AgJHE1 gene and siRNA that interferes with cotton aphid pesticide metabolism, and their application in cotton aphid resistance management.

Abstract

This invention discloses the AgJHE1 gene and siRNA that interferes with the pesticide metabolism of cotton aphids, as well as their applications in the management of cotton aphid resistance, belonging to the field of biotechnology. The invention utilizes RNAi technology to interfere with the expression of the AgJHE1 gene, specifically designed siRNA based on the cotton aphid AgJHE1 gene. Results show that the siRNA enters the cotton aphid cells and triggers an RNAi cascade reaction, interfering with the expression of the target gene. Under pesticide stress, cotton aphids fed with siRNA showed a significantly increased sensitivity to the pesticide trifluralin. The siRNA provided by this invention can interfere with the detoxification and metabolic processes of cotton aphids to pesticides. This research contributes to understanding the metabolic resistance mechanism of cotton aphids and provides a new target for integrated pest management of resistant pests. The research results have significant theoretical and practical implications for the sustainable management of cotton aphids.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to the AgJHE1 gene and siRNA that interferes with cotton aphid pesticide metabolism, and their application in cotton aphid resistance management. Background Technology

[0002] cotton aphids Aphis gossypii Glover, also known as the melon aphid, is a major pest of agricultural and forestry crops belonging to the Cucurbitaceae, Malvaceae, Solanaceae, and Rutaceae families. It damages host plants through direct feeding, virus transmission, and honeydew contamination, causing severe economic losses. For a long time, control methods for cotton aphids have been relatively simple, mainly relying on chemical control. However, due to the short lifespan and rapid population growth of cotton aphids, especially under the selective pressure of high doses of insecticides, they have developed high levels of resistance to organophosphates, carbamates, pyrethroids, and neonicotinoids.

[0003] Juvenile hormone esterase JHE belongs to the carboxylesterase family and is the most important juvenile hormone JH degrading enzyme in most insects. It can highly recognize and decompose JH into juvenile hormone acids, thereby disrupting JH levels in insects by promoting or inhibiting their activity. This affects molting, pupation, and reproduction, leading to developmental disorders, loss of viability, or reduced fertility. Recent studies have found that JHE may be related to insect resistance, with overexpression of JHE in Bt Cry1Ac-resistant strains of diamondback moth. PxJHE Knocking down the AgJHE1 gene via RNAi increased the diamondback moth's tolerance to the Cry1Ac protein. Previous studies found that the AgJHE1 gene was significantly upregulated in trifluralin-resistant cotton aphid strains compared to susceptible strains. The AgJHE1 gene could serve as a novel pesticide target for cotton aphid control. Therefore, by interfering with the expression of the key JHE gene and increasing the cotton aphid's sensitivity to insecticides, the goal of controlling cotton aphids can be achieved.

[0004] Due to its high specificity and low production cost, RNAi has been applied as a novel method in agricultural pest control. siRNA (Small Interfering RNA) is a double-stranded RNA of 20 to 25 nucleotides in length. It achieves gene silencing by selectively targeting specific genes through complementary base pairing, leading to efficient and specific degradation of mRNA. Compared to dsRNA, siRNA is more stable in the insect digestive system and more effectively triggers the RNAi pathway in insects.

[0005] In view of this, the present invention is hereby proposed. Summary of the Invention

[0006] The purpose of this invention is to provide the AgJHE1 gene and siRNA that interferes with the metabolism of cotton aphid insecticides, as well as their application in the management of cotton aphid resistance, in order to solve the problems existing in the prior art.

[0007] To achieve the above objectives, the present invention provides the following solution: One of the technical solutions of this invention is the application of silencing or knocking out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluralin. The AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0008] The second technical solution of the present invention is the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria that silence or knock out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluoropyrimidine, wherein the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0009] The third technical solution of the present invention is a method for improving the sensitivity of cotton aphids to trifluorophenylpyrimidine, which includes the step of inhibiting the expression of the AgJHE1 gene in cotton aphids; the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0010] The fourth technical solution of the present invention is a product for controlling cotton aphids, the product containing a nucleotide sequence capable of silencing the AgJHE1 gene; the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0011] Based on the above technical solution, the present invention has the following technical effects: This invention reveals that, compared to dsRNAs, using siRNA to silence cotton aphid target genes is a more precise and effective method. siRNAs possess advantages such as specificity, high efficiency, and high stability. This invention identifies a siRNA with outstanding silencing efficiency against the cotton aphid AgJHE1 gene, significantly interfering with the regulation of AgJHE1 gene sensitivity to the insecticide trifluralin. This siRNA holds promise for field application in improving the control efficacy of insecticides against cotton aphids and has excellent application prospects. Attached Figure Description

[0012] Figure 1 The relative expression level of the AgJHE1 gene after feeding with siRNA.

[0013] Figure 2 To assess the sensitivity of cotton aphids to trifluoropyrimidine after siRNA-mediated inhibition of AgJHE1 expression. Detailed Implementation

[0014] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0015] Enhanced metabolic capacity mediated by detoxification enzymes is a major reason for the development of resistance in pest populations. Research by the inventors revealed that the expression level of the AgJHE1 gene in resistant cotton aphids to trifluralin was significantly higher than in susceptible strains, and AgJHE1 was significantly correlated with cotton aphid resistance to trifluralin. This invention reduces the cotton aphid's metabolic capacity for insecticides by silencing the AgJHE1 gene, thereby increasing its sensitivity to pesticides. This is of great significance for the efficient and green control of cotton aphids, especially resistant cotton aphids.

[0016] This invention provides the application of silencing or knocking out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluralin. The AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0017] In some specific implementation schemes, small interfering RNA is used to silence or knock out the AgJHE1 gene.

[0018] In some specific implementations, the sense strand of the small interfering RNA is the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.8.

[0019] This invention also provides the application of recombinant vectors, expression cassettes, transgenic cell lines or recombinant bacteria that silence or knock out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluoropyrimidine, wherein the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0020] This invention also provides a method for improving the sensitivity of cotton aphids to trifluorophenylpyrimidine, including the step of inhibiting the expression of the AgJHE1 gene in cotton aphids; the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0021] In some specific implementations, the inhibition is achieved by feeding cotton aphids or introducing siRNA targeting the AgJHE1 gene.

[0022] This invention also provides a product for controlling cotton aphids, the product containing a nucleotide sequence capable of silencing the AgJHE1 gene; the AgJHE1 gene has the NCBI gene number XM_050208220.1.

[0023] In some specific implementations, the product comprises a nucleotide sequence as shown in SEQ ID NO. 7-8.

[0024] This invention provides the cotton aphid juvenile hormone lipase gene AgJHE1, and siRNA that interferes with cotton aphid insecticide metabolism, along with their application in cotton aphid resistance management. The siRNA, designed based on the cotton aphid AgJHE1 gene, is introduced into cotton aphids, effectively using RNAi technology to interfere with AgJHE1 gene expression. Results show that the siRNA enters the cotton aphid cells and triggers an RNAi cascade reaction, interfering with the expression of the target gene. Under insecticide stress, cotton aphids fed with siRNA showed a significantly increased sensitivity to the insecticide trifluralin. The siRNA provided by this invention can interfere with the cotton aphid's detoxification and metabolic processes related to insecticides. This research contributes to understanding the metabolic resistance mechanism of cotton aphids and provides a new target for integrated pest management of resistant pests. The research results have significant theoretical and practical implications for the sustainable management of cotton aphids.

[0025] Example 1 Cloning of the AgJHE1 gene of cotton aphid 1. Using the transcriptome of the cotton aphid resistant and susceptible strains that have been tested in our laboratory, and combined with NCBIBlast and other programs, the AgJHE1 gene (XM_050208220.1) of the cotton aphid was identified.

[0026] Based on the found sequence, primers for amplifying the AgJHE1 gene of cotton aphid were designed using primer design software, and amplification primers were designed with β-actin as the internal reference gene (KF018928.1). The primer sequences are shown in Table 1.

[0027] Table 1 PCR Primers

[0028] 2. The tested aphid-sensitive strain SS was a long-term indoor rearing population collected in July 2017 from cotton fields at the Hebei Academy of Agricultural and Forestry Sciences Institute of Plant Protection farm. The collected aphids were brought indoors and, without any pesticide exposure, were hydroponically grown on cotton seedlings and continuously reared in aphid cages. The trifluralin-resistant aphid strain TFMR was obtained after 24 generations of continuous screening of the sensitive strain, yielding a resistance multiple of 34.77. Both the resistant and sensitive strains were reared on cotton seedlings under laboratory conditions: temperature (20±1)℃, relative humidity 60%-70%, L:D = 16h:8h, using indoor-grown cotton seedlings.

[0029] 3. RNA Extraction: Collected SS and TFMR strains were ground into powder using liquid nitrogen. For each treatment, 0.5 mL of TransZol Up and 0.1 mL of RNA Extraction Agent were added, and the mixture was repeatedly pipetted and aspirated. The mixture was vortexed at room temperature for 5 min, centrifuged at 10000g at 4℃ for 15 min, and 200 μL of colorless aqueous phase was transferred to a new centrifuge tube. An equal volume of anhydrous ethanol was added, and the mixture was gently inverted and added to a centrifuge column. The column was centrifuged at 12000×g at room temperature for 30 s, and the supernatant was discarded. 500 μL of LCB9 was added, and the column was centrifuged at 12000g at room temperature for 30 s, and the supernatant was discarded. This process was repeated once. Then, 500 μL of WB9 (pre-treated with anhydrous ethanol) was added, and the column was centrifuged at 12000g at room temperature for 30 s, and the eluent was discarded. This process was repeated once, followed by centrifugation at 12000g at room temperature for 2 min to remove residual ethanol. Place the centrifuge column into an enzyme-free centrifuge tube, add 50 μL of RNase-free water to the center of the column, incubate at room temperature for 1 min, then centrifuge at 12000g for 1 min at room temperature to elute RNA. Take a portion to determine the RNA concentration, and store the remainder at -80℃.

[0030] 4. Synthesis of first-strand cDNA First-strand cDNA was synthesized from the extracted total RNA using the TransScript One-step gDNA Removal and cDNA Synthesis SuperMix reverse transcription kit, following its instructions. The preparation system is shown in Table 2.

[0031] Table 2 cDNA Synthesis

[0032] Add each component according to Table 2, mix gently, incubate at 42°C for 15 min, heat at 85°C for 5 s to inactivate TransScript®RT / RI and gDNA Remover, and store at -80°C in an ultra-low temperature freezer.

[0033] The cDNA sequence is identical to the reference gene AgJHE1 (XM_050208220.1) sequence mentioned earlier.

[0034] Example 2 Design and preparation of target gene siRNA The green fluorescent protein (GFP) gene (MK078653.1) was selected as the control gene. Starting from the AUG start codon of the mRNA, the "AA" doublet sequence was searched, and two different siRNA sequences were generated through NCBI verification to screen for the optimal target site. These were named siRNA1- AgJHE1 siRNA2- AgJHE1 The siRNA was synthesized by Sangon Biotech (Shanghai) Co., Ltd.

[0035] siRNA1- AgJHE1 Chain of Justice: 5'-GGTTCTTAGTTGGAACACC-3' (SEQ ID NO.5); Antisense chain: 5'-GGTGTTCCAACTAAGAACC-3' (SEQ ID NO.6).

[0036] siRNA2- AgJHE1 Chain of Justice: 5'-GCGACGAGACCAATTCTTC-3' (SEQ ID NO.7); Antisense chain: 5'-GAAGAATTGGTCTCGTCGC-3' (SEQ ID NO.8).

[0037] siGFP positive strand: 5'-GGGCGATGCCACCTACGGC-3' (SEQ ID NO.9); Antisense chain: 5'-GCCGTAGGTGGCATCGCCC-3' (SEQ ID NO.10).

[0038] siRNA was prepared according to the method described in the T7 RiboMAX™ Express RNAi System (P1700) kit. The sample loading system is shown in Table 3. After that, the DNA template was removed, the siRNA was annealed and purified, and the synthesized siRNA was stored in a -80°C freezer.

[0039] Table 3 siRNA Synthesis

[0040] Example 3 RNA interference Three hundred third-instar nymphs of a trifluorophenylpyrimidine-resistant strain were placed in 50 mL centrifuge tubes. The tubes were sealed with a sealing film (sterilized in a clean bench for 15 min), and nutrient solution containing 100 ng / μL of siRNA was added to the film. The solution was then sealed again with the sealing film. An equal volume of nutrient solution containing 100 ng / μL of siGFP served as a negative control. The bottom and walls of the centrifuge tubes were wrapped with aluminum foil, while the top was sealed with a light-transmitting structure. The aphids in the centrifuge tubes were placed in a light incubator at (20±2)℃, 65% relative humidity, and a photoperiod of 16 L:8 D (feed end facing upwards). After 20 h, they were re-fed with fresh siRNA feed. Samples were taken for 3 consecutive days after 20 h, with 90 aphids from each treatment, and stored at -80℃. Total RNA was then extracted and cDNA was reverse transcribed. The target gene was detected by quantitative real-time PCR. AgJHE Changes in siRNA expression levels at different interference times.

[0041] Using the cDNA obtained in Example 1 as a template, RT-qPCR was performed using ChamQ Universal SYBR qPCR Master Mix (Vazyme Nanjing, China) and gene-specific primers (Table 1). The qPCR reaction system is shown in Table 4.

[0042] Table 4. Real-time PCR reaction system

[0043] Reaction program: 94℃ pre-denaturation for 30 s; 94℃ denaturation for 5 s, 60℃ annealing extension for 30 s, 40 cycles.

[0044] Then apply 2 to the obtained Ct value. -△△Ct The data were processed using the method described by ΔCt = (ΔCt target gene - ΔCt internal reference gene). SPSS and Microsoft Excel software were used for data analysis. One-way analysis of variance (ANOVA) was used to compare the relative expression levels of the AgJHE1 gene across treatments, and Tukey's method was used for multiple comparisons.

[0045] The results are as follows Figure 1As shown, compared with the control group (siRNA-GFP), feeding with siRNA1-AgJHE1 and siRNA2-AgJHE1 significantly reduced the transcription level of the AgJHE1 gene in cotton aphids from 24 to 72 hours post-feeding. Among them, siRNA2-AgJHE1 showed higher interference efficiency, reducing AgJHE1 expression by 60.83% (t = 14.34, p = 0.0007), 30.45% (t = 14.25, p = 0.0018), and 44.54% (t = 21.82, p = 0.0002) at 24, 48, and 72 hours post-feeding, respectively. It significantly inhibited the expression of the AgJHE1 gene in cotton aphids. Subsequent experiments will use siRNA2-AgJHE1.

[0046] Example 4 Changes in the sensitivity of cotton aphids to trifluoperidine after AgJHE1 gene silencing Third-instar nymphs of the trifluralin-resistant cotton aphid strain were fed with siGFP nutrient solution and siRNA2-AgJHE1 nutrient solution, respectively. After 20 h at standard rearing temperature, they were fed a second batch of fresh siRNA nutrient solution. Sensitivity testing was then conducted 20 h later. Trifluralin was added to the nutrient solution to dilute it to 5 mg / L to test the sensitivity of cotton aphids to trifluralin after the target gene was silenced. Each treatment consisted of 30 aphids, with 3 biological replicates. Mortality was checked after 48 h; the absence of a normal reaction when the aphid was lightly touched with a brush was considered the criterion for death.

[0047] After feeding siRNA2-AgJHE1, the mortality rate increased to 68.33% under treatment with 5 mg / L trifluoropyrimidine compared to the control (38.33%). Figure 2 This significantly increased the cotton aphid's sensitivity to trifluoropyrimidine.

[0048] The above results indicate that introducing siRNA that inhibits the AgJHE1 gene in cotton aphids into larvae increases the sensitivity of cotton aphids to trifluorophenylpyrimidine by suppressing AgJHE1 gene expression. The siRNA2-AgJHE interference effect of this invention is outstanding and shows promise for combined application with insecticides in the field to control cotton aphids, especially resistant cotton aphids, thereby improving insecticide control efficacy, reducing pesticide dosage, and protecting the ecological environment. It has excellent application prospects.

[0049] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. The application of silencing or knocking out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluralin, characterized in that, The AgJHE1 gene has the NCBI gene number XM_050208220.

1.

2. The application according to claim 1, characterized in that, Small interfering RNA was used to silence or knock out the AgJHE1 gene.

3. The application according to claim 2, characterized in that, The sense strand of the small interfering RNA is the nucleotide sequence shown in SEQ ID NO.7, and the antisense strand is the nucleotide sequence shown in SEQ ID NO.

8.

4. The application of recombinant vectors, expression cassettes, transgenic cell lines, or recombinant bacteria that silence or knock out the AgJHE1 gene in improving the sensitivity of cotton aphids to trifluralin, characterized in that, The AgJHE1 gene has the NCBI gene number XM_050208220.

1.

5. A method for improving the sensitivity of cotton aphids to trifluorophenylpyrimidine, characterized in that, The method includes the step of inhibiting the expression of the AgJHE1 gene in cotton aphids; the AgJHE1 gene has the NCBI gene number XM_050208220.

1.

6. The method according to claim 5, characterized in that, The inhibition is achieved by feeding cotton aphids or introducing siRNA targeting the AgJHE1 gene.

7. A product for controlling cotton aphids, characterized in that, The product contains a nucleotide sequence capable of silencing the AgJHE1 gene; the AgJHE1 gene has the NCBI genotype number XM_050208220.

1.

8. The product according to claim 7, characterized in that, The product comprises nucleotide sequences as shown in SEQ ID NO. 7~8.

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