CN118497206A - DsRNA and pesticide compound for preventing and controlling bactrocera dorsalis - Google Patents
DsRNA and pesticide compound for preventing and controlling bactrocera dorsalis Download PDFInfo
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- CN118497206A CN118497206A CN202410954059.8A CN202410954059A CN118497206A CN 118497206 A CN118497206 A CN 118497206A CN 202410954059 A CN202410954059 A CN 202410954059A CN 118497206 A CN118497206 A CN 118497206A
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- bactrocera dorsalis
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01P—BIOCIDAL, PEST REPELLANT, PEST ATTRACTANT OR PLANT GROWTH REGULATORY ACTIVITY OF CHEMICAL COMPOUNDS OR PREPARATIONS
- A01P7/00—Arthropodicides
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Abstract
The invention provides a target gene segment, dsRNA and pesticide compound for controlling bactrocera dorsalis. The target gene segment is a target gene segment of a bactrocera dorsalis park in gene; the invention also discloses dsRNA transcribed from the target gene segment; the dsRNA and the star-shaped cationic nano-carrier SPc are incubated together, and the injection method is used for RNAi application of the bactrocera dorsalis park gene, so that the mortality rate of the bactrocera dorsalis and the sensitivity to chlorfenapyr are obviously increased under the condition of ensuring the interference efficiency. The invention further discloses an RNA pesticide compound composed of dsRNA and SPc, and biological assay results show that the RNA pesticide compound has good effect on reducing the chlorfenapyr resistance of the bactrocera dorsalis and can delay the development of the chlorfenapyr resistance.
Description
Technical Field
The invention belongs to the technical field of pest control, and in particular relates to a pesticide composition for controlling bactrocera dorsalisAnd (3) pesticide compound.
Background
Bactrocera dorsalis (L.) Kuntze() Belongs to diptera%) Bactrocera genus) Fruit fly genus) Is a worldwide quarantine pest, and damages up to 250 fruits and vegetables, such as citrus, mango, banana, papaya and the like. The imago of the bactrocera dorsalis spawns into the fruits, and the larvae hatch and eat the pulp, so that the fruits drop or rot, and the quality and the yield of the fruits are seriously affected. The bactrocera dorsalis has the characteristics of wide host range, strong fertility, strong flying capability, high migration speed and the like, so that the bactrocera dorsalis is difficult to control. Therefore, the development of green prevention and control measures for the bactrocera dorsalis is of great significance.
The chlorfenapyr, the name chlorfenapyr, the removal and the special attack are carried out by natural antibiotics) The modified pesticide has stomach toxicity and contact killing effect on various pests, has the characteristics of no cross resistance with other pesticides, low dosage, long lasting period, high efficiency, broad spectrum and the like, and is widely applied to the field of agricultural production. Chlorfenapyr is a precursor insecticide, prepared byOxidative removal of N-ethoxymethyl groups to form N-dealkylated metabolitesAs uncouplers of oxidative phosphorylation, they disrupt the proton gradient across the intragranular membrane of the line and thus disrupt the ability of mitochondria to produce ATP, leading to death of the affected cells and ultimately to death of the insect.
The chlorfenapyr is used for a long time, so that the problems of poor insecticidal effect, drug resistance and the like can be generated. To solve this problem, a potential solution is RNA interference (RNA INTERFERENCE,) Techniques.Refers to double-stranded RNA in eukaryotes (double STRAND RNA,) The phenomenon of silencing target genes by inducing target mRNA degradation is now commonly used in insect gene function research, typically by introducing exogenous sourcesOr a small interfering RNA (SMALL INTERFERING RNA,) To inhibit target gene expression.Is considered as a promising environment-friendly pest control technology, and is expected to realize sustainable pest control by interfering the expression of key genes for the growth and development of insects.
However, how to getThe technology is associated with the prevention and treatment of the bactrocera dorsalis by using the chlorfenapyr, so that the bactrocera dorsalis is sensitive to the chlorfenapyr, the administration amount of the chlorfenapyr is reduced, the killing power of the chlorfenapyr to the bactrocera dorsalis is improved, and the problem of drug resistance in long-time use is avoided. The present application aims to solve this technical problem.
Disclosure of Invention
When the pest is stressed by the chlorfenapyr, the chlorfenapyr breaks the membrane potential of mitochondria to damage the mitochondriaGenes are essential for mitochondrial homeostasis and are involved in the process of clearing damaged mitochondria.
Based on this, the applicant creatively proves thatThe gene is a potential gene target point throughTechnique silencingThe gene can raise the sensitivity of Drosophila melanogaster to chlorfenapyr and the insecticidal effect of chlorfenapyr.
Therefore, one aspect of the application provides a method for controlling Bactrocera dorsalisThe saidThe sequence is shown as SEQ ID NO. 1.
Further, the saidConsists of the sequence as shown in SEQ ID NO:2, and the DNA sequence shown in the sequence 2 is transcribed.
In another aspect, the present application provides a pesticidal composition for controlling bactrocera dorsalis, the pesticidal composition comprisingThe saidThe sequence is shown as SEQ ID NO. 1.
Further, the pesticide complex also comprises a cationic nano-carrier. Common cationic nano-carriers can be used forDelivery of, for example, cationic liposomes, lipid nanoparticles) Gold nanoparticles, calcium carbonate nanoparticles, and the like.
Preferably, a star cation synthesized in advance by the applicant is selected) The nanometer carrier is prepared from the nanometer material,Has the advantages of low cost, high biological safety, high water solubility, strong nucleic acid binding capacity, high delivery efficiency and the like, and can be used for preparing the novel nucleic acidAnd a great potential in the efficient delivery of pesticides. Thus, by Bactrocera dorsalisThe gene being obtained from a targetAnd using nano-carriersEfficient delivery, facilitating liftingThe stability and the insecticidal effect of the chlorfenapyr on the bactrocera dorsalis. Specific preparation method of star-shaped cationic nano-carrier, and reference is made to patent literature。
In another aspect, the present application provides a target gene fragment effective in controlling bactrocera dorsalis, said gene fragmentA gene; by passing throughGene preparation. That is, the application provides a method for preventing and controlling bactrocera dorsalis, which can inhibit bactrocera dorsalisThe gene expression substance is introduced into the bactrocera dorsalis, so that the bactrocera dorsalis is more sensitive to chlorfenapyr.
Further, the method for inhibiting bactrocera dorsalisThe gene expression substance is; The saidThe sequence is shown as SEQ ID NO. 1.
The application provides a method for preventing and controlling bactrocera dorsalis, which comprises the steps of injecting the pesticide compound into the bactrocera dorsalis; the bactrocera dorsalis is an adult.
Further, the said is carried out before injectionMixing and incubating with the cationic nano-carrier for a period of time, typically 15 minutes, to obtain a mixture; the mixture was injected into the abdomen of bactrocera dorsalis.
Further, the saidThe mass ratio of the cationic nano-carrier to the cationic nano-carrier is 1:1, the injection quantity is at least 2 mug of each bactrocera dorsalis。
The method for preventing and controlling the bactrocera dorsalis comprises preventing and controlling the bactrocera dorsalis, preventing and controlling invasion and diffusion of the bactrocera dorsalis and inhibiting the bactrocera dorsalisExpression of the genes.
Another aspect of the invention provides a method for producing a composition from Bactrocera dorsalisControl of bactrocera dorsalis transcribed from a target gene segment of a gene，A kind of electronic deviceIs double-stranded RNA, and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is shown as SEQ ID No.1, and the nucleotide sequence of the antisense strand is the reverse complementary sequence of SEQ ID No. 1.
Wherein, the sequence is shown in SEQ ID No.1The sense strand nucleotide sequence of (a) is as follows:
UCGUUUGGCCAGAAAACCAUUACAAACAGUUUAAGUAUUUAUAUUAAAACCAACACUGGUAGAACGUUAUCUGUGAAUUUAGAACCACAAUGGGACAUAAAAAAUGUCAAGGAAAUCGUUGCACCGCAAUUAGGUUUGCAACCCGAAGAAGUGAAAAUCAUCUUCGCUGGUAAAGAAUUGAGUGAUGCAACAACCAUACAGGAAUGUGAUUUGGGUCAACAAAGUAUAUUACACGCGAUACGGAGUCGUCCUCAAGCGCAACGACAACGGUUACAAUCCACAGUAAUGGAGGAAGAACCGCAAGAUGUUAUGAACAUAUCUACUUCUACUGGUAGAGCAACACCUUCCCCGACAGAAGAACCAUCAAAACCUUUAUGCGAAACAUUGGUAGAUUUACAACUGCUGAGCGAAGAACG
One aspect of the present invention provides a target gene segment for controlling bactrocera dorsalis, which is bactrocera dorsalis The target gene segment of the gene, the double-stranded DNA sense strand sequence of which is shown in SEQ ID No. 2.
Wherein, the nucleotide sequence shown in SEQ ID No.2 is as follows:
TCGTTTGGCCAGAAAACCATTACAAACAGTTTAAGTATTTATATTAAAACCAACACTGGTAGAACGTTATCTGTGAATTTAGAACCACAATGGGACATAAAAAATGTCAAGGAAATCGTTGCACCGCAATTAGGTTTGCAACCCGAAGAAGTGAAAATCATCTTCGCTGGTAAAGAATTGAGTGATGCAACAACCATACAGGAATGTGATTTGGGTCAACAAAGTATATTACACGCGATACGGAGTCGTCCTCAAGCGCAACGACAACGGTTACAATCCACAGTAATGGAGGAAGAACCGCAAGATGTTATGAACATATCTACTTCTACTGGTAGAGCAACACCTTCCCCGACAGAAGAACCATCAAAACCTTTATGCGAAACATTGGTAGATTTACAACTGCTGAGCGAAGAACG
another aspect of the invention is to control the bactrocera dorsalis Mixing with nano carrier to obtain RNA pesticide compound.
Preferably, the saidIs prepared by an in vitro transcription method or an in vivo vector expression method; the in vitro transcription method comprises the following steps: by means of beltPrimers for promoter sequencesAmplification ofTranscription synthesis of gene fragment with amplified product as template。
Preferably, the carrier is a star cationic polymer.
Preferably, the method comprises the steps of,And (3) withThe mass ratio of the composite is 1:1,The total amount of (2) is at least 2. Mu.g.
Further, it willWith nano-carriersMixing, vibrating and mixing uniformly to obtain the nano-composite.
Preferably, willWith nano-carriersAfter mixing, the nanocomposite was injected into the abdomen of the adult bactrocera dorsalis using microinjection.
Preferably, after 48 h injections, an in-house bioassay experiment was performed on the back panel spot of the adult bactrocera dorsalis.
The application has the following beneficial effects:
1) The application creatively discovers the target gene for improving the sensitivity of the bactrocera dorsalis to the chlorfenapyr, namely A gene; silencing the gene can effectively improve the sensitivity of the bactrocera dorsalis to the chlorfenapyr and reduce the using amount of the chlorfenapyr, thereby solving the problem of drug resistance caused by long-time and large-dose use of the pesticide;
2) To silence Genes creatively transcribeExperiments prove that theIs guided into the body of the bactrocera dorsalis and can effectively silenceA gene;
3) For the purpose of Star cationic nanocarriers were developed that can be efficiently deliveredAvoid the degradation of the fruit fly in the body of the fruit fly, and improve the delivery efficiency.
Drawings
Fig. 1: injection of fruit fly adultsRear part (S)The relative expression of gene, the sign indicates the difference significance) (Significant difference analysis uses independent sample T test, and inter-group difference analysis usesMultiple comparative analysis), the data in the graph are mean ± standard error, as in fig. 2 and 3.
Fig. 2: injection ofMortality of adult Bactrocera dorsalis from the back 48 h orange.
Fig. 3: mortality of adult bactrocera dorsalis after drop-in of chlorfenapyr.
Detailed Description
The present invention will be further described with reference to the following examples, which are conventional, unless otherwise indicated. The test materials used in the examples described below, unless otherwise specified, were purchased from conventional biochemical reagent stores. It will be understood by those skilled in the art that various changes and substitutions can be made in the details and form of the invention without departing from the spirit and scope of the invention, but these modifications and substitutions are intended to be within the scope of the invention.
The nano-carrier in the following example is star cationic nano-carrier) The preparation method of the star-shaped cationic nano-carrier is disclosed in patent literature。
Example 1 Bactrocera dorsalisGene of geneIs prepared from
1. Test Bactrocera dorsalis
The Bactrocera dorsalis used in the study is a laboratory indoor breeding population, larvae and adults are fed by artificial feed developed in a laboratory, the breeding temperature is (26+/-1), the relative humidity is 60% -70%, and the photoperiod is。
2. Synthesis
UsingKit [ ]Nanjing Norvigator Biotech Co., ltd., nanjing) to extract total RNA of fruit fly adults, usingReverse transcription kitTiangen Biochemical technologies Co., ltd., beijing). From the following componentsDatabase acquisition of Bactrocera dorsalisGene sequence [ (] ) In the following（ ) Design of target coding region on websitePrimer and additionPromoter sequences, designed simultaneouslyA kind of electronic deviceThe primer and the primer sequence are shown in Table 1. Using the above primers, cDNA was used as a templateThe amplification is carried out,The product is recovered and purified after verification by agarose gel electrophoresis. To be purifiedThe product is taken as a template according to Kit [ ]Nanjing Norvigator Biotech Co., ltd., nanj) manual in vitro transcription synthesis and purificationVerification by 1% agarose gel electrophoresisQuality is detected by an ultraviolet spectrophotometerIs stored at-80 ℃ after split charging.
Synthesized by the methodComprises two kinds, one isAs a control group, the non-endogenous gene enhanced fluorescent protein of the targeted bactrocera dorsalis is providedA kind of electronic deviceHas no interference effect on the gene of the Bactrocera dorsalis, and is used for comparingEfficiency and chlorfenapyr biological measurement effect; the other group isThe treatment group refers to targeting of the endogenous gene of Bactrocera dorsalisA kind of electronic device. Two groups ofThe synthesis method is the same, except that the primers are different. Wherein the method comprises the steps ofGroup and method for producing the sameThe primer sequences used in the sets are specifically shown in Table 1. Above-mentionedGene of geneIs double-stranded and consists of a sense strand and an antisense strand, wherein the nucleotide sequence of the sense strand is SEQ ID No.3, and the nucleotide sequence of the antisense strand is the reverse complement of SEQ ID No. 3.The target gene segment of the gene, the double-stranded DNA sense strand sequence of which is shown as SEQ ID No. 4.
Wherein, the nucleotide sequence of SEQ ID No.3 is as follows:
GUUCACCUUGAUGCCGUUCUUCUGCUUGUCGGCCAUGAUAUAGACGUUGUGGCUGUUGUAGUUGUACUCCAGCUUGUGCCCCAGGAUGUUGCCGUCCUCCUUGAAGUCGAUGCCCUUCAGCUCGAUGCGGUUCACCAGGGUGUCGCCCUCGAACUUCACCUCGGCGCGGGUCUUGUAGUUGCCGUCGUCCUUGAAGAAGAUGGUGCGCUCCUGGACGUAGCCUUCGGGCAUGGCGGACUUGAAGAAGUCGUGCUGCUUCAUGUGGUCGGGGUAGCGGCUGAAGCACUGCACGCCGUAGGUCAGGGUGGUCACGAGGGUGGGCCAGGGCACGGGCAGCUUGCCGGUGGUGCAGAUGAACUUCAGGGUCAGCUUGCCGUAGGUGGCAUCGCCCUCGCCCUCGCCGGACACGCUGAACUUGUG
the nucleotide sequence depicted in SEQ ID No.4 is as follows:
GTTCACCTTGATGCCGTTCTTCTGCTTGTCGGCCATGATATAGACGTTGTGGCTGTTGTAGTTGTACTCCAGCTTGTGCCCCAGGATGTTGCCGTCCTCCTTGAAGTCGATGCCCTTCAGCTCGATGCGGTTCACCAGGGTGTCGCCCTCGAACTTCACCTCGGCGCGGGTCTTGTAGTTGCCGTCGTCCTTGAAGAAGATGGTGCGCTCCTGGACGTAGCCTTCGGGCATGGCGGACTTGAAGAAGTCGTGCTGCTTCATGTGGTCGGGGTAGCGGCTGAAGCACTGCACGCCGTAGGTCAGGGTGGTCACGAGGGTGGGCCAGGGCACGGGCAGCTTGCCGGTGGTGCAGATGAACTTCAGGGTCAGCTTGCCGTAGGTGGCATCGCCCTCGCCCTCGCCGGACACGCTGAACTTGTG
Example 2 Application in controlling bactrocera dorsalis
1. Injection of
Selecting adult Bactrocera dorsalis with eclosion for about 5 days as experimental material, and experimental settingControl groupTreatment groups. Put a volume of 25 into a disposable plastic cupAdding proper amount of adult feed (the mass ratio of sucrose to soybean peptone is 3:1) and a lump of soaked cotton into the sauce cup. The preparation of example 1Or (b)Diluting to a concentration ofThen with a concentration of 60 mg +.Nano-carrier of (a)According to 1:1 into a centrifuge tube with 200 mu L, and standing. Placing the bactrocera dorsalis after being anesthetized by carbon dioxide on a carbon dioxide anesthetic plate, and using a microinjection instrumentUSA) 0.4 μl of the mixture was injected into the abdomen of each adultAndAbout 2 μg each), a total of 100 were injected, 20 adults were placed in each plastic cup, finally covered with a layer of gauze over the mouth of the cup, fastened with rubber bands, marked, and the dead bactrocera dorsalis was held out with forceps after 12h injections (probably because the wound created by the injection resulted in death of a small portion of bactrocera dorsalis).
2. Detection of RNA interference efficiency
12 Adults were randomly selected in each of the 24h and 48 h groups after injection, of which 4 (2 females and 2 males) were one biological repeat, for a total of 3 biological repeats, toFor comparison, proceedAnd (5) detecting. After quick freezing with liquid nitrogen, the liquid nitrogen is usedExtracting total RNA by using a kitThe kit was reverse transcribed into cDNA using 1. Mu.g of total RNA as a template, and then usedKit [ ]Nanjing Norvigator Biotech Co., ltd., nanj) in real-time fluorescence quantificationInstrument @) Go onThe test was performed with 3 technical replicates per sample,The total system was 10. Mu.L: 2×5. Mu L forward and reverse primer ]) 0.2. Mu.L each, 1. Mu.L of cDNA, 3.6 μL。 The procedure is 30 s at 95℃and 10 s at 95℃for 30 s at 60℃for 40 cycles. Specific amplification is ensured according to the melting curve. Real-time fluorescent quantitationResults utilizationAnalysis of the method). UsingFor comparison, bactrocera dorsalisGene [ (B/C)Accession number: ) As reference genes, the primer sequences are shown in Table 1.
The results are shown in FIG. 1. As can be seen from the figures: and (3) withIn comparison with the control group,In treated groups of Bactrocera dorsalisThe expression level of the gene was significantly reduced at 48 h.
3. Chlorfenapyr bioassay after RNA interference
Mortality of bactrocera dorsalis was counted at 48 h post injection and the results are shown in figure 2, from which it can be seen that: and (3) withIn comparison with the control group,Mortality of bactrocera dorsalis in the treated group increased significantly at 48 h. This illustratesThe interference of the gene has obvious inhibition effect on the survival of the bactrocera dorsalis, and is a potential target gene for green prevention and control of the bactrocera dorsalis.
48 H from after injectionControl groupIn the treatment group, 60 adults were randomly selected, 20 (10 females and 10 males) were each biological replicates, and 3 biological replicates were all selected. Placing the fruit fly after being anesthetized by carbon dioxide on a carbon dioxide anesthetic plate, and dripping 1 mu L of the fruit fly with the concentration of 106 to the backboard of each adult by using a microsyringeChlorfenapyr (acetone as solvent). The treated bactrocera dorsalis is placed in a plastic cup, marked, and the mortality is counted after 24 h and 48 h respectively.
The results are shown in FIG. 3. As can be seen from the figures: and (3) withIn comparison with the control group,Mortality of bactrocera dorsalis in the treated group increased significantly at both 24h and 48 h. This illustrates the use of the inventionA kind of electronic deviceEffectively interfere with the fruit flyThe gene can increase the sensitivity of the bactrocera dorsalis to chlorfenapyr, and can be used for preventing and controlling the bactrocera dorsalis.
TABLE 1 primer sequences
* Lower case letter representationsA promoter sequence.
Claims (10)
1. Control of bactrocera dorsalisCharacterized in that the following stepsThe sequence is shown as SEQ ID NO. 1.
2. The method according to claim 1Characterized in that the following stepsConsists of the sequence as shown in SEQ ID NO:2, and the DNA sequence shown in the sequence 2 is transcribed.
3. An RNA pesticide complex for controlling Bactrocera dorsalis, characterized in that the RNA pesticide complex comprises the composition according to claim 1。
4. A pesticide composite as set forth in claim 3 wherein said pesticide composite further comprises a cationic nanocarrier.
5. A pesticide composite as set forth in claim 4 wherein saidThe mass ratio of the cationic nano-carrier to the cationic nano-carrier is 1:1.
6. A method for preventing and controlling Bactrocera dorsalis is characterized in that Bactrocera dorsalis is inhibitedThe gene expression substance is introduced into the bactrocera dorsalis, so that the bactrocera dorsalis is more sensitive to chlorfenapyr.
7. The method of claim 6, wherein the inhibiting bactrocera dorsalis isThe gene expression substance is; The saidThe sequence is shown as SEQ ID NO. 1.
8. A method for controlling bactrocera dorsalis, characterized in that the pesticide compound according to any one of claims 3 to 5 is injected into bactrocera dorsalis; the bactrocera dorsalis is an adult.
9. The method of claim 8, wherein the step of injecting is preceded byMixing and incubating with a cationic nano-carrier for a period of time to obtain a mixture; the mixture was injected into the abdomen of bactrocera dorsalis.
10. The method according to any one of claims 8 or 9, wherein theThe mass ratio of the cationic nano-carrier to the cationic nano-carrier is 1:1, the injection quantity is at least 2 mug of each bactrocera dorsalis。
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| CN107771181A (en) * | 2015-06-16 | 2018-03-06 | 先锋国际良种公司 | To prevent and treat the composition of insect pest and method |
| CN108949762A (en) * | 2018-08-02 | 2018-12-07 | 中国农业大学 | The dsRNA of wing development related gene optomotor-blind and its application in control of insect |
| US20200181639A1 (en) * | 2017-05-01 | 2020-06-11 | Donald Danforth Plant Science Center | Rnai approach for crop pest protection |
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| CN107771181A (en) * | 2015-06-16 | 2018-03-06 | 先锋国际良种公司 | To prevent and treat the composition of insect pest and method |
| US20200181639A1 (en) * | 2017-05-01 | 2020-06-11 | Donald Danforth Plant Science Center | Rnai approach for crop pest protection |
| CN108949762A (en) * | 2018-08-02 | 2018-12-07 | 中国农业大学 | The dsRNA of wing development related gene optomotor-blind and its application in control of insect |
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