CN102605093B - Method for identifying molecule of athetis lepigone - Google Patents
Method for identifying molecule of athetis lepigone Download PDFInfo
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Abstract
The invention relates to a deoxyribose nucleic acid (DNA) sequence for identifying the type of an insect, in particular to the DNA sequence of new corn insect athetis lepigone and a method for identifying a molecule of the athetis lepigone. The invention provides the DNA sequence of a cytochrome oxidase (COI) gene of the athetis lepigone. The DNA sequence comprises the sequence shown as SEQ ID No: 1 or the sequence with at least 99 percent of sequence identity of the sequence shown as SEQ ID No: 1. The DNA sequence and the method have the effect that whether an insect is the athetis lepigone or not can be quickly and accurately judged by comparing the COI gene sequence of the detected insect and the sequence provided by the invention, and a basis for taking a corresponding efficient control measure in a field is provided.
Description
Technical field
The present invention relates to the DNA sequence dna of caste qualification, be specifically related to DNA sequence dna and the molecular assay method thereof of corn New harmful insect 2 committee noctuid.
Background technology
2 committee noctuid [Athetis lepigone (
1860)] be the de novo a kind of Agricultural pests in China's summer corn producing region.
Record according to pertinent data, domestic early than finding 2 committee's noctuids harm summer corn seedlings in Shijiazhuang Region, Hebei Province in July, 2005, bore moth basal part of stem, cause the withered heart of seedling to be wilted dead, or bite Zea mays root broken, cause lodging, cause the serious disconnected ridge that is short of seedling, this belongs to reported first at home.It is reported, within 2010,2 committee noctuids generally occur in farmland productivity in south-center of Hebei Province summer corn district, 2011 in Hebei, Henan, Shandong, Shanxi, Anhui, Jiangsu etc. economizes and breaks out, occurring area reaches more than 3,000 ten thousand mu, minority plot is reseeded or is ruined kind, is in recent years rare in Maize Production serious insect pest.
Because 2 committee noctuids are the kainogenesis insects just causing people to pay close attention in recent years, to the understanding of this insect not enough, there is lighter place, people can not note observing, just think cutworm, be unaware of this is a kind of New harmful insect at all, and in the serious area of generation, professional of agriculture and peasant are through examining, also often it can be thought by mistake be cutworm, incur loss through delay or employ prophylactico-therapeutic measures improperly, cause damage to production, be directed to this, on the basis that existing morphological specificity describes, object of the present invention is exactly the gene fragment that will provide a kind of 2 committee noctuids, for the precise Identification of 2 committee noctuids.
The Mitochondrial Genome Overview (mtDNA) of organism strictly observes matrilinear inheritance, there is genetic recombination hardly, the heritable variation in population and between population can be reflected comprehensively, and its structure is simple, evolutionary rate is fast, thus the research (Norgate et al., 2009) exploring population structure and genetic diversity is widely used in.Wherein Terminal oxidase C subunit (COI) gene differentiate in vertebrates and invertebrates kind, be all used widely in the research such as population genetic diversity and Molecular Phylogeny and Evolution (Novo et al., 2010; Feng et al., 2011; Xu et al., 2011).COI gene can be used in the research each monoid Phylogenetic Relationships of insect and evolution and verifies traditional classification system, set up natural system and the evolutionary relationship of species, contribute to the difference qualification of species, find cryptic species, study species differentiation and genetic diversity (Liang Xia etc., 2011; Chi Yu etc., 2010; Yang Baoshan etc., 2009; Collet et al., 2006).
Summary of the invention
Object of the present invention is exactly easily 2 committee noctuids and cutworm are obscured in actual production, provides the gene fragment of a kind of 2 committee noctuids, for the precise Identification of 2 committee noctuids.
The invention provides a kind of DNA sequence dna of 2 committee noctuid COI genes, it comprises the sequence (GenBank accession number JQ395055) shown in following SEQ ID NO:1 or has the sequence of at least 99% sequence iden with sequence shown in SEQ ID NO:1.
The present invention also provides a kind of molecular assay method of 2 committee's noctuid kinds, it is characterized in that comprising the steps:
(1) insect sample is collected: the sample of collection is put in 95% ethanol ,-20 DEG C of preservations;
(2) extraction of genomic dna: extract insect genes group DNA according to animal tissues/cellular genome rapid extraction test kit, be stored in-20 DEG C for subsequent use;
(3) amplification of COI gene, clone, order-checking and sequential analysis, wherein the pcr amplification primer of COI gene order: upstream primer/downstream primer 5-GGTCAACAAATCATAAAGATATTGG-3/5-TAAACTTGAGGGTGACCAAAAAAT CA-3;
(4) sequencing result shows that ZB1 ~ ZB17 is 2 different haplotypes of committee noctuid, all reaches more than 99% with ZB1 (JQ395055) similarity.
In molecular assay method of the present invention, the concrete steps of COI gene amplification are:
The PCR reaction system of 20 μ L: pcr amplification total reaction system is 20 μ L, wherein template DNA 2.0 μ L, each 1 μ L of upstream and downstream primer (concentration 10 μMs), 10 × PCR buffer 2.0 μ L, dNTP 1.6 μ L (2.5mM), enzyme 0.3U, ultrapure water are supplied;
Response procedures: 94 DEG C, 5min; 35 circulations (94 DEG C, 45s, 54 DEG C, 45s, 72 DEG C, 1min); 72 DEG C extend 5min; Agarose gel electrophoresis with 1% detects, and reclaims object band.
In molecular assay method of the present invention, according to the step on TaKaRa test kit, the PCR primer of cutting glue recovery is connected with PMD19-T carrier, the bacterium liquid of positive colony is checked order.
In molecular assay method of the present invention, Sequencher_v4.1.4 software is first utilized to read the sequence of order-checking, observe peak value repeatedly to proofread, utilize DNAMAN_v6, ClustalX1.81 software to carry out the comparison of multisequencing homology to the sequence nucleotide sequence shown in the sequence recorded and JQ395055; In addition, according to MEGA5.05 achievement model, with noctuid black cutworm (Agrotis ipsilon) COI gene as outer group, build haplotype genealogical tree by contiguous method, and utilize p-distance to calculate genetic distance between each haplotype; Result show result show with JQ395055 similarity up to more than 99% can think that this insect is 2 noctuids of entrusting.
The invention still further relates to the purposes being used for above-mentioned molecular assay method to distinguish fast discriminating 2 committee noctuid.
The invention still further relates to a kind of primer for the identification of 2 committee's noctuid kinds, it is characterized in that sequence is: 5-GGTCAACAAATCATAAAGATATTGG-3/5-TAAACTTGAGGGTGACCAAAAAAT CA-3.
The invention still further relates to the test kit comprising above-mentioned primer.
The invention has the beneficial effects as follows: COI gene order and the sequence provided by the invention of detected insect are compared, can judge whether this insect is 2 committee noctuids, implements corresponding effective prophylactico-therapeutic measures provide foundation for field fast and accurately.
Accompanying drawing explanation
The sequence of 2 committee noctuid COI gene fragments of sequence table SEQ ID NO:1. separating clone of the present invention.
Fig. 1: example one, (N-J) evolutionary tree of Hebei, Henan, Shandong, the different haplotype of committee of 2, Shanxi Si Sheng 13 cities and counties noctuid different population COI
(N-J) evolutionary tree of the different haplotype of Fig. 2: example two, committee of 2, Hebei 6 cities and counties noctuid different population COI
Embodiment
Embodiment 1
1. collect insect sample
In 8 months, entrust a noctuid Sheng phase, the lamp respectively at Hebei, Henan, Shandong, Shanxi Si Sheng 13 cities and counties lures adult in the first tenday period of a month 2, guarantees that institute's sample thief is 2 committee noctuids, be then put in 95% ethanol ,-20 DEG C of preservations through adult characteristic feature.
The Information Monitoring of 2, table 1 committee armyworm population sample
Table?1Collecting?data?and?individual?number?of?Athelis?lepigone
2. the extraction of genomic dna
Extract insect genes group DNA according to the animal tissues/cellular genome rapid extraction test kit (SK8252) of Shanghai Sheng Gong biotechnology company limited, be stored in-20 DEG C for subsequent use.
The amplification of 3.COI gene, clone, order-checking and sequential analysis
(1) amplification of COI gene
The pcr amplification primer of COI gene order: upstream primer/downstream primer 5-GGTCAACAAATCATAAAGATATTGG-3/5-TAAACTTGAGGGTGACCAAAAAAT CA-3
The PCR reaction system of 20 μ L: pcr amplification total reaction system is 20 μ L, wherein template DNA 2.0 μ L (about 50ng), each 1 μ L of upstream and downstream primer (concentration 10 μMs), 10 × PCR buffer 2.0 μ L, dNTP 1.6 μ L (2.5mM), enzyme 0.3U, ultrapure water are supplied.
Response procedures: 94 DEG C, 5min; 35 circulations (94 DEG C, 45s, 54 DEG C, 45s, 72 DEG C, 1min); 72 DEG C extend 5min.Agarose gel electrophoresis with 1% detects, and reclaims object band.
(2) choning and sequencing of COI gene
According to the step on TaKaRa test kit (D104A), the PCR primer of cutting glue recovery is connected with PMD19-T carrier, the bacterium liquid of positive colony is served Hai Shenggong order-checking.
(3) sequential analysis
First utilize Sequencher_v4.1.4 software to read the sequence of order-checking, observe peak value and repeatedly proofread, utilize DNAMAN_v6, ClustalX1.81 software to carry out the comparison of multisequencing homology to the sequence recorded.The achievement model be applicable to is selected according to the models provided in MEGA5.05, with noctuid black cutworm (Agrotis ipsilon) COI gene as outer group, build haplotype genealogical tree by contiguous method, and utilize p-distance to calculate genetic distance between each haplotype.
4. experimental result
(1) gene order:
ZB1:
AACATTATATTTTATTTTTGGTATTTGAGCTGGAATAGTAGGAACTTCCCTAAGATTATTAATTCGAGCAGAATTAGGAAATCCTGGATCTTTAATTGGAGATGACCAAATTTATAATACTATTGTTACAGCTCATGCATTTATTATAATTTTTTTTATGGTAATACCTATTATAATTGGAGGATTTGGAAATTGATTAGTACCTTTAATATTAGGAGCCCCAGATATAGCTTTTCCTCGAATAAATAATATAAGTTTTTGACTTCTTCCCCCCTCTTTAACTTTACTTATTTCAAGAAGAATTGTAGAAAATGGAGCAGGTACAGGATGAACTGTTTATCCTCCTCTTTCTTCTAATATCGCTCATGGAGGTAGATCTGTTGATTTAGCAATTTTTTCATTACATTTAGCTGGTATTTCTTCTATTTTAGGAGCTATTAATTTTATTACTACAATTATTAATATACGATTAAATAGACTATCATTTGATCAAATACCTTTATTTATTTGAGCTGTAGGAATTACTGCATTTTTACTATTATTATCATTACCTGTTTTAGCTGGAGCTATTACTATACTTTTAACGGATCGAAATTTAAATACTTCTTTTTTTGATCCTGCAGGAGGGGGAGACCCAATTTTATATCAACATTTATTT
ZB1 ~ ZB13 is 2 different haplotypes of committee noctuid, GenBank accession number: JQ395055 ~ JQ395067.The ZB1 frequency of occurrences is the highest, therefore selects ZB1 to regard the mark of 2 committee noctuids.
(2) homologous sequence comparison
138 2 committee noctuids are compared with JQ395055 respectively, and similarity all reaches more than 99%, and is less than 92% with the similarity of cutworm COI gene fragment (No. GenBank is GU438723.1).(see table 2).
(3) phylogenetic analysis
Between the haplotype of 2 committee noctuid COI genes, the distance of ZB13 and ZB6 is 0.026 farthest, to be starkly lower than between 2 committee noctuids and an outer group of mean people cutworm closest range 0.114 (between ZB6 and WQ) between COI haplotype and to see Fig. 1 and table 3.
The variant sites of 2, table 2 committee noctuid sample COI haplotype and and black cutworm (WQ) between variant sites
Table?2Site?of?variation?in?COI?gene?sequence?between?Athelis?lepigone?and?Agrotis?ipsilon
52, table 3 committee noctuid different population COI haplotypes do not correct p distance
Table?3p-distance?of?17COI?haplotypes?among?different?populations?of?Athelis?lepigone
5. reference:
Collet?T,Ferreira?KM,Arias?MC,Soares?AEE,Del?Lama?MA，2006.Genetic?structure?of?Africanized?honeybee?populations(Apis?mellifera?L.)from?Brazil?and?Uruguay?viewed?through?mitochondrial?DNA?COI-COII?patterns.Heredity，97:329-335.
Chi Y, Wang SD, Zhang CT, 2010.Advance of Diptera based on mitochondrial COI gene.Entomotsxonomia, 32 (S1): 71-78. [Chi Yu, Wang Shidi, Zhang Chuntian, 2010. based on the dipteral insect progress [J] of mitochondrial COI gene. Entomotaxonomia, 32 (S1): 71-78]
Feng?YW,Li?Q,Kong?LF,Zheng?XD,2011.DNA?barcoding?and?phylogenetic?analysis?of?pectinidae(Mollusca:Bivalvia)based?on?mitochondrial?COI?and?16S?rRNA?genes.Molecular?Biology?Reports,38:291－299.
Liang RX, Wang ZY, He KL, Cong B, Li J, 2011.Genetic diversity of geographic populations of Monolepta hieroglyphica (Motschulsky) (Coleoptera:Chrysomelidae) from North China estimated by mitochondrial CO II gene sequences.Acta Entomologica Sinica, 54 (7): 828-837. [Liang Xia, Wang Zhenying, He Kang comes, from refined, Li Jing, 2011. based on the genetic diversity Journal of Sex Research of the Monolepta hieroglyphica north of China geographical population of mtDNA-COⅡ gene sequence. insect journal, 54 (7): 828-837]
Novo?M,Almodovar?A,Fernandez?R,Trigo?D,Cosin?DJD,2010.Cryptic?speciation?of?hormogastrid?earthworms?revealed?by?mitochondrial?and?nuclear?data.Molecular?Phylogenetics?and?Evolution,56:507－512.
Norgate?M,Chamings?J,Pavlova?A,Bull?JK,Murray?ND,Sunnucks?P,2009.Mitochondrial?DNA?Indicates?Late?Pleistocene?Divergence?of?Populations?of?Heteronympha?merope,an?Emerging?Model?in?Environmental?Change?Biology.Public?library?of?science?one,4(11):1-13.
Xu?ZH,Chen?JL,Cheng?DF,2011.Genetic?Variation?Among?the?Geographic?Population?of?the?grain?aphid,Sitobion?avenae(Hemiptera:Aphididae)in?China?Inferred?from?Mitochondrial?COI?Gene?Sequence.Agricultural?Sciences?in?China,10(7):1041－1048.
Yang BS, Hou QJ, Wang H, Li XS, Jiang DF, Liu YQ, Qin L, 2009.Different geographic populations of Caligula japonica COI gene sequences variation and genetic.Acta Entomologica Sinica, 52 (4): 406-412. [Yang Baoshan, Hou Qingjun, Wang Huan, Li Xisheng, Jiang Defu, Liu Yanqun, Qin Li, 2009. variation of different geographic populations dictyoploca japonica butler COI gene order and genetic variation and genetic differentiation. insect journal, 52 (4): 406-412]
Embodiment 2
1. collect insect sample
Late August, the lamp respectively at Hebei 6 cities and counties lures adult, according to adult feature after expert statement, is put in 95% ethanol ,-20 DEG C of preservations.
The Information Monitoring of table 4 sample
Table?4Collecting?data?of?insect
2. the extraction of genomic dna
Extract insect genes group DNA according to the animal tissues/cellular genome rapid extraction test kit (SK8252) of Shanghai Sheng Gong biotechnology company limited, be stored in-20 DEG C for subsequent use.
The amplification of 3.COI gene, clone, order-checking and sequential analysis
(1) amplification of COI gene
The pcr amplification primer of COI gene order: upstream primer/downstream primer 5-GGTCAACAAATCATAAAGATATTGG-3/5-TAAACTTGAGGGTGACCAAAAAAT CA-3
The PCR reaction system of 20 μ L: pcr amplification total reaction system is 20 μ L, wherein template DNA 2.0 μ L (about 50ng), each 1 μ L of upstream and downstream primer (concentration 10 μMs), 10 × PCR buffer 2.0 μ L, dNTP 1.6 μ L (2.5mM), enzyme 0.3U, ultrapure water are supplied.
Response procedures: 94 DEG C, 5min; 35 circulations (94 DEG C, 45s, 54 DEG C, 45s, 72 DEG C, 1min); 72 DEG C extend 5min.Agarose gel electrophoresis with 1% detects, and reclaims object band.
(2) choning and sequencing of COI gene
According to the step on TaKaRa test kit (D104A), the PCR primer of cutting glue recovery is connected with PMD19-T carrier, the bacterium liquid of positive colony is served Hai Shenggong order-checking.
(3) sequential analysis
First utilize Sequencher_v4.1.4 software to read the sequence of order-checking, observe peak value and repeatedly proofread, utilize DNAMAN_v6, ClustalX1.81 software to carry out the comparison of multisequencing homology to the sequence recorded and this sequence (JQ395055).The achievement model be applicable to is selected according to the models provided in MEGA5.05, with noctuid black cutworm (Agrotis ipsilon) COI gene as outer group, build haplotype genealogical tree by contiguous method, and utilize p-distance to calculate genetic distance between each haplotype.
4. experimental result
There are again four kinds of new haplotype (ZB14 ~ ZB17) GenBank accession number: JQ395068 ~ JQ395071 in these 2 committee noctuids, but with JQ395055 similarity all more than 99%.(see table 5)
The variant sites of 2, table 5 committee noctuid sample COI haplotype and and black cutworm (WQ) between variant sites
Table?5Site?of?variation?in?COI?gene?sequence?between?Athelis?lepigone?and?Agrotis?ipsilon
The distance of sibship between 2 committee noctuids and cutworm can be seen clearer, more intuitively by Fig. 3 and table 6.
2, table 6 committee noctuid different population COI haplotype does not correct p distance
Table?6p-distance?of?17COI?haplotypes?among?different?populations?of?Athelis?lepigone
COI gene fragment and the molecular assay method thereof of 2 committee noctuids of the present invention are described by specific embodiment.Those skilled in the art can use for reference the link such as content appropriate change insect-catching mode of the present invention, extraction DNA condition and realize other object corresponding, its relevant change does not all depart from content of the present invention, all similar replacements and change will become apparent to those skilled in the art that and be all deemed to be included within scope of the present invention.
Claims (3)
1. a molecular assay method for 2 committee's noctuid kinds, is characterized in that, comprise the steps:
(1) insect sample is collected: the sample of collection is put in 95% ethanol ,-20 DEG C of preservations;
(2) extraction of genomic dna: extract insect genes group DNA according to animal tissues/cellular genome rapid extraction test kit, be stored in-20 DEG C for subsequent use;
(3) amplification of COI gene, clone, order-checking and sequential analysis, wherein the pcr amplification primer of COI gene order: upstream primer/downstream primer 5 '-GGTCAACAAATCATAAAGATATTGG-3 '/5 '-TAAACTTGAGGGTGACCAAAAAATCA-3 ';
The concrete steps of described COI gene amplification are:
The PCR reaction system of 20 μ L: pcr amplification total reaction system is 20 μ L, wherein the upstream primer of template DNA 2.0 μ L, concentration 10 μMs and each 1 μ L of downstream primer, dNTP1.6 μ L, the enzyme 0.3U of 10 × PCR buffer 2.0 μ L, 2.5mM, ultrapure water are supplied;
Response procedures: 94 DEG C, 5min; 35 circulations: 94 DEG C, 45s, 54 DEG C, 45s, 72 DEG C, 1min; 72 DEG C extend 5min; Agarose gel electrophoresis with 1% detects, and reclaims object band;
Check order, first utilize Sequencher_v4.1.4 software to read the sequence of order-checking, observe peak value and repeatedly proofread, utilize DNAMAN_v6, ClustalX1.81 software to carry out the comparison of multisequencing homology to the sequence shown in the sequence recorded and SEQ ID NO:1; In addition, according to MEGA5.05 achievement model, with noctuid black cutworm (Agrotis ipsilon) COI gene as outer group, build haplotype genealogical tree by contiguous method, and utilize p-distance to calculate genetic distance between each haplotype; Result show with SEQ ID NO:1 similarity up to more than 99% can think that this insect is 2 noctuids of entrusting.
2. the molecular assay method of 2 committee's noctuid kinds according to claim 1, is characterized in that:
According to the step on TaKaRa test kit, the PCR primer of cutting glue recovery is connected with PMD19-T carrier, the bacterium liquid of positive colony is checked order.
3. method according to claim 1 is used for the purposes distinguishing discriminating 2 committee noctuid fast.
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| CN103131699B (en) * | 2013-02-25 | 2014-06-11 | 中国农业科学院植物保护研究所 | Pair of tuta absoluta specific SS-COI primers, and rapid PCR detection method and kit |
| CN103266174B (en) * | 2013-05-22 | 2014-12-10 | 青岛农业大学 | PCR (Polymerase Chain Reaction) primer and identification method of athetis lepigone and sibling species thereof |
| CN103952463B (en) * | 2013-07-16 | 2016-01-13 | 中国农业大学 | A kind of test kit identifying 5 kinds of flat ostomatids of common grain storage based on special primer |
| CN103451184B (en) * | 2013-09-18 | 2015-08-05 | 中国农业科学院植物保护研究所 | Green bugs specificity SS-COI primer, test kit containing this primer and detection method thereof |
| CN104651351B (en) * | 2014-07-28 | 2017-12-12 | 岳巧云 | DNA amplification bar code total length method and kit from collection drying sarcophagid sample |
| CN104164433B (en) * | 2014-09-03 | 2016-09-14 | 扬州大学 | For expanding the specific primer of lepidopteran insects mitochondrial COI gene |
| CN104498608B (en) * | 2014-12-20 | 2016-06-29 | 福建农林大学 | Utilize the method that SNP marker differentiates the anti-Chalk characteristic of disease shape of bee colony |
| CN111269968B (en) * | 2020-03-13 | 2023-03-21 | 华南师范大学 | PCR-RFLP-based method for rapidly identifying Spodoptera frugiperda |
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Effective date of registration: 20161031 Address after: 050035 Shijiazhuang City, Hebei Province East Development Zone, Mt. Hengshan street, No. 162, millet, by the end of 113 Patentee after: Grain Institute, Provincial Academy of Agriculture and Forestry, Hebei Provinee Address before: 050035 Hebei city in Shijiazhuang province Mt. Hengshan East Development Zone No. 162 South Street Room 113 millet Patentee before: Dong Zhiping |
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