Application of an apple yellow aphid Cmb4b gene and its dsRNA in controlling apple yellow aphid

Abstract

The present invention provides an application of an apple yellow aphid Cmb4b gene and its dsRNA in preventing and controlling apple yellow aphids. The provided apple yellow aphid Cmb4b gene has an amino acid sequence of a protein encoded by it as SEQ ID NO: 2; and a nucleotide sequence as shown in SEQ ID NO: 1. The present invention also provides a dsRNA, which can reduce the expression level of the apple yellow aphid gene. In another aspect, the present invention also provides a biological product for preventing and controlling apple yellow aphids, wherein dsRNA and matrine are added, and the 72h mortality rate of the apple yellow aphid is 90%, and the biological control of the apple yellow aphid can be achieved efficiently. The present invention takes the apple yellow aphid as the research object, screens a target gene with a high lethality rate based on RNAi technology, obtains a dsRNA interference fragment, and provides a new target for the biological control of the apple yellow aphid.

Description

Technical Field

[0001] The present invention belongs to the field of biological pesticide manufacturing and application, and specifically relates to an apple yellow aphid Cmb4b gene and a dsRNA thereof for use in preventing and controlling apple yellow aphid. Background Art

[0002] Apple yellow aphid (Aphis citricolavander Goot), also known as Spiraea aphid, belongs to the Aphididae family of the order Hemiptera and is widely distributed in my country. It is one of the important pests in my country's apple orchards. It mainly gathers in the form of nymphs and adults on the young leaves of various Rosaceae plants such as apple, crabapple, pear, and hawthorn to suck juice, harming the new shoots, buds, and leaves of apple trees, reducing the quality and yield of fruits. For a long time, it has relied on chemical pesticides for prevention and control, and there is a lack of effective biological control agents.

[0003] RNA interference (RNAi) is a post-transcriptional gene regulation phenomenon in which double-stranded RNA (dsRNA) mediates specific silencing of target genes. RNAi is an inherent conservative mechanism of eukaryotic organisms to resist the invasion of exogenous nucleic acids (viruses, transposons, etc.), and is ubiquitous in eukaryotic organisms such as mammals, plants, insects, nematodes, etc. Based on the principle of RNAi, small RNA molecules can be designed to silence key genes (called "lethal genes") in the life activities of pests and pathogens. When RNA molecules enter the cells of pests or pathogens, they can silence the "lethal genes" of pests and pathogens, causing the pests to die or stop growing, thereby achieving the purpose of preventing and controlling pests and diseases. Therefore, RNAi technology can be used to develop biological pesticides.

[0004] Domestic and foreign researchers have screened more than 10 lethal target genes by delivering artificial dsRNA to aphids through injection, feeding, dripping and transgenic plants. Indoor biological tests of these target genes show that most of them cause a mortality rate of about 40-60% when used alone, but with the assistance of nanocarriers and surfactants, the mortality rate of some genes can reach more than 80%. However, relevant research on apple aphids, a major pest of fruit trees, has not been carried out. Summary of the invention

[0005] The invention provides an apple yellow aphid Cmb4b (Charged multivesicular body protein4b) gene and application of the dsRNA thereof in preventing and controlling the apple yellow aphid, thereby making up for the deficiencies of the prior art.

[0006] The present invention first provides an apple yellow aphid gene, the amino acid sequence of the protein encoded by the gene is as follows:

[0007] MSFLGKLFGSKKEEKGPSTEDAIQKLRSTEEMLIKKQEFLEKKIEQEVAIAKKNGTTNKRAALQALKRKKRYEQQLAQIDGTMLTIEQQREALEGANTNTAVLTTMKTAADAL KSAHQNMNVDDVHNMMDDIAESQDLSKEISEAISNPVAFGTDVDEDELQKELEELEQEELDKELLNTGKTPVHDLPTPAVPTFEPSGRGKAKTKAEEDDDEMKELANWAS(SEQ ID NO: 2);

[0008] A specific nucleotide sequence of the apple yellow aphid gene is shown in SEQ ID NO: 1:

[0009] (SEQ ID NO: 1).

[0010] The present invention also provides a dsRNA, which can reduce the expression level of the apple yellow aphid gene, and the dsRNA binds to the 201bp-692bp of the nucleotide sequence shown in SEQ ID NO: 1;

[0011] Furthermore, a specific sequence of the dsRNA is shown in SEQ ID NO: 3;

[0012] GAGCTGCATTGCAAGCATTAAAGCGTAAAAAACGATATGAACAACAATTAGCGCAAATTGATGGTACTATGTTAACTATTGAACAACAGCGAGGCGTTAGAAGGTGCTAACACAAACACAGCAGTATTAACTACGATGAAAACTGCTGCGGATGCACTTAAATCAGCTCATCAAAATATGAATGTCGATGATGTTCACAATATGATGGATGATATAGCAGA GTCACAAGATTTATCAAAAGAAATATCAGAAGCTATTTCAAATCCAGTTGCATTTGGAACTGATGTAGATGAGGATGAATTACAAAAAGAATTGGAAGAGCTAGAACAAGAAGAATTGGACAAAGAATTGTTGAATACAGGCAAGACACCTGTTCACGATTTGCCAACACCTGCTGTGCCAACATTTGAGCCCAGTGGCCGAGGAAAAGCAAAAACCAA (SEQ IDNO: 3).

[0013] The present invention also provides a method for controlling yellow apple aphids, which uses the above-mentioned dsRNA to control yellow apple aphids.

[0014] In another aspect, the present invention provides a biological product for preventing and controlling yellow apple aphids. The dsRNA is used in combination with matrine, and the mortality rate within 72 hours is 90%, thereby achieving biological control of yellow apple aphids.

[0015] Furthermore, the biological product is a spray insecticide.

[0016] The present invention takes apple yellow aphid as the research object, screens target genes with high lethality based on RNAi technology, thereby obtaining dsRNA with strong specificity, green safety, genetic stability and difficulty in drug resistance, and provides a new biological control technology for the comprehensive management of apple yellow aphid. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : A graph showing the aphid production of apple yellow aphid after dropletization of dsGFP and dsCmb4b;

[0018] Figure 2 : Figure of the mortality of apple yellow aphid after dropletization of dsGFP and dsCmb4b;

[0019] Figure 3 : A graph showing the aphid production of apple yellow aphid after dropletization of dsGFP and dsCmb4b;

[0020] Figure 4 : Figure of the mortality of apple yellow aphid after dropletization of dsGFP and dsCmb4b;

[0021] Figure 5 : A graph showing the aphid production of apple yellow aphid after spraying with dsGFP and dsCmb4b;

[0022] Figure 6 : Graph of the mortality of apple yellow aphid after spraying with dsGFP and dsCmb4b. DETAILED DESCRIPTION

[0023] The invention detects whether RNAi interference will affect physiological indicators such as aphid yield and mortality of apple yellow aphid, and combines transcriptome analysis of apple yellow aphid to determine that the dsCmb4b gene has the best effect after interference from energy metabolism genes (V-type proton ATPase subunit d, ATPD), protein transport-related genes (Coatomer subunit beta), neurotransmitter genes (Synapsin), snf7 homologous genes (Charged multivesicular body protein 4b, Cmb4b) and the like. Compared with a control group (dsGFP), the aphid yield in 72 hours is reduced by 35%, and the mortality rate is as high as 78%. Moreover, the aphid yield and mortality rate after RNAi interference gradually increase with time.

[0024] The present invention is described in detail below in conjunction with specific embodiments and drawings.

[0025] Example 1: Amplification of the dsCmb4b gene of the yellow apple aphid

[0026] Based on the analysis of the transcriptome of apple aphid, the primers were designed using Primer 5 software after the gene sequence was determined (Table 1). The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. 20 adult apple aphids were taken, and the total RNA was extracted using the Novozyme kit, and then cDNA was obtained using the TaKaRa reverse transcription kit with 1 μg of total RNA as a template. Then the apple aphid Cmb4b gene fragment was amplified using cDNA as a template. The PCR reaction system was 25 μL: 2×Taq Plus Master MixII (Dye Plus) 12.5 μL, cDNA 1.0 μL, upstream primer 0.5 μL, downstream primer 0.5 μL, DEPC-treated H2O 10.5 μL. The PCR reaction conditions were: 94℃4min: 94℃30s, 55℃30s, 72℃1:20min, 35 cycles: 72℃10min, and finally stored at 4℃. The PCR product was detected by 1% agarose gel electrophoresis and then recovered using the Novozyme gel recovery kit. The target band was connected to the PMD19-T vector, and the connection product was finally transformed into the E. coli strain. The strain with the correct sequencing was stored in glycerol. The plasmid was extracted from the correct strain using a kit, and after measuring the concentration, it was stored at -20°C.

[0027] The results determined that the full-length sequence of the apple yellow aphid gene Cmb4b was 672 bp, the specific nucleotide sequence was shown in SEQ ID NO: 1, and the amino acid sequence of the protein encoded by it was shown in SEQ ID NO: 2.

[0028] Table 1: Gene cloning specific primer sequence list

[0029]

[0030] Example 2: Synthesis of dsRNA of dsCmb4b gene

[0031] 1. Synthesis of dsCmb4b gene

[0032] Primers for synthesizing dsRNA of the dsCmb4b gene and the control GFP gene were designed (see Table 2).

[0033] dsRNA specific primers were designed using the E-RNAi (E-RNAi Webservice (dkfz.de)) website, and T7 promoter sequences were added to the 5' ends of the upstream primer and the downstream primer. A total of three specific primers were designed and screened using the following experimental method. The 72h mortality rate of dsCmb4b (F1, R1) 442bp was 78%; the 72h mortality rate of dsCmb4b (F2, R2) 378bp was 28%; the 72h mortality rate of dsCmb4b (F3, R3) 358bp was 30%; therefore, dsCmb4b (F1, R1) 442bp had the best effect, and the primer used in the following experiments was dsCmb4b (F1, R1) 442bp.

[0034] Table 2: dsRNA specific primer information

[0035]

[0036] Refer to the instructions of TranscriptAid T7 High Yield transcription Kit to synthesize dsRNA for RNAi experiment. The specific steps are as follows:

[0037] 1. RNA extraction

[0038] (1) Extraction of total RNA from apple aphid

[0039] Total RNA was extracted using the instructions of the Novozyme Total RNA Extraction Kit. The specific steps for RNA extraction are as follows:

[0040] ① Homogenization: Take 10-20 mg of fresh tissue and add 500 μL Buffer RL1 (with β-mercaptoethanol / DTT added), and grind the tissue thoroughly and evenly using an electric homogenizer.

[0041] ② Transfer the treated homogenate to the gDNA-Filter Columns (the gDNA-Filter Columns have been placed in the collection tube), centrifuge at 12,000rpm (13,400×g) for 2 minutes. Discard the gDNA-Filter Columns and keep the supernatant in the collection tube.

[0042] ③ Add 1.6 times the volume of Buffer RL2 (with anhydrous ethanol added) to the supernatant in the collection tube and mix gently.

[0043] ④ Transfer the mixed solution from step 2 to RNAPure Columns (RNAPure Columns have been placed in the collection tube), centrifuge at 12,000 rpm (13,400 x g) for 1 min, and discard the waste liquid.

[0044] ⑤ After discarding the waste liquid, put the RNAPure Columns adsorption column back into the collection tube, add all the remaining liquid into the adsorption column, centrifuge at 12,000rpm (13,400xg) for 1min, and discard the waste liquid.

[0045] ⑥ Add 500 μl Buffer RW1 to RNAPure Columns, centrifuge at 12,000 rpm (13,400 x g) for 1 min, and discard the waste liquid.

[0046] ⑦ Add 700 μl Buffer RW2 (with anhydrous ethanol) to RNAPure Columns, centrifuge at 12,000 rpm (13,400 x g) for 1 min, and discard the waste liquid.

[0047] ⑧Repeat step 6.

[0048] ⑨ Place the RNAPure Columns adsorption column back into the collection tube and centrifuge at 12,000rpm (13,400xg) for 2 minutes to completely remove the ethanol residue in the RNAPure Columns.

[0049] ⑩ Transfer the adsorption column to a new RNase-free Collection Tubes 1.5ml centrifuge tube, and drop 50-200μl of RNase-free ddHzO into the center of the adsorption column. Leave it at room temperature for 2

[0050] min, and centrifuge at 12,000 rpm (13,400 x g) for 1 min to elute RNA.

[0051] The elution volume of RNase-free ddH2O should not be less than 50μl. A volume that is too small will affect the elution efficiency. To increase the total RNA yield, RNase-free ddH2O can be preheated to 65℃ in advance, and the RNA elution solution can be added back to the RNAPureColumns purification column, placed at room temperature for 2 minutes, centrifuged at 12,000rpm (13,400xg) for 1 minute, and the RNAPureColumns are discarded.

[0052] The extracted Total RNA can be used directly in downstream experiments or stored at -80℃.

[0053] 2. Synthesis of cDNA

[0054] The cDNA was synthesized by reverse synthesis using a kit from TaKaRa. The genomic DNA digestion reaction was performed on ice in accordance with the reaction system established in Table 3 below.

[0055] Table 3: Genomic DNA digestion reaction system

[0056]

[0057] Reaction conditions: 42°C, 2 min

[0058] The reverse transcription reaction was set up according to Table 4 below and performed on ice.

[0059] Table 4: Reverse transcription reaction system

[0060]

[0061]

[0062] The reaction conditions were: 37°C, 15 min, 85°C, 5 s. The reaction solution was stored at -20°C for later use.

[0063] 3. Synthesis of dsRNA

[0064] The dsRNA-specific primers were designed using the E-RNAi (E-RNAi Webservice (dkfz.de)) website, and the T7 promoter sequence was added to the 5' end of the upstream primer and the downstream primer, as shown in Table 1. The primers were synthesized by Beijing Qingke Biotechnology Co., Ltd. The plasmid synthesized in the above steps was used as a DNA template and the designed primers for PCR amplification. The amplification system of the Cmb4b gene was 2×Taq Plus Master Mix II (Dye Plus 12.5μL, 0.5μL each of the upstream and downstream primers, 1μL of template, and 10.5μL of ddH2O. The PCR reaction procedure was: 94℃ pre-denaturation for 3min, 94℃ denaturation for 30s, 55℃ annealing for 30s, 35 cycles, and 72℃ extension for 1min. The amplification system of the GFP gene was 2×Taq Plus Master Mix II (Dye Plus 12.5μL, 0.5μL each of the upstream and downstream primers, 1μL of template, and ddH2O 10.5 μL. The PCR reaction program was: 94°C pre-denaturation for 3 min, 94°C denaturation for 30 s, 55°C annealing for 30 s, 5 cycles, 94°C denaturation for 30 s, 65°C denaturation for 30 s, 72°C extension for 30 s, 35 cycles, then 72°C for 10 min, and finally stored at 10°C. The PCR product was detected using 1% agarose gel and then purified by gel recovery. The dsRNA was synthesized using the TranscriptAidT7 High Yield transcription Kit. The mixed system is shown in Table 5.

[0065] Table 5: dsRNA synthesis system

[0066]

[0067] After the above reaction is completed, add 2μL DNaseⅠ to the mixture and incubate at 37℃ for 15min, then add 2μL EDTA and incubate at 65℃ for 10min; transfer to a 1.5mL centrifuge tube, add 115μL DEPC-treated H2O and 3M sodium Acetute Solution and mix well; add 150μL 1:1 water-saturated phenol / chloroform (75 μL each), then add 300 μL chloroform, shake to mix, and centrifuge at 12000g, 4°C for 10 min; transfer the supernatant to a new 1.5 mL centrifuge tube, add 350 μL anhydrous ethanol, mix, and place at -20°C for 2 h; centrifuge the 1.5 mL centrifuge tube at 12000g, 4°C for 10 min, pour out the supernatant, and keep the precipitate; add 500 μL of ice-cold 75% ethanol to the precipitate, centrifuge at 7500g, 4°C for 5 min, pour out the supernatant, absorb as much as possible, and dry the precipitate for 10 min; add 50 μL RNase-Free H2O, measure the concentration, and run electrophoresis detection (Table 6).

[0068] Table 6: Purity test results of dsRNA

[0069] name Concentration (μg / ml) A260 / A280 dsCmb4b 1000 2.12 dsGFP 1000 2.10

[0070] Example 3: Determination of RNAi effect of dsCmb4b gene using the spotting method

[0071] In this example, 0.4 μL of dsRNA complex was dropped onto the abdomen of the apple yellow aphid by a Hamilton PB600-1 repeating dispenser to measure the phenotypic results of dsGFP and dsCmb4b (i.e., dsRNA). The specific experimental process is as follows:

[0072] The nanocarrier was mixed with dsRNA (dsCmb4b and dsGFP) at the recommended mass ratio of 1:1 (the final concentration of the nanocarrier and dsRNA was 500ng / μL), and then 0.6% volume of surfactant was added (the nanocarrier and surfactant were provided by Professor Shen Jie of China Agricultural University). After standing for 30 minutes, 0.4μL of dsRNA complex was dropped on the abdomen of aphids using a Hamilton PB600-1 repeat dispenser. The experiment set up 5 biological replicates, 20 aphids in each biological replicate, and a total of 100 aphids. Aphids were placed in a culture dish with 1% agar medium and fresh apple leaves for feeding, and cultured at a temperature of 25℃ and a relative humidity of 30%. The aphid production and mortality were observed daily for a total of 72 hours.

[0073] The results showed that (such as Figure 1 and Figure 2As shown in the figure, compared with the control, the aphid production of dsCmb4b was most significantly reduced by 35% at 72h, and the mortality rate reached 78% at 72h; the mortality rate of the control dsGFP was 20% at 72h.

[0074] Example 4: Using the drip method to determine the effect of the combined use of dsCmb4b gene and matrine

[0075] The nanocarrier was mixed with dsRNA (dsCmb4b and dsGFP) at the recommended mass ratio of 1:1 (the final concentration of the nanocarrier and dsRNA was 500ng / μL), and then 0.6% volume of surfactant was added. After standing for 15 minutes, 1000 times of matrine was added. After standing for 15 minutes, 0.4μL of dsRNA complex was dropped on the abdomen of aphids using a Hamilton PB600-1 repeat dispenser. The experiment was repeated for 5 times, with 20 nymphs in each biological repeat, for a total of 100 aphids. Aphids were placed in a culture dish with agar medium and fresh apple leaves were placed for feeding, and cultured at a temperature of 25°C and a relative humidity of 30%. The aphid production and mortality were observed daily for a total of 72 hours.

[0076] The results showed that (such as Figure 3 and Figure 4 As shown in the figure, compared with the control, the aphid production of dsCmb4b was most significantly reduced by 40% at 72h, and the mortality rate reached 90% at 72h; the mortality rate of the control dsGFP was 20% at 72h.

[0077] Example 5: Determination of the effect of combined use of dsCmb4b gene and matrine using the spray method

[0078] In this embodiment, the drip method in Example 4 is replaced by a spray method. The nanocarrier and dsRNA (dsCmb4b and dsGFP) are mixed at the recommended mass ratio of 1:1 (the final concentration of the nanocarrier and dsRNA is 500ng / μL), and then a 0.6% volume of surfactant is added. After standing for 15 minutes, 2% of 1000 times matrine is added, and the dsRNA complex is sprayed on apple leaves and aphids using a small spray pot. The experiment has 5 biological replicates, 20 nymphs in each biological replicate, and a total of 100 aphids. Aphids are placed in a culture dish with agar culture medium and fed with fresh apple leaves, and cultured at a temperature of 25°C and a relative humidity of 30%. The aphid production and mortality rate are observed daily for a total of 72 hours.

[0079] The results showed that (such as Figure 5 and Figure 6 As shown in the figure, compared with the control, the aphid production of dsCmb4b was most significantly reduced by 40% at 72h, and the mortality rate reached 90% at 72h; the mortality rate of the control dsGFP was 20% at 72h.

[0080] It can be seen from Examples 3, 4 and 5 that both implementation methods can achieve a mortality rate of dsCmb4b gene against apple yellow aphids, with the mortality rate reaching up to 90%, and have extremely high application prospects.

Claims

1. A dsRNA, characterized in that The dsRNA can reduce the expression level of the apple yellow aphid gene, the amino acid sequence of the protein encoded by the apple yellow aphid gene is SEQ ID NO: 2, and the sequence of the dsRNA is SEQ ID NO:

3.

2. Use of the dsRNA according to claim 1 in controlling apple yellow aphid.

3. Use of the dsRNA according to claim 1 in the preparation of biological products for controlling apple yellow aphid.

4. A biological product for preventing and controlling apple yellow aphids, characterized in that: The biological product contains the dsRNA according to claim 1.

5. The biological product according to claim 4, characterized in that Matrine is also added into the biological product.

6. The biological product according to claim 4, characterized in that The biological product is a sprayable preparation.

Images