CN110734985B - Method for identifying cotton aphidiidae and stipes equinus punctatus - Google Patents
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Abstract
The invention provides a method for identifying cotton aphidiidae and stigmatis nodorum braconid wasps, and belongs to the technical field of PCR detection. According to the analysis of the conserved regions of the genome sequences of the cotton aphidiidae and the stigmatis nodorum, the invention designs a specific primer pair for detecting the cotton aphidiidae and the stigmatis nodorum, the nucleotide sequences of the specific primer pair are respectively shown as SEQ ID NO.1-2 and SEQ ID NO.3-4, and the sequences of the positive amplification products are respectively shown as SEQ ID NO.5 and 6. By adopting the specific primer pair, whether the DNA to be detected is the Aphidius gossypii or Aphis fabae nodosa can be judged through the sequence of the amplification product. The method disclosed by the invention is high in sensitivity, good in specificity and high in detection speed, can be used for identifying or distinguishing the cotton aphid braconid bees and the stigmatis nodorum braconid bees, and provides an effective means support for accurately researching the relationship between the parasitic wasps and the cotton aphids and utilizing the parasitic wasps to control the cotton aphids.
Description
Technical Field
The invention relates to the technical field of PCR detection, in particular to a method for identifying cotton aphid hornworm (Lysiphlobia japonica Ashmead) and stipe myrtle (Lysiphllebus fabarum Marshall).
Background
The cotton aphid cocoon bee (Lysiphlobia japonica Ashmead) is the dominant parasitic natural enemy of the important pest cotton aphid in the cotton field, is the parasitic bee of the cotton aphid occupying the absolute dominance in the northern cotton field, and can effectively suppress the population quantity of the cotton field aphid in the early summer. Oncorhynchus nodus (Lysiphlebus fabarum Marshall) is the dominant parasitic natural enemy of soybean aphids in soybean fields, and meanwhile, the Oncorhynchus nodus is also often hosted by cotton aphids. Because of the high-efficiency and specific parasitic characteristics of aphidiidae, the aphidiidae becomes an important means for controlling the agricultural and forestry pests. The resource utilization of the aphidiidae has important significance for preventing and controlling the pests in agriculture and forestry. Because they are closely related to those belonging to the family hymenoptera, Aphididae. Both have similar morphology and parasitic habits. Meanwhile, because the individual is tiny, the two are difficult to distinguish in practical application.
According to research, the human eyes can not distinguish the cotton aphid bracon larvae from the stigmatis nodorum larvae and pupae, even imagoes, and can distinguish the cotton aphid bracon larvae from the stigmatis nodorum larvae and pupae according to wing veins of wings only in the imagoes stage. Because parasitic wasps are various in types, the relationship between the parasitic wasps and the cotton aphids cannot be accurately researched, and the control effect of different parasitic wasps on the cotton aphids cannot be accurately evaluated. Because of the extremely close external shapes of the cotton aphidiidae and the stigmatis nodorum, how to distinguish the cotton aphidiidae from the stigmatis nodorum is a difficult problem which always puzzles entomologists. Therefore, the development of a rapid and accurate identification method for the cotton aphidiidae and the stigmatis arboricius is of great significance to production and scientific research.
Disclosure of Invention
The invention aims to make up for the defects of the prior art and provides a method for identifying cotton aphid hornworm (Lysiphlobia japonica Ashmead) and stipe myrtle (Lysiphllebus fabarurum Marshall).
According to the invention, through analyzing genome sequences of cotton aphidiidae and stigmatis arboricius (the mitochondrial similarity of the two cocoons is 99.8%, species specificities of the cotton aphidiidae, the stigmatis arboricius and other species of bees are also considered), a conserved region is searched, a difference target sequence is searched in a mitochondrial gene sequence, a conserved region with specificity is found, and specific primer pairs for detecting the cotton aphidiidae and the stigmatis arboricius are designed through elaborative design and screening aiming at the region, wherein nucleotide sequences of the specific primer pairs are respectively shown as SEQ ID NO.1-2 and SEQ ID NO. 3-4.
The invention firstly provides a specific primer combination for identifying cotton aphid hornworm (Lysiphlobia japonica Ashmead) and stigmatoceles granulosus (Lysiphlibus fabarurum Marshall), which contains primers with nucleotide sequences shown as SEQ ID NO.1-2 and SEQ ID NO. 3-4.
The specific primer pair for identifying or detecting cotton aphid hornet (Lysiphlobia japonica Ashmead) also belongs to the protection scope of the invention, and the primer pair comprises 2 primers, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 1-2.
Meanwhile, a specific primer pair for identifying or detecting the Oncorhynchus fabius Marshall also belongs to the protection scope of the invention, wherein the primer pair comprises 2 primers, and the nucleotide sequence of the primer pair is shown as SEQ ID NO. 3-4.
Further, the invention provides a kit or a detection reagent containing the specific primer combination or the specific primer pair.
The invention provides application of the specific primer combination in identifying cotton aphid hornworm (Lysiphlobia japonica Ashmead) and/or stipes fabarum Marshall.
The invention provides application of the specific primer combination or the specific primer pair containing the primers shown in SEQ ID NO.1-2 in preventing and treating cotton aphid pests by using parasitic wasps, and selects parasitic wasps capable of parasitizing cotton aphids by identifying whether the parasitic wasps are cotton aphid braconid wasps or stipes nodularis.
The invention also provides application of the specific primer combination in preventing and treating soybean aphid pests by using parasitic wasps, and parasitic wasps capable of parasitizing soybean aphids are selected by identifying whether the parasitic wasps are cotton aphid braconid wasps or stipes nodosum braconid wasps.
The invention provides a method for identifying cotton aphid hornworm (Lysiplebia japonica Ashmead) and stipe myrtle (Lysiplebus fabarum Marshall), which takes DNA of a parasitic wasp sample to be detected as a template, respectively applies the specific primer combination, uses a PCR method for amplification, sequences amplification products, and judges results according to sequence length or specific sequence.
On the other hand, the invention also provides a method for detecting cotton aphid hornworm (Lysiphlobia japonica Ashmead), which takes the DNA of a parasitic wasp sample to be detected as a template, applies a specific primer pair shown in SEQ ID NO.1-2, performs amplification by using a PCR method, sequences amplified products, and judges the result according to the sequence length or specific sequence.
The invention also provides a method for detecting the stipes nodorum japonicum Marshall, which takes the DNA of a parasitic wasp sample to be detected as a template, applies a specific primer pair shown in SEQ ID NO.3-4, amplifies by using a PCR method, sequences amplified products and judges the result according to the sequence length or the specific sequence.
In the embodiment of the present invention, the PCR reaction system is:
the PCR conditions in the PCR method described in the examples of the present invention were: pre-denaturation: 94 ℃ for 2 min; 30s at 94 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.
In the method, if the length of the amplification product is 261bp, the parasitic wasp to be detected is cotton aphid hornet (Lysiphlebia japonica Ashmead); if the length of the amplification product is 310bp, the parasitic wasp to be detected is stipes faberi (Lysiphlebus fabarum Marshall).
Another judgment standard of the method is that if the amplification product has a sequence shown as SEQ ID No.5, the parasitic wasp to be detected is Aphidius gossypii (Lysiphlobia japonica Ashmead); if the amplification product has a sequence shown as SEQ ID NO.6, the parasitic wasp to be detected is stigmatocystus granulosus (Lysiplebus fabarum Marshall).
The two judgment standards can be used in combination to ensure the accuracy of the detection result.
The invention provides application of the method in agricultural production and aphid disease monitoring.
The method disclosed by the invention is high in sensitivity, good in specificity and high in detection speed, can be used for identifying or distinguishing the cotton aphid braconid bees and the stigmatis nodorum braconid bees, and provides an effective means support for accurately researching the relationship between the parasitic wasps and the cotton aphids and utilizing the parasitic wasps to control the cotton aphids. The invention has at least the following beneficial effects: (1) the operation is simple. The DNA extraction and PCR hybridization technology used by the invention is the traditional molecular biology technology, the technology is mature, and the detection sensitivity is high. (2) The application is wide. Can be used for identifying cotton aphid braconid bees and legume aphid braconid bees in any period and insect state. (3) The sensitivity and the accuracy are high. The sensitivity detection line of the method is 0.515 ng/mu L, and the Aphidius gossypii and Aphidius somniferus are distinguished according to different DNA sequences, so that the accuracy rate can reach 100%.
Drawings
FIG. 1 is a diagram showing the results of the detection of the specificity of the primers of the present invention, wherein lane 1. Oesophagostoma japonicum Choreid; 2. cotton aphid hornet; 3. cotton aphid; 4. aphidius gifuensis; 5. myzus guanshuang (Aphidius avenae); 6. aphid vespids; 7. aphid-derived golden wasp; 8. soybean aphidiidae; 9. aphid hornet; 10. aphid wasp with thin waist and long neck; 11. red lateral osmia immatura; 12. amaurus brevifolius (Meadowrue) Sing. Lane 1 band 310bp, lane 2 band 261 bp.
FIG. 2 is a diagram of an electrophoresis set of example 2 of the present invention. The template in lanes 1-12 is DNA of Aphidius gossypii, wherein lanes 1-6 correspond to the specific primer pair shown in SEQ ID NO.1-2, and the amplification result has a 261bp band; lanes 7-12 correspond to the specific primer pairs shown in SEQ ID NO. 3-4; the template in lanes 13-24 is DNA of Aphis fabae, wherein lanes 13-18 correspond to the specific primer pair shown in SEQ ID NO.1-2, lanes 19-24 correspond to the specific primer pair shown in SEQ ID NO.3-4, and the amplification result has a 310bp band.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention.
Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1 design and screening of primers for identifying Aphidiidae and Aphidiidae
According to the invention, through analyzing genome sequences of Aphidiidae and Aphidius stigmatis fabri, searching for conserved regions, and searching for differential target sequences in mitochondrial gene sequences, specific conserved regions are found, specific primer pairs for detecting Aphidiidae and Aphidius stigmatis fabri are designed through elaborate design and screening aiming at the regions, the process of screening optimal primers is provided in the embodiment, 3 pairs of a plurality of candidate primers designed by software are randomly selected for effect verification with primers to be protected in the application, and the alternative primers are as follows:
(1) candidate primer 1
Seq1-F 5’-AATATTGGGATTGTCAATAAGTT-3’
Seq1-R 5’-TCCTATAATTGATGAGATACCAG-3’
Seq2-F 5’-GGAGGATTTGGAAATTGAT-3’
Seq2-R 5’-GATCTATTTTAATATTATAAGGACG-3’
(2) Candidate primer 2
Seq1-F 5’-CTGGTGTGGGTACAGGTTG-3’
Seq1-R 5’-TTATACGGACGTATATTAAAAATAG-3’
Seq2-F 5’-TAATAGTATTGTTACAGCTCATGC-3’
Seq2-R 5’-CAGCAACACCTCTATGTCCTA-3’
(3) Candidate primer 3
Seq1-F 5’-TCCTGATATAGCTTTCCCTC-3’
Seq1-R 5’-ATCACCACCTCCAGCAA-3’
Seq2-F 5’-CTCCAGATATGGCTTTTCC-3’
Seq2-R 5’-CAGCAGTAATTAACACTGACCAA-3’
(4) Candidate primer 4
Seq1-F 5’-AGAATTGTCACTGCTCATGC-3’
Seq1-R 5’-TGCTACCCCTCTATGACCT-3’
Seq2-F 5’-ATGAACAGTTTATCCACCTTTA-3’
Seq2-R 5’-AGGATCTCCTCCACCAGC-3’
Extracting whole genome DNA of cotton aphid braconid bees and stigmatis nodorum braconid bees by adopting animal tissue cells and Trizol reagent; sequentially adding reaction reagents into a PCR reaction tube according to the table 1, and flicking and uniformly mixing by fingers; after short-time centrifugation, putting the mixture into a PCR instrument; the PCR reaction was run. The reaction procedure is as follows: denaturation at 94 deg.C for 2 min; 35 times of cyclic amplification reaction (denaturation at 94 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30 s); extension at 72 ℃ for 10 min. And (4) taking out the PCR reaction tube after the reaction is finished, and carrying out electrophoresis detection on the PCR reaction product. Sterile water was used as a template for a blank PCR reaction system. Weighing agarose according to the concentration of 18g/L, adding the agarose into 1 XTAE buffer solution, heating and dissolving to prepare agarose solution, pouring the agarose solution onto an electrophoresis plate after cooling slightly, inserting a comb plate, solidifying the agarose solution into gel in a greenhouse, and slightly pulling the comb plate vertically upwards. Mixing 5 mu L of PCR product with 3 mu L of sample adding buffer solution, adding the mixture into sample application air, adding DNA marker into one sample application hole, and switching on a power supply to carry out electrophoresis under the condition of 2V/cm-5V/cm. And after the electrophoresis is finished, taking out the agarose gel, and placing the agarose gel on a gel imager for imaging. The size of the amplified band was estimated according to DNA molecular weight standards.
TABLE 1 PCR reaction System
The sensitivity of the specific candidate primer 1, the specific candidate primer 2 and the specific primer 3 is respectively 8.525 ng/. mu.L, 10.125 ng/. mu.L and 1.625 ng/. mu.L, which are all lower than that of the candidate primer 4(0.515 ng/. mu.L), so the sensitivity of the candidate primer 4 is the best.
In addition, the specificity of the 4 groups of candidate primers is verified, and the specificity detection is performed on the stipes nodularis, the gossypium hirsutum, the sulzer braconid wasps, the eurotium cristatum, the aphid vespid, the aphid pan, the soybean aphid braconid wasps, the aphid vespid, the aphid loins and girlfulb braconid wasps, the middle red lateral groove braconid wasps and the wide shoulder aeschynomencus, so that the candidate primer 4 is the best in specificity, only the stipes nodularis hirsutum and the gossypium hirsutum can be amplified, amplified bands are obviously different, and no amplified bands appear on other braconid wasps. See fig. 1.
According to the invention, after a plurality of primer pairs which aim at the same target and have weaker specificity and sensitivity are abandoned, a candidate primer 4, namely the following specific primer is selected as a primer combination for detecting cotton aphidiidae and stigmatis arboricius.
Seq1-F 5’-AGAATTGTCACTGCTCATGC-3’(SEQ ID NO.1)
Seq1-R 5’-TGCTACCCCTCTATGACCT-3’(SEQ ID NO.2)
Seq2-F 5’-ATGAACAGTTTATCCACCTTTA-3’(SEQ ID NO.3)
Seq2-R 5’-AGGATCTCCTCCACCAGC-3’(SEQ ID NO.4)
After PCR amplification and product sequencing, the cotton aphidiidae and the stigmatis nodosa obtain different DNA sequences: aphidius gossypii sequence: SEQ ID NO.5, Oesophagostoma leguminosa sequence: and comparing the SEQ ID NO.6 to identify the aphidiidae or the stigmatis nodorum.
Example 2 application of the method of the invention
1. And identifying the cotton aphidiidae and the stigmatis nodorum braconidae which are separately bred.
Taking 12 heads of cotton aphidiidae and stigmatis nodorum pupa and imago from different families respectively, wherein the 12 heads are known as the cotton aphidiidae or the stigmatis nodorum. The DNA samples of the 12 parasitic wasps are respectively detected by adopting the PCR method and the PCR system in the embodiment 1 and using SEQ ID NO.1-2 and SEQ ID NO. 3-4. The result of the PCR product electrophoresis identification is shown in figure 2, and the length of the amplification product of the parasitic wasp to be detected, namely cotton aphid cocoon bee (Lysiphlobia japonica Ashmead), is 261 bp; the parasitic wasp to be detected is a stipes faberi (Lysiphlebus fabarum Marshall), and the length of an amplification product is 310 bp.
Sequencing the amplification product, and finding that the parasitic wasp to be detected, which is the amplification product and corresponds to the sequence shown in SEQ ID NO.5, is a cotton aphid cocoon (Lysiphlobia japonica Ashmead); the amplification product is a sequence shown in SEQ ID NO.6, and the corresponding parasitic wasp to be detected is stigmatophagus granulosus (Lysiplebus fabarum Marshall). The method of the invention has the accuracy rate of 100 percent.
2. And (3) identifying the cotton aphidiidae and the stigmatis nodorum braconid wasps which are bred in a mixed mode.
The stiff aphids and adults of insects (unknown cotton aphidiidae and stigmatis nodosa) were collected 12 heads each.
The DNA samples of the 24 parasitic wasps are respectively detected by adopting the PCR method and system of the embodiment 1 and using SEQ ID NO.1-2 and SEQ ID NO. 3-4.
Aiming at the 24 DNA detection results of the unknown cotton aphid cocoon bee and the unknown stigmatis nodorum, an electrophoresis chart of an amplification product shows that 14 strips correspond to 261bp in length, and 10 strips correspond to 310bp in length.
All 261bp bands and 310bp bands are recovered and sent to a company for sequencing, and the sequences of all 261bp bands are found to correspond to SEQ ID NO.5, and the sequences of all 310bp bands are found to correspond to SEQ ID NO. 6.
Sequencing the amplification product, and finding that the parasitic wasp to be detected, which is the amplification product and corresponds to the sequence shown in SEQ ID NO.5, is a cotton aphid cocoon (Lysiphlobia japonica Ashmead); the amplification product is a sequence shown in SEQ ID NO.6, and the corresponding parasitic wasp to be detected is stigmatophagus granulosus (Lysiplebus fabarum Marshall). The method of the invention has the accuracy rate of 100 percent.
Although the invention has been described in detail hereinabove with respect to a general description and specific embodiments thereof, it will be apparent to those skilled in the art that modifications or improvements may be made thereto based on the invention. Accordingly, such modifications and improvements are intended to be within the scope of the invention as claimed.
Sequence listing
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Claims (3)
1. Specific primer combination for identifying Aphidius gossypii (Aphidius gifuensis Koch.) (Lysiphlebia japonicaAshmead) and/or gloeostereum stipitis (Aphidius multifiliis)Lysiphlebus fabarumMarshall), wherein the specific primer combination consists of primers with nucleotide sequences shown as SEQ ID NO.1-2 and SEQ ID NO. 3-4.
2. Identifying Aphidius gossypii (A) GossypiiLysiphlebia japonicaAshmead) and Amycophorus somniferus (Aphidius somniferus)Lysiphlebus fabarumMarshall) is characterized in that DNA of a parasitic wasp sample to be detected is taken as a template, specific primer combinations are respectively applied,amplifying by using a PCR method, sequencing an amplification product, and judging a result according to the sequence length or a specific sequence; the specific primer combination consists of primers with nucleotide sequences shown as SEQ ID NO.1-2 and SEQ ID NO. 3-4;
if the length of the amplification product is 261bp, the parasitic wasp to be detected is cotton aphid hornet: (A)Lysiphlebia japonicaAshmead); if the length of the amplification product is 310bp, the parasitic wasp to be detected is stigmariurus cuneatus (A) (bee)Lysiphlebus fabarumMarshall); or
If the amplification product has a sequence shown as SEQ ID NO.5, the parasitic wasp to be detected is Aphidius gossypii (Aphidius) gossypii: (Abelmoschus)Lysiphlebia japonicaAshmead); if the amplification product has a sequence shown as SEQ ID NO.6, the parasitic wasp to be detected is stigmatis nodorum (Aphidius avenae) (II)Lysiphlebus fabarum Marshall）。
3. The method of claim 2, wherein the PCR conditions in the PCR method are: pre-denaturation: 94 ℃ for 2 min; 30s at 94 ℃, 30s at 50 ℃, 30s at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.
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