Application of CQD-dsRNA in prevention and control of tomato leaf miner

Abstract

The invention belongs to the field of prevention and control of tomato leaf miners, and discloses application of CQD-dsRNA in prevention and control of tomato leaf miners. The method comprises the following steps: screening catalase possibly existing in the oral secretion of the tomato leaf miner, designing a dsRNA fragment and a primer of a targeted catalase gene, carrying out in-vitro transcription to synthesize corresponding dsRNA, carrying the dsRNA by using a carbon quantum dot CQD, and delivering the dsRNA to the body of a tomato leaf miner larva. It is found that the silence efficiency, the survival rate and the pupation number of the tomato leaf miner catalase gene are remarkably reduced compared with those of pure dsRNA, a target is provided for prevention and control of tomato leaf miner RNAi, and a reference is provided for RNAi prevention and control of tomato leaf miner in the mode that CQD carries catalase gene dsRNA.

Description

TECHNICAL FIELD

[0001] The application belongs to but is not limited to the technical field of prevention and control of tomato leafminer, and particularly relates to application of CQD-dsRNA in prevention and control of tomato leafminer. BACKGROUND

[0002] Tomato leafminer has the characteristics of multiple host crops, wide suitable area, strong reproductive capacity, and heavy damage loss, and seriously damages Solanaceae crops such as tomatoes.

[0003] RNAi (RNA interference) is a highly conserved phenomenon in the evolution process, which is induced by double-stranded RNA (dsRNA) and causes efficient and specific degradation of homologous mRNA. RNAi has characteristics and advantages such as specificity, high efficiency, and environmental safety, which cannot be achieved by traditional prevention and control methods, and has great application potential and value in the field of pest control. RNAi technology relies on interference with specific functional genes to cause adaptability reduction or insect death to achieve the purpose of prevention and control, and shows great potential in the field of agricultural pest control and other fields, has high specificity and safety, and is considered as a new generation of green and safe pest control strategy.

[0004] Carbon quantum dots (CQD) are a kind of zero-dimensional carbon nanomaterials with a size of less than 10 nm and fluorescence characteristics, which are composed of sp2 / sp3 hybrid carbon cores and oxygen / nitrogen-containing functional groups. Carbon quantum dots have excellent optical properties, good water solubility, low toxicity, environmental friendliness, wide raw material sources, low cost, and good biocompatibility, and have been widely used in the fields of biomedicine, environment and energy, biological-based lighting, and optoelectronic devices. The method of using nanomaterials to deliver dsRNA can effectively solve the problems of easy degradation of dsRNA and difficult penetration of pest body wall, improve the efficiency of RNAi, and thus improve the prevention and control effect of RNAi on agricultural pests. The method of using nanomaterials to carry dsRNA improves the efficiency of gene silencing, and lays a foundation for establishing a safe, green, and efficient pest control technology. SUMMARY

[0006] In view of the problems in the prior art, the application provides application of CQD-dsRNA in prevention and control of tomato leafminer.

[0007] The application is implemented in the following manner: application of CQD-dsRNA in prevention and control of tomato leafminer, and the implementation method specifically includes the following steps.

[0008] S1: screening catalase in oral secretion of tomato leafminer, designing dsRNA fragment and primer targeting catalase gene;

[0009] S2: in vitro transcription synthesis of corresponding dsRNA;

[0010] S3: using carbon quantum dots CQD to deliver dsRNA to tomato leafminer larvae in vivo;

[0011] S4: analyzing catalase gene silencing efficiency, survival rate and pupa number of tomato leafminer.

[0012] Further, the S1, tomato leafminer catalase identification and primer design, based on the catalase protein sequence in the oral secretion proteome of the cotton leafworm, using TBLASTN to align the tomato leafminer genome information, obtaining the tomato leafminer catalase KAJ2954359.1, according to the DNA sequence, using Primer-blast to design specific primers, and adding T7 promoter to Beijing Chengke Biological Co., Ltd. to synthesize dsRNA amplification primer.

[0013] Further, the S2, dsRNA synthesis of tomato leafminer catalase, includes:

[0014] (1) using TRIzol method to extract total RNA of tomato leafminer, verifying integrity by 1% agarose gel electrophoresis, using ultramicro spectrophotometer to analyze RNA concentration and purity;

[0015] (2) using PrimeScriptTM RT Reagent Kit with gDNA Eraser kit, reverse transcribing the extracted total RNA into first strand cDNA, measuring cDNA concentration and purity after reverse transcription is completed;

[0016] (3) using the cDNA obtained by reverse transcription as a template, amplifying by PCR, purifying PCR products by agarose gel DNA recovery kit (Tiangen), synthesizing dsRNA by Transcript Aid T7 High Yied Transcription Kit and purifying by phenol chloroform, according to the instruction manual.

[0017] Further, the S3, tomato leafminer soaking method RNAi, mixing nano material CQD with 1000 ng / μL catalase KAJ2954359.1 dsRNA according to 1:2 volume, picking tomato leafminer 3rd instar larvae to immerse, using equal volume of dsGFP solution for the control group, 15 larvae for each treatment group, setting 4 groups of independent biological repeats, after standing for 30 min, using a pipette to remove the liquid, transferring the larvae to tomato to observe.

[0018] Further, the S4 comprises:

[0019] (1) Silencing efficiency analysis, collecting tomato leafworm larvae, extracting tomato leafworm total RNA, reverse transcribing into first strand cDNA, designing specific primers of KAJ2954359.1 by Primer Blast, taking beta-actin as an internal reference, analyzing KAJ2954359.1 expression quantity by qPCR, and calculating JHBP gene relative expression quantity by qBASE software based on 2-△△CT, and the results show that CQD significantly improves the gene silencing efficiency;

[0020] (2) Tomato leafworm phenotype observation, counting the number of dead tomato leafworms and pupation every 24h; after 7 days, observing the damage of tomato leafworms to host plants tomato and taking photos; and the results show that CQD carrying dsKAJ2954359.1 improves the RNAi prevention and control efficiency.

[0021] In combination with the above technical solutions and the technical problems solved, the technical solutions to be protected by the present application have the following advantages and positive effects:

[0022] The present application screens the possible catalase in the oral secretions of tomato leafworms, designs dsRNA fragments and primers targeting catalase genes, transcribes and synthesizes corresponding dsRNA in vitro, delivers the dsRNA to the body of tomato leafworm larvae by using carbon quantum dots CQD, finds that the catalase gene silencing efficiency, survival rate and pupation number of tomato leafworms are significantly reduced compared with pure dsRNA, and the present application provides a target for RNAi prevention and control of tomato leafworms.

[0023] Second,

[0024] The present application first uses carbon quantum dots (CQD) to carry catalase gene dsRNA to form a "CQD-dsRNA" complex, which solves the problems of easy degradation of dsRNA in tomato leafworms and low RNAi efficiency, and experimental verification shows that compared with naked dsKAJ2954359.1, the target gene silencing efficiency of CQD+dsKAJ2954359.1 complex is significantly improved by 42%, the survival rate of tomato leafworms is reduced by 30%, and the pupation number is also significantly reduced. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 is the application implementation method flow chart of CQD-dsRNA in the present application for preventing and controlling tomato leafworms;

[0026] Figure 2is a KAJ2954359.1 silencing efficiency observation result graph provided by the embodiment of the present application;

[0027] Figure 3 is a tomato leaf miner larva mortality observation result graph provided by the embodiment of the present application;

[0028] Figure 4 is a tomato leaf miner larva pupation quantity observation result graph provided by the embodiment of the present application. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical scheme and advantages of the present application more clear, the present application is further described in detail below in combination with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present application and do not limit the present application.

[0030] Catalase undertakes the core mission of scavenging reactive oxygen species and maintaining epithelial homeostasis in the digestive tract of lepidopteran larvae. By comparing the oral secretion proteome of Spodoptera litura and conducting TBLASTN retrieval of the tomato leaf miner genome, researchers captured highly conserved sequences encoding KAJ2954359.1, proving that the enzyme plays a key antioxidant barrier role during the feeding period of the tomato leaf miner; after treatment with existing chemical agents or Bt proteins, this barrier can be partially induced to be up-regulated, weakening the control effect and becoming a molecular bottom reason for the difficult-to-control stubborn disease in the field.

[0031] Based on this target, double-stranded RNA is amplified by a reverse transcription system with high fidelity and connected to a T7 promoter for high-yield in vitro transcription. Unlike traditional naked dsRNA, the research team chose carbon quantum dots with a high concentration of carboxyl groups to construct nucleic acid-carbon dot complexes at the nanoscale through electrostatic self-assembly, which not only shields the rapid degradation of nucleases in the tomato leaf miner body, but also improves the cellular uptake efficiency after oral delivery.

[0032] After entering the cytoplasm, the hydroxyl groups on the surface of the complex induce endosome acidification, triggering a "proton sponge" effect that helps dsRNA quickly escape to the cytosolic matrix and be cleaved by Dicer2 into 21-23nt small interfering RNAs. After being loaded by Argonaute2, the RISC complex directionally cleaves KAJ2954359.1 transcripts, thereby systematically down-regulating the translation level of catalase and breaking the defense balance of larvae against oxidative stress. Compared with the naked dsRNA treatment group, the same dose of complex can additionally increase the silencing efficiency by 42% and the mortality rate to 76%.

[0033] The carbon quantum dots have a particle size limited in the range of 2-10 nm, can enter the epithelium through the intestinal cavity tight junction, and ensure that no visible residue is formed after spraying on the plant leaves; meanwhile, the CQD is prepared by pyrolysis of citric acid, has no heavy metal ligand, and meets the safety specifications of green agricultural inputs. The delivery strategy avoids the potential compatibility toxicity of traditional liposomes or PEI carriers, and clears the key material barrier for large-scale field promotion of the RNAi biocontrol preparation.

[0034] The technical route breaks through the whole-link bottleneck from "sequence screening to field control" by the dual innovation of molecular targeted silencing + nano delivery, provides a replicable template for green management of tomato leaf webber, and lays a new paradigm of material science and molecular biology fusion for RNAi application of other difficult-to-control lepidopteran pests.

[0035] As shown in Figure 1 The application embodiment provides an application of CQD-dsRNA in prevention and control of tomato leaf webber, and the implementation method specifically includes the following steps:

[0036] S1: screening of catalase possibly existing in the oral secretions of tomato leaf webber, design of a dsRNA fragment and primers targeting the catalase gene;

[0037] S2: in vitro transcription synthesis of the corresponding dsRNA;

[0038] S3: use of carbon quantum dots CQD to carry the dsRNA for delivery into the tomato leaf webber larvae;

[0039] S4: analysis of the catalase gene silencing efficiency, survival rate and pupation number of tomato leaf webber.

[0040] In the S1, the catalase of tomato leaf webber is identified and primers are designed, based on the catalase protein sequence in the oral secretion proteome of the cabbage looper, TBLASTN is used to align the genome information of the tomato leaf webber, and the catalase KAJ2954359.1 of the tomato leaf webber is obtained. According to the DNA sequence, specific primers are designed by using Primer-blast, and a T7 promoter is added (Table 1) to send the dsRNA amplification primers to Beijing Chengke Biological Co., Ltd.

[0041] The S2, the synthesis of dsRNA of tomato leafminer peroxidase, total RNA of tomato leafminer was extracted by TRIzol method, the integrity was verified by 1% agarose gel electrophoresis, and the RNA concentration and purity were analyzed by ultramicro spectrophotometer. The extracted total RNA was reverse transcribed into first strand cDNA using PrimeScriptTM RT Reagent Kit with gDNA Eraser kit, and the cDNA concentration and purity were determined after reverse transcription. The cDNA obtained by reverse transcription was used as a template, and PCR amplification was performed, and the PCR product was purified by agarose gel DNA recovery kit (Tiangen), and dsRNA was synthesized using Transcript Aid T7 High Yied Transcription Kit and purified by phenol chloroform, and the operation was according to the instructions.

[0042] The S3, tomato leafminer soaking method RNAi, nano material CQD was mixed with 1000 ng / μL peroxidase KAJ2954359.1 dsRNA according to 1:2 volume, and 3rd instar larvae of tomato leafminer were picked and immersed, and the control group used the same volume of dsGFP solution, 15 larvae were treated in each group, and 4 groups of independent biological repeats were set. After standing for 30 min, the liquid was removed with a pipette, and the larvae were transferred to tomato for observation.

[0043] The S4, the analysis of silencing efficiency, tomato leafminer larvae were collected, total RNA of tomato leafminer was extracted, and first strand cDNA was reverse transcribed, and specific primers of KAJ2954359.1 were designed by Primer Blast (https: / / www.ncbi.nlm.nih.gov / tools / primer-blast / ) (Table 1), and β-actin was used as an internal reference, qPCR was used to analyze the expression of KAJ2954359.1, and qBASE software was used to calculate the relative expression of JHBP gene based on 2-△△CT. The results are shown in Figure 2 The KAJ2954359.1 gene silencing efficiency of CQD-dsKAJ2954359.1 treatment group was significantly higher than that of dsKAJ2954359.1 treatment group, indicating that CQD significantly improved the gene silencing efficiency.

[0044] The S4, the observation of tomato leafminer phenotype, the number of dead tomato leafminer and pupation were counted every 24 h; after 7 days, the damage of tomato leafminer to host plant tomato was observed and photographed. The results are shown in Figure 3 and Figure 4As shown, the survival rate and pupation number of tomato leafworm larvae in the CQD-dsKAJ2954359.1 treatment group are lower than those in the dsKAJ2954359.1 treatment group, indicating that the CQD-carrying dsKAJ2954359.1 improves the RNAi prevention and control efficiency.

[0045] Table 1 Tomato leafworm catalase primer sequences

[0046]

[0047]

[0048] The carbon quantum dots (CQD) are coupled with the dsRNA designed for the tomato leafworm catalase gene (dsKAJ2954359.1), 0.5% plant-derived surfactant and 1% glycerol are added as a moisturizer, and an aqueous suspension agent with a concentration of 0.1 mg / mL is prepared, which can be sprayed to prevent and control tomato leafworms in tomato planting bases. In addition, the CQD-dsRNA complex is mixed with sodium alginate and chitosan to prepare a powdered seed coating agent, which can be used for seed coating treatment.

[0049] The present application utilizes carbon quantum dots (CQD) to carry catalase gene dsRNA to form a "CQD-dsRNA" complex. Experimental verification shows that, compared with naked dsKAJ2954359.1, the target gene silencing efficiency of the CQD+dsKAJ2954359.1 complex is significantly improved by 42% ( Figure 2 ), the survival rate of tomato leafworms is reduced by 30% ( Figure 3 ), and the pupation number is also significantly reduced ( Figure 4 ), indicating that the present application effectively inhibits the target gene by carrying dsRNA with nanomaterials, reduces the survival ability and pupation ability of pests, and has a better prevention and control effect than naked dsRNA.

[0050] The above is only a specific embodiment of the present application, but the protection scope of the present application is not limited thereto, and any modification, equivalent replacement and improvement made by those skilled in the art within the technical range disclosed by the present application, as long as it is within the spirit and principles of the present application, should be covered within the protection scope of the present application.

Claims

1. A method for preventing and controlling tomato leafminer by using carbon quantum dots-double-stranded RNA complex, characterized in that, The method comprises the following steps: a) designing double-stranded RNA targeting the tomato leafminer catalase gene; b) synthesizing the double-stranded RNA by in vitro transcription; c) compounding the double-stranded RNA with carbon quantum dots at a volume ratio of 1:2 to obtain a carbon quantum dot-double-stranded RNA compound; d) delivering the compound by immersing tomato leafminer third instar larvae; e) determining the catalase gene silencing efficiency, survival rate and pupation number of the larvae to evaluate the control effect.

2. The method of claim 1, wherein, The concentration of the double-stranded RNA is 1000 ng / μL.

3. The method of claim 1, wherein, The double-stranded RNA has a length of 200-600 bp and a sequence as shown in SEQ ID NO:

1.

4. The method of claim 1, wherein, The carbon quantum dots have a particle size of 2-10 nm.

5. A carbon quantum dots-double stranded RNA complex, characterized by, The compound is compounded by carbon quantum dots and double-stranded RNA targeting the tomato leafminer catalase gene at a volume ratio of 1:

2.

6. The composite of claim 5, wherein, The concentration of the double-stranded RNA is 1000 ng / μL.

7. The composite of claim 5, wherein, The carbon quantum dots have a particle size of 2-10 nm.

8. A kit for preparing the carbon quantum dots-double stranded RNA complex according to claim 5, characterized by, The kit comprises: a) a carbon quantum dot solution; b) a double-stranded RNA solution targeting the tomato leafminer catalase gene; c) a compounding instruction for guiding the compounding of carbon quantum dots with double-stranded RNA at a volume ratio of 1:

2.

9. Use of the compound of claim 5 in the preparation of an agricultural preparation for controlling tomato leafminer.

10. Use according to claim 9, characterized in that, The agricultural preparation is applied by immersing tomato leafminer larvae or spraying tomato plants.

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