Application of dsRNA in controlling cyantraniliprole-resistant whitefly

Abstract

The invention provides an application of dsRNA in preventing and controlling cyantraniliprole-resistant whitefly, belonging to the technical field of pesticides; the dsRNA is ds <h2 style=";text-align:left;direction:ltr">UGT352B2 、ds <h2 style=";text-align:left;direction:ltr"> UGT352F1 One or more of these, by feeding whiteflies ds <h2 style=";text-align:left;direction:ltr"> UGT352B2 and / or ds <h2 style=";text-align:left;direction:ltr"> UGT352F1 , which can interfere with the expression of UGT genes in whiteflies, thereby weakening the whiteflies' own detoxification ability of cyantraniliprole and reducing the whiteflies' resistance to cyantraniliprole. This has laid a good theoretical and application foundation for the development of new cyantraniliprole-resistant whitefly control methods.

Description

Technical Field

[0001] The invention relates to the technical field of pesticides, and in particular to application of dsRNA in preventing and controlling cyantraniliprole-resistant whiteflies. Background Art

[0002] As a worldwide agricultural pest, Bemisia tabaci belongs to the family Aleyrodidae in the order Hemiptera. It is a piercing-sucking pest that poses a significant threat to agricultural production activities. Cyantraniliprole (CYA), the first diamide insecticide with high activity against piercing-sucking pests, can bind to the nicotinic acid receptors in insects, causing endoplasmic reticulum Ca 2+ Large amounts of release lead to an imbalance of calcium ions inside and outside the cells, ultimately causing the death of the insect.

[0003] UDP-glycosyltransferase (UGT) is a multifunctional superfamily enzyme system found in all organisms in nature (including plants, animals, bacteria, and fungi). It can catalyze the binding of originally lipophilic exogenous substances and endogenous substances with glycosyl donors to generate hydrophilic glycosides, thereby effectively excreting exogenous substances from the body. On the one hand, UGT can protect the organism from being damaged by exogenous substances. On the other hand, UGT can effectively regulate the internal environmental balance of the organism, and therefore plays a key role in the growth and development of animals and plants. In recent years, studies have found that UGT-catalyzed glycosylation of exogenous compounds occupies an important position in the insect detoxification metabolic system. Insects can respond to the toxic effects of certain insecticides by regulating the expression abundance of certain UGT genes. It is currently known that UGT is involved in the development of insect resistance to insecticides such as organophosphates, pyrethroids, neonicotinoids, and amides.

[0004] At present, Bemisia whitefly has developed obvious resistance to cyantraniliprole. In order to control cyantraniliprole-resistant Bemisia whitefly in agricultural production, it is necessary to actively find new methods for controlling Bemisia whitefly. Summary of the invention

[0005] In view of the above problems, the present invention discovered and confirmed that the UGT gene family UGT352B2 , UGT352F1 Related to the resistance of Bemisia tabaci to cyantraniliprole, when Bemisia tabaci feeds UGT352B2 , UGT352F1 dsRNA (ds UGT352B2 、ds UGT352F1 ), the resistance to cyantraniliprole was significantly reduced.

[0006] The technical solution of the present invention is as follows:

[0007] A use of dsRNA in controlling cyantraniliprole-resistant whitefly, wherein the dsRNA is ds UGT352B2 、ds UGT352F1 One or more of; wherein, the ds UGT352B2 The nucleotide sequence of ds UGT352F1 The nucleotide sequence is shown in SEQ ID NO.17.

[0008] Preferably, the application method is to feed ds UGT352B2 and / or ds UGT352F1 , reducing the resistance of Bemisia whitefly to cyantraniliprole.

[0009] Preferably, the feeding ds UGT352B2 and / or ds UGT352F1 The method is: use a mixture containing ds UGT352B2 and / or ds UGT352F1 Whiteflies were fed with sucrose water.

[0010] Preferably, the concentration of sucrose in the sucrose water is 0.17-0.23 mg / L.

[0011] Preferably, the concentration of sucrose in the sucrose water is 0.2 mg / L.

[0012] Preferably, the ds UGT352B2 or ds UGT352F1 The concentration in sucrose water is 250ng / µL.

[0013] Beneficial effects:

[0014] The present invention discovered and confirmed the UGT gene family UGT352B2 , UGT352F1 Involved in the resistance of Bemisia tabaci to cyantraniliprole. UGT352B2 , UGT352F1 dsRNA (ds UGT352B2 、ds UGT352F1 ), the resistance of whitefly to cyantraniliprole can be reduced; the present invention lays a good theoretical and application foundation for the development of new cyantraniliprole-resistant whitefly control methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 To determine the UGT enzyme activity in different whitefly species;

[0016] Figure 2 The results of determination of UGT enzyme kinetic parameters in different whitefly species populations;

[0017] Figure 3 The UGT gene expression profile in transcriptome sequencing;

[0018] Figure 4 The relative expression of related UGT genes in different whitefly species;

[0019] Figure 5 For feeding ds UGT352B2 、ds UGT352F1 、ds UGT352B2 +ds UGT352F1 、ds UGT352B3 Afterwards, the expression level of the corresponding gene in Bemisia tabaci changed;

[0020] Figure 6 For feeding ds UGT352B2 、ds UGT352F1 、ds UGT352B2 +ds UGT352F1 、ds UGT352B3 After that, the concentration-mortality curve of Bemisia tabaci to cyantraniliprole. DETAILED DESCRIPTION

[0021] The following is explained in conjunction with specific embodiments:

[0022] Source of experimental materials:

[0023] Cyantraniliprole (94% technical): purchased from FMC;

[0024] 5-Nitrouracil: purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0025] Sulfinpyrazone: purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0026] UDP-glucose: purchased from Shanghai MacLean Biochemical Technology Co., Ltd.;

[0027] α-Naphthol: purchased from Shanghai Yuanye Biotechnology Co., Ltd.;

[0028] Trizol reagent: purchased from Thermo Fisher Scientific;

[0029] TruSeq Stranded mRNA LTSample Prep Kit: purchased from Illumina;

[0030] Evo M-MLV reverse transcription premix kit: purchased from Acridine Biotechnology Co., Ltd.

[0031] SYBR Green Pro Taq HS: purchased from Accurium Biotechnology Co., Ltd.

[0032] PrimeScript™ II 1st Strand cDNA Synthesis Kit: purchased from Bio-Rad Biotechnology Co., Ltd.

[0033] ApexHF HS DNA Polymerase: purchased from Apex Biotech;

[0034] TranscriptAid T7 High Yield Transcription Kit: purchased from Thermo Fisher Scientific;

[0035] PrimeScript™ RT reagent Kit with gDNA eraser: purchased from Bio-Rad Biotechnology Co., Ltd.

[0036] The primers involved in the examples were all commissioned to be synthesized by Qingke Biotechnology Co., Ltd.

[0037] The resistance levels of the laboratory sensitive population QS and cyantraniliprole-resistant populations QR, SG19, WF20, and SG20 to cyantraniliprole involved in the examples are shown in Table 1 below:

[0038] Table 1. Resistance levels of whitefly populations

[0039]

[0040] Example 1: Screening of related UGT genes

[0041] 1. Effects of UGT inhibitors on cyantraniliprole resistance levels in different whitefly populations

[0042] First, the median lethal concentration of cyantraniliprole to different whitefly populations (QS, QR, SG19, WF20, and SG20) was determined by the agar moisturizing leaf dip method: 1.0638 g of cyantraniliprole (94% stock solution) was dissolved in dimethyl sulfoxide (DMSO) to prepare 20 mL of 5 × 10 4The mother solution was prepared by gradient dilution with 0.5‰ Triton X-100 solution (solvent is deionized water) to five concentrations to obtain cyantraniliprole working solution. The concentrations after dilution were 200 mg / L, 100 mg / L, 33.3 mg / L, 11.1 mg / L, and 3.7 mg / L, respectively. X-100 solution was used as the blank group; the cotton true leaves were cut into circular leaves with a diameter of 2.5 cm, and the circular leaves were immersed in the above-mentioned cyantraniliprole working solution of different concentrations and the blank group for 10 seconds; after immersion, the circular leaves were taken out and dried in the air for 15 minutes, and then placed with the back side down on a 1.0% (0.01g / mL) agar bed in a 50mL centrifuge tube cap for feeding whiteflies; one side of the centrifuge tube was closed by an insect-proof net, and the other side was sealed by the centrifuge tube cap containing the circular leaves, and 15 adult whiteflies were placed in the centrifuge tube; after 48 hours, the mortality rate of whiteflies was counted, and the concentration and mortality rate were input into PoloPlus software (Windows 11) for data analysis to obtain LC 50 and its 95% confidence interval.

[0043] Then, the effect of UGT inhibitors on the cyantraniliprole resistance level of different whitefly populations (QS, QR, SG19, WF20, SG20) was determined: the UGT inhibitors sulfinpyrazone (Sul) and 5-nitrouracil (5-Nul) were dissolved in acetone to obtain sulfinpyrazone solution and 5-nitrouracil solution with a concentration of 400 mg / L, respectively; cyantraniliprole was diluted with sulfinpyrazone solution and 5-nitrouracil solution to the above-mentioned cyantraniliprole working solution concentration, and then the effect of adding UGT inhibitors on the resistance level was determined by the agar moisturizing leaf dip method according to the same steps as above.

[0044] The measurement results are shown in Table 2 below:

[0045] Table 2. Effects of UGT inhibitors on cyantraniliprole resistance levels in different whitefly populations

[0046]

[0047] Wherein, No. represents the number of whiteflies involved in the test; Slope±SE represents the standard error of the regression slope; χ 2 (df) represents the chi-square test statistic; SR represents the synergistic factor of UGT inhibitors; RR represents the resistance factor of resistant populations. The above data are calculated and generated by PoloPlus software.

[0048] As shown in Table 2, the addition of UGT inhibitors 5-nitrouracil or sulfinpyrazone can reduce the resistance level of different whitefly populations to cyantraniliprole and increase the toxicity of cyantraniliprole to whitefly. Among them, 5-nitrouracil and sulfinpyrazone showed the highest synergistic ratio against SG20 population, with synergistic ratios of 2.93 times and 2.31 times, respectively. The above results indicate that UGT may be involved in the resistance of whitefly to cyantraniliprole.

[0049] 2. Determination of UGT enzyme activity in different whitefly populations

[0050] UDP-glycosyltransferase (UGT) has the function of catalyzing the conversion of the model substrate α-naphthol into α-naphthyl glucoside.

[0051] PBS buffer (pH 7.8) was used as the lysis solution. Different populations (QS, QR, SG19, WF20, SG20) of whiteflies were ground under the same conditions, and then centrifuged at 4°C and 12000g for 15 min. The supernatant was taken to obtain the enzyme solution. MgCl2, UDP-glucose, and fresh enzyme solution were added to a 1.5mL centrifuge tube in sequence, and then water was added to 960µL, so that the final concentration of MgCl2 was 10mM and the final concentration of UDP-glucose was 1mM to obtain a mixed system. The mixed system was dispensed into an ELISA plate at 240µL / well, and finally 10µL of α-naphthol solution with a final concentration of 250µM was added and mixed evenly to obtain an enzyme reaction system. Infinite The enzyme reaction system was continuously monitored for 15 minutes with a 200Pro microplate reader under the conditions of 287nm excitation wavelength, 335nm emission wavelength, and 5nm slit width to detect the fluorescence value of the enzyme reaction system; at the same time, the reaction system without α-naphthol was used as a negative control to determine the background fluorescence value. A standard curve of α-naphthyl glucoside-fluorescence value was drawn, and the content of α-naphthyl glucoside in the enzyme reaction system was calculated according to the standard curve, and then the enzyme activity of UGT was calculated according to the content of α-naphthyl glucoside; 3 groups of biological replicates were set for each whitefly population.

[0052] Among them, the specific calculation method of UGT enzyme activity is: according to the standard curve of α-naphthyl glucoside-fluorescence value, the fluorescence value of the enzyme reaction system is substituted into the standard curve to obtain the content of α-naphthyl glucoside; the α-naphthyl glucoside content is divided by the monitoring time, and then divided by the protein content of the measured sample to obtain the enzyme activity value in the unit of "pmol / min / mg".

[0053] The results of the test are as follows Figure 1 As shown. Figure 1It can be seen that compared with the UGT enzyme activity value in the whitefly population QS, the UGT enzyme activity in the whitefly populations QR, SG19, WF20, and SG20 was significantly increased. Specifically, the UGT enzyme activity of QR, SG19, WF20, and SG20 was approximately 2.4 times, 2.9 times, 2.9 times, and 3.7 times that of QS, respectively.

[0054] 3. Determination of UGT enzyme kinetic parameters

[0055] The Michaelis-Menten equation is an equation that expresses the relationship between the starting rate of an enzymatic reaction and the substrate concentration. In this experiment, the steady-state kinetic parameters of the UGT enzyme with α-naphthol as the substrate were determined based on the Michaelis-Menten equation. The Michaelis constant ( Km ) and the maximum reaction rate ( Vmax ). The specific steps are as follows:

[0056] The mixed system was prepared according to the above-mentioned UGT enzyme activity assay method; the mixed system was dispensed into the ELISA plate at 240 μL / well, and finally 10 μL of α-naphthol solution of different concentrations was added and mixed evenly to obtain the enzyme reaction system; wherein the final concentrations of the α-naphthol solution were 160 μM, 80 μM, 40 μM, 20 μM, 10 μM, 5 μM and 2.5 μM, respectively.

[0057] According to the above enzyme activity determination method, the fluorescence values ​​of different populations (QS, QR, SG19, WF20, SG20) of whitefly samples were measured in different concentrations of α-naphthol solution. The obtained series of data were input into GraphPad Prism 8 software (Windows 11), and the obtained data were fitted with the Michaelis-Menten equation to obtain the fluorescence values ​​of different whitefly populations. Km Value and Vmax value.

[0058] The fitting results of the Michaelis equation are as follows Figure 2 As shown, Figure A, Figure B, Figure C, Figure D, and Figure E respectively represent the results of the determination of UGT enzyme kinetic parameters in the whitefly populations QS, QR, SG19, WF20, and SG20. Figure 2 We can get: Km There was no significant difference in the values ​​between the QS population and the four resistant populations (QR, SG19, WF20, and SG20). Km The value results are between 11.01 and 16.12; compared with the QS population, Vmax The values ​​were significantly increased in the four resistant populations (QR, SG19, WF20, and SG20). VmaxThe values ​​were approximately 2.4, 2.8, 2.8 and 3.0 times that of QS, respectively. The above results show that the catalytic efficiency of UGT in the whitefly populations QS, QR, SG19, WF20 and SG20 did not change significantly, but the increase in UGT content in the four resistant populations (QR, SG19, WF20 and SG20) significantly enhanced the UGT in the resistant populations. Vmax value.

[0059] 4. RNA-Seq

[0060] Adults of different whitefly populations (QS, QR, SG19, WF20, and SG20) were collected, and three biological replicates were set for each whitefly population. Total RNA of whiteflies was extracted using Trizol reagent and digested with DNase, and its integrity was measured by Agilent 2100 Bioanalyzer (Agilent Technologies). Samples with RNA integrity values ​​of 7 or above were constructed using the TruSeq Stranded mRNA LTSample Prep Kit and sequenced on the Illumina sequencing platform (Hiseq-2500).

[0061] Raw reads containing poly-N and low-quality reads were removed by Trimmomatic software, and the filtered reads were mapped to the reference genome (NCBI bioproject PRJNA347299) using HISTAT 2 software. Fragments per kilobase of transcript per million mapped reads (FRKM) and read counts were calculated by Bowite2 and eXpress software, respectively. Differentially expressed UGT transcripts with P values ​​< 0.05 and fold changes of more than 1.5 were selected as candidate genes using the estimateSizeFactors and nbinomTest functions in DESeq software.

[0062] The candidate genes were compared with the literature "Genome-Wide Identification and Expression Analysis of Udp-Glucuronosyltransferases in the Whitefly Bemisia tabaci(Gennadius) (Hemiptera: Aleyrodidae). Int J Mol Sci. 2020 Nov 11;21(22):8492.doi: 10.3390 / ijms21228492. PMID: 33187355; PMCID: PMC7697561.》, a total of 71 UGT genes were matched, of which 68 UGT genes were expressed in all five populations. The specific details of the 68 UGT genes are as follows: Figure 3 shown.

[0063] Depend on Figure 3 It can be seen that 19 UGT genes (in red boxes) were overexpressed by more than 1.5 times in the four resistant populations (QR, SG19, WF20, SG20) compared with the QS population, among which 9 UGT genes (indicated by red squares) were overexpressed by more than 2 times in the four resistant populations; the 9 UGT genes are: UGT352A1 , UGT352B2 , UGT352B3 , UGT352F1 , UGT352K1 , UGT352Q5 , UGT352Q6 , UGT356A2 , UGT361A1 The nucleotide sequences are shown in SEQ ID NOs.1~9 respectively.

[0064] 5. Verification of the relative expression levels of 9 UGT genes in different whitefly species

[0065] Fifty adults of different whitefly populations (QS, QR, SG19, WF20, SG20) were collected, and four biological replicates were set for each whitefly population. Total RNA of whitefly adults was extracted using Trizol reagent and digested with DNase. 1000 ng of total RNA was reverse transcribed using EvoM-MLV reverse transcription premix kit. Reverse transcription was performed according to the kit instructions to synthesize the first-strand cDNA, which was used as a template for real-time fluorescence quantitative PCR (qPCR) reaction. Succinate dehydrogenase complex A (SDHA) and heat shock protein 40 (HSP40) were used as two internal reference genes for mRNA expression analysis; three biological replicates were performed for each treatment.

[0066] The qPCR reaction system (20 μL) was as follows: SYBR Green Pro Taq HS 10 μL, Primer-F (10 μM) 0.5 μL, Primer-R (10 μM) 0.5 μL, cDNA 2 μL, and ddH2O 7 μL.

[0067] The qPCR amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 34 s, and 40 cycles.

[0068] After the qPCR reaction, the Ct values ​​of 9 UGT genes and internal reference genes were collected and analyzed using 2 -ΔΔCt The data were processed by relative quantification method, and the relative expression differences of UGT genes among different whitefly populations were calculated using the expression levels in the sensitive population QS as a benchmark.

[0069] The results are as follows Figure 4 shown by Figure 4 It can be seen that compared with the QS population, UGT352B2 The gene was more than 10 times more expressed in the two resistant populations WF20 and SG20. UGT352F1 The gene was expressed more than 4 times in the three resistant populations SG19, WF20, and SG20. UGT352B2 , UGT352F1 The functions of these two UGT genes were subsequently verified.

[0070] Embodiment 2: UGT352B2 , UGT352F1 Effects of genes on cyantraniliprole resistance in Bemisia tabaci

[0071] 1. Synthetic dsRNA

[0072] (1) Total RNA from SG20 population of whitefly was extracted using Trizol reagent, and the first-strand cDNA was synthesized using PrimeScript™ II 1stStrand cDNA Synthesis Kit. ApexHF HS DNA Polymerase was used to amplify the cDNA using cDNA as a template. UGT352B2 , UGT352F1 and UGT352B3 gene fragments.

[0073] Among them, amplification UGT352B2 The primers used for the gene fragments are shown in SEQ ID NOs. 10-11, respectively. UGT352F1 The primers used for the gene fragments are shown in SEQ ID NOs. 12-13, respectively. UGT352B3 The primers used for the gene fragments are shown in SEQ ID NOs. 14-15;

[0074] The PCR amplification system (50 μL) was: ApexHF HS DNA Polymerase 25 μL, Primer-F 1 μL, Primer-R 1 μL, cDNA template 2 μL, and ddH2O 21 μL.

[0075] The PCR amplification program was as follows: pre-denaturation at 94°C for 1 min; denaturation at 94°C for 15 s, annealing at 55°C for 15 s, and extension at 72°C for 10 s, for a total of 35 cycles; and 72°C for 1 min.

[0076] (2) Synthesized using step (1) UGT352B2 , UGT352F1 or UGT352B3 The gene fragments were used as templates and synthesized using the TranscriptAid T7 High Yield Transcription Kit. UGT352B2 , UGT352F1 and UGT352B3 Double-stranded RNA (dsRNA);

[0077] The dsRNA synthesis system is: 5×TranscriptAid Reaction Buffer 5μL, ribonucleotide (A / G / C / U, 100mM) 2μL each, DNA template 1μg, enzyme-free water to 20μL, and mix thoroughly.

[0078] The dsRNA synthesis system was incubated at 37°C for 4 h. After the incubation, 2 μL of DNase I was added and the mixture was incubated in a 37°C water bath for 15 min. 2 μL of 0.5 M EDTA was added and the mixture was incubated at 65°C for 10 min. Enzyme-free water was added to make up to 500 μL, and 200 μL of chloroform was added to mix gently and allowed to stand for 10 min. The mixture was centrifuged at 12000 g and 4°C for 15 min and the supernatant was taken. 200 μL of chloroform was added to the supernatant and the mixture was centrifuged and the supernatant was taken. 1 / 10 volume of sodium acetate solution (3 M, pH 5.3) was added to the supernatant. 5.2) Add 2.5 times the volume of ethanol to the supernatant and place at -80℃ for 2.5h; centrifuge at 12000g and 4℃ for 30min, discard the supernatant, add 1mL of 75% ethanol solution, and resuspend the precipitate; centrifuge at 10000g and 4℃ for 10min, discard the supernatant, wait for the remaining ethanol in the tube to dry, and add 200μL of enzyme-free water to dissolve, and obtain the target product ds UGT352B2 、ds UGT352F1 and ds UGT352B3 The nucleotide sequences are shown in SEQ ID NOs.16~18 respectively.

[0079] At the same time, the dsRNA (ds GFP ) was used as a control group for subsequent RNA interference (RNAi) experiments.

[0080] 2. RNAi

[0081] ds UGT352B2 、ds UGT352F1 、dsUGT352B2 +ds UGT352F1 、ds UGT352B3 and ds GFP Dissolve them separately in 20% (0.2 mg / L) sucrose water to make the final concentration of each dsRNA 250 ng / µL (when dissolving ds UGT352B2 and ds UGT352F1 When steaming sugar water, ds UGT352B2 and ds UGT352F1 The final concentrations of the two compounds were 250 ng / µL, respectively), to obtain feeding solutions; the feeding solutions were added into feeding devices, which were made of 50 mL centrifuge tubes, the sides and bottom of the tubes were closed by insect-proof nets, and the top was sealed by centrifuge tube caps; the feeding solutions were separated from about 500 adult whiteflies of the SG20 population by a polytetrafluoroethylene membrane, and the mouthparts of the whiteflies could pierce the polytetrafluoroethylene membrane to absorb the feeding solution on the inner side of the membrane; after feeding for 72 hours, the surviving whiteflies were collected for subsequent qPCR experiments and bioassay experiments.

[0082] 3. qPCR

[0083] (1) Detection of surviving whiteflies by qPCR UGT352B2 , UGT352F1 and UGT352B3 Relative expression levels:

[0084] Total RNA from surviving whiteflies after RNAi was extracted using Trizol reagent, and then reverse transcribed using PrimeScript™ RTreagent Kit with gDNA eraser. Reverse transcription was performed according to the kit instructions to synthesize first-strand cDNA. Succinate dehydrogenase complex A (SDHA) and heat shock protein 40 (HSP40) were used as two internal reference genes for mRNA expression analysis. Each treatment was repeated three times, using 2 -ΔΔCt Relative quantitative analysis of RNAi in Bemisia tabaci UGT352B2 , UGT352F1 and UGT352B3 The relative expression of

[0085] The qPCR reaction system (20 μL) was as follows: TB Green Advantage Premix 10 μL, Primer-F 0.5 μL, Primer-R 0.5 μL, cDNA 2 μL, and ddH2O 7 μL.

[0086] The qPCR amplification program was as follows: pre-denaturation at 95°C for 30 s; denaturation at 95°C for 5 s, annealing and extension at 60°C for 30 s, and 40 cycles.

[0087] The qPCR test results of each gene are shown in Figure 5As shown, Figure A shows feeding ds UGT352B2 back UGT352B2 The relative expression of UGT352F1 back UGT352F1 The relative expression of UGT352B2+ ds UGT352F1 back UGT352B2 and UGT352F1 The relative expression of D is shown in Figure 5. UGT352B3 back UGT352B3 The relative expression of .

[0088] Depend on Figure 5 As shown in Figure A, when taking ds UGT352B2 Afterwards, the whitefly UGT352B2 The expression level of ds UGT352F1 Afterwards, the whitefly UGT352F1 The expression level of ds UGT352B2 and ds UGT352F1 Afterwards, the whitefly UGT352B2 The expression level of UGT352F1 The expression level of ds UGT352B3 Afterwards, the whitefly UGT352B3 The expression level of WT WT was significantly reduced to 0.61 times that of the control group.

[0089] The above results showed that the target gene dsRNA was successfully knocked down in whiteflies after feeding for 72 hours. UGT352B2 , UGT352F1 and UGT352B3 expression level.

[0090] 4. Verification UGT352B2 , UGT352F1 and UGT352B3 Changes in the resistance level of Bemisia tabaci to cyantraniliprole after knockdown

[0091] The median lethal concentration of cyantraniliprole to the whitefly population (SG20) after RNAi was obtained by the agar moisturizing leaf dip method: 1.0638 g of cyantraniliprole (94% stock solution) was dissolved in dimethyl sulfoxide (DMSO) to prepare 20 mL of 5 × 10 4The mother solution was prepared by gradient dilution with 0.5‰ Triton X-100 solution (solvent is deionized water) to five concentrations to obtain cyantraniliprole working solution. The concentrations after dilution were 200 mg / L, 100 mg / L, 33.3 mg / L, 11.1 mg / L, and 3.7 mg / L, respectively. X-100 solution was used as a blank group; cotton true leaves were cut into circular leaves with a diameter of 2.5 cm, and the circular leaves were immersed in the above-mentioned cyantraniliprole working solution of different concentrations and the blank group for 10 seconds; after immersion, the circular leaves were taken out and dried in the air for 15 minutes, and then placed with the back side down on a 1.0% (0.01g / mL) agar bed in a 50mL centrifuge tube cap for feeding whiteflies; one side of the centrifuge tube was closed by an insect-proof net, and the other side was sealed by the centrifuge tube cap containing the circular leaves, and 15 surviving SG20 whiteflies fed with dsRNA were placed in the centrifuge tube; after 48 hours, the mortality rate of whiteflies was counted, and the concentration and mortality rate were input into the PoloPlus software for data analysis to obtain LC 50 and its 95% confidence interval.

[0092] The results of the test are as follows Figure 6 As shown, Figure A shows feeding ds UGT352B2 Concentration-mortality curve of cyantraniliprole in Bemisia tabaci. Figure B shows the concentration-mortality curve of cyantraniliprole in Bemisia tabaci fed with ds UGT352F1 Concentration-mortality curve of cyantraniliprole in Bemisia tabaci. Figure C shows the concentration-mortality curve of cyantraniliprole in Bemisia tabaci fed with ds UGT352B2+ ds UGT352F1 Concentration-mortality curve of cyantraniliprole in Bemisia tabaci. Figure D shows the concentration-mortality curve of cyantraniliprole in Bemisia tabaci fed with ds UGT352B3 Concentration-mortality curve of Bemisia tabaci to cyantraniliprole.

[0093] Depend on Figure 6 Figure A shows that the same as feeding ds GFP Compared with the control group, whiteflies fed with ds UGT352B2 After feeding, the resistance level to cyantraniliprole decreased significantly. Specifically, UGT352B2 LC of cyantraniliprole in Bemisia tabaci 50 Compared with the control group, it was reduced by 49.1%. As shown in Figure B, the intake of ds UGT352F1 LC of cyantraniliprole in Bemisia tabaci 50 Compared with the control group, it was reduced by 46.0%; from Figure C, it can be seen that the intake of ds UGT352B2 +ds <h2 style=";text-align:left;direction:ltr">UGT352F1 LC of cyantraniliprole in Bemisia tabaci 50 Compared with the control group, it was reduced by 61.1%; from Figure D, it can be seen that the intake of ds <h2 style=";text-align:left;direction:ltr"> UGT352B3 LC of cyantraniliprole in Bemisia tabaci 50 There was no significant difference compared with the control group.

[0094] The above results showed that two key UGT genes ( <h2 style=";text-align:left;direction:ltr"> UGT352B2 , <h2 style=";text-align:left;direction:ltr"> UGT352F1 ) is involved in the resistance of Bemisia tabaci to cyantraniliprole. <h2 style=";text-align:left;direction:ltr"> UGT352B2 , <h2 style=";text-align:left;direction:ltr"> UGT352F1 After the expression level of was reduced, the resistance of whitefly to cyantraniliprole was significantly reduced, which provides a new idea for the prevention and control of cyantraniliprole-resistant whitefly.

Claims

1. Use of dsRNA in controlling cyantraniliprole-resistant whiteflies, It is characterized in that The dsRNA is ds UGT352B2 、ds UGT352F1 One or more of; wherein, the ds UGT352B2 The nucleotide sequence of ds UGT352F1 The nucleotide sequence is shown in SEQ ID NO.

17.

2. The use according to claim 1, It is characterized in that The application method is to feed ds UGT352B2 and / or ds UGT352F1 , reducing the resistance of Bemisia whitefly to cyantraniliprole.

3. The use according to claim 2, It is characterized in that The feeding ds UGT352B2 and / or ds UGT352F1 The method is: use a mixture containing ds UGT352B2 and / or ds UGT352F1 Whiteflies were fed with sucrose water.

4. The use according to claim 3, It is characterized in that The concentration of sucrose in the sucrose water is 0.17-0.23 mg / L.

5. The use according to claim 3, It is characterized in that The concentration of sucrose in the sucrose water is 0.2 mg / L.

6. The use according to claim 3, It is characterized in that The ds UGT352B2 or ds UGT352F1 The concentration in sucrose water is 250ng / µL.

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