CN111567566B - Aphid control agent, RNAi and beauveria bassiana combined application aphid control method - Google Patents

Aphid control agent, RNAi and beauveria bassiana combined application aphid control method Download PDF

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CN111567566B
CN111567566B CN202010468312.0A CN202010468312A CN111567566B CN 111567566 B CN111567566 B CN 111567566B CN 202010468312 A CN202010468312 A CN 202010468312A CN 111567566 B CN111567566 B CN 111567566B
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王进军
牛金志
张君
尚峰
叶超
张永军
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Abstract

The invention relates to the technical field of pest control and genetic engineering, and discloses an aphid control agent: dsRNA containing aphid and beauveria bassiana spore; also discloses a method for controlling aphids by combining RNAi and beauveria bassiana, which is to treat plants with the aphid control agent to control aphids. The invention develops a novel aphid controlling agent by combining the biocontrol fungus and the RNAi technology, the aphid controlling agent has an obvious synergistic mechanism, can overcome the defect of controlling aphids by singly using the fungus or the RNAi technology, and the aphid controlling capacity can reach 80 percent. The aphid control agent can be directly used for carrying out spray treatment on crops in the field to effectively control aphids. The method has wide application range, can mix the corresponding dsRNA with the spore suspension of the beauveria bassiana for spray treatment only by synthesizing the dsRNA of different target genes of the aphid, and can be widely applied to aphids such as pea aphid, peach aphid, brown orange aphid and the like.

Description

Aphid control agent, RNAi and beauveria bassiana combined application aphid control method
Technical Field
The invention relates to the technical field of pest control and genetic engineering, in particular to a method for controlling aphids by combining an aphid control agent, RNAi and beauveria bassiana.
Background
The damage of aphids to plants includes both direct feeding and virus transmission. At present, the harm and drug resistance of aphids are increasingly serious in production, and the development of novel aphid control strategies and technologies is urgently needed. Beauveria bassiana Bassiaana is a biological control microorganism with wide application, has the characteristics of wide host range, strong control toxicity and the like, and has great application potential in pest control. Among them, Beauveria bassiana is most widely used in corn borers and pine caterpillars.
RNA interference (RNAi), an important gene silencing means widely used in recent years, refers to a highly conserved phenomenon during evolution, induced by double-stranded RNA (dsRNA), that specifically degrades homologous RNA with high efficiency, and specifically degrades mRNA of a corresponding sequence through the mediation of dsRNA. Since the expression of a specific gene can be specifically inhibited using the RNAi technology, the technology has been widely used in the fields of exploring gene functions and gene therapy. The RNAi technology has the potential of being developed into a new generation of pesticide due to the advantages of strong specificity, no pollution, multiple gene targets and the like.
At present, beauveria bassiana or RNAi technology is generally adopted to control pests independently, and the problem of slow effect of fungus exists when beauveria bassiana is used independently. The effect of one control method used alone is poor, and no report of combining the two methods for controlling aphids is found.
Disclosure of Invention
The invention aims to solve the problems and provides a method for controlling aphids by combining an aphid control agent, RNAi and beauveria bassiana.
In order to achieve the purpose, the invention adopts the technical scheme that:
an aphid control agent comprises dsRNA of aphid and beauveria bassiana spore.
The aphid control agent is a mixed solution of dsRNA solution of aphids and beauveria bassiana spore suspension.
Preferably, the dsRNA of the aphid refers to dsRNA or a plurality of dsRNA combinations corresponding to one or more genes of gene4714, gene15782 and gene20844 of the aphid.
Preferably, the aphid is a pea aphid or a green peach aphid or a brown orange aphid.
Further preferably, the dsRNA is Apds15782 or Apds20844 or Apds4714+ Apds15782 or Apds15782+ Apds20844 or Apds4714+ Apds20844 or Mpds4714+ Mpds15782 or Acds4714+ Acds 15782.
A method for controlling aphids by combining RNAi and beauveria bassiana is to treat plants to control aphids by adopting any one of the above aphid control agents.
Preferably, the aphid control agent is used for carrying out spray treatment on plants to control aphids.
The invention has the beneficial effects that: the invention develops a novel aphid controlling agent by combining the biocontrol fungus and the RNAi technology, the aphid controlling agent has an obvious synergistic mechanism, can overcome the defect of controlling aphids by singly using the fungus or the RNAi technology, and the aphid controlling capacity can reach 80 percent. The aphid control agent can be directly used for carrying out spray treatment on crops in the field to effectively control aphids. The method has wide application range, can mix the corresponding dsRNA with the spore suspension of the beauveria bassiana for spray treatment only by synthesizing the dsRNA of different target genes of the aphid, and can be widely applied to aphids such as pea aphid, peach aphid, brown orange aphid and the like.
Drawings
FIG. 1 is a survival rate curve of the pea aphids in the combined aphid control experiment of the pea aphids by single gene interference of Apgene4714, Apgene20844 and Apgene15782 and Beauveria bassiana.
FIG. 2 is a pea aphid survival rate curve of a pea aphid multi-gene interference and beauveria bassiana combined aphid control experiment.
FIG. 3 shows the result of experiments for verifying beauveria bassiana infection by preserving moisture and culturing of a dead body of a pea aphid.
FIG. 4 is a survival rate curve of the pea aphids in the experiment of combining single gene interference of the pea aphids Apgene14740, Apgene14741, Apgene16968 and Apgene15105 with beauveria bassiana.
FIG. 5 is a survival rate curve of myzus persicae in a combined aphid control experiment of myzus persicae double-gene interference and beauveria bassiana.
FIG. 6 is a survival rate curve of brown orange aphid in the experiment of combining interference of brown orange aphid double genes and beauveria bassiana.
Detailed Description
The invention is further illustrated by the following examples, which are not intended to be limiting.
The experimental procedures in the following examples are conventional unless otherwise specified.
The main reagent sources are as follows:
2 XTAQAQASTER Mix (near shore protein science and technology limited, China)
Figure GDA0003253197120000021
reagent (Invitrogen corporation, USA), gel recovery and purification kit (Takara corporation, Japan)
TranscriptAId T7 High Yield transformation Kit (Thermo fisher Scientific, USA), comprising:
5×TranscrptAid Reaction Bμffer、TranscriptAid Enzyme Mix。
PrimeScriptTMRT Reagent Kit (Takara, Japan)
Example 1
Based on the sequencing data of the Piper pisum transcriptome, the gene expression conditions of 3 time points of 0h, 36h and 72h of Beauveria bassiana infection are analyzed, and the differential expression genes at different time points are analyzed in a comparison manner. After 567 differential genes are obtained by sequencing the transcriptome of the pea aphid infected with the beauveria bassiana Bb07, NR annotation is carried out on the differential expression genes, and the results show that the genes related to immunity and metabolism are mostly up-regulated and down-regulated in 36h vs 0h and 72h vs 36 h. Thus, a total of 7 target genes were screened: apgene4714, Apgene14740, Apgene14741, Apgene16968, Apgene15105, Apgene20844, and Apgene 15782. Wherein Apgene4714 is an unidentified gene and is significantly up-regulated in both 36h and 72 h; apgene14740 and Apgene14741 are cathepsin genes, KEGG analysis shows that they are involved in lysosomal pathways; apgene16968 is an epidermal protein gene, and when Beauveria bassiana infects insects, the insect epidermis is a first barrier; apgene15105 belongs to cytochrome P450 family gene, Beauveria bassiana infects the insect and will produce the toxin, the insect needs to resist and infect through detoxifying; apgene20844 is a zinc finger protein gene, and is related to the osmotic pressure of insects; apgene15782 is a trypsin-like serine protease gene involved in insect immune responses.
Example 2 Combined aphid control of Gene interference of Piper pisum and Beauveria bassiana
Synthesis of pea aphid dsRNA
The method comprises the following steps:
1) according to transcriptome data, an aphid online database (https:// biopaa. geneuest. org/is/aphidbase /) was used to obtain the aphid pea (academic name: acyrthosporin pisum) Apgene4714, Apgene20844 and Apgene15782 genes, and Apgene4714, Apgene20844 and Apgene15782 gene specific primers containing a T7 promoter are designed, wherein the upstream primer sequences and the downstream primer sequences are respectively as follows:
T7-Apgene4714-S1(SEQ ID NO:1):
TAATACGACTCACTATAGGGAAGACACCTAGTGGCTTGT；
T7-Apgene4714-A1(SEQ ID NO:2):
TAATACGACTCACTATAGGGTTGCAGCAGTGTTCAGATT；
T7-Apgene20844-S1(SEQ ID NO:3)：
TAATACGACTCACTATAGGGGGCATTTAGCAAATCATAC；
T7-Apgene20844-A1(SEQ ID NO:4)：
TAATACGACTCACTATAGGGCTGGTCGAGAATACACTTT；
T7-Apgene15782-S1(SEQ ID NO:5)：
TAATACGACTCACTATAGGGCCGAAGACCAGAAGAAGAT；
T7-Apgene15782-A1(SEQ ID NO:6)：
TAATACGACTCACTATAGGGCGACTCTATCCGAAACTCC。
2) by using
Figure GDA0003253197120000041
Extracting total RNA of the pea aphids by the agent according to the instruction, and then utilizing a reverse transcription kit PrimeScriptTMRT Reagent Kit according to the instructions for 1 u g total RNA reverse transcription into cDNA, reverse transcription system is 20 u l, which includes: total RNA 1. mu.l (ca. 1. mu.g), 5 XPrimeScriptTM Bμffer 4μl，Prime ScriptRT Enzyme Mix I 1μl，Oligo dT Primer(50μM)1μl，Random 6mers(100μM)1μl，RNase Free ddH2O12. mu.l. Using the obtained cDNA, gene Apgene4714, gene Apgene20844, and gene Apgene15782 were amplified:
the PCR reaction conditions of the Apgene4714 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min. A total of 25. mu.l of PCR reaction was included: mu.l of cDNA template with concentration of 1000 ng/. mu.l, 1. mu.l of upstream and downstream primers with concentration of 0.15-0.25. mu.M, 12.5. mu.l of 2 XTaq Master Mix and RNase Free ddH2O 9.5μl。
The PCR reaction conditions of the Apgene20844 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 50 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min. The PCR reaction system is consistent with that of the Apgene4714 gene.
The PCR reaction conditions of the Apgene15782 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min. The PCR reaction system is consistent with that of the Apgene4714 gene.
3) Purifying and recycling the PCR product in the step 2) by using a glue recycling and purifying Kit to be used as a Transcription template for synthesizing dsRNA, and transcribing, synthesizing and purifying by using an in vitro synthesis RNA Kit Transcription Aid T7 High Yield Transcription Kit according to an instruction so as to obtain a dsRNA solution of the gene of the aphis pisum; the dsRNA synthesis reaction system is 20 mu l, and comprises: PCR recovery product 2.5. mu.l (about 1. mu.g), 5 × Transcriptaid Reaction B. mu.ffer 4. mu.l, ATP/CTP/GTP/μm TP Mix 8. mu.l at a concentration of 100mM, Transcriptaid Enzyme Mix 2. mu.l and DEPC-sequenced H2O3.5 μ l; the reaction conditions are as follows: incubate at 37 ℃ for 6 h.
Meanwhile, the dsRNA solution of GFP (green fluorescent protein) synthesized and purified by the method is used as a negative control, and the upstream primer sequence and the downstream primer sequence used for amplifying the GFP gene in the step 2) are respectively as follows:
GFP-ds-T7F(SEQ ID NO:7)：
TAATACGACTCACTATAGGGCAGTTCTTGTTGAATTAGATG；
GFP-ds-T7R(SEQ ID NO:8)：
TAATACGACTCACTATAGGGTTTGGTTTGTCTCCCATGATG。
the dsRNA sequences of the obtained pea aphid Apgene4714, Apgene20844 and Apgene15782 genes are as follows:
dsRNA sequence of the Apgene4714 gene (Apds4714, SEQ ID NO: 9):
AAGACACCTAGTGGCTTGTATGAATCATCCGCTCATCGATATTTATTTTGTCCTATAAAAGAGCAAGTCTAACACTCCGTAACACATAGTCTACCTTACAATTGTCGAAATAACTCTATCAACTCACAATTCAAGAGTTAACGTTTAAACAACTACAACATCAACATGAATCGTAACTTGTCTTTGTTTCTTTTGGTCGTCGCAGTGGTTTTGCTGGTCGCAGCCACAACGATCGATGCAGAGTGTCGCTGGCTCGACTGCCATGCACATTCGGCCGGCGACTGGTGCAACATTCTTGGACCCGGCTGGAAGGTGAAGAATTGGCGACGGTGTAACGGATTGCTTGGAAAATCTGAACACTGCTGCAA。
dsRNA sequence of Apgene20844 gene (Apds20844, SEQ ID NO: 10):
GGCATTTAGCAAATCATACTAAGGCCCATGCCAGAAAAAATTCATATATATGTGATATCTGTAATAAAGTTTTTGATCAAAAATGGTATTTAAAATGCCATATGAGGACACATACTGCAGAAAAATCTTATAAATGTGATATTTGTAATAAACTATATTCTCGAAAAGATAGTTTAAAAACTCATAAAAAGATACATATTGGAGAAAAGACATTTAAATGTGATGTCTGTGGTAAAGGATTTTATCAAGCACAACAATTAAGAGGCCACGTGAGAACACATACTGGAGAAAAACCGTATCAATGTGAAATCTGTGGTAGATTATTTAATCAAACATCAAATTTAAGAAACCACAGAATTACACATACTGGAATAAAGTCGTATTCATGTGACATCTGTAATAAAGTGTATTCTCGACCAG。
dsRNA sequence of Apgene15782 gene (Apds15782, SEQ ID NO: 11):
CCGAAGACCAGAAGAAGATTATGTGAGCAAGAGTGCAAGAAATTGTAATGCTTTCCCAGAAAGTATTAGCACTAATAATCATCTTGATATATGTAAGTTCGTTGCTTTAGAACCAATAGTATGCTGTCCGGCGATGACTAATGAACCGTTTATAGAAAAACCAGAAAGTAATAATGAAAATTCAATATCCGCCGATAAAAAATGTCATGATTATTTCAAATTAAAGCACAAAATTCAAAGTGAGGTGCCATTTGAGTCTCAGTTACCTCCGTTGCCAGTTGTGGGTGGCATACCGGCTAACATAAAACAGTTTCCACATATGGCGCTGATAGGCTATGGTGACACCACAGCAGATGGAGAGGATTGGAGATGCGGAGGTTCGCTGATAAGCGAAAGATGGATATTATCTGCAGCGCACTGTCAACAGAGTTCTGGGAATCTGGTGGCTCGTTGGGTTCGCCTCGGAGTTTCGGATAGAGTCG。
dsRNA sequence of GFP gene (dsGFP, SEQ ID NO: 12):
CAGTTCTTGTTGAATTAGATGGCGATGTTAATGGGCAAAAATTCTCTGTCAGTGGAGAGGGTGAAGGTGATGCAACATACGGAAAACTTACCCTTAATTTTATTTGCACTACTGGGAAGCTACCTGTTCCATGGCCAACACTTGTCACTACTTTCTCTTATGGTGTTCAATGCTTCTCAAGATACCCAGATCATATGAAACAGCATGACTTTTTCAAGAGTGCCATGCCCGAAGGTTATGTACAGGAAAGAACTATATTTTACAAAGATGACGGGAACTACAAGACACGTGCTGAAGTCAAGTTTGAAGGTGATACCCTTGTTAATAGAATCGAGTTAAAAGGTATTGATTTTAAAGAAGATGGAAACATTCTTGGACACAAAATGGAATACAACTATAACTCACATAATGTATACATCATGGGAGACAAACCAAA。
preparation of beauveria bassiana spore suspension
Culturing Beauveria bassiana: beauveria bassiana strain Bb07 (which is a premium for the biocontrol microorganism innovation team of the biological technology center of southwest university and is stored in the laboratory) separated from the green peach aphids is cultured by adopting a PDA (personal digital assistant) culture medium under the conditions that the temperature is 25 +/-1 ℃ and the relative humidity is 75-80%.
Taking a plurality of 15ml nuclease-free centrifuge tubes, a plurality of 1.5ml nuclease-free centrifuge tubes, a plurality of filter gauzes, 1 spore scraper, 1 forceps, 0.05% Tween 80100 ml and 1 box of each of the yellow and blue tips of the nuclease-free centrifuge tubes, then placing the centrifuge tubes in an autoclave, sterilizing at 120 ℃ for 15-20min, and using within 1-2 hours after sterilization. The autoclave sterilized instrument was placed in a clean bench and sterilized with the bench for 10-15 minutes, and then hands were wiped with 75% alcohol. Taking a proper amount of 0.05% Tween 80 into a 15ml nuclease-free centrifuge tube, then taking a proper amount of beauveria bassiana cultured for 10-14 days into the 0.05% Tween 80, washing spores from hyphae by a vortex shaking instrument, and dissolving the spores in the 0.05% Tween 80; filtering hypha and culture medium residues from the fully-vibrated spore suspension by using gauze, and leaving the spore suspension; and then proportionally diluting the spore suspension (5 times/10 times), counting by using a blood counting chamber, averaging 3 times of counting each time, finally calculating the spore concentration of the original beauveria bassiana spore suspension according to a formula, and diluting to the required concentration for later use.
Thirdly, inoculating the mixed solution of the dsRNA and the beauveria bassiana spore suspension to the pea aphids by using a spray tower
The method comprises the following steps:
(1) diluting the previously synthesized dsRNA to the desired concentration, and diluting the resulting suspension of beauveria bassiana spores to 2X 109Spores/ml. The following experimental groups were set up:
0.05% Tween 80 group: 1ml of 0.05% Tween 80+1ml of nuclease-free water;
bb07 group: 1ml spore suspension (2X 10)9Spores/ml Bb07) +1ml nuclease-free water;
dsGFP panel: 1ml of 0.05% Tween 80+1ml of dsGFP (2004 ng/. mu.l);
dsGFP + Bb 07: 1ml dsGFP (2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds4714 group: 1ml of 0.05% Tween 80+1ml of Apds4714(2004 ng/. mu.l);
apds4714+ Bb 07: 1ml Apds4714(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds20844 group: 1ml of 0.05% Tween 80+1ml of Apds20844(2004 ng/. mu.l);
apds20844+ Bb 07: 1ml Apds20844(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds15782 group: 1ml of 0.05% Tween 80+1ml of Apds15782(2004 ng/. mu.l);
apds15782+ Bb 07: 1ml Apds15782(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
Apds4714+Apds15782：1ml 0.05％Tween 80+500μl Apds4714(4008ng/μl)+500μl Apds15782(4008ng/μl)；
Apds4714+Apds20844：1ml 0.05％Tween 80+500μl Apds4714(4008ng/μl)+500μl Apds20844(4008ng/μl)；
Apds15782+Apds20844：1ml 0.05％Tween 80+500μl Apds15782(4008ng/μl)+500μl Apds20844(4008ng/μl)；
apds4714+ Apds15782+ Bb 07: 1ml spore suspension (2X 10)9Spores/ml Bb07) + 500. mu.l Apds4714(4008 ng/. mu.l) + 500. mu.l Apds15782(4008 ng/. mu.l);
Apds4714+apds20844+ Bb 07: 1ml spore suspension (2X 10)9Spores/ml Bb07) + 500. mu.l Apds4714(4008 ng/. mu.l) + 500. mu.l Apds20844(4008 ng/. mu.l);
apds15782+ Apds20844+ Bb 07: 1ml spore suspension (2X 10)9Spores/ml Bb07) + 500. mu.l Apds15782(4008 ng/. mu.l) + 500. mu.l Apds20844(4008 ng/. mu.l).
(2) Preparing experimental pea aphids: taking a clean disposable culture dish, shearing a sticky part, upwards fixing the sticky region of the sticky note at the bottom of the culture dish, picking 40 pea aphids to become aphids, and upwards fixing the backs of the pea aphids to the sticky region of the sticky note. 3 dishes per experimental group.
(3) Spray treatment and feeding: taking 2ml of the prepared experimental group solutions respectively, placing a culture dish adhered with the green pea aphids on a tray of a Potter spray tower, and performing spray treatment by using a Potter spray tower (Potter of a Burgard precision laboratory spray tower in UK); standing for 30min, taking down the treated aphids from the culture dish, transferring the aphids to fresh broad bean seedling plants in a yarn cage, feeding the aphids under the conditions of 25 +/-1 ℃, humidity of 85 +/-10% and illumination of 14 h/10 h in darkness, and recording the survival state of the aphids to 168h every 12 h. The yarn cage is a self-made yarn cage which is formed by PVC materials and gauze and is covered on each broad bean seedling potting shell to prevent the pea aphids from escaping.
Fourthly, survival rate and phenotype observation:
1. single-gene interference of pea aphids and beauveria bassiana Bb07 combined aphid control evaluation
As shown in fig. 1, Apds4714+ Bb07, Apds15782+ Bb07 and Apds20844+ Bb07 did not advance the time point of death compared with Bb07, but in 168h cumulative mortality, Apds15782+ Bb07(p is 0.0471) and Apds20844+ Bb07(p is 0.0106) were significantly different from Bb07, and Apds4714+ Bb07 was not significantly different from Bb07(p is 0.3175), but had a synergistic trend.
2. Aphid control assessment combining polygenic interference of pea aphids and beauveria bassiana Bb07
As shown in fig. 2, after 12h, the treatment groups Apds4714+ Apds15782+ Bb07 and Apds4714+ Apds20844+ Bb07 began to die, and after 24h, the treatment groups Apds4714+ Apds15782+ Bb07, Apds15782+ Apds20844+ Bb07 and Apds4714+ Apds20844+ Bb07 all had lower survival rates than the treatment group Bb07, and after 168h, the cumulative survival rates of the treatment groups Apds4714+ Apds15782+ Bb07, Apds15782+ Apds20844+ Bb07, Apds14 + Apds20844+ Bb07, Bb07 and dsGFP + Bb07 were as follows: 17.903%, 39.339%, 23.256%, 46.215% and 42.742%, it was found from the data that on the one hand the method of RNAi beauveria bassiana can make aphid lethal more quickly, and finally the cumulative survival rate is lower (i.e. the death rate is higher), and the death rate is increased by 28.3% compared with the beauveria bassiana alone. Meanwhile, the insect corpses are subjected to moisture preservation culture, and found that hyphae are grown on the surfaces of the insect corpses (as shown in figure 3), so that the pea aphids are proved to be infected and killed by beauveria bassiana.
The combination effect of Apds4714+ Apds15782+ Bb07 screened from the Aphis victoria is optimal, so that a combined aphid control evaluation test is also carried out on the Aphis citricola and the Myzus persicae.
Example 3 pea aphid other Gene interference test
The pea aphids Apgene14740, Apgene14741, Apgene16968 and Apgene15105 genes were also tested according to the method of example 2, the corresponding dsrnas were synthesized, and the experimental combinations were set:
apds14740 group: 1ml of 0.05% Tween 80+1ml of Apds14740(2004 ng/. mu.l);
apds14740+ Bb 07: 1ml Apds14740(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds14741 group: 1ml of 0.05% Tween 80+1ml of Apds14741(2004 ng/. mu.l);
apds14741+ Bb 07: 1ml Apds14741(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds15105 group: 1ml of 0.05% Tween 80+1ml of Apds15105(2004 ng/. mu.l);
apds15105+ Bb 07: 1ml Apds15105(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07);
apds16968 group: 1ml of 0.05% Tween 80+1ml of Apds16968(2004 ng/. mu.l);
Apds16968+Bb07：1ml Apds16968(2004 ng/. mu.l) +1ml spore suspension (2X 10)9Spores/ml Bb 07).
As shown in FIG. 4, the survival rate of the pea aphids cannot be reduced remarkably by interfering Apgene14740, Apgene14741, Apgene16968 and Apgene15105 genes or controlling the aphids by combining with beauveria bassiana Bb 07.
Example 4 Combined aphid control of two-gene interference of Myzus persicae and Beauveria bassiana
Synthesis of Myzus persicae dsRNA
Synthesizing dsRNA of genes Mpgene4714 and Mpgene15782 of the myzus persicae according to the method for synthesizing the dsRNA of the pisum sativum in the example 2: specific primers of Mpgene4714 and Mpgene15782 genes, wherein the sequences of the upstream primer and the downstream primer are respectively as follows:
T7-Mpgene4714-S1(SEQ ID NO:13):
TAATACGACTCACTATAGGGACTGCCATGCACATTCAGC；
T7-Mpgene4714-A1(SEQ ID NO:14):
TAATACGACTCACTATAGGGTTAAACGAGTCGTAGTGTCTT；
T7-Mpgene15782-S1(SEQ ID NO:15):
TAATACGACTCACTATAGGGCCCATTTGTCTCAACTCAG；
T7-Mpgene15782-A1(SEQ ID NO:16):
TAATACGACTCACTATAGGGTTCCAGGTGTATCCTTGTC。
the PCR reaction conditions of the Mpgene4714 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 60 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min.
The PCR reaction conditions of the Mpgene15782 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 53 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min.
The remaining steps (including PCR reaction system, dsRNA synthesis reaction system, reaction conditions, etc.) were the same as in example 2. The dsRNA sequence of the Mpgene4714 gene is (Mpds4714, SEQ ID NO: 17):
ACTGCCATGCACATTCAGCCGGCAACTGGTGCAACATACTCGGACCCGGCTGGAAGGTCAAAACTTGGAGACGTTGTAACGGTTTGCTGGGAAAATCTGAACAGTGCTGCAAATGATAACATTTTTTAGTGTTTATTTTTTTCGTATACTTATAACTCTGTATGTTTATATACCTACATATTTGATACCAGTGCATACGATATGTTGTAAGACACTACGACTCGTTTAA。
the dsRNA sequence of the Mpgene15782 gene is (Mpds15782, SEQ ID NO: 18):
CCCATTTGTCTCAACTCAGATCCATATTTAACACCAATAAAACAGATAGTTACTGGTTGGGGAAGAATTTCAACGGCTGGACCACTTAGTGATAATTTGTTAAAAGTGGATTTGGATATTTTTCCGGTGAATCGGTGTAATGAAAGTTATTTTTCATATAATAATCAAAAATTACGGTTTGGCATACTACCCGACAGCATGATATGTGCGGGTTCGTTTGATGGTGAAAGAGACGGTTGTTCGGTACGAACTATGAAAGTTGGTGATTCAGGAGGTCCACTTCAATTAGAACATGTCAACTACGGGGGCATGTATACACAATACGGGATTACATCTTTTGGGAAATTTTGTGCTGACAAGGATACACCTGGAA。
second, gene interference and beauveria bassiana combined aphid control
Beauveria bassiana spore suspension is prepared according to the method in the example 2, and a plurality of experimental group solutions are prepared to spray the green peach aphids by using a Potter spray tower. The experimental combinations were as follows:
Mpds4714+Mpds15782：1ml 0.05％Tween 80+500μl Mpds4714(4008ng/μl)+500μl Mpds15782(4008ng/μl)；
mpds4714+ Mpds15782+ Bb 07: 1ml spore suspension (2X 10)9Spore/ml Bb07) + 500. mu.l Mpds4714(4008 ng/. mu.l) + 500. mu.l Mpds15782(4008 ng/. mu.l).
The results of the two-gene interference of myzus persicae combined with Bb07 are shown in FIG. 5, and the 168h cumulative survival rates of the 4 control groups (0.05% Tween 80, dsGFP group, Bb07 and dsGFP + Bb07) were normal, and were 88.0%, 87.5%, 55.0% and 57.5%, respectively. In Mpds4714+ Mpds15782, the survival rate of the green peach aphids is reduced at 72h, and the cumulative survival rate at 168h is 69.0%; mpds4714+ Mpds15782 showed significant difference compared to the dsGFP group (p ═ 0.0196). In Mpds4714+ Mpds15782+ Bb07, the survival rate of Myzus persicae is reduced at 48h, the cumulative survival rate at 168h is 12.5%, and compared with the Bb07 group, the initial death time of aphid is consistent, but the cumulative survival rate is reduced by 42.5%. The Mpds4714+ Mpds15782+ Bb07 showed a very significant difference compared to the Bb07 group (p ═ 0.0004).
Example 5 Brown orange aphid double-gene interference and Beauveria bassiana combined control aphid
Synthesis of brown orange aphid dsRNA
dsRNA of the brown orange aphid Acgene4714 and Acgene15782 genes was synthesized according to the method for synthesizing the dsRNA of the pea aphid in example 2: specific primers of genes of the antigen 4714 and the antigen 15782, wherein the sequences of the upstream primer and the downstream primer are respectively as follows:
T7-Acgene4714-S1(SEQ ID NO:19):
TAATACGACTCACTATAGGGATGAATCGCAACTTGTCTA；
T7-Acgene4714-A1(SEQ ID NO:20):
TAATACGACTCACTATAGGGCCCAACAATCCGTTACACC；
T7-Acgene15782-S1(SEQ ID NO:21):
TAATACGACTCACTATAGGGCCGATAATGGCTAATGGAC；
T7-Acgene15782-A1(SEQ ID NO:22):
TAATACGACTCACTATAGGGTTGTAAAGCGAAGGTGGTT。
the PCR reaction conditions of the antigen 4714 gene are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min.
The PCR reaction conditions of the gene Acgene15782 are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 30s, annealing at 55 ℃ for 30s, and extension at 72 ℃ for 30s for 35 cycles; extension was carried out at 72 ℃ for 10 min.
The remaining steps (including PCR reaction system, dsRNA synthesis reaction system, reaction conditions, etc.) were the same as in example 2.
The dsRNA sequence of the Acgene4714 gene is (Acds4714, SEQ ID NO: 23):
ATGAATCGCAACTTGTCTATGTTTCTTTTGGTCGCCGCGTTGGTTTTGCTGGTTGCAACCACAATGATCGATGCTGAGTGCCGCTGGCTGGACTGCCACGCACATTCAGCTGGCGACTGGTGCAACATACTTGGACCCGGTTGGAGGATCAAATCTTGGCGTCGGTGTAACGGATTGTTGGG。
the dsRNA sequence of the gene Acgene15782 is (Acds15782, SEQ ID NO: 24):
CCGATAATGGCTAATGGACCGTTAATTGCAGAAAATAATAAAGACAATTTAATAACTGTCGATAAAAAATGTAATGAATATTTCAAATTAGCGAATAGAATTCAATCTAAAACTATATCGAGTATTCAGTCTGCTGATGATGGTCCGTATTCAGCTGTGGGTGGTACACCAGCTAACATAAAGCAGTTTCCACACATGGCACTAATAGGATTTGGTGAAAACACAGAAGATAGTGAGGATTGGAGATGTGGGGGCTCGCTGATCAGCGAAAGGTGGATATTGACGGCAGCGCACTGTCAACAAACGAATTCAGGGAGTATGGCTCGTTGGGCTCGCCTCGGAGTTACAGACAGAGTCATTAATAAAGACAGTATAGTTCAGCCAAAAGACTATCGAATAATGCATCATGAAATACATCCTGATTACAAACCACCTTCGCTTTACAA。
second, gene interference and beauveria bassiana combined aphid control
Beauveria bassiana spore suspension is prepared according to the method in the example 2, and a plurality of experimental group solutions are prepared to spray the brown orange aphid by using a Potter spray tower.
The results of brown orange aphid double gene interference combined with Bb07 are shown in FIG. 6, and the 168h cumulative survival rates of 4 control groups (0.05% Tween 80, dsGFP group, Bb07 and dsGFP + Bb07) are normal, and are 90.2%, 89.9%, 71.1% and 70.7%, respectively. In the cases of the Nos 4714 and 15782, the survival rate of the brown orange aphids is reduced at 24h, and the cumulative survival rate at 168h is 78.3%; the Acds4714+ Acds15782 showed no significant difference from the dsGFP group (p. 0.1691). In Ac4714+ Acds15782+ Bb07, survival rate of orange brown aphid is reduced at 24h, while cumulative survival rate at 168h is 19.0%, compared with Bb07 group, aphid death time is consistent, but cumulative survival rate is reduced by 52.1%; acds4714+ Acds15782+ Bb07 showed very significant difference compared to Bb07(p ═ 0.0006).
Example 6 field application
Referring to the method of example 2, a mixed solution of dsRNA + Bb07 of the pea aphid is prepared, and a plant to be controlled by the pea aphid is sprayed, wherein the mixed solution of dsRNA + Bb07 can be one of the following combinations: apds4714+ Bb07, Apds15782+ Bb07, Apds20844+ Bb07, Apds4714+ Apds15782+ Bb07, Apds15782+ Apds20844+ Bb07, Apds4714+ Apds20844+ Bb 07.
Secondly, referring to the method of example 4, a mixed solution of the peach aphid dsRNA + Bb07 is prepared, and the plant to be controlled with the peach aphid is sprayed, wherein the mixed solution of the dsRNA + Bb07 can adopt Mpds4714+ Mpds15782+ Bb 07.
Thirdly, referring to the method of example 5, a mixed solution of dsRNA + Bb07 of the brown orange aphid is prepared, and the plant to be controlled with the brown orange aphid is sprayed, wherein the mixed solution of dsRNA + Bb07 can adopt Acds4714+ Acds15782+ Bb 07.
Sequence listing
<110> university of southwest
<120> novel aphid control agent, RNAi and beauveria bassiana combined application aphid control method
<160> 24
<170> PatentIn version 3.5
<210> 1
<211> 39
<212> DNA
<213> Artificial sequence
<400> 1
taatacgact cactataggg aagacaccta gtggcttgt 39
<210> 2
<211> 39
<212> DNA
<213> Artificial sequence
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taatacgact cactataggg ttgcagcagt gttcagatt 39
<210> 3
<211> 39
<212> DNA
<213> Artificial sequence
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taatacgact cactataggg ggcatttagc aaatcatac 39
<210> 4
<211> 39
<212> DNA
<213> Artificial sequence
<400> 4
taatacgact cactataggg ctggtcgaga atacacttt 39
<210> 5
<211> 39
<212> DNA
<213> Artificial sequence
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taatacgact cactataggg ccgaagacca gaagaagat 39
<210> 6
<211> 39
<212> DNA
<213> Artificial sequence
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taatacgact cactataggg cgactctatc cgaaactcc 39
<210> 7
<211> 41
<212> DNA
<213> Artificial sequence
<400> 7
taatacgact cactataggg cagttcttgt tgaattagat g 41
<210> 8
<211> 41
<212> DNA
<213> Artificial sequence
<400> 8
taatacgact cactataggg tttggtttgt ctcccatgat g 41
<210> 9
<211> 368
<212> DNA
<213> Artificial sequence
<400> 9
aagacaccta gtggcttgta tgaatcatcc gctcatcgat atttattttg tcctataaaa 60
gagcaagtct aacactccgt aacacatagt ctaccttaca attgtcgaaa taactctatc 120
aactcacaat tcaagagtta acgtttaaac aactacaaca tcaacatgaa tcgtaacttg 180
tctttgtttc ttttggtcgt cgcagtggtt ttgctggtcg cagccacaac gatcgatgca 240
gagtgtcgct ggctcgactg ccatgcacat tcggccggcg actggtgcaa cattcttgga 300
cccggctgga aggtgaagaa ttggcgacgg tgtaacggat tgcttggaaa atctgaacac 360
tgctgcaa 368
<210> 10
<211> 420
<212> DNA
<213> Artificial sequence
<400> 10
ggcatttagc aaatcatact aaggcccatg ccagaaaaaa ttcatatata tgtgatatct 60
gtaataaagt ttttgatcaa aaatggtatt taaaatgcca tatgaggaca catactgcag 120
aaaaatctta taaatgtgat atttgtaata aactatattc tcgaaaagat agtttaaaaa 180
ctcataaaaa gatacatatt ggagaaaaga catttaaatg tgatgtctgt ggtaaaggat 240
tttatcaagc acaacaatta agaggccacg tgagaacaca tactggagaa aaaccgtatc 300
aatgtgaaat ctgtggtaga ttatttaatc aaacatcaaa tttaagaaac cacagaatta 360
cacatactgg aataaagtcg tattcatgtg acatctgtaa taaagtgtat tctcgaccag 420
<210> 11
<211> 482
<212> DNA
<213> Artificial sequence
<400> 11
ccgaagacca gaagaagatt atgtgagcaa gagtgcaaga aattgtaatg ctttcccaga 60
aagtattagc actaataatc atcttgatat atgtaagttc gttgctttag aaccaatagt 120
atgctgtccg gcgatgacta atgaaccgtt tatagaaaaa ccagaaagta ataatgaaaa 180
ttcaatatcc gccgataaaa aatgtcatga ttatttcaaa ttaaagcaca aaattcaaag 240
tgaggtgcca tttgagtctc agttacctcc gttgccagtt gtgggtggca taccggctaa 300
cataaaacag tttccacata tggcgctgat aggctatggt gacaccacag cagatggaga 360
ggattggaga tgcggaggtt cgctgataag cgaaagatgg atattatctg cagcgcactg 420
tcaacagagt tctgggaatc tggtggctcg ttgggttcgc ctcggagttt cggatagagt 480
cg 482
<210> 12
<211> 436
<212> DNA
<213> Artificial sequence
<400> 12
cagttcttgt tgaattagat ggcgatgtta atgggcaaaa attctctgtc agtggagagg 60
gtgaaggtga tgcaacatac ggaaaactta cccttaattt tatttgcact actgggaagc 120
tacctgttcc atggccaaca cttgtcacta ctttctctta tggtgttcaa tgcttctcaa 180
gatacccaga tcatatgaaa cagcatgact ttttcaagag tgccatgccc gaaggttatg 240
tacaggaaag aactatattt tacaaagatg acgggaacta caagacacgt gctgaagtca 300
agtttgaagg tgataccctt gttaatagaa tcgagttaaa aggtattgat tttaaagaag 360
atggaaacat tcttggacac aaaatggaat acaactataa ctcacataat gtatacatca 420
tgggagacaa accaaa 436
<210> 13
<211> 39
<212> DNA
<213> Artificial sequence
<400> 13
taatacgact cactataggg actgccatgc acattcagc 39
<210> 14
<211> 41
<212> DNA
<213> Artificial sequence
<400> 14
taatacgact cactataggg ttaaacgagt cgtagtgtct t 41
<210> 15
<211> 39
<212> DNA
<213> Artificial sequence
<400> 15
taatacgact cactataggg cccatttgtc tcaactcag 39
<210> 16
<211> 39
<212> DNA
<213> Artificial sequence
<400> 16
taatacgact cactataggg ttccaggtgt atccttgtc 39
<210> 17
<211> 229
<212> DNA
<213> Artificial sequence
<400> 17
actgccatgc acattcagcc ggcaactggt gcaacatact cggacccggc tggaaggtca 60
aaacttggag acgttgtaac ggtttgctgg gaaaatctga acagtgctgc aaatgataac 120
attttttagt gtttattttt ttcgtatact tataactctg tatgtttata tacctacata 180
tttgatacca gtgcatacga tatgttgtaa gacactacga ctcgtttaa 229
<210> 18
<211> 373
<212> DNA
<213> Artificial sequence
<400> 18
cccatttgtc tcaactcaga tccatattta acaccaataa aacagatagt tactggttgg 60
ggaagaattt caacggctgg accacttagt gataatttgt taaaagtgga tttggatatt 120
tttccggtga atcggtgtaa tgaaagttat ttttcatata ataatcaaaa attacggttt 180
ggcatactac ccgacagcat gatatgtgcg ggttcgtttg atggtgaaag agacggttgt 240
tcggtacgaa ctatgaaagt tggtgattca ggaggtccac ttcaattaga acatgtcaac 300
tacgggggca tgtatacaca atacgggatt acatcttttg ggaaattttg tgctgacaag 360
gatacacctg gaa 373
<210> 19
<211> 39
<212> DNA
<213> Artificial sequence
<400> 19
taatacgact cactataggg atgaatcgca acttgtcta 39
<210> 20
<211> 39
<212> DNA
<213> Artificial sequence
<400> 20
taatacgact cactataggg cccaacaatc cgttacacc 39
<210> 21
<211> 39
<212> DNA
<213> Artificial sequence
<400> 21
taatacgact cactataggg ccgataatgg ctaatggac 39
<210> 22
<211> 39
<212> DNA
<213> Artificial sequence
<400> 22
taatacgact cactataggg ttgtaaagcg aaggtggtt 39
<210> 23
<211> 182
<212> DNA
<213> Artificial sequence
<400> 23
atgaatcgca acttgtctat gtttcttttg gtcgccgcgt tggttttgct ggttgcaacc 60
acaatgatcg atgctgagtg ccgctggctg gactgccacg cacattcagc tggcgactgg 120
tgcaacatac ttggacccgg ttggaggatc aaatcttggc gtcggtgtaa cggattgttg 180
gg 182
<210> 24
<211> 446
<212> DNA
<213> Artificial sequence
<400> 24
ccgataatgg ctaatggacc gttaattgca gaaaataata aagacaattt aataactgtc 60
gataaaaaat gtaatgaata tttcaaatta gcgaatagaa ttcaatctaa aactatatcg 120
agtattcagt ctgctgatga tggtccgtat tcagctgtgg gtggtacacc agctaacata 180
aagcagtttc cacacatggc actaatagga tttggtgaaa acacagaaga tagtgaggat 240
tggagatgtg ggggctcgct gatcagcgaa aggtggatat tgacggcagc gcactgtcaa 300
caaacgaatt cagggagtat ggctcgttgg gctcgcctcg gagttacaga cagagtcatt 360
aataaagaca gtatagttca gccaaaagac tatcgaataa tgcatcatga aatacatcct 420
gattacaaac caccttcgct ttacaa 446

Claims (6)

1. An aphid control agent, which is characterized in that: dsRNA (double-stranded ribonucleic acid) containing aphids and beauveria bassiana spores, wherein the aphids refer to pea aphids or peach aphids or brown orange aphids, and the dsRNA corresponding to the pea aphids is a geneApds15782 orApds20844 orApds4714 orApds4714 + Apds15782 orApds15782 + Apds20844 orApds4714 + Apds20844, wherein the dsRNA corresponding to the myzus persicae is a geneMpds4714 + Mpds15782, wherein the dsRNA corresponding to the brown orange aphid is a geneAcds4714 + AcdsRNA of ds 15782; the geneApds15782、Apds20844、ApThe dsRNA sequence of ds4714 is shown as SEQ ID NO 11, SEQ ID NO 10 and SEQ ID NO 9 in sequence, and the geneMpds4714、MpThe dsRNA sequence of ds15782 is shown as SEQ ID NO 17 and SEQ ID NO 18 in sequence, and the geneAcds4714、AcThe dsRNA sequence of ds15782 is shown as SEQ ID NO. 23 and SEQ ID NO. 24 in sequence.
2. An aphid control agent according to claim 1, wherein: the aphid control agent is a mixed solution of dsRNA solution of aphids and beauveria bassiana spore suspension.
3. An aphid control agent according to claim 2, wherein: the aphid controlling agent is obtained by mixing the dsRNA solution and the beauveria bassiana spore suspension according to any one of the following mixture ratios:
(1) 1ml of 2004 ng/. mu.lApds15782 dsRNA +1ml concentration 2X 109Spore/ml beauveria bassiana spore suspension;
(2) 1ml of 2004 ng/. mu.lApds20844 dsRNA +1ml concentration 2X 109Spore/ml beauveria bassiana spore suspension;
(3) 1ml of 2004 ng/. mu.lApds4714 dsRNA +1ml concentration 2X 109Beauveria bassiana spore suspension with spores/ml；
(4) 1ml of 2X 109Spore/ml Beauveria bassiana spore suspension and 500 mul of concentration 4008 ng/mulApds4714 dsRNA + 500. mu.l concentration 4008 ng/. mu.lApds15782 dsRNA；
(5) 1ml of 2X 109Spore/ml Beauveria bassiana spore suspension and 500 mul of concentration 4008 ng/mulApds15782 dsRNA + 500. mu.l concentration 4008 ng/. mu.lApds20844 dsRNA；
(6) 1ml of 2X 109Spore/ml Beauveria bassiana spore suspension and 500 mul of concentration 4008 ng/mulApds4714 dsRNA + 500. mu.l concentration 4008 ng/. mu.lApds20844 dsRNA；
(7) 1ml of 2X 109Spore/ml Beauveria bassiana spore suspension and 500 mul of concentration 4008 ng/mulMpds4714 dsRNA + 500. mu.l concentration 4008 ng/. mu.lMpds15782 dsRNA。
4. A method for controlling aphids by combining RNAi and beauveria bassiana is characterized in that: the aphid controlling agent of any one of claims 1 to 3 is used to treat plants for controlling aphids.
5. The method of claim 4, wherein: the aphid control agent of any one of claims 1 to 3 is used for spraying plants to control aphids.
6. Use of an aphid control agent according to any of claims 1 to 3 for controlling aphids of the pea or green peach or brown orange.
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