected in the field (15 worms in total, wherein the numbers 11 and 12 are samples of different tissues of the same worm body, and the specific information of each sample is shown in Table 1), and a strip with the size of 658bp is detected. M is 2000plus DNA marker.
TABLE 1 sampling information Table
| Sample numbering | Testing insect status | Test tissue | Collection time (year/month) | Collecting |
| 1 | Egg | All are | 2015/11 | Larva of a |
| 2 | Larva of a living creature | Chest foot | 2015/11 | Larva of a |
| 3 | Larva of a living creature | Chest foot | 2015/05 | Larva of a |
| 4 | Larva of a living creature | Chest foot | 2015/05 | Larva of a |
| 5 | Larva of a living creature | Chest foot | 2015/09 | Larva of a |
| 6 | Larva of a living creature | Chest foot | 2015/05 | Larva of a |
| 7 | Larva of a living creature | Chest foot | 2015/09 | Larva of a |
| 8 | Pupa | Body wall | 2015/05 | Larva of a |
| 9 | Pupa | Body wall | 2015/07 | Larva of a |
| 10 | Pupa | Body wall | 2015/09 | Larva of a |
| 11 | Imago | Chest foot | 2015/07 | Larva of a |
| 12 | Imago | Pectoral muscle | 2015/07 | Larva of a |
| 13 | Imago | Wing base | 2015/10 | Larva of a |
| 14 | Imago | Chest foot | 2015/05 | Larva of a |
| 15 | Cordyceps sinensis runt worm | Body wall | 2015/11 | Cordyceps sinensis runt |
| 16 | Cordyceps sinensis runt worm | Chest foot | 2016/03 | Cordyceps sinensis runt worm |
FIG. 3 is the electrophoresis identification picture of the PCR amplification result of the Xuefeng Cordyceps sinensis Bombyx Batryticatus CO I gene of the invention. The numbering is as follows: lane 1-3 shows the CO I gene of wild three-headed Cordyceps sinensis runt collected in the field, and the detected band is about 658bp in size. M is 2000plus DNA marker.
Detailed Description
Example 1
1. Collection and preservation of giant furuncle bat moth specimen
Collecting larvae and Bombyx Batryticatus specimen in field, storing the larvae in 75% ethanol, naturally drying Bombyx Batryticatus specimen, placing into a sealed bag containing silica gel, and storing in a specimen box; pupa and adult are obtained by breeding field collected larva, pupa is stored in 75% alcohol, and adult is frozen and stored in centrifugal tube at-20 deg.C; eggs are laid by the raised adults and stored in an artificial climate chamber at 8 ℃.
2. Pretreatment of test samples
Taking out 15 of the stored giant furuncle bat moth specimens in different insect states, carefully taking down a small amount of corresponding tissues by using tweezers, transferring the tissues into an EP (EP) tube, taking one tissue per tube, and carrying out unique numbering (the number 1 is a single-seed egg, 2-7 are the pectoral feet of six-head larvae respectively, 8-10 are pupal wall tissues, 11 and 12 are the pectoral feet and pectoral muscle parts of the same adult respectively, 13 is the wing base part of one adult, 14 is the pectoral foot of one adult, and 15 and 16 are the body wall and the pectoral foot parts of two-head cordyceps sinensis stiff worms respectively) (see Table 1 in detail) for DNA extraction. The rest of the pool is kept for species identification.
3. DNA template preparation
Extracting sample genome DNA by using a blood tissue cell genome extraction kit (DP304, Hunan Oracle biomedical Co., Ltd.) and storing at-20 deg.C for later use.
4. Primer synthesis
The primers used in this example were as follows:
forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3'
Reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3'
5. PCR amplification
The PCR reaction system of example 1 is shown in Table 2.
TABLE 2 PCR reaction System for CO I gene of giant furuncle moth (25ul system)
| Composition (I) | Volume (ul) |
| |
3 |
| |
1 |
| |
1 |
| 2×Mastermix | 12.5 |
| ddH2O | 7.5 |
The reaction procedure of this example is shown in Table 3.
TABLE 3 PCR reaction procedure for CO I gene of giant furuncle swift moth
The PCR product was stored at 4 ℃ until use.
6. PCR amplification product detection
5ul of PCR amplification product was electrophoresed on 1% agar gel (130V for 10 min). The gel imaging system detects, and the electrophoresis pattern of the amplification product of the sample No. 1-16 is shown in figure 2. It can be seen that 16 samples of example 1 can specifically amplify the product of about 658 bp.
7. Gene sequence determination
And (3) purifying and sequencing a product obtained by PCR amplification by a biological company (a sequencing primer is the same as a primer used by PCR), and correcting and splicing the obtained gene sequence to obtain the target gene sequence. Sequencing results show that the homology of 16 samples and the gene sequence SEQ ID NO.1 is 98.7-100%, so that the 16 samples with different insect states can be confirmed to belong to the giant furuncle bat moth.
Example 2
1. Selection and preservation of samples
Collecting the Xuefeng cordyceps sinensis runt worms in the field, drying the Xuefeng cordyceps sinensis runt worms, putting the Xuefeng cordyceps sinensis runt worms into a sealed bag filled with silica gel, and storing the Xuefeng cordyceps sinensis runt worms in a specimen box.
2. Pretreatment of samples
Taking out three preserved Xuefeng Chinese caterpillar fungus stiff worm specimens, carefully taking down the Chinese caterpillar fungus stiff worm body walls with the size of sesame grains by using forceps, moving the Chinese caterpillar fungus stiff worm body walls into an EP (EP) tube, and carrying out unique numbering (the number 1-3 is the body wall of three-head Xuefeng Chinese caterpillar fungus stiff worms collected in the field respectively) for DNA extraction. The rest part is continuously stored for standby.
3. DNA template preparation
Extracting sample genome DNA by using a blood tissue cell genome extraction kit (DP304, Hunan Oracle biomedical Co., Ltd.) and storing at-20 deg.C for later use.
4. Primer synthesis
The primers used in this example were as follows:
forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3'
Reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3'
5. PCR amplification
The PCR reaction system of this example is shown in Table 4.
TABLE 4 Xuefeng Cordyceps muscardine CO I gene PCR reaction system (25ul system)
The reaction procedure of this example is shown in Table 5.
TABLE 5 PCR reaction procedure for Xuefeng Cordyceps sinensis Bombyx Batryticatus CO I gene
The PCR product was stored at 4 ℃ until use.
6. PCR amplification product detection
5ul of PCR amplification product was electrophoresed on 1% agar gel (130V for 10 min). And (3) detecting by a gel imaging system, wherein an electrophoresis pattern is shown in an attached figure 3, and 1-3 are amplification products of DNA extracted from the body wall of the three-headed cordyceps sinensis stiff worm respectively. It can be seen that the three-headed Cordyceps sinensis runt of example 2 can also specifically amplify a product of about 658 bp.
7. Gene sequence determination
And (3) purifying and sequencing a product obtained by PCR amplification by a biological company (a sequencing primer is the same as a primer used by PCR), and correcting and splicing the obtained gene sequence to obtain the target gene sequence. The sequencing result shows that the homology of the three samples and the gene sequence SEQ ID NO.1 is 98.9-99.7%, so that the cordyceps hosts of the three snowpeak cordyceps stiff worm samples can be confirmed to be the giant furuncle bat moths.
Sequence listing
<110> university of Master in Hunan
<120> DNA barcode standard gene sequence of giant furuncle moth and application thereof
<160>3
<170>PatentIn version 3.5
<210>1
<211>658
<212>DNA
<213> DNA of giant furuncle moth
<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 (2)
1. A DNA barcode standard detection gene of giant furuncle moth [ Endoclita davidi ], which is characterized in that the barcode standard detection gene is a CO I gene, and the sequence of the CO I gene is shown as SEQ ID NO. 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。
2. a molecular identification method of giant furuncle swift moths comprises the following steps:
1) separating and extracting DNA from the tissue of the giant furuncle swift moth to be detected;
2) amplifying CO I genes of the giant furuncle moth by using the DNA extracted in the step 1) as a template and a pair of primers, namely a forward primer 5'-ATTCAACCAATCATAAAGATATTGG-3' and a reverse primer 5'-TAAACTTCTGGATGTCCAAAAAATCA-3' through polymerase chain reaction;
3) taking a proper amount of the CO I gene amplified by the polymerase chain reaction in the step 2), carrying out agarose electrophoresis separation, observing by using an ultraviolet lamp, judging a result according to the size of an amplification product, specifically amplifying a band of about 658bp, and sending the band to a biological company for sequencing;
4) according to a sequencing result, when the homology of the sequence obtained in the step 3) and the CO I gene sequence shown in SEQ ID NO.1 of claim 1 is more than 98%, the tissue to be detected in the step 1) is obtained to belong to the giant furuncle bat moth;
in the step 1), the megafuruncle and bat moth tissues to be detected are megafuruncle and bat moth tissues in different insect states, wherein the different insect states comprise eggs, larvae, pupae, imagoes and cordyceps batryticatus;
when the worm state is worm grass stiff worm, in the step 4), when the homology of the gene sequence is more than 98%, the host of the worm grass fungus is the giant furuncle bat moth.
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Non-Patent Citations (3)
| Title |
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| GenBank: KT780172.1;Yang,X.等;《Genbank》;20160402;第1-7页 * |
| GenBank: KY928030.1;Zhou,Q.等;《Genbank》;20171226;第1页 * |
| 疖蝙蛾生物学特性的初步研究;赵锦年 等;《林业科学》;19880229;第24卷(第1期);第101-105页 * |
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