Coil sequence of cosmophagum concurrens and its application

Abstract

The application discloses a Cotesia melitaearum COI gene and application thereof. The sequence of a Cotesia melitaearum COI gene (Mman) Microplitis manilae mitochondrial cytochrome oxidase subunit 1, Mman COI is shown in SEQ ID NO. 1. The application provides a Cotesia melitaearum COI Microplitis manilae COI COI Microplitis manilae COI COI gene sequence, and designs specific amplification primers based on the sequence, and provides a precise and stable molecular biology identification method, which can be used for molecular identification of the bee and close relatives, different residues of the bee or whether successful parasitization in a host body. The disclosure and application of the gene sequence provide great convenience for rapid and accurate identification of the Cotesia melitaearum and early parasitization success detection of the parasitic bee.

Description

Technical Field

[0001] This invention belongs to the field of species identification and biological detection, specifically relating to the Manila lateral groove braconid wasp. helm Gene( MmanCOI ) and its applications. Background Technology

[0002] Manila side groove braconid wasp Microplitis manilae It is the fall armyworm. Spodoptera frugiperda Spodoptera litura Spodoptera litura Beet armyworm Spodoptera exigua Parasitic wasps that parasitize the larval stage of major agricultural pests such as noctuid moths play an important role in the biological control of pests.

[0003] There are hundreds of thousands of parasitic wasps in nature. Most of them are small in size, and the larvae of species in the same family are very similar in appearance. In the absence of high-precision instruments and non-related professional researchers, classification and identification are extremely difficult. Furthermore, there are many unknown species and synonyms of the same species, making the classification and identification of parasitic wasps very challenging.

[0004] mitochondrial cytochrome oxidase subunit 1 helm Genes are among the most commonly used markers in molecular systematics, and a portion of a gene is often used to infer phylogenetic relationships. Furthermore, helm The 5' region of the gene, consisting of approximately 640 nucleotides, has been used as a unique identifier for many species, currently primarily to aid in the accurate identification of specimens and the discovery of new species. This gene can be used for the identification of parasitic wasp species.

[0005] Parasitic wasps inject their embryos into the host, using the host to provide nutrients for reproduction. Therefore, the success of parasitic wasp parasitism is an important indicator for pest control. Current methods of identification include: in the early stages of parasitism, dissecting the host under a microscope to find the parasitic wasp embryos, but the wasp dies simultaneously; in the later stages, researchers rely on their experience in raising the host to visually observe the host's condition and changes in body wall color to determine success, which prolongs the biological experiment cycle and has low accuracy. Summary of the Invention

[0006] The purpose of this invention is to provide a method for the rapid and accurate detection of *Parasitic lateral groove* wasps. helm sequence.

[0007] Manila lateral groove braconid wasps for rapid and accurate detection according to specific embodiments of the present invention. helm The gene sequence is shown in SEQ ID NO.1: CAACATTTATTTTGATTTTTTGGTCATCCAGAGGTTTATATTTTAATTTTACCTGGATTTGGAATTATTTCACATATAATTTTTAATGAAAGAGGAAAGAAAGAAACATTTGGTTTTATTAGGTATAATTTATGCTATATTAACAATTGGATTTTTAGGATTTATTGTTTGAGCTCATCATATATTTACAGTTGGAATAGATGTTGATACACGAGCTTATT TTACATCAGCAACTATAATTATTGCTGTTCCAACAGGAATTAAAATTTTTAGTTGAATAGCAACATTTAGAGGGGTAAAATTAATAAATAATTTAAATGTTTTATGATCAATAGGATTTATTTTTTTTATTTACATTAGGAGGTTTAACAGGTGTTGTTTTATCTAATTCATCAGTTGATGTAGTTTTACATGATACATATTATGTTGTAGCTCATTTTCAT TATGTATTATCTATAGGAGCTGTATTTGCTATTTTAGGTGGTTTTATTTATTGATATCCATTATTTTTTGGATTTATAATTAATGAAAAATTTTTAAAAGTTCATTTTTATTTAACTTTTATTGGTGTTAATTTAATTTTTTTCCACAACATTTTTTAGGTTTAGGAGGAATACCTCGTCGTTATTCGGATTATCCTGATATATATCTAATATGAAATAT TTTTTCTTCAATTGGTTCATTAATTACTTTAATTAGAGTAATTTTATTTATTTTTATTATATGAGAAGGAATATTAATAAATCGTATTATTTTATTTATTAAATATATAAATTCTTCAATTGAATGAATTCAAATATATCCACCAAAGGATCATAGATATGATGAATCAAGGTATATTTTAATTGGAAAAAAATTCTAATATGGCAGATTAGTGCATTGGA The present invention also provides the above-mentioned Manila lateral groove braconid wasp. helm Sequence-specific primers, wherein the primer sequences are as follows: MmanCOI-F: 5'-ATTTACAGTTGGAATAGATG-3'; MmanCOI-R: 5'-GATAATCCGAATAACGAC-3'.

[0008] The present invention also provides a method for identifying Manila lateral groove parasitic wasps, the method comprising the step of performing PCR amplification of DNA in tissues of the parasitic wasp to be tested using the specific primers described herein.

[0009] A method for identifying Manila lateral groove braconid wasps according to a specific embodiment of the present invention includes the following steps: (1) After grinding the tissue of the parasitic wasp to be tested, DNA was extracted; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the specific primers described in claim 1; (3) When a bright band of 455 bp is present in the agarose gel electrophoresis, the parasitic wasp to be tested is identified as Manila lateral groove parasitic wasp.

[0010] Preferably, in step (2), the PCR amplification program is as follows: reaction conditions are 98 °C pre-denaturation for 2 min; 98 °C denaturation for 10 s, 55 °C annealing for 30 s, 68 °C extension for 6 s, 35 cycles; 68 °C extension for 5 min; and storage at 4 °C.

[0011] On the other hand, the present invention also provides a method for detecting Manila parasitic noctuid host plants by Braconidia spp., the method comprising the step of performing PCR amplification of DNA in the noctuid host tissue using the specific primers described above.

[0012] A method for detecting Manila parasitic moths (Noctuidae) by the *Braconidae* parasitic wasp according to a specific embodiment of the present invention includes the following steps: (1) After grinding the tissue of the Noctuidae host, DNA was extracted; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the specific primers described above; (3) When a bright band of 455 bp is present in the agarose gel electrophoresis, it is determined that the Manila parasitoid wasp has successfully parasitized the Noctuidae host.

[0013] The host plants of the Noctuidae family include fall armyworm, beet armyworm and / or beet armyworm.

[0014] Preferably, in step (2), the PCR amplification program is as follows: reaction conditions are 98 °C pre-denaturation for 2 min; 98 °C denaturation for 10 s, 55 °C annealing for 30 s, 68 °C extension for 6 s, 35 cycles; 68 °C extension for 5 min; and storage at 4 °C.

[0015] Meanwhile, those skilled in the art have observed the aforementioned Manila lateral groove parasitic wasps. helm Based on the sequence, a kit for identifying Manila lateral groove braconid wasps can be prepared according to common knowledge in the field.

[0016] The beneficial effects of this invention are: This invention discloses for the first time the Manila lateral groove braconid wasp. helm The sequence is then used to identify the parasitic wasp species using PCR technology, which is more accurate and convenient than morphological identification. It can also detect whether parasitism has been successful, providing accurate results and a basis for subsequent biological and molecular biological experiments, saving time and costs. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0018] Figure 1 The image shows gel electrophoresis images of DNA amplification products from different parasitic wasps; the samples in the lanes are, in order: marker; 1: Manila lateral groove braconid wasp; 2: Ichthyophthirius multifiliis wasp; 3: Trichogramma multifiliis wasp; Figure 2 Gel electrophoresis images showing DNA amplification products from different hosts and tissue remains of *Lactuca manilae*. Lane samples are as follows: marker; 1: *Lactuca manilae* parasitized *Betweenia spp.*; 2: *Lactuca manilae* parasitized *Spodoptera litura*; 3: *Lactuca manilae* parasitized *Pseudomonas manilae*; 4: female *Lactuca manilae*; 5: male *Lactuca manilae*; 6: head tissue of *Lactuca manilae*; 7: thorax tissue of *Lactuca manilae*; 8: abdominal tissue of *Lactuca manilae*. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be described in detail below. Obviously, the described embodiments are merely some embodiments of this invention, and not all embodiments. Based on the embodiments of this invention, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0020] Example 1: Obtaining Manila lateral groove braconid wasps helm sequence 1. DNA extraction The specific steps for using the TSINGKE Animal DNA Extraction Kit (Universal) are as follows: (1) Take 5 adult Manila parasitic wasps and place them in a 1.5 ml centrifuge tube. Add liquid nitrogen and grind them thoroughly. After grinding, add 200 μl physiological saline, 200 μl Buffer gA1 and 20 μl proteinase K, vortex and incubate at 65℃ for 2 h.

[0021] (2) Centrifuge the sample from step (1) at 12,000×g for 5 min and transfer the supernatant to a new 1.2ml 96-well plate.

[0022] (3) Add 10 μl of animal genome magnetic beads and an equal volume of anhydrous ethanol to the supernatant, cover with a silicone pad, shake for 1 min, shake at 800 rpm for 5 min with a horizontal shaker, then insert the 96-well plate into the magnetic rack and magnetically hold it for 1 min, during which time you can shake to mix.

[0023] (4) After adsorption, discard the waste liquid and tap it lightly on absorbent paper.

[0024] (5) Add 500 μl Buffer PW, cover with a silicone pad, vortex for 30 s, insert the 96-well plate into the magnetic holder, magnetically attract for 1 min, discard the waste liquid, and gently tap on absorbent paper.

[0025] (6) Add 500 μl wash buffer, cover with silicone pad, vortex for 30s, insert the 96-well plate into the magnetic holder, magnetically attract for 1 min, discard waste liquid, and gently tap on absorbent paper.

[0026] (7) Repeat step (6) once, centrifuge to 500 rpm.

[0027] (8) After drying the alcohol on the 96-well plate on the ultra-clean workbench, add 30 μl of sterile water to wash it off, cover it with a sealing film, shake it vigorously for 30 s, bathe it in a 65 ℃ water bath for 5 min, centrifuge it to 1000 rpm, insert the 96-well plate into the magnetic rack, and let it adsorb for 1 min.

[0028] 2. The extracted DNA sample was diluted appropriately and used as a PCR template. Amplification was performed using the Chintech 1×TSE101 Gold Mix. The components of the amplification system are as follows: 1.1×T3 super PCR mix 35ul; helm -F(10P) 2ul; helm -R(10P) 2ul; 1 μL DNA template; use helm The universal primer is: helm -F CAACATTTATTTTGATTTTTTGG; helm -RTCCAATGCACTAATCTGCCATATTA.

[0029] The PCR reaction system and temperature were as follows: 98℃, 2 min; 98℃, 10 s, 55℃, 20 s, 68℃, 7 s for a total of 35 cycles; 68℃, 5 min; stored at 4℃.

[0030] 3. Sequencing Sequencing the amplified PCR products yields... helm sequence.

[0031] Example 2 examines the specificity and sensitivity of primers. 1. DNA extraction.

[0032] The specific steps for using the TSINGKE Animal DNA Extraction Kit (Universal) are as follows: (1) Take appropriate amounts of insect tissue (samples are: 3 Manila lateral sulphur wasps; 3 ichneumon wasps; 5 Trichogramma wasps; a single beet armyworm parasitized by Manila lateral sulphur wasps for 24 h; a single beet armyworm parasitized by Manila lateral sulphur wasps for 24 h; a single fall armyworm parasitized by Manila lateral sulphur wasps for 24 h; 3 female Manila lateral sulphur wasps; 3 male Manila lateral sulphur wasps; head tissue of 5 Manila lateral sulphur wasps; thorax tissue of 5 Manila lateral sulphur wasps; abdominal tissue of 5 Manila lateral sulphur wasps) and place them in a 1.5 ml centrifuge tube. Add liquid nitrogen and grind thoroughly. After grinding, add 200 μl of physiological saline, 200 μl of Buffer gA1 and 20 μl of proteinase K, vortex and incubate at 65 ℃ for 2 h.

[0033] (2) Centrifuge the sample from step (1) at 12,000×g for 5 min and transfer the supernatant to a new 1.2ml 96-well plate.

[0034] (3) Add 10 μl of animal genome magnetic beads and an equal volume of anhydrous ethanol to the supernatant, cover with a silicone pad, shake for 1 min, shake at 800 rpm for 5 min with a horizontal shaker, then insert the 96-well plate into the magnetic rack and magnetically hold it for 1 min, during which time you can shake to mix.

[0035] (4) After adsorption, discard the waste liquid and tap it lightly on absorbent paper.

[0036] (5) Add 500 μl Buffer PW, cover with a silicone pad, vortex for 30 s, insert the 96-well plate into the magnetic holder, magnetically attract for 1 min, discard the waste liquid, and gently tap on absorbent paper.

[0037] (6) Add 500 μl wash buffer, cover with silicone pad, vortex for 30s, insert the 96-well plate into the magnetic holder, magnetically attract for 1 min, discard waste liquid, and gently tap on absorbent paper.

[0038] (7) Repeat step (6) once, centrifuge to 500 rpm.

[0039] (8) After drying the alcohol on the 96-well plate on the ultra-clean workbench, add 30 μl of sterile water to wash it off, cover it with a sealing film, shake it vigorously for 30 s, bathe it in a 65 ℃ water bath for 5 min, centrifuge it at 1000 rpm, and then insert the 96-well plate into the magnetic rack and let it adsorb for 1 min.

[0040] 2. PCR amplification (1) Amplification of DNA using primers MmanCOI-F 5' ATTTACAGTTGGAATAGATG 3'; MmanCOI-R 5' GATAATCCGAATAACGAC 3'; (2) The extracted DNA sample was diluted appropriately and used as a PCR template. It was amplified using the Qingke 1×TSE101 Gold Mix. The components of the amplification system are as follows: 1.1×T3 super PCRmix 35 µl; MmanCOI-F (10P) 2 µl; MmanCOI-R (10P) 2 µl; 1 µl of DNA template; (3) The above amplification system was amplified according to the following amplification program: 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 6 s, 35 cycles; 68℃ extension for 5 min; 4℃ storage.

[0041] 3. Electrophoresis detection The amplified PCR products were subjected to agarose gel electrophoresis (2 µl sample + 6 µl bromophenol blue), using DL2000 DNA marker (Takara) at 300 V for 12 min to obtain the identification gel image.

[0042] The results are as follows Figure 1 , 2As shown, the presence of certain substances in the electrophoresis pattern can be determined by... MmanCOI Gene fragments are used to determine whether the insect is a Manila lateral groove parasitoid wasp or whether the host pest has been successfully parasitized by the wasp.

[0043] (4) Perform first-generation sequencing on the PCR products. The sequencing results and splicing results are shown in SEQ ID NO.4: ATTTACAGTTGGAATAGATGTTGATACACGAGCTTATTTTACATCAGCAACTATAATTATTGCTGTTTCCAACAGGAATTAAAATTTTTAGTTGAATAGCAACATTTAGAGGGGTAAAATTAATAAATAATTTAAATGTTTTATGATCAATAGGATTTATTTTTTATTTACATTAGGAGGTTTAACAGGTGTTGTTTTATCTAATTCATCAGTTGATGTAGTTTTAC ATGATACATATTATGTTGTAGCTCATTTTCATTATGTATTATCTATAGGAGCTGTATTTGCTATTTTAGGTGGTTTTATTTATTGATATCCATTATTTTTTGGATTTATAATTAATGAAAAATTTTTAAAAGTTCATTTTTATTTAACTTTTATTGGTGTTAATTTAATTTTTTTCCACAACATTTTTTAGGTTTAGGAGGAATACCTCGTCGTTATTCGGATTATC The amplified product was associated with Manila lateral groove braconid wasp. helm Partial sequence consistency.

[0044] The bands in the attached figure are single and clear, and the ability to detect even trace amounts of insect tissue demonstrates strong sensitivity and specificity.

[0045] The above description is merely a specific embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any variations or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed in the present invention should be included within the scope of protection of the present invention. Therefore, the scope of protection of the present invention should be determined by the scope of the claims.

Claims

1. A method for identifying Manila parasitic wasps, characterized in that, The method includes the step of PCR amplification of DNA from the tissue of the parasitic wasp to be tested using primers specific to the COI sequence of the Manila lateral groove braconid wasp. The primer sequence is as follows: SEQ ID NO.2:5'-ATTTACAGTTGGAATAGATG-3'; SEQ ID NO. 3: 5'-GATAATCCGAATAACGAC-3'.

2. The method for identifying Manila lateral groove braconid wasps according to claim 1, characterized in that, The method includes the following steps: (1) After grinding the tissue of the parasitic wasp to be tested, DNA was extracted; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the specific primers described above; (3) When a bright band of 455bp is present in the agarose gel electrophoresis, the parasitic wasp to be tested is identified as Manila lateral groove parasitic wasp.

3. The method for identifying Manila lateral groove braconid wasps according to claim 2, characterized in that, In step (2), the PCR amplification program is as follows: reaction conditions are 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 6 s, 35 cycles; 68℃ extension for 5 min; storage at 4℃.

4. A method for detecting the parasitic host of the Manila lateral groove wasp, characterized in that, The method includes the step of PCR amplification of DNA in the host tissue of the Noctuidae family using primers with a specific COI sequence of the Manila lateral groove braconid wasp; The primer sequence is as follows: SEQ ID NO.2:5'-ATTTACAGTTGGAATAGATG-3'; SEQ ID NO. 3: 5'-GATAATCCGAATAACGAC-3'.

5. The method for detecting the host plant of the Manila lateral furrow wasp parasitizing Noctuidae moths according to claim 4, characterized in that, The method includes the following steps: (1) After grinding the tissue of the Noctuidae host, DNA was extracted; (2) Using the DNA obtained in step (1) as a template, perform PCR amplification using the specific primers described above; (3) When a bright band of 455bp is present in the agarose gel electrophoresis, it is determined that the Manila parasitoid wasp has successfully parasitized the Noctuidae host.

6. The method for detecting the host plant of the Manila lateral furrow wasp parasitizing Noctuidae moths according to claim 5, characterized in that, The host plants of the Noctuidae family include fall armyworm, beet armyworm, or cotton bollworm.

7. The method for detecting the host plant of the Manila lateral furrow wasp parasitizing the Noctuidae family according to claim 5, characterized in that, In step (2), the PCR amplification program is as follows: reaction conditions are 98℃ pre-denaturation for 2 min; 98℃ denaturation for 10 s, 55℃ annealing for 30 s, 68℃ extension for 6 s, 35 cycles; 68℃ extension for 5 min; storage at 4℃.

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