Method for preventing and treating aphis citricola by dsRNA fusion fragment

Abstract

The invention aims to provide a method for preventing and treating aphis citricola by using a dsRNA fusion fragment, and the dsRNA fusion fragment is screened and constructed, so that the aphis citricola can be effectively prevented and treated. The method comprises the following steps: firstly, providing a dsRNA fragment for preventing and treating aphis citricola, wherein the provided dsRNA fragment can interfere with the expression quantity of dsCmb4b or dsSynapsin genes through RNAi (Ribonucleic Acid Interference) treatment; wherein the nucleotide sequence of the dsRNA fragment for interfering the Cmb4b gene is SEQ ID NO: 1, and the nucleotide sequence of the dsRNA for interfering the Syn gene is SEQ ID NO: 2. The invention also provides a dsRNA fusion fragment, and the nucleotide sequence of the dsRNA fusion fragment is SEQ ID NO: 5. According to the invention, the aphis citricola which seriously damages fruit trees is taken as a research object, a target gene fusion fragment with high fatality rate is obtained based on an RNAi technology and a technology of fusing a plurality of dsRNA small fragments, and a new thought and a target are provided for biological prevention and control of the aphis citricola. The established method can efficiently prevent and treat the aphis citricola and reduce diseases and insect pests in the apple breeding process.

Description

Technical Field

[0001] The invention belongs to the fields of biotechnology and agricultural applications, and particularly relates to a method for preventing and controlling apple yellow aphids by utilizing a dsRNA fusion fragment. Background Art

[0002] Apple yellow aphid ( Aphis citricolavonder ) is a serious agricultural pest in apple orchards, causing huge economic losses every year. In order to reduce the use of chemical pesticides, exploring new, efficient, precise and safe prevention and control strategies has become an important direction of current research. RNA interference (RNAi) technology is a biological control strategy based on gene silencing. In recent years, it has shown broad application prospects in the field of agricultural pest control. This technology induces the RNAi pathway in insects through exogenous feeding or spraying of double-stranded RNA (dsRNA), specifically inhibiting the expression of target genes, thereby interfering with the growth and development of pests and even causing their death. Compared with traditional chemical pesticides, RNAi has a high target specificity and low toxicity to non-target organisms. At the same time, it can avoid the pollution of the ecological environment caused by chemical pesticide residues.

[0003] However, while RNAi has demonstrated promising insecticidal effects against a wide range of agricultural pests, its application in Hemipteran insects like aphids still faces numerous challenges, including RNAi inefficiency, optimizing target gene selection, and dsRNA stability and delivery efficiency. Studies have shown that dsRNA fragments from different genes can exhibit significant differences in their ability to induce RNAi, with fragment length, secondary structure, and sequence characteristics all influencing their efficiency. Therefore, optimizing dsRNA fragment design and improving RNAi efficiency remain key areas of research in RNAi biocontrol. Summary of the Invention

[0004] The purpose of the present invention is to provide a method for controlling apple yellow aphids by using dsRNA fusion fragments, and the dsRNA fusion fragments are screened and constructed, thereby effectively controlling apple yellow aphids.

[0005] The present invention first provides a dsRNA segment for controlling apple yellow aphids. The provided dsRNA segment can interfere with the expression of Charged multivesicular body protein 4b (Cmb4b) or Synapsin (Syn) genes through RNAi treatment. The nucleotide sequence of the dsRNA fragment that interferes with the Cmb4b gene is as follows: GAGCTGCATTGCAAGCATTAAAGCGTAAAAAACGATATGAACAACAATTAGCGCAAATTGATGGTACTATGTTAACTATTGAACAACAGCGAGAGGCGTTAGAAGGTGCTAACACAAACACAGCAGTATTAACTACGATGAAAACTGCTGCGGATGCACTTAAATCAGCTCATCAAAATATGAATGTCGATGATGTTCACAATATGATGGATGATATAGCAGAGTCACAAGATTTATCAAAAGAAATATCAGAAGCTATTTCAAATCCAGTTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAA (SEQ ID NO: 1), The nucleotide sequence of the dsRNA for interfering with the Synapsin gene is as follows: TTGGTTCAGCGATGTTGGAAGAAACCGAAGTTCTAGAAAGATATAAATTTTGGTTGGATTCTGTGGCAGAACTTTTTGGTGGTTTGGACATTTTGTCATTGGAGGTAGTTGTCTCTAAAGACGGAATAGAACAAATCATTGGTGTTAATGACTCTGCATTATCTTTGCTGGGAACTCAACAAGAAGAAGATAGGAAATATATTTTCGATTTGATCATGGAAAAATTAGAAACACAAGTTTTGGCAAATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACCAATCTAGTCAACCGGTGAAAGCAAATCTAAGTCGTCAAGGTTCTATCATTTCAGCTCCCACTCCAGTT (SEQ ID NO: 2), Furthermore, the sequence of the dsRNA fragment for interfering with the Cmb4b gene is as follows: TTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAA (SEQ ID NO: 3), Furthermore, the nucleotide sequence of the dsRNA used to interfere with the Synapsin gene is as follows: ACACAAGTTTTGGCAAATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACCAATCTAGTCAACCGGTGAAAGCAAATCTAAGTCGTCAAGGTTCTATCATTTCAGCTCCCACTCCAGTT (SEQ ID NO: 4), The present invention also provides a dsRNA fusion fragment for interfering with the two target fragments, the specific sequence of which is as follows: TTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAAACACAAGTTTTGGCA AATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACATCTAGTCAACCGGTGAAAGCAAATCTAAGTCGTCAAGGTTCTATCATTTCAGCTCCCACTCCAGTT (SEQ ID NO: 5); The present invention also provides an application of the dsRNA fusion fragment, which is an application as an apple aphid-killing dsRNA fragment.

[0006] This study focuses on the apple aphid, a serious pest of fruit trees. Using RNAi technology and the fusion of multiple small dsRNA fragments, a target gene fusion fragment with high lethality was obtained, providing new ideas and targets for the biological control of the apple aphid. The developed method is highly effective in controlling the apple aphid, achieving green pest control in apple production. BRIEF DESCRIPTION OF THE DRAWINGS

[0007] Figure 1 : In Example 1, the dsCmb4b gene and the dsSyn gene were mixed to determine the effect of the mixed gene on apple The effect diagram of yellow aphids, where Figure A is the aphid yield diagram after dsCmb4b + dsSyn mixed drip treatment, and Figure B is the mortality diagram after dsCmb4b + dsSyn mixed drip treatment; Figure 2 : In Example 2 and Example 3, dsRNA fragments of genes of different lengths were designed, wherein Figure A is a small dsRNA fragment of the dsCmb4b gene, and Figure B is a small dsRNA fragment of the dsSyn gene; Figure 3 : Graph showing the aphid production and mortality of various dsRNA fragments of the dsCmb4b gene measured by the spotting method in Example 4, wherein Graph A shows the aphid production and Graph B shows the mortality; Figure 4 : Graph showing the aphid production and mortality of various dsRNA fragments of the dsSyn gene measured by the spot method in Example 4, wherein Graph A shows the aphid production and Graph B shows the mortality; Figure 5 : Example 5 shows the effect of low-concentration dsRNA fragments on apple yellow aphids, wherein Figure A shows the aphid production of each dsRNA fragment of the dsCmb4b gene, Figure B shows the mortality rate of each dsRNA fragment of the dsCmb4b gene, Figure C shows the aphid production of each dsRNA fragment of the dsSyn gene, and Figure D shows the mortality rate of each dsRNA fragment of the dsSyn gene; Figure 6 : The effect of direct mixing and dripping of dsC2 + dsS2 on apple yellow aphids in Example 6, wherein Figure A shows the aphid yield and Figure B shows the mortality rate; Figure 7 : Roadmap for overlapping PCR fusion methods, Figure 8 : Graph showing the effect of 250 ng / µL of the lethal gene on the yellow apple aphid in Example 8, where Panel A shows aphid production and Panel B shows mortality; Figure 9 : The effect of dripping 500ng / μL of the lethal gene on the yellow apple aphid in Example 8, wherein Figure A shows the aphid yield and Figure B shows the mortality rate; Figure 10: Relative expression levels of apple yellow aphid after dripping 500 ng / μL of the lethal gene dsRNA in Example 9, wherein (A) dsC1 fragment, (B) dsC2 fragment, (C) dsC3 fragment, (D) dsS1 fragment, (E) dsS2 fragment, (F) (D) dsS3 fragment; Figure 11 : Relative expression levels of different concentrations of lethal gene dsRNA after dripping into apple yellow aphid in Example 9, where (A) is 500 ng / µL, and (B) is 250 ng / µL. DETAILED DESCRIPTION

[0008] The present invention screened and obtained a 442bp apple yellow aphid lethal gene dsCmb4b and a 428bp dsSyn fragment. In order to further improve the insecticidal activity of dsRNA, small fragments of dsRNA were constructed and formed into dsRNA fusion fragments, thereby improving the lethality to apple yellow aphid.

[0009] The present invention is described in detail below with reference to the embodiments and accompanying drawings.

[0010] Example 1: Effect of Mixed Use of dsCmb4b Gene and dsSyn Gene dsRNA on Apple Yellow Aphid The nucleotide sequence of the dsRNA target fragment dsCmb4b of the Cmb4b gene is as follows: GAGCTGCATTGCAAGCATTAAAGCGTAAAAAACGATATGAACAACAATTAGCGCAAATTGATGGTACTATGTTAACTATTGAACAACAGCGAGGCGTTAGAAGGTGCTAACACAAACACAGCAGTATTAACTACGATGAAAACTGCTGCGGATGCACTTAAATCAGCTCATCAAAATATGAATGTCGATGATGTTCACAATATGATGGATGATATAGCA GAGTCACAAGATTTATCAAAAGAAATATCAGAAGCTATTTCAAATCCAGTTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAA, The nucleotide sequence of the Synapsin gene dsRNA target fragment dsSyn is as follows: TTGGTTCAGCGATGTTGGAAGAAACCGAAGTTCTAGAAAGATATAAATTTTGGTTGGATTCTGTGGCAGAACTTTTTGGTGGTTTGGACATTTTGTCATTGGAGGTAGTTGTCTCTAAAGACGGAATAGAACAAATCATTGGTGTTAATGACTCTGCATTATCTTTGCTGGGAACTCAACAAGAAGAAGATAGGAAATATATTTTCGATTTGAT CATGGAAAAATTAGAAACACAAGTTTTGGCAAATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACCAATCTAGTCAACCGGTGAAAGCAAATCTAAGTCGTCAAGGTTCTATCATTTCAGCTCCCACTCCAGTT, The dsRNA of the dsCmb4b gene (442 bp) and the dsSyn gene (428 bp) were mixed with the star-shaped polymeric cationic (SPc) nanocarrier at the recommended mass ratio of 1:1 to prepare the dsRNA complex (final concentration of 500 ng / μL), and then 0.6% volume of laundry detergent surfactant was added. Figure 1 There was no significant difference in aphid production between mixed and single dripping. The mortality rates of dsCmb4b+dsSyn, dsSyn, dsCmb4b, and dsGFP at 72 h were 56%, 37.1%, 76.62%, and 18.4% respectively.

[0011] Example 2: Design of small dsRNA fragments targeting the Cmb4b gene according to Figure 2 Primers for dsRNA of different lengths of Cmb4b gene and control GFP gene were designed (see Table 1). dsRNA was synthesized according to the instructions of TranscriptAid T7 High Yield transcription Kit for use in RNAi experiments.

[0012] Table 1: dsRNA specific primers

[0013] The names and nucleotide sequences of the designed dsRNA fragments targeting dsCmb4b are as follows: dsC1(239bp): AGCTGCATTGCAAGCATTAAAGCGTAAAAAACGATATGAACAACAATTAGCGCAAATTGATGGTACTATGTTAACTATTGAACAACAGCGAGAGGCGTTAGAAGGTGCTAACACAAACACAGCAGTATTAACTACGATGAAAACTGCTGCGGATGCACTTAAATCAGCTCATCAAAATATGAATGTCGATGATGTTCACAATATGATGGATGATATAGCAGAGTCACAAGATTTATCAA; dsC2(172bp): TTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAA; dsC3(195bp): GCAGAGTCACAAGATTTATCAAAAGAAATATCAGAAGCTATTTCAAATCCAGTTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAG。

[0014] Primer5 was used to design dsRNA multi-fragment-specific primers. T7 promoter sequences were added to the 5' ends of the upstream and downstream primers, as shown in Table 1. Primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. PCR amplification of the Cmb4b gene was performed using the full-length gel-recovered product of the gene clone as a template and the designed primers. The amplification system consisted of 12.5 μL of 2× Taq Plus MasterMix II, 0.5 μL of each upstream and downstream primer, 1 μL of template, and 10.5 μL of ddH2O. The PCR reaction program was as follows: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 35 cycles, and 72°C extension for 1 min. The GFP gene amplification system consisted of 2× Taq Plus Master Mix II (12.5 μL of Dye Plus, 0.5 μL of each upstream and downstream primer, 1 μL of template, and 10.5 μL of ddH2O). The PCR reaction program was as follows: 94°C pre-denaturation for 3 min, 5 cycles of 94°C denaturation for 30 s, 55°C annealing for 30 s, 35 cycles of 94°C denaturation for 30 s, 65°C denaturation for 30 s, and 72°C extension for 30 s, followed by 72°C for 10 min, and finally storage at 10°C. The PCR product was detected on a 1% agarose gel and purified by gel recovery. dsRNA synthesis was performed using the TranscriptAid T7 High Yield transcription kit. The mixed system was as follows: Reagent volume (μL) 5×Transcript Aid Reaction Buffer 4.0 ATP / CTP / GTP / UTP 8.0 Template cDNA 1 μg Transcript Aid Enzymix 2.0 DEPC-treated H2O Up to 20.0 Mix well and centrifuge; incubate at 37°C overnight; white flocs will be visible during the reaction.

[0015] After the reaction is complete, add 2 μL DNase I to the mixture, incubate at 37°C for 15 min, then add 2 μL EDTA and incubate at 65°C for 10 min; transfer to a 1.5 mL centrifuge tube, add 115 μL DEPC-treated H2O and 15 μL 3M sodium Acetute Solution and mix well; add 150 μL 1:1 water-saturated phenol / chloroform (75 μL each) to the 1.5 mL centrifuge tube, then add 300 μL chloroform, shake and mix well, centrifuge at 12,000 g, 4°C for 10 min; transfer the supernatant to a new 1.5 mL centrifuge tube, add 1 ml of anhydrous ethanol, mix well, and place at -20°C for 2 h; centrifuge the 1.5 mL centrifuge tube at 12,000 g, 4°C for 10 min, discard the supernatant, and retain the precipitate; add 500 μL ice-cold 75% ethanol to the precipitate, centrifuge at 7,500 g, 4°C for 5 min, discard the supernatant, aspirate as much as possible, and dry the precipitate for 10 min; add 50 μL RNase-Free H2O, measure the concentration, and run electrophoresis to confirm that the preparation is complete.

[0016] Example 3: Design of small dsRNA fragments targeting the Synapsin gene according to Figure 2 Primers for dsRNA of varying lengths targeting the dsSyn gene were designed (see Table 2). Following the instructions for the TranscriptAidT7 High Yield transcription Kit, dsRNA was synthesized for use in RNAi experiments. Specific experimental procedures were the same as above.

[0017] Table 2: dsRNA specific primer sequence list

[0018] The names and nucleotide sequences of the designed dsRNA fragments targeting the dsSyn gene are as follows: dsS1 (252 bp) CGATGTTGGAAGAAACCGAAGTTCTAGAAAGATATAAATTTTGGTTGGATTCTGTGGCAGAACTTTTTGGTGGTTTGGACATTTTGTCATTGGAGGTAGTTGTCTCTAAAGACGGAATAGAACAAATCATTGGTGTTAATGACTCTGCATTATCTTTGCTGGGAACTCAACAAGAAGAAGATAGGAAATATATTTTCGATTTGATCATGGAAAAATTAGAAACACAAGTTTTGGCAAATATGGGTCTTCAAG; dsS2 (198 bp) ACACAAGTTTTGGCAAATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACCATCTAGTCAACCGGTGAAAGCAAATCTAAGTCGTCAAGGTTCTATCATTTCAGCTCCCACTCCAGTT; dsS3 (238 bp) GCATTATCTTTGCTGGGAACTCAACAAGAAGAAGATAGGAAATATATTTTCGATTTGATCATGGAAAAATTAGAAACACAAGTTTTGGCAAATATGGGTCTTCAAGCAGGTTCAGGTATCACCCAAACACAAGAAGAAGAACCACCAGCTATTCCGGCTAGACGAGATTCTTTGGTTTCAGAGAGCAGCGTCACCAGCGGACACCATCTAGTCAACCGGTGAAAGCAAATCTAAGTC.

[0019] Example 4: Spot-drip method to determine the effects of different lengths of dsCmb4b and dsSyn genes In this example, 0.4 μL of the dsRNA complex was dropped onto the abdomen of the apple yellow aphid using a Hamilton PB600-1 repeating dispenser to measure the phenotypic results of the dsGFP, dsCmb4b, and dsSyn dsRNA fragments (i.e., dsRNA). The specific experimental process is as follows: Star-shaped polycationic (SPc) nanocarriers were mixed with dsRNAs (dsCmb4b, dsSyn, and dsGFP) at the recommended mass ratio of 1:1 (final concentrations of both nanocarriers and dsRNA were 500 ng / μL). A 0.6% volume of laundry detergent surfactant was then added. After 30 minutes of stagnation, 0.4 μL of the dsRNA complex was dripped onto the abdomen of aphids using a Hamilton PB600-1 repeating dispenser. Five biological replicates were performed, with 20 aphids per replicate, for a total of 100 aphids. Aphids were reared on fresh apple leaves in Petri dishes containing agar medium at 25°C and 50% relative humidity. Aphid production and mortality were observed daily for 72 hours.

[0020] The results showed that (such as Figure 3 and Figure 4 (As shown), compared to the control, aphid production for each dsRNA fragment was not significantly different at 24 and 48 hours. However, significant differences were observed for dsC3 and dsS1 at 72 hours. Mortality was highest at 72 hours, with dsC1, dsC2, and dsC3 mortality rates of 49.5%, 55.5%, and 44.65%, respectively. The mortality rate for the GFP control was 16%. The mortality rates for dsS1, dsS2, and dsS3 were 49%, 59%, and 40.9%, respectively. The mortality rate for the GFP control was 12.1%. Therefore, dsC2 and dsS2 had the highest mortality rate.

[0021] Example 5: Effects of low concentrations of dsCmb4b and dsSyn genes on the growth and development of apple aphids Star-shaped polycationic (SPc) nanocarriers were mixed with dsRNA (dsCmb4b, dsSyn, and dsGFP) at the recommended mass ratio of 1:1 (final concentration of both nanocarriers and dsRNA was 250 ng / μL), and 0.6% by volume of laundry detergent surfactant was added, following the same procedure as above.

[0022] The results showed that (such as Figure 5 As shown in the figure, compared with the control, there was no significant difference in the aphid production of each dsRNA fragment of dsCmb4b and dsSyn at 24 h and 48 h. At 72 h, there were significant differences in the aphid production of dsS1, dsS2, and dsS3. The mortality rate was highest at 72 h, with the mortality rates of dsC1, dsC2, and dsC3 being 39%, 41.6%, and 37%, respectively, while the mortality rate of the control GFP was 18%; the mortality rates of dsS1, dsS2, and dsS3 were 38.5%, 47%, and 35%, respectively, while the mortality rate of the control GFP was 16%. This shows that lowering the concentration significantly reduced the mortality rate of apple yellow aphid.

[0023] Example 6: Effect of direct mixing and dripping of small fragments of dsC2 and dsS2 on apple yellow aphids Star-shaped polymeric cationic (SPc) nanocarriers were mixed with dsRNA (dsC2 + dsS2 and dsGFP) at the recommended mass ratio of 1:1 (final concentrations of both nanocarriers and dsRNA were 500 ng / μL), and 0.6% volume of laundry detergent surfactant was added, following the same procedures as above.

[0024] The results showed that (such as Figure 6 As shown in the figure, the aphid production of dsC2 + dsS2 was significantly different from that of the control at 72 h, and the mortality rate of dsC2 + dsS2 was as high as 42% at 72 h; the mortality rate of the control dsGFP was 17% at 72 h.

[0025] Example 7: Effect of Fusion ds (C2 + S2) Assay on Apple Yellow Aphid The results of the previous experiments showed that the dsC2 and dsS2 fragments were more effective. In this case, the overlapping PCR method (such as Figure 7 (as shown) The two fragments dsC2 and dsS2 were connected, and the original bases of the downstream primer of dsC2 and the upstream primer of dsS2 were complementary.

[0026] 1. The first step is to use the primers dsC2 and dsS2 in Table (3) to perform PCR amplification and gel recovery.

[0027] 2. In the second step, the dsC2 upstream primer and dsS2 downstream primer in Table (3) were used, and the full-length gel recovery products of dsC2 and dsS2 in the previous step were used as templates for PCR amplification of the ds (C2 + S2) gene. The amplification system was 2 × Taq Plus Master Mix II (Dye Plus 12.5 μL, 0.5 μL each of upstream and downstream primers, 1 μL template, 10.5 μL ddH2O. The PCR reaction program was as follows: 94 ° C pre-denaturation for 3 min, 94 ° C denaturation for 30 s, 55 ° C annealing for 30 s, 35 cycles, 72 ° C extension for 1 min, and finally stored at 4 ° C. The PCR product was detected using 1% agarose gel and then purified by gel recovery. 3. Synthesis of ds(C2+S2) using in vitro transcription method dsRNA synthesis was performed using the Transcript Aid T7 High Yield transcription Kit, and the specific implementation method was the same as above.

[0028] Table 3: dsRNA overlapping PCR primers

[0029] Example 8: Determination of the effects of different concentrations of ds (C2 + S2) on apple yellow aphids In this example, 0.4 μL of a dsRNA complex was dropped onto the abdomen of an apple yellow aphid using a Hamilton PB600-1 repeating dispenser to measure the phenotypic results of dsGFP and ds(C2+S2) (i.e., dsRNA). The specific experimental procedure was as follows: a star-shaped polycationic (SPc) nanocarrier was mixed with dsRNA (dsC2+S2 and dsGFP) at the recommended mass ratio of 1:1 (final concentrations of the nanocarrier and dsRNA were 500 ng / μL and 250 ng / ul, respectively), and then 0.6% by volume of a laundry detergent surfactant was added. The specific implementation method was the same as above.

[0030] The results showed that (such as Figure 8After the final concentration of 250 ng / µL of dsRNA complex was dropped, the aphid production of ds (C2 + S2) was not significantly different at 24 h and 48 h compared with the control, but there was a significant difference at 72 h; the mortality rate was as high as 60% at 72 h; the mortality rate of the control dsGFP was 15% at 72 h (as shown in Figure 2). Figure 8 and Figure 9 shown). Figure 9 As shown in the figure, after the droplet of dsRNA complex with a final concentration of 500 ng / µL, the aphid production of ds(C2+S2) had no significant difference at 24 h compared with the control, but there were significant differences at 48 h and 72 h. The mortality rate was as high as 84% ​​at 72 h; the mortality rate of the control dsGFP was 17% at 72 h.

[0031] The results of measuring different concentrations of ds (C2 + S2) showed that the lethality rate of overlapping PCR connection was higher than that of direct mixing or single use.

[0032] Example 9: Real-time fluorescence quantitative PCR detection of the expression level of lethal dsRNA fragments After 24 hours of dripping the lethal gene dsRNA fragment, samples were collected, RNA was extracted and reverse transcribed into cDNA, and the expression level of the lethal gene in the apple yellow aphid was detected by qPCR. The reaction system was as follows: Reagent volume (μL) 2×RealStar Green Fast Mixture 5.0 Forward Primer 0.25 Reverse Primer 0.25 cDNA 1.0 dd H2O Up to 10.0 The reaction conditions were 95°C pre-denaturation for 30 s, 95°C denaturation for 5 s, 60°C annealing and extension for 30 s, and 40 cycles; the reaction temperature was increased at a rate of 0.6°C / s.

[0033] Compared with the control dsGFP, the expression levels of dsCmb4b, dsSyn and each fragment were significantly decreased. As shown in Figure (10), the silencing efficiencies of dsC1, dsC2 and dsC3 were 42.2%, 80% and 45.4%, respectively; and as shown in Figure (18), the silencing efficiencies of dsS1, dsS2 and dsS3 were 74.3%, 47.1% and 52%, respectively.

[0034] As shown in Figures (11A and 11B), the silencing efficiency of 500 ng / ul of ds (C2 + S2) on the Cmb4b gene was 61.6%, and the silencing efficiency on the Synapsin gene was 71.7%; as shown in Figures (11C and 11D), the silencing efficiency of 250 ng / ul of ds (C2 + S2) on the Cmb4b gene was 50.9%, and the silencing efficiency on the Synapsin gene was 60.7%.

[0035] The above results indicate that the dsRNA and fusion fragment constructed by the present invention, as well as the established RNAi method, can effectively kill the apple yellow aphid.

Claims

1. A dsRNA fragment for controlling apple yellow aphid, characterized in that: The dsRNA fragment can interfere with the expression of dsCmb4b or dsSynapsin gene through RNAi treatment, wherein the nucleotide sequence of the dsRNA fragment interfering with Cmb4b gene is SEQ ID NO: 1, and the nucleotide sequence of the dsRNA for interfering with Syn gene is SEQ ID NO:

2.

2. The dsRNA fragment according to claim 1, wherein The nucleotide sequence of the dsRNA fragment used to interfere with the Cmb4b gene is shown in SEQ ID NO:

3.

3. The dsRNA fragment according to claim 1, wherein The nucleotide sequence of the dsRNA fragment used to interfere with the dsSynapsi gene is shown in SEQ ID NO:

4.

4. Use of the dsRNA fragment according to any one of claims 1 to 3 in the preparation of a product for killing apple yellow aphid.

5. A dsRNA fusion fragment, characterized in that The dsRNA fusion fragment is constructed by connecting the dsRNA fragments according to claim 1.

6. The dsRNA fusion fragment according to claim 5, wherein The nucleotide sequence of the dsRNA fusion fragment is SEQ ID NO:

5.

7. Use of the dsRNA fusion fragment according to claim 5 in the preparation of a product for killing apple yellow aphid.

8. An RNAi product for killing apple yellow aphids, characterized in that: The product comprises the dsRNA segment according to any one of claims 1 to 3 and / or the dsRNA fusion segment according to claim 5.

9. A method for preventing and controlling apple yellow aphids, characterized in that: The method is to use the dsRNA fragment according to any one of claims 1 to 3 to reduce the expression level of the dsCmb4b or dsSynapsin gene in apple yellow aphid.

10. The method according to claim 9, wherein The method is to use the dsRNA fusion fragment according to claim 5 to reduce the expression level of the dsCmb4b or dsSynapsin gene in apple yellow aphid.

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