CN117050998B - Application of miRNA in preventing and controlling cyantraniliprole-resistant bemisia tabaci - Google Patents
Application of miRNA in preventing and controlling cyantraniliprole-resistant bemisia tabaci Download PDFInfo
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Abstract
The invention provides an application of miRNA (micro ribonucleic acid) in preventing and controlling cyantraniliprole-resistant bemisia tabaci, and belongs to the technical field of pesticides, wherein the miRNA is miR-276-3p, the nucleotide sequence of the miRNA is shown as SEQ ID NO.1, and the miR-276-3p can regulate and control the resistance of the bemisia tabaci to cyantraniliprole by regulating and controlling the CYP6CX3 expression level in the bemisia tabaci; the invention also provides a method for regulating and controlling the expression quantity of miR-276-3p in the bemisia tabaci body by feeding bemisia tabaci, which lays a good theoretical and application foundation for developing a novel method for preventing and controlling the bemisia tabaci with the cyantraniliprole resistance.
Description
Technical Field
The invention relates to the technical field of pesticides, in particular to application of miRNA in preventing and controlling cyantraniliprole-resistant bemisia tabaci.
Background
Bemisia tabaci belongs to the family of Bemisia tabaci, is a worldwide agricultural pest, can harm more than 400 plants and transmits more than 200 viruses. Cyantraniliprole as the first broad spectrum bisamide insecticide, can cause endoplasmic reticulum Ca by binding to and activating ryanodine receptors in insects 2+ The large amount of release causes unbalance of intracellular and extracellular calcium ions, the calcium ions and troponin are combined to cause muscle contraction, and finally the death of insects is caused, but the bemisia tabaci has obvious drug resistance to cyantraniliprole at present. In our previous studies, it was found that overexpression of the cytochrome P450 gene CYP6CX3 was involved in resistance of bemisia tabaci to cyantraniliprole, and therefore, upregulation of the P450 gene might be the basic resistance mechanism of bisamide insecticides; however, we are now working on P45The regulatory mechanism of the 0 gene is poorly understood, and the role of mirnas in regulating CYP6CX3 and in resistance of bemisia tabaci to cyantraniliprole is not clear.
miRNA (microRNA) is an endogenous non-coding single-stranded microRNA that can degrade or inhibit expression of a target gene by base-pairing with the mRNA of the target gene. miRNA can be involved in the resistance of pests to bisamide pesticides by regulating the expression of a target or detoxification enzyme, for example, miR-7a and miR-8519 can up-regulate the expression of a ryanodine receptor (target of bisamide pesticides), so that the resistance of plutella xylostella to chlorantraniliprole (a bisamide drug variety) is increased; the miRNA can also regulate and control the gene expression of the metabolic enzyme, so that the resistance of pests to pesticides is increased, for example, miR-2b-3p can regulate and control the up-regulated expression of CYP9F2, so that the resistance of plutella xylostella to chlorantraniliprole is increased; miR-2-13-71 cluster can participate in the resistance of culex pallidum to deltamethrin by regulating and controlling the expression of CYP9J35 and CYP325BG 3.
miR-276 is a miRNA which is ubiquitous in insects, for example miR-276 has high expression abundance in anopheles gambiae and aedes aegypti; miR-276b-3p can participate in the occurrence of sperm of the oriental fruit fly by regulating and controlling the expression of orb gene; at present, a miR-276-3p regulation resistance related gene and application thereof in regulating resistance of pests to pesticides are not reported yet.
In order to overcome the high drug resistance of the bemisia tabaci to the cyantraniliprole insecticide, improve the control effect of the bemisia tabaci and reduce the use amount of field insecticide, a new method for controlling the bemisia tabaci with the cyantraniliprole resistance is necessary to be searched.
Disclosure of Invention
Aiming at the defects of the prior art, the invention discovers that miR-276-3p can further participate in the drug resistance of bemisia tabaci to cyantraniliprole by regulating and controlling the expression of CYP6CX3, and provides a method for regulating and controlling the expression level of miR-276-3p in bemisia tabaci body, thereby laying a good theory and application foundation for developing a new cyantraniliprole-resistant bemisia tabaci control method.
The technical scheme of the invention is as follows:
the application of miRNA in preventing and controlling cyantraniliprole-resistant bemisia tabaci is characterized in that the miRNA is miR-276-3p, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1.
Preferably, the miRNA is applied to preventing and controlling the cyantraniliprole-resistant bemisia tabaci, and the application method is to regulate and control the miR-276-3p expression quantity in the bemisia tabaci.
The invention discovers that miR-276-3p can inhibit the expression of gene CYP6CX3 through the base complementation pairing with target gene mRNA, thereby reducing the resistance of bemisia tabaci to cyantraniliprole; therefore, the expression of CYP6CX3 can be regulated and controlled by regulating and controlling the expression quantity of miR-276-3p in the bemisia tabaci, so that the resistance of the bemisia tabaci to cyantraniliprole is regulated and controlled.
Preferably, the method for regulating and controlling the miR-276-3p expression in the bemisia tabaci is to add miR-276-3p analogue or miR-276-3p inhibitor into the bemisia tabaci.
Preferably, the nucleotide sequence of the miR-276-3p analogue is shown as SEQ ID NO. 2.
Preferably, the nucleotide sequence of the miR-276-3p inhibitor is shown as SEQ ID NO. 3.
Resistance of bemisia tabaci to cyantraniliprole can be reduced by adding miR-276-3p analogue into bemisia tabaci; resistance of bemisia tabaci to cyantraniliprole can be increased by adding a miR-276-3p inhibitor to bemisia tabaci.
Preferably, the method for adding miR-276-3p analogue or miR-276-3p inhibitor into bemisia tabaci comprises the following steps: bemisia tabaci is fed with sucrose water containing miR-276-3p analogues or containing miR-276-3p inhibitors.
Preferably, the concentration of sucrose in the sucrose water is 0.17-0.23 mg/L.
Preferably, the concentration of sucrose in the sucrose water is 0.2mg/L.
Preferably, the miR-276-3p analogue or miR-276-3p inhibitor is present in the sucrose solution at a concentration of 20 mu M.
A method for regulating and controlling the expression quantity of miRNA in organism is to feed the organism with sucrose water containing the analogue of the target miRNA or the inhibitor of the target miRNA.
Preferably, the concentration of sucrose in the sucrose water is 0.17-0.23 mg/L.
The beneficial effects are that:
the invention discovers and proves that miR-276-3p can regulate and control the expression of CYP6CX3 so as to regulate and control the resistance of bemisia tabaci to cyantraniliprole, and provides a method for regulating and controlling the expression quantity of miR-276-3p in bemisia tabaci, thereby laying a good theory and application foundation for developing a novel cyantraniliprole-resistant bemisia tabaci control method.
Drawings
FIG. 1 shows the expression levels of CYP6CX3 in Bemisia tabaci after cyantraniliprole treatment;
FIG. 2 shows the expression levels of 10 related miRNAs in Bemisia tabaci after cyantraniliprole treatment;
FIG. 3 is the effect on miR-276-3p and CYP6CX3 expression levels in bemisia tabaci after feeding miR-276-3p agomir and miR-276-3p antagomir;
FIG. 4 shows bemisia tabaci mortality after different concentrations of cyantraniliprole are treated to ingest miR-276-3p agomir and miR-276-3p antagomir;
FIG. 5 is a schematic representation of the binding sites for CYP6CX3 and miR-276-3 p;
FIG. 6 is a graph showing that a dual luciferase expression system verifies the binding capacity of miR-276-3p to CYP6CX 3.
Detailed Description
Experimental material sources:
cyantraniliprole (94% of original drug): purchased from FMC under accession number PD20140321;
trizol: purchased from Invitrogen, USA, cat: 410411;
Mir-X TM miRNA First-Strand Synthesis Kit: purchased from Takara, japan, cat: 638315;
PrimeScript TM RT reagent Kit with gDNA eraser: purchased from Takara, japan, cat: RR047A;
miR-276-3p analogs (miR-276-3 p agomir) and miR-276-3p inhibitors (miR-276-3 p antagomir), negative control of miR-276-3p agomir and Negative control of miR-276-3p antagomir: are synthesized by Shanghai Ji Ma Gene company;
pmirplo vector: purchased from Promega, cat No. E1330;
endotoxin removal plasmid extraction kit: purchased from TIANGEN under the designation DP118;
cell transfection kit by calcium phosphate method: purchased from bi yun tian, cat No. C0508;
dual luciferase assay substrate: firefly luciferin, cat# 103404-75-7; renilla fluorescein, cat No. 55779-48-1; all purchased from source foliar organisms;
biochemical incubator: purchased from Heal Force, model HF90 (HT);
multifunctional enzyme-labeled instrument: purchased from TECAN, model INFINITE E PLEX;
fluorescent quantitative PCR instrument: purchased from BIO RAD, model 184-5096;
the primers involved in the examples were all prepared by the same order of synthesis by the department of Prinsepia.
The bemisia tabaci populations involved in the examples are laboratory sensitive populations QS, cyantraniliprole culling resistant populations SG19 and QR, and the various population resistance levels are shown in table 1 below:
TABLE 1 Bemisia tabaci population resistance level
The following description is made in connection with specific embodiments:
example 1
1. Method for treating bemisia tabaci by using pesticide cyantraniliprole and searching genes related to resistance of cyantraniliprole
The specific operation steps are as follows:
(1) The lethal medium concentration of cyantraniliprole on bemisia resistant populations (SG 19 and QR) was obtained by the agar-wet leaf dipping method: first, 1.0638g of cyantraniliprole (94% stock solution) was dissolved in dimethyl sulfoxide (DMSO) to prepare 20mL, 5X 10 4 Then, carrying out gradient dilution on the mother liquor to five concentrations by using 0.5 per mill of Triton X-100 solution (the solvent is deionized water), wherein the diluted concentrations are 120mg/L, 60mg/L, 30mg/L, 15mg/L and 7.5mg/L respectively, and 0.5 per mill of Triton X-100 solution is used as a control group; cotton is put intoCutting the true leaves into round leaves with the diameter of 2.5cm, and respectively soaking the round leaves in the cyantraniliprole with different concentrations and the control group for 10s; taking out the round leaves after soaking, drying for 15min in air, and placing the round leaves with the back face downwards on a 1.0% (0.01 g/mL) agar bed in a 50mL centrifugal tube cover for feeding bemisia tabaci; one side of the centrifuge tube is sealed by an insect-proof net, the other side of the centrifuge tube is sealed by the centrifuge tube cover containing the round blades, and 15 SG19 or QR adults with three days are placed in each centrifuge tube; counting bemisia tabaci mortality after 48 hours, and inputting the concentration and mortality into PoloPlus analysis data to obtain LC 50 And 95% confidence intervals thereof;
(2) Prediction of related mirnas: based on the full-length sequence of CYP6CX3 mRNA (NCBI nucleic acid database: XM 019062419) and miRNA transcriptome data (NCBI Sequence Read Archive database: SRP 076077), miRNAs likely to bind to CYP6CX3 are predicted by two miRNA prediction software RNAhybrid (Bielefeld BioInformatics Service, german) and RNA22 (Computational Medicine Center, USA); 10 relevant miRNAs are predicted, and are respectively: miR-2b-1-5p, miR-184-5p, miR-190-3p, miR-1000-3p, miR-305-5p, miR-87b-5p, miR-2765-3p, miR-276-3p, miR-bantam and miR-2a-1-5p;
(3) Detecting the expression levels of CYP6CX3 and 10 related miRNAs in bemisia tabaci by a real-time fluorescence quantitative PCR method: LC obtained in step (1) 50 Inducing and treating Bemisia tabaci SG19 and QR three-day-old adults as test groups, taking Bemisia tabaci on which 0.5 per mill of Triton X-100 solution is taken as a control group in the step (1), collecting the surviving Bemisia tabaci and the Bemisia tabaci of the same control group in the test groups after 48 hours, and extracting total RNA by using a Trizol method respectively; by Mir-X TM miRNA First-Strand Synthesis Kit and PrimeScript TM RT reagent Kit with gDNA eraser reverse transcribing miRNA and mRNA respectively, the reverse transcription being performed according to the kit instructions; using a succinic dehydrogenase complex A (SDHA) and a heat shock protein 40 (HSP 40) as two reference genes for mRNA expression analysis, and using a U6 gene as the reference gene for miRNA expression analysis, wherein three groups of repetition are arranged for each gene;
wherein, qPCR reaction system (20. Mu.L) is as follows: TB Green Advantage Premix: 10. Mu.L; primer-F: 0.5. Mu.L;Primer-R：0.5μL；cDNA：2μL；ddH 2 O：7μL；
the miRNA qPCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 10s; denaturation at 95℃for 5s, annealing at 60℃for 20s and extension for 40 cycles;
the mRNA qPCR amplification procedure was as follows: pre-denaturation at 95 ℃ for 30s; denaturation at 95℃for 5s, annealing at 60℃for 34s and extension for 40 cycles;
wherein, the upstream primer and the downstream primer used for detecting CYP6CX3 are respectively shown in SEQ ID NO. 4-5; the upstream primer sequences for detecting 10 related miRNAs are respectively shown as SEQ ID NO. 6-15, and the downstream primers uniformly adopt Mir-X TM Primer mRQ' Primer in miRNA First-Strand Synthesis Kit;
by 2 -ΔΔ The Ct method is used for analyzing the relative expression conditions of CYP6CX3 and 10 related miRNAs in bemisia tabaci after cyantraniliprole treatment, and the results are respectively shown in figures 1 and 2; as can be taken from fig. 1, after the cyantraniliprole treatment, the expression level of CYP6CX3 in SG19 and QR populations is significantly increased by 2.24 times and 1.78 times respectively compared with the control group; as can be seen from fig. 2, only miR-276-3p was significantly inhibited in both SG19 and QR-resistant populations among 10 related mirnas, showing the opposite expression profile to CYP6CX 3.
From FIGS. 1 and 2, it can be preliminarily deduced that the gene CYP6CX3 is related to the resistance of the bemisia tabaci to cyantraniliprole, and the expression level is influenced by miR-276-3p.
2. Regulating and controlling miR-276-3p expression level in bemisia tabaci by feeding method
The specific operation steps are as follows:
(1) Dissolving miR-276-3p agomir and miR-276-3p antagomir in 20% (0.2 mg/L) sucrose water respectively to obtain a final concentration of 20 mu M, and obtaining a feeding solution; feeding liquid is respectively added into a feeding device, the feeding device is made of a 50mL centrifuge tube, the periphery and the bottom of the tube are sealed by an insect-proof net, and the top end of the tube is sealed by a centrifuge tube cover; separating the feeding liquid from the bemisia tabaci adults through the polytetrafluoroethylene film, and enabling the bemisia tabaci mouthparts to pierce the polytetrafluoroethylene film and absorb the feeding liquid at the inner side of the film;
(2) Introducing 500 SG19 bemisia tabaci two-day-old adults into the feeding device in the step (1), feeding for 48 hours, collecting 200 surviving bemisia tabaci to extract RNA, and respectively measuring the expression levels of miR-276-3p and CYP6CX3 in bemisia tabaci bodies fed with miR-276-3pagomir and miR-276-3p antagomir by adopting a real-time fluorescent quantitative PCR method (the same method is adopted);
the results are shown in FIG. 3, wherein Panel A shows Bemisia tabaci fed with miR-276-3p agomir and a negative control group (Negative control of miR-276-3p agomir); panel B shows Bemisia tabaci fed with miR-276-3p antagomir and a negative control group (Negative control of miR-276-3p antagomir); wherein the negative control group feed is a chemically modified miRNA solubilized in 20% (0.2 mg/L) sucrose solution (the chemically modified miRNA is provided by Ji Ma gene); as can be obtained from fig. 3, the expression level of miR-276-3p in bemisia tabaci after feeding miR-276-3p agomir is obviously increased, and the expression level of CYP6CX3 is obviously reduced, which is specifically expressed as follows: when the expression level of miR-276-3p is increased by 5.51 times, the expression level of CYP6CX3 is reduced to 0.56 times; the expression level of miR-276-3p in bemisia tabaci after miR-276-3p antagomir is fed is obviously reduced, and the expression level of CYP6CX3 is obviously increased, which is specifically expressed as follows: when miR-276-3p expression level is reduced by 0.014 times, CYP6CX3 expression level is increased to 2.23 times; this shows that miR-276-3p can be effectively over-expressed or knocked down in bemisia tabaci by a feeding method, so that the expression of CYP6CX3 can be regulated and controlled;
(3) Collecting the bemisia tabaci which is over-expressed with miR-276-3p and the bemisia tabaci which is knocked down with miR-276-3p and is obtained in the step (2) for subsequent experiments.
Example 2
1. Study of the Effect of overexpression or knock-down of miR-276-3p on resistance to Bemisia tabaci cyantraniliprole
The specific operation steps are as follows:
(1) Measuring the lethal medium concentration of cyantraniliprole by an agar moisture leaf dipping method from the bemisia tabaci which is over-expressed with miR-276-3p and the bemisia tabaci which is knocked down with miR-276-3p and is collected in the example 1, and referring to the example 1 for a specific method;
(2) Drawing a concentration-mortality relation graph according to the detection result in the step 1, wherein the result is shown in fig. 4, and the graph A is bemisia tabaci (miR-276-3 p agomir) and a negative control group (Negative control of miR-276-3p agomir) which are over-expressed by miR-276-3 p; panel B shows bemisia tabaci (miR-276-3 p antagomir) knocked down miR-276-3p and a negative control group (Negative control of miR-276-3p antagomir);
as can be seen from fig. 4, when bemisia tabaci overexpressed miR-276-3p, mortality was significantly higher after 15mg/L, 60mg/L, and 120mg/L of cyantraniliprole treatment than in the negative control group; when the bemisia tabaci miR-276-3p is knocked down, the death rate is obviously lower than that of a negative control group after being treated by cyantraniliprole of 30mg/L, 60mg/L and 120 mg/L; further analysis of LC under different treatments 50 Value change: compared with a negative control group, miR-276-3p overexpression leads to LC of cyantraniliprole 50 Reduced from 86.10mg/L to 51.52mg/L, and LC was enabled after miR-276-3p was knocked down 50 Increasing from 68.19mg/L to 111.98mg/L;
in conclusion, the over-expression of miR-276-3p reduces the resistance of bemisia tabaci to cyantraniliprole, and the knocking-down of miR-276-3p increases the resistance of bemisia tabaci to cyantraniliprole.
2. Research on molecular mechanism of miR-276-3p affecting resistance of bemisia tabaci to cyantraniliprole
The binding capacity of miR-276-3p and CYP6CX3 is verified by a double-luciferase expression system, and the specific operation steps are as follows:
(1) Vector construction
Amplifying the 1302 th to 1660 th bases of CYP6CX3-WT-F (SEQ ID NO. 16) and CYP6CX3-WT-R (SEQ ID NO. 17) by using primers to obtain an amplification product, carrying out double digestion on the amplification product by NheI and Xbal, connecting the amplification product with a PnirGLO vector which is also digested by NheI and Xbal, carrying out conversion, picking positive clones for sequencing identification, extracting plasmids from positive clones which are sequenced correctly by using an endotoxin plasmid extraction kit, and obtaining PnirGLO-WT plasmids; wherein the 1445 th to 1453 th bases of CYP6CX3 are complementary sequences of miR-276-3p, the complementary sequences are shown in figure 5, the primer CYP6CX3-MUT-F (SEQ ID NO. 18) and CYP6CX3-MUT-R (SEQ ID NO. 19) are utilized to obtain an amplification product of the reverse complementary sequences of the 1445 th to 1453 th bases of CYP6CX3 by a fusion PCR method, and the reverse complementary sequences are used as mutation sequences to replace the 1445 th to 1453 th bases of CYP6CX3 to be connected with a Pmir GLO vector according to the same digestion method, so as to obtain a Pmir GLO-MUT mutant plasmid;
the fusion PCR method comprises the following specific steps: (1) using Pmir GLO-WT as a template, and using primers CYP6CX3-WT-F and CYP6CX3-MUT-R to amplify to obtain an amplified product P1; using Pmir GLO-WT as a template, and using primers CYP6CX3-MUT-F and CYP6CX3-WT-R to amplify to obtain an amplified product P2; (2) recovering amplified products P1 and P2 by using a glue respectively; (3) using P1 and P2 as templates, amplifying with primers CYP6CX3-WT-F and CYP6CX3-WT-R to obtain amplified products, and obtaining reverse complementary sequences of the 1445 th-1453 th bases of CYP6CX 3;
(2) Plasmid transfection of 293T cells
Transfection was performed with reference to instructions for use of the calcium phosphate method cell transfection kit, when 293T cells in six well plates grew to 80-90%, 2 μg of plasmid and miRNA were added per well, the final concentration of miRNA was 100nM; wherein the plasmid is a Pmir GLO empty vector, pmir GLO-WT or Pmir GLO-MUT, the miRNA is miR-276-3p Agomir (Agomir) or negative control (NC of Agomir, miRNA subjected to chemical modification and provided by Ji Ma gene company), and the specific scheme is as follows: pmirGLO empty vector + Agomir, pmirGLO-WT + Agomir, pmirGLO-MUT + Agomir, pmirGLO empty vector +NC of agomir, pmirGLO-WT +NC of agomir, pmirGLO-MUT +NC of agomir; after 6 hours of incubation, the complete culture medium is replaced to continue culturing for 48 hours, and transfected cells are obtained;
(3) Collecting cells for luciferase activity detection
The activities of firefly luciferase and Renilla luciferase were determined by reference to the Cold spring harbor laboratory Manual, and the specific procedures were as follows: after the transfected cells from each well were washed twice with PBS, 1mL of cell lysate (25 mM glycylglycine, 1% Triton X-100, 15mM MgSO) was added 4 And 4mM EGTA), lysing the cells for 5 minutes to obtain a cell lysate; centrifuging 12000g of cell lysate at 4 ℃ for 5 minutes, and taking supernatant as enzyme solution; mu.L of enzyme solution, 120. Mu.L of luciferase assay buffer (15 mM potassium phosphate, 25mM glycylglycine, 15mM MgSO) 4 4mM EGTA and 2mM ATP) and 80. Mu.L luciferase solution (0.2 mM coelenterazine or D-sodium fluorescein salt, 15mM MgSO 4 25mM glycylglycine and 4mM EGTA) was gently mixed in a 96-well plate, and the enzyme activity was measured at room temperature using a multifunctional microplate reader, and the enzyme activities were measured by firefly luciferase and Renilla luciferaseTo evaluate the relative luciferase activity and thus the binding capacity of miR-276-3p to CYP6CX3 bases 1445-1453;
the results are shown in FIG. 6 (where the different letters indicate significant differences between treatments), when 293T cells were co-transfected with PmirGLO, agomir and Pmir GLO, NC of agomir, their relative luciferase activities were 1.15+ -0.02 and 1.44+ -0.05, respectively, indicating that miR-276-3p had no major effect on firefly luciferase expression in empty vector; when 293T cells were co-transfected with Pmir GLO-WT (i.e., pmir GLO vector comprising CYP6CX3 region sequence 1445-1453), agomir, their relative luciferase activity was significantly reduced to 0.56+ -0.09, only 43.08% of co-transfected Pmir GLO-WT, NC of agonir (1.30+ -0.19); when 293T cells co-transfected with Pmir GLO-MUT (i.e., pmir GLO vector comprising CYP6CX3 mutant sequence), agomin, their relative luciferase activity (1.21.+ -. 0.08) was similar to that of co-transfected with Pmir GLO-MUT, NC of agonir (1.29.+ -. 0.10);
the results in FIG. 6 show that Agomir binds to mRNA base complementary to the target sequence (base sequence 1445-1453 of CYP6CX 3) in PnirGLO-WT to inhibit gene expression, resulting in decreased relative luciferase activity.
In conclusion, the invention discovers and verifies that miR-276-3p can regulate and control the expression of CYP6CX3 so as to regulate and control the resistance of bemisia tabaci to cyantraniliprole, analyzes a molecular mechanism of miR-276-3p affecting the resistance of bemisia tabaci to cyantraniliprole, and lays a good theory and application foundation for developing a novel cyantraniliprole-resistant bemisia tabaci control method.
Claims (9)
1. The application of miRNA in preventing and controlling cyantraniliprole-resistant bemisia tabaci is characterized in that the miRNA is miR-276-3p, and the nucleotide sequence of the miRNA is shown as SEQ ID NO. 1.
2. The use of claim 1, wherein the method of use is over-expression of miR-276-3p in bemisia tabaci.
3. The use of claim 2, wherein the means for over-expressing miR-276-3p in bemisia tabaci is by adding a miR-276-3p analog to bemisia tabaci; the nucleotide sequence of the miR-276-3p analogue is shown as SEQ ID NO. 2.
4. The use of claim 3, wherein the method of adding the miR-276-3p analog to bemisia tabaci comprises: bemisia tabaci was fed with sucrose water containing miR-276-3p analogues.
5. The use according to claim 4, wherein the sucrose concentration in sucrose water is between 0.17 and 0.23mg/L.
6. The use according to claim 4, wherein the sucrose concentration in sucrose water is 0.2mg/L.
7. The use of claim 4, wherein the miR-276-3p analog is at a concentration of 20 μm in sucrose water.
8. A method for regulating and controlling miR-276-3p expression quantity in bemisia tabaci, which is characterized in that the bemisia tabaci is fed by sucrose water containing miR-276-3p analogue or miR-276-3p inhibitor; the nucleotide sequence of the miR-276-3p analogue is shown as SEQ ID NO.2, and the nucleotide sequence of the miR-276-3p inhibitor is shown as SEQ ID NO. 3.
9. A method according to claim 8, wherein the sucrose concentration in sucrose water is between 0.17 and 0.23mg/L.
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