Secretory protein RpSP4043 from the bee-margin stink bug and its application in the prevention and control of soybean "green syndrome"

Abstract

The present invention relates to the dsRNA synthesis of a secretory protein gene RpSP4043 of a sphenotype stink bug and application of the gene in the prevention and control of soybean "greening syndrome" disease.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, and particularly to the synthesis of dsRNA of a secretory protein RpSP4043 of a schizont, the introduction of the dsRNA into the schizont, and the application of the gene in preventing and controlling soybean diseases related to "greening syndrome." Background Art

[0002] In recent years, soybean "greening syndrome" has exploded across my country's soybean-producing regions, becoming particularly severe in the Huanghuai and Haihe regions. This syndrome manifests as the plant's stems, leaves, and pods remaining green and refusing to age at maturity, resulting in pods that are not full or underdeveloped kernels that are shriveled or deformed.

[0003] The pedestris bug (Riptortus pedestris) is a common piercing-sucking pest in soybean fields, primarily obtaining nutrients by continuously sucking plant and seed sap with its piercing-sucking mouthparts. Continuous feeding by the bug can lead to stunted growth or even death of soybean plants, and affected soybean seeds often fail to develop normally, ultimately resulting in reduced soybean yields. Currently, the control of the bug relies primarily on conventional pesticides, including 1% emamectin benzoate emulsifiable concentrate (emamectin benzoate), 2.5% lambda-cyhalothrin emulsifiable concentrate, and 50% sulfoxaflor water-dispersible granules. However, the widespread use of traditional pesticides not only creates serious environmental problems but also leads to the development of pesticide resistance in the bug, making effective control more difficult and detrimental to the sustainable development of agriculture. Therefore, developing new technologies to control the bug is a promising approach to address the current overuse of chemical pesticides and a necessary solution to the soybean "greening" disease.

[0004] While biological control methods are effective and environmentally friendly, they are slow to take effect, have poor results, and are significantly affected by the surrounding environment. Transgenic plants can effectively control pests, but over time they can induce increased pest resistance, exacerbate the damage caused by other non-target pests, and their safety remains controversial. Since its discovery in plants in 1990, the RNA interference (RNAi) phenomenon has rapidly developed, with the first report of successful silencing of target gene expression in cultured mammalian cells in 2001. RNAi technology involves the introduction of small double-stranded RNA molecules to specifically degrade or inhibit target gene mRNA expression, thereby inhibiting or shutting down specific genes. RNAi offers advantages such as high efficiency, high specificity, and ease of use, making it an ideal system for species-specific pest control. RNAi technology is widely used in the insect field, and reports of its use in pest control are common. Case studies of controlling pests such as the corn rootworm, cotton bollworm, and beet armyworm have demonstrated the feasibility of RNAi technology as a new pest control method. In addition, in recent years, researchers have used RNA interference technology to conduct relevant functional studies using Riptortus pedestris as experimental materials. For example, TOMOKO IKENO's team studied the role of the circadian clock gene Clock in the circadian rhythm of Riptortus pedestris and the photoperiodic regulation of reproductive diapause (TOMOKO IKENO et al., 2013), and Jang's team found that Duox expression is tracheal-specific, and downregulating this gene through RNAi interference led to the collapse of its respiratory system (Seonghan Jang et al., 2021); however, these studies are more mechanistic studies and are not very applicable. At present, there is still a lack of more effective and environmentally friendly pest control technologies for Riptortus pedestris; the many problems with existing control technologies have forced people to seek better methods for the control of Riptortus pedestris.

[0005] At present, although relevant studies have reported that the bee stink bug is closely related to the occurrence of soybean "greening", the key pathogenic factors are still unclear, and there have been no reports at home and abroad on the use of RNAi technology to control the bee stink bug, thereby reducing the occurrence of soybean "greening". Based on the above defects in the existing technology, the inventors of this patent established an RNA interference system for the bee stink bug and a mutant of the RpSP4043 gene of the bee stink bug by targeting the secretory protein RpSP4043 of the bee stink bug, thereby reducing the pathogenicity of the bee stink bug to soybeans and alleviating the occurrence of soybean "greening". Based on the experimental model of soybean "greening" established indoors, the results showed that silencing the RpSP4043 gene can significantly reduce the pathogenicity of the bee stink bug on soybean plants, thereby achieving the purpose of preventing and controlling soybean "greening".

[0006] Compared with existing technologies, the present invention has the following beneficial effects: The dsRNA of the RpSP4043 gene obtained by the present invention, when introduced into the schizont bug to silence the RpSP4043 gene, significantly reduces the pathogenicity of the schizont bug to soybeans, alleviating the symptoms of soybean "greening syndrome," as specifically manifested by the restoration of the pod shrivelling rate and growth period to levels not significantly different from those of control plants. The present invention clarifies the important role of the RpSP4043 gene in soybean damage by the schizont bug, providing a theoretical basis and practical strategy for the prevention and control of soybean "greening syndrome." Summary of the Invention

[0007] The present invention clones a partial sequence of the secretory protein RpSP4043 from the schizont, obtains dsRNA of the RpSP4043 gene through in vitro synthesis, and introduces it into the schizont. Silencing the RpSP4043 gene significantly reduces the survival rate of the schizont on soybean plants, thereby achieving the purpose of controlling the schizont.

[0008] In one aspect, the present invention relates to a gene for a secretory protein RpSP4043 of a bee-margin stink bug, the nucleotide sequence of which is as shown in SEQ ID NO.1 or nucleotides having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO.1 or consisting of the nucleotide sequence thereof;

[0009] In another aspect, the present invention relates to a gene for a secretory protein RpSP4043 of a bee-margin stink bug, the amino acid sequence of which is the amino acids shown in SEQ ID NO. 8 or amino acids having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity with the amino acids shown in SEQ ID NO. 8, or consisting thereof;

[0010] In some embodiments, the RpSP4043 nucleotide sequence is as follows (SEQ ID NO. 1):

[0011] ATGAAATCTATACTCTGTCTAGTACTTGTCATTTATGCAATAGACGCCATTTTCGGTGTCTCAGTCGTCATCAACAACCATAAGGTCTACGTGGACGAAAAGCCCGTCGACCTGTCCAAGCTCGAAGTGGTGGAAGACACCGAGTTCGCTACCCGCTACCTCCTCCATTCGGGTGACACCAACATCACCATCACCAAGAGCAAGTT CGGCGATGCCATATCCGTCTCCTCCTCCTCTCACATGACCCCCGAGGAACTCGCTAAGGCTAAGGAGTTCAGTAAGAAGCTCTCCGAAAAGATTGAACAAAACGTCAAGAACATCCAGAAGCAAGTCGAAGACCAGATAAAGAACATTCAAAAGAGTATCCAGGAAAATTTGAAAAAATCATTCAGTTTTTTCCCATTTTTCTAA;

[0012] The amino acid sequence of RpSP4043 is as follows (SEQ ID NO.8):

[0013] MKSILCLVLVIYAIDAIFGVSVVINNHKVYVDEKPVDLSKLEVVEDTEFATRYLLHSGDTNITITKSKFGDAISVSSSSHMTPEELAKAKEFSKKLSEKIEQNVKNIQKQVEDQIKNIQKSIQENLKKSFSFFPFF

[0014] On the other hand, the present invention also relates to a method for cloning the RpSP4043 gene, comprising the following steps:

[0015] (1) The salivary glands of adult bee-edge bugs were collected, total RNA was extracted using the Trizol method, and the first-strand cDNA was obtained by reverse transcription;

[0016] (2) using the cDNA of the bee-edge stink bug obtained in step (1) as a template, and performing PCR amplification using the upstream and downstream primers shown in the sequences SEQ ID NO.2 and SEQ ID NO.3;

[0017] (3) The gene fragment obtained by the above amplification was connected to the cloning vector pBinGFP, transformed into Escherichia coli DH5α, and cultured on LB solid medium containing kanamycin to obtain a monoclonal colony containing the gene fragment.

[0018] (4) A single clone was selected and expanded in LB liquid medium containing kanamycin, and the plasmid was extracted and DNA sequenced to obtain Bin-RpSP4043 containing the target sequence shown in SEQ ID NO. 1;

[0019] (5) Using the Bin-RpSP4043 plasmid as a template, PCR amplification was performed using the upstream primer shown in SEQ ID NO.4 and the downstream primer shown in SEQ ID NO.5 to obtain a large number of RpSP4043 gene fragments containing the T7 promoter.

[0020] In another aspect, the present invention relates to a method for synthesizing dsRNA of the RpSP4043 gene, comprising the following steps:

[0021] (1) Synthesize dsRNA using the gene fragment obtained by PCR amplification as a template. The reaction system is as follows: 10× Reaction Buffer 2μl, ATP solution 2μl, UTP solution 2μl, CTP solution 2μl, GTP solution 2μl, T7 RNA Polymerase 2μl, DNA template 1μg, RNase-free H2O to 20μl, mix well, and react at 37℃ for 8h.

[0022] (2) Add 1 μl of DNase to the system and react at 37°C for 15 min;

[0023] (3) Denature the reaction sample at 65°C for 5 min, determine the dsRNA concentration, and determine the RNA quality by 1% agarose gel electrophoresis;

[0024] (4) Store the remaining dsRNA at -80°C for future use.

[0025] On the other hand, the present invention also relates to a method for introducing ds RpSP4043 into the bee-margin stink bug to alleviate soybean "greening syndrome":

[0026] (1) Prepare an injection solution using RNase-free H₂O, diluting the dsRNA synthesized in step (1) of the RpSP4043 gene dsRNA synthesis method to a final concentration of 4000 ng / ml. Select adult insects of the genus R. punctatus (male and female insects are not distinguished) 3-10 days after eclosion, inject dsRNA into them using a microinjector, and place them in an insect cage for 24 hours for subsequent inoculation experiments.

[0027] (2) The early-maturing soybean variety "Mengdou 16" from northern China was selected as the soybean material. The soybean plants at the beginning of pod formation were placed in insect-proof nets of uniform specifications (35 cm × 35 cm × 115 cm). The soybeans at the beginning of pod formation were inoculated with RpSP4043 and GFP dsRNA-transfected stink bugs, respectively, at a density of 5 per plant, and the insects were fed for 14 days. The number of adult insects in the net was counted and the eggs were removed every day. After feeding for 14 days, the insects were removed. When the plants in the blank control group entered the peak grain-filling stage, the agronomic traits of the plants in each treatment group were counted, including the number of pods per plant and the shrunken pod rate. When the soybean plants entered the maturity stage, the number of days of the growth period of the plants in each treatment group was counted.

[0028] (3) The salivary glands of the bee-margined stink bugs were taken out of the insect cage, and total RNA was extracted and reverse transcribed to obtain cDNA. The expression level of RpSP4043 in the samples was detected by fluorescent quantitative PCR using primers shown in SEQ ID NO.6 and SEQ ID NO.7. -ΔΔCt The relative expression level of the RpSP4043 gene in the schizont was calculated by the method, the differences among different groups were compared, and the silencing efficiency of the RpSP4043 gene was evaluated.

[0029] On the other hand, the present invention also relates to a dsRNA that specifically inhibits the expression of the RpSP4043 gene, which is synthesized by the aforementioned method;

[0030] In some embodiments, the present invention also relates to an in vitro cell containing the dsRNA;

[0031] In some embodiments, the present invention also relates to a pharmaceutical composition comprising the dsRNA and a pharmaceutically acceptable carrier;

[0032] In some embodiments, the carrier is a nanomaterial carrier.

[0033] In some embodiments, the present invention also relates to the use of the dsRNA and related pharmaceutical compositions in the preparation of pesticides;

[0034] On the other hand, the present invention also relates to a method for controlling the bee stink bug, comprising spraying the dsRNA or a pharmaceutical composition comprising the dsRNA on plants;

[0035] In some embodiments, the present invention also provides a method for alleviating or preventing soybean "greening disease", which comprises spraying the dsRNA or pharmaceutical composition on soybean plants.

[0036] The present invention has achieved at least the following beneficial effects:

[0037] The present invention obtains dsRNA encoding the RpSP4043 gene, which is then introduced into the beetle to silence the RpSP4043 gene. This significantly reduces the beetle's pathogenicity to soybeans, alleviating the "greening" symptoms of soybeans, as evidenced by the return of pod shrivelling rate and growth period to levels not significantly different from those of control plants. This invention clarifies the important role of the RpSP4043 gene in soybean damage by the beetle, providing a theoretical basis and practical strategy for the prevention and control of soybean "greening." BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 : Experimental effect diagram of dsRNA-treated bee-margin stink bug after feeding.

[0039] A: The “green syndrome” phenotype of the dsRNA-treated bee-margin stink bug after feeding.

[0040] B: Detail of the pod after feeding by the dsRNA-treated bee-margin stink bug.

[0041] C: Statistical graph of the soybean pod shrinkage rate after feeding by dsRNA-treated bee-margin stink bugs.

[0042] D: Statistical diagram of soybean ripening cycle after feeding by dsRNA-treated bee-margin stink bugs.

[0043] Figure 2 : Relative expression level of RpSP4043 gene after dsRNA treatment. Example

[0044] Example 1 Cloning of the RpSP4043 gene fragment of the bee stink bug

[0045] 1.1 Extraction of total RNA from the salivary glands of the bee-margined stink bug

[0046] (1) Obtain salivary glands of the bee-margin bug by microscopic dissection and transfer to a pre-chilled RNase-free centrifuge tube. Immediately add 1 ml of Trizol reagent to a homogenizer, thoroughly crush the tissue, and place on ice.

[0047] (2) Add 200 μL of chloroform using an RNase-free pipette tip, shake vigorously for 15 seconds, and let stand on ice for 8-10 minutes;

[0048] (3) 4°C, 12000 rpm, 20 min, the mixture was separated into layers, and the upper aqueous phase was transferred to a new 1.5 ml centrifuge tube;

[0049] (4) Add an equal amount of isopropanol, mix thoroughly by inversion, and precipitate the RNA at -20°C for 2 h;

[0050] (5) Incubate at 4°C, 12,000 rpm for 20 min. The precipitate in the tube is RNA. Discard the supernatant and add 500 μL of 75% anhydrous ethanol to suspend and wash the precipitate. Incubate at 4°C, 12,000 rpm for 5 min. Discard the supernatant. Repeat twice.

[0051] (6) Place on ice for 5 min to evaporate the ethanol;

[0052] (7) Add 30 μL RNase-free H2O and dissolve the precipitate at 4°C for 2 h.

[0053] (8) Store the RNA in a -80°C freezer.

[0054] 1.2 RpSP4043 gene cloning

[0055] (1)Use Total RNA extracted in the previous step was reverse transcribed to cDNA using the III qRT Super Mix (vazyme, China) kit: 1500 ng of total RNA was added to 2 μl of 4× gDNA wiper mix, and the volume was made up to 8 μl with RNase-free HO. Mix by pipetting and incubate at 42°C for 3 minutes. Then, 2 μl of 5X HiScript II qRT Super Mix II was added. Mix by pipetting and incubate at 50°C for 15 minutes and 85°C for 5 seconds. The product was stored at -20°C until needed.

[0056] (2) Using the obtained cDNA as a template, PCR amplification was performed using the primers shown in SEQ ID NO. 2 and SEQ ID NO. 3.

[0057] Upstream primer: ACGAGCTGTACAAGGGTACCATGAAATCTATACTCTGTCT (SEQ ID NO. 2);

[0058] Downstream primer: GCGGACTCTAGTTCATCTAGATTAGAAAAATGGGAAAAAAC (SEQ ID NO. 3);

[0059] The PCR reaction system is as follows: 50 μL: cDNA 1 μL, 1.5 μL each of 10 μmol / L upstream and downstream primers, 15 μL ddH2O, 1 μL TaqDNA Polymerase, 25 μL 2x PCR Buffer, and 5 μL dNTP Mixture.

[0060] The PCR reaction program was as follows: 95°C, 3 min; 95°C, 30 s, 58°C, 30 s, 72°C, 30 s, 35 cycles; 72°C, 10 min;

[0061] (3) The gene fragment obtained by amplification was ligated into the cloning vector pBinGFP, transformed into Escherichia coli DH5α, and cultured on LB solid medium containing kanamycin to obtain a monoclonal colony containing the gene fragment;

[0062] (4) A single clone was selected and expanded in LB liquid medium containing kanamycin, and the plasmid was extracted and DNA sequenced to obtain Bin-RpSP4043 containing the sequence shown in SEQ ID NO. 1;

[0063] (5) Using the Bin-RpSP4043 plasmid as a template, PCR amplification was performed using the upstream primer shown in SEQ ID NO. 4 and the downstream primer shown in SEQ ID NO. 5 to obtain a large number of RpSP4043 gene fragments containing the T7 promoter;

[0064] Upstream primer: TAATACGACTCACTATAGGGATGAAATCTATACTCTGTCT (SEQ ID NO. 4)

[0065] Downstream primer: TAATACGACTCACTATAGGGGGAAAAAACTGAATGATTTT (SEQ ID NO. 5).

[0066] Example 2: dsRNA synthesis of RpSP4043 gene

[0067] (1) Synthesize dsRNA using the gene fragment obtained by PCR amplification as a template. The reaction system is as follows: 10× Reaction Buffer 2μl, ATP solution 2μl, UTP solution 2μl, CTP solution 2μl, GTP solution 2μl, T7 RNA Polymerase 2μl, DNA template 1μg, RNase-free H2O to 20μl, mix well, and react at 37℃ for 8h.

[0068] (2) Add 1 μl of DNase to the system to eliminate DNA in the system and react at 37°C for 15 min;

[0069] (3) Denature a portion of the reaction sample at 65°C for 5 min, determine the dsRNA concentration, and determine the RNA quality by 1% agarose gel electrophoresis;

[0070] (4) Store the remaining ds RpSP4043 RNA at -80°C for future use.

[0071] Example 3: Effect of dsRNA targeting RpSP4043 gene on the expression of RpSP4043 in the bee-margin stink bug

[0072] 3.1 Microinjection of dsRNA

[0073] (1) Using RNase-free H2O to prepare an injection solution, dilute the dsRNA synthesized in Example 2 to a final concentration of 4000 ng / ml;

[0074] (2) Select adult bee-margin stink bugs (male and female) 3-10 days after emergence and inject them at the junction of the hind legs and thorax using a microinjector. Each insect is injected with 1 μL of injection solution and placed in an insect cage for 24 hours for the inoculation experiment.

[0075] 3.2 Insect inoculation experiment

[0076] (1) In this experiment, the early-maturing soybean variety "Mengdou 16" from northern China was selected as the soybean material. Two soybean plants were placed in each pot at the beginning of the pod stage in a 20-mesh insect-proof net measuring 35 cm × 35 cm × 115 cm.

[0077] (2) The injected bee stink bugs were placed in an insect net at a density of 5 per plant, with a total of 10 per pot. The plants were placed in an insect net without bee stink bugs as the negative control group, and the non-injected adults were placed in the same proportion as the experimental group as the positive control. Three replicates were set for each treatment. The number of adults in the net was counted daily. If the number of insects decreased due to death or other reasons, they were supplemented accordingly. After 14 days of feeding, the insects were removed. When the plants in the negative control group entered the peak grain-filling stage, the agronomic traits of the plants in each treatment group were counted, including the number of pods per plant and the pod-shrinking rate. When the soybean plants entered the maturity stage, the number of days of the growth period of the plants in each treatment group was counted.

[0078] The experimental results show that (see Figure 1 AD): Compared with the positive control plants, silencing the expression of the RpSp4043 gene of the bee-margin stink bug reduced its expression and significantly alleviated the soybean "green disease" symptoms caused by it, mainly manifested in a significant decrease in the number of pods per plant, but a significant decrease in the rate of bulging and shrunken pods (CK: 18.3%; dsGFP: 58.3%; RpSp4043: 28.3%), and a significant reduction in the number of days in the reproductive period (CK: 63d; dsGFP: 76d; RpSp4043: 67d), which showed no significant differences compared with the negative control plants.

[0079] Note: CK refers to soybean plants that have not been treated and eaten by the bee-edge bugs.

[0080] Example 4: RpSP4043 gene silencing efficiency evaluation experiment

[0081] The specific steps are as follows:

[0082] Total RNA was extracted from the salivary glands of the insect caged beetles, and cDNA was obtained by reverse transcription. The expression level of the RpSP4043 gene was detected by quantitative real-time PCR (qRT-PCR) using the primers shown in (SEQ ID NO. 6) and (SEQ ID NO. 7). The relative expression level of the RpSP4043 gene in the beetle was calculated using the 2-ΔΔCt method. The differences between different groups were compared to evaluate the efficiency of RpSP4043 gene silencing.

[0083] Upstream primer: CGCTAAGGCTAAGGAGTTCAGTAA (SEQ ID NO. 6)

[0084] Downstream primer: CTTTATCTGGTCTTCGACTTGCTT (SEQ ID NO. 7)

[0085] The qRT-PCR reaction system was as follows: 10 μL: 1.5 μg cDNA, 0.3 μL each of 10 μmol / L upstream and downstream primers, 5 μL SYBR qPCR Master Mix, and ddH2O to 10 μL.

[0086] The qRT-PCR reaction program was as follows: 95°C, 3 min; 95°C, 15 s, 60°C, 15 s, 72°C, 20 s, 40 cycles.

[0087] The experimental results show that (see Figure 2 ):14 days after dsRNA injection, the relative expression level of the RpSP4043 gene in the bee stink bug was 0.00969, indicating a high silencing efficiency.

[0088] The preferred embodiments of the present invention have been described in detail above. It should be understood that a person skilled in the art can make numerous modifications and variations based on the concepts of the present invention without inventive effort. Therefore, any technical solution that can be derived by a person skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A method for alleviating or preventing soybean "greening syndrome" by spraying soybean plants with dsRNA targeting the RpSP4043 gene, wherein the dsRNA is synthesized as follows: (1) The salivary glands of adult bee-edge bugs were collected, total RNA was extracted using the Trizol method, and the first-strand cDNA was obtained by reverse transcription; (2) using the cDNA of the bee-edge stink bug obtained in step (1) as a template, and performing PCR amplification using the upstream and downstream primers shown in SEQ ID NO.2 and SEQ ID NO.3; (3) The gene fragment obtained by amplification was ligated into the cloning vector pBinGFP, transformed into Escherichia coli DH5α, and cultured on LB solid medium containing kanamycin to obtain a monoclonal colony containing the gene fragment; (4) A single clone was selected and expanded in LB liquid medium containing kanamycin, and the plasmid was extracted and DNA sequenced to obtain Bin-RpSP4043 containing the sequence shown in SEQ ID NO. 1; (5) Using the Bin-RpSP4043 plasmid as a template, PCR amplification was performed using the upstream primer shown in SEQ ID NO. 4 and the downstream primer shown in SEQ ID NO. 5 to obtain the RpSP4043 gene fragment containing the T7 promoter; (6) Synthesize dsRNA using the gene fragment obtained by PCR amplification in step (5) as a template. The reaction system is as follows: 10× Reaction Buffer 2 μl, ATP solution 2 μl, UTP solution 2 μl, CTP solution 2 μl, GTP solution 2 μl, T7 RNA Polymerase 2 μl, DNA template 1 μg, RNase-free H2O to 20 μl, mix well, and react at 37°C for 8 h. (7) Add 1 μl of DNase to the system and react at 37°C for 15 min; (8) Denature the sample after the reaction at 65°C for 5 min, measure the dsRNA concentration, and determine the RNA quality by 1% agarose gel electrophoresis; (9) Store the remaining dsRNA at -80°C for future use.

2. Use of a dsRNA targeting the RpSP4043 gene in the preparation of a medicament for alleviating or preventing soybean "greening syndrome", wherein the dsRNA synthesis steps are as follows: (1) The salivary glands of adult bee-edge bugs were collected, total RNA was extracted using the Trizol method, and the first-strand cDNA was obtained by reverse transcription; (2) using the cDNA of the bee-edge stink bug obtained in step (1) as a template, and performing PCR amplification using the upstream and downstream primers shown in SEQ ID NO.2 and SEQ ID NO.3; (3) The gene fragment obtained by amplification was ligated into the cloning vector pBinGFP, transformed into Escherichia coli DH5α, and cultured on LB solid medium containing kanamycin to obtain a monoclonal colony containing the gene fragment; (4) A single clone was selected and expanded in LB liquid medium containing kanamycin, and the plasmid was extracted and DNA sequenced to obtain Bin-RpSP4043 containing the sequence shown in SEQ ID NO. 1; (5) Using the Bin-RpSP4043 plasmid as a template, PCR amplification was performed using the upstream primer shown in SEQ ID NO. 4 and the downstream primer shown in SEQ ID NO. 5 to obtain the RpSP4043 gene fragment containing the T7 promoter; (6) Synthesize dsRNA using the gene fragment obtained by PCR amplification in step (5) as a template. The reaction system is as follows: 10× Reaction Buffer 2 μl, ATP solution 2 μl, UTP solution 2 μl, CTP solution 2 μl, GTP solution 2 μl, T7 RNA Polymerase 2 μl, DNA template 1 μg, RNase-free H2O to 20 μl, mix well, and react at 37°C for 8 h. (7) Add 1 μl of DNase to the system and react at 37°C for 15 min; (8) Denature the sample after the reaction at 65°C for 5 min, measure the dsRNA concentration, and determine the RNA quality by 1% agarose gel electrophoresis; (9) Store the remaining dsRNA at -80°C for future use.

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