DsRNA targeting tetranychus urticae bunching protein gene CS640 and application

Abstract

The invention discloses dsRNA (double-stranded ribonucleic acid) targeting a tetranychus urticae bunchy protein gene CS640 and application of the dsRNA. The dsRNA consists of a nucleotide sequence as shown in SEQ ID No.6 and a reverse complementary sequence of the nucleotide sequence as shown in SEQ ID No.6. Specific dsRNA is synthesized and delivered into tetranychus urticae eggs by a drop method, so that the development process of the tetranychus urticae can be effectively blocked, and a remarkable lethal effect is caused; and the synthesized specific dsRNA is harmless to the natural enemy organism neoseiulus barkeri, and has good safety. Therefore, the gene has important application value as a mite RNAi prevention and control target.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, specifically relating to a dsRNA targeting the two-spotted spider mite bundle protein gene CS640 and its application. Background Technology

[0002] RNA interference (RNAi) refers to the highly efficient and specific degradation of homologous mRNA induced by double-stranded RNA (dsRNA), ultimately leading to the silencing of target genes. Because this process inhibits the protein synthesis directed by the target gene, it is also known as gene silencing. Its high specificity and efficiency make it considered an ideal method for pest control. In developing RNAi control strategies, the selection of target genes is crucial: ideal target genes should have a significant lethal effect on the target organism while possessing high specificity to ensure safety against natural enemies and non-target organisms such as mammals.

[0003] The two-spotted spider mite (Tetranychus urticae) is a global pest mite that primarily feeds on sap by congregating on the undersides of leaves as nymphs and adults, causing imbalances in plant growth. The two-spotted spider mite is small, highly adaptable, reproduces rapidly, is resilient, and has a diverse diet, feeding on a wide range of host species. It is a polyphagous mite that often causes severe yield reductions in legumes, fruit trees, and vegetables. Currently, chemical control is the primary method for managing the two-spotted spider mite, which has led to increasing resistance to chemical pesticides. Therefore, exploring safe and effective control targets and methods is of great significance. Summary of the Invention

[0004] The technical problem to be solved by this invention is: how to provide a highly efficient dsRNA for controlling two-spotted spider mites, thereby solving the problem that two-spotted spider mites are difficult to control due to their resistance to chemical drugs.

[0005] The technical solution of the present invention is: a dsRNA, which is a double-stranded RNA composed of the nucleotide sequence shown in SEQ ID No. 6 and its reverse complementary sequence.

[0006] Biological agents containing the aforementioned dsRNA.

[0007] The application of the aforementioned dsRNA or biological agents in the control of two-spotted spider mites.

[0008] Furthermore, the method of prevention and control involves spraying the leaves of the two-spotted spider mite host with the dsRNA described in claim 1 or the biological agent described in claim 2, thereby causing the two-spotted spider mite eggs to die.

[0009] Compared with the prior art, the present invention has the following beneficial effects:

[0010] 1. The lethality of the dsRNA of the present invention reaches 100%, which is significantly higher than that of existing similar products and methods.

[0011] 2. This invention enables dsRNA to directly contact mite eggs to block their development. Mite eggs are usually placed statically on leaves, and this characteristic facilitates the widespread application of the product. Attached Figure Description

[0012] Figure 1 The lethal effect of silencing the TuCS640 gene on the egg stage of Tetranychus taeniorhynchus.

[0013] Figure 2 Lethal phenotype of the silent two-spotted spider mite CS640 gene.

[0014] Figure 3 Safety assessment of dsTuCS640 against Neoseui babesi.

[0015] Figure 4 Survival rate of Neoseii bark after treatment with dsTuCS640. Detailed Implementation

[0016] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the experimental materials used in the following examples were all purchased from commercial channels.

[0017] 1. Gene sequence of Tetranychus CS640 and primer design used

[0018] The open reading frame sequence of the two-spotted spider mite CS640 gene is shown in SEQ ID No. 1, containing 1944 bases and encoding 648 amino acids. Two pairs of primers were designed based on its sequence to synthesize the full-length CS640 gene and dsRNA, respectively. The full-length primers and dsRNA primers are shown in Table 1.

[0019] Table 1. Information on the dsRNA and qPCR primer sequences of the two-spotted spider mite CS640 gene.

[0020]

[0021] 2. dsRNA synthesis targeting the CS640 gene of the two-spotted spider mite

[0022] 1) Total RNA extraction and cDNA preparation from Tetranychus taeniorhynchus

[0023] Two hundred to three hundred adult two-spotted spider mites, aged 3-5 days, were selected for total RNA extraction. The procedure was strictly performed according to the instructions for the BGMG CS640-1t Total RNA / DNA Extraction Kit provided by BIOGROUND. The total RNA concentration and OD were determined using a nucleic acid concentration analyzer. 260 / 280 Value, concentration greater than 100 ng / μL, OD 260 / 280 Samples with values ​​between 1.9 and 2.1 can be used for further testing.

[0024] Total RNA integrity was assessed using 1.0% agarose gel electrophoresis. Electrophoresis conditions were: 150 V for 25 min, followed by observation and photography using a gel imaging system. A clear band structure (28S band and 18S band) with a 28S:1 band brightness ratio of approximately 2:1 indicated good RNA integrity.

[0025] Total RNA was reverse transcribed into cDNA using Promega's GoScript™ Reverse Transcription System, which served as a template for amplifying the full-length CS640 gene sequence. The procedure was performed according to the kit instructions.

[0026] 2) Synthesize the full-length CS640 gene of the two-spotted spider mite.

[0027] The full-length fragment of the Tetranychus CS640 gene was amplified by PCR using the designed full-length primers. The PCR reaction system and conditions are shown below:

[0028] Table 2 PCR reaction system

[0029]

[0030]

[0031] After the reaction was complete, 1.0% agarose gel electrophoresis was performed under the conditions described above. The electrophoresis products were recovered using the Biomed Multifunction DNA Purification Kit, following the manufacturer's instructions. Using the recovered electrophoresis products as templates, ligation products were synthesized using the Promega pGEM®-T Easy Vector System Ⅰ. Transformation was performed using Biomed DH5α competent cells, following the manufacturer's instructions. After amplifying the correctly sequenced colonies, plasmids were extracted using the Biomed HighPure Rapid Mini plasmid Kit.

[0032] 3) Amplify dsRNA targeting the CS640 gene of the two-spotted spider mite.

[0033] Referring to primers SEQ ID No. 4 and SEQ ID No. 5 for the dsRNA fragment, the dsRNA fragment was amplified by PCR. The PCR reaction system and conditions are shown below:

[0034] Table 3 PCR reaction system

[0035]

[0036]

[0037] After the reaction was completed, agarose gel electrophoresis was performed, and the electrophoretic products were recovered using the Biomed Multifunction DNA Purification Kit. Using the recovered products as templates, dsRNA was synthesized using the Thermoscientific TranscriptAid T7 High Yield Transcription Kit. The final synthesized dsRNA was diluted with enzyme-free water to 2000 ng / μL. The synthesized dsRNA consists of the nucleotide sequence shown in SEQ ID No. 6 and its reverse complementary sequence.

[0038] 3. Interference with the expression of the CS640 gene during the egg stage of the two-spotted spider mite using the leaf disc drip method.

[0039] Fresh cowpea leaves were cut into squares approximately 2×2 cm to create fresh leaf dishes. Ten 3-5 day old female adult mites were selected from each leaf and removed 24 hours after they laid eggs. The number of eggs on each leaf was counted to ensure that there were 15-20 eggs per leaf. Each egg was treated with 0.3 μL (approximately 2000 ng / μL) of dsRNA solution by spot dripping twice a day for 5 consecutive days. A water treatment was used as a control group. The mortality rate and phenotype of the eggs were observed and recorded until all eggs hatched or died.

[0040] 4. Lethal effect of silencing the CS640 gene in the two-spotted spider mite

[0041] Treatment of *Tetranychus spp.* eggs with dsRNA targeting the CS640 gene synthesized in vitro using a drip method resulted in shrunken, cloudy eggs that failed to hatch normally, effectively blocking the egg stage development of *Tetranychus spp.* This demonstrates that silencing the expression of the CS640 gene in *Tetranychus spp.* has a significant lethal effect on its egg stage (see Table 4). Figure 1 .

[0042] Table 4. Lethal effect of dsTucs640 on the egg stage of Tetranychus taeniorhynchus.

[0043]

[0044] 5. Lethal phenotype

[0045] Lethal phenotype of the silenced two-spotted spider mite CS640 gene, as follows Figure 2 As shown, the typical phenotype of the egg is characterized by wrinkled, cloudy, and sunken egg skin. Since this gene is primarily involved in the development of the nervous system during the early stages of biological development, interference with its expression may affect the neural development of the two-spotted spider mite, leading to egg failure to hatch. This indicates that silencing the expression of the two-spotted spider mite's CS640 gene during the egg stage can effectively block the mite's developmental process.

[0046] 6. dsTuCS640 Security Assessment

[0047] Fresh cowpea leaves were cut into squares approximately 2×2 cm to create fresh leaf dishes. Fifteen 3-5 day old female adult mites were collected from each leaf. After laying eggs for 24 hours, the leaves were removed. The number of eggs on each leaf was counted, ensuring 25-30 eggs per leaf. Each egg was treated with 0.3 μL (approximately 2000 ng / μL) of dsRNA solution via spot dripping, twice daily for 5 consecutive days. Ten to fifteen *Neoseiuyes barkei* mites were also collected from each cowpea leaf. The condition of *Neoseiuyes barkei* after feeding on *Tetranychus two-spotted* eggs was observed and recorded.

[0048] Table 5. Safety assessment of dsTuCS640 against predatory mites.

[0049]

[0050] The results are as follows Figure 3 , Figure 4 As shown in Table 5, the eggs of the two-spotted spider mite become concave after being ingested, while the life activities of the neoseii bark mite are normal.

Claims

1. A dsRNA, which is a double-stranded RNA consisting of the nucleotide sequence shown in SEQ ID No. 6 and its inverse complementary sequence.

2. A biological agent containing the dsRNA of claim 1.

3. The application of the dsRNA of claim 1 or the biological agent of claim 2 in the control of two-spotted spider mites.

4. The application according to claim 3, characterized in that, The method of prevention and control is to spray the dsRNA of claim 1 or the biological agent of claim 2 onto the leaves of the two-spotted spider mite host, thereby killing the two-spotted spider mite eggs.

Images