Feeding device for diaphorina citri RNA interference experiment
By designing an RNAi tube and closed system suitable for citrus psyllids, and combining it with a 20% sucrose/fluorescent dye feeding solution, the problem that existing artificial rearing devices for citrus psyllids are not suitable for dsRNA interference was solved, achieving the effects of simplified operation, reduced cost, and improved RNA interference efficiency.
Patent Information
- Application Number
- CN202422852939.2
- Authority / Receiving Office
- CN · China
- Patent Type
- Utility models(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-22
- Publication Date
- 2025-11-07
- Estimated Expiration
- 2034-11-22
AI Technical Summary
In the existing technology, the artificial feeding feed and equipment for citrus psyllids are not suitable for RNA interference using dsRNA, which leads to complicated operation, time-consuming and expensive operation, thus limiting the application of RNAi in the control of citrus psyllid pests.
A feeding device comprising an RNAi tube, a parafilm feeding solution covering membrane, and an opaque aluminum foil tube sleeve was designed. The closed system consisting of a transparent feeding tube, an opaque tube plug, and a parafilm sealing membrane, combined with a 20% sucrose/fluorescent dye feeding solution, simulates the feeding habits of the citrus psyllid to achieve efficient delivery of dsRNA.
This method simplifies RNA interference procedures, reduces costs and time, and improves the efficiency of RNA interference. It is suitable for stable rearing of citrus psyllids and RNA interference experiments, providing an efficient and convenient experimental environment.
Smart Images
Figure CN223515556U_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The utility model belongs to the field of insect RNA interference experiment, concretely relates to a kind of rearing device for citrus psylla RNA interference experiment. BACKGROUND
[0002] RNA interference is a highly conserved gene regulation mechanism in evolution, double-stranded RNA induces efficient and specific degradation of homologous messenger RNA, this phenomenon is first discovered in Caenorhabditis elegans, and later confirmed in fungi, fruit flies, Arabidopsis, trypanosomes, hydra, planarian, zebrafish and other eukaryotes, in agricultural pest control, RNAi can be used to develop dsRNA-based biological pesticides, these dsRNA can specifically silence key genes in pests, leading to developmental arrest and even death, therefore, RNAi shows great potential in green pest management. Although RNAi has great application potential in theory, it still faces some challenges in practical application, such as the stability of dsRNA in the environment, the lack of efficient delivery methods and the possible non-target effect, in addition, the short duration and instability of RNAi efficiency are particularly prominent in some pests, which leads to the short duration and instability of RNAi efficiency, especially in some Lepidoptera and Hemiptera and small-sized pests, which limits the development and practice of RNAi-based pest management strategies, and even limits the functional identification of insect genes. In order to overcome these challenges, researchers are working to improve the design and production of dsRNA, as well as to develop new efficient delivery systems, in pest control, the phenomenon of dsRNA-mediated RNAi is becoming more and more popular, methods to introduce dsRNA into insects include injection and feeding, but there are limitations such as low delivery efficiency and slow action.
[0003] Based on the above, gene / drug delivery systems containing nanoparticles have been widely used in the pharmaceutical field, for example, a star-shaped polycation (SPc)-based transdermal dsRNA delivery system constructed by Shen Jie's team at China Agricultural University, for efficient gene silencing and pest control in soybean aphids, the SPc-based transdermal dsRNA delivery system greatly promotes the development and practice of RNAi-based pest control strategies, SPc can load dsRNA, and the best fusion is achieved when the mass ratio of dsRNA to SPc is 1:1, the morphological characteristics change from chain-like dsRNA to spherical dsRNA-nanoparticle complex, nanomaterials effectively protect dsRNA from nuclease degradation, significantly improve penetration efficiency, in addition, they penetrate the epidermis into the insect gut and promote their translocation across the cell membrane, thereby achieving effective gene silencing.
[0004] The artificial feeding feed of the citrus psylla in the prior art is usually Litsea cubeba, and the artificial feeding method, feed and device are not suitable for RNA interference of the citrus psylla by using dsRNA. Content of the utility model
[0005] In order to overcome that the artificial feeding feed of the citrus psylla in the prior art is usually Litsea cubeba, and the artificial feeding method, feed and device are not suitable for RNA interference of the citrus psylla by using dsRNA.
[0006] The technical scheme of the utility model is: a feeding device for RNA interference experiment of citrus psylla, including RNAi pipe, parafilm feeding liquid covering film and light-proof aluminum foil pipe sleeve, the RNAi pipe includes transparent feeding pipe, light-proof pipe plug with air hole and parafilm sealing film, the parafilm sealing film and light-proof pipe plug cover the upper end and lower end of the transparent feeding pipe respectively, the parafilm feeding liquid covering film covers on the parafilm sealing film and forms the space of placing feeding liquid main body between the parafilm sealing film, the parafilm sealing film is placed with feeding liquid main body, the light-proof aluminum foil pipe sleeve is sleeved outside the transparent feeding pipe, and the height is slightly lower than the transparent feeding pipe, and the citrus psylla is placed in the transparent feeding pipe.
[0007] As preferred, the transparent feeding pipe is acrylic plastic pipe.
[0008] As preferred, the outer diameter of the transparent feeding pipe is 35mm, the inner diameter is 33mm, and the height is 60mm.
[0009] As preferred, the transparent feeding pipe and the light-proof pipe plug are connected through detachable buckle.
[0010] As preferred, the light-proof pipe plug with air hole is plastic pipe plug.
[0011] As preferred, the thickness of the parafilm sealing film and the parafilm feeding liquid covering film is about 0.3mm.
[0012] As preferred, the air hole of the light-proof pipe plug is covered by gauze.
[0013] The utility model has the advantages of:
[0014] 1, through the observation result of 20% sucrose / fluorescent dye feeding liquid main body under microscope, artificial feed can enter the insect body by feeding, and the utility model effectively improves the problems of complex operation, time consumption and high cost of RNA interference operation by using feeding method for smaller insects, and lays the foundation for further overcoming the insect damage of citrus psylla in the future. Brief description of drawings
[0015] Fig. 1 A perspective view of the feeding device for Diaphorina citri RNA interference experiment is shown.
[0016] Fig. 2 A bottom view of the light-tight tube plug for Diaphorina citri RNA interference experiment is shown.
[0017] Fig. 3 A cross-sectional view of the buckle for Diaphorina citri RNA interference experiment is shown.
[0018] Reference signs: 1-feeding liquid main body, 2-parafilm feeding liquid covering film, 3-parafilm sealing film, 4-transparent feeding tube, 5-light-tight tube plug, 6-light-tight aluminum foil tube sleeve, 7-vent hole, 8-gauze, 9-buckle. DETAILED DESCRIPTION
[0019] When conducting Diaphorina citri RNA interference experiments, a suitable feeding device is needed to ensure the healthy growth and stable reproduction of the wood lice, and to effectively carry out RNA interference treatment. The following are some points to consider when designing and using the feeding device for RNA interference experiments:
[0020] 1. Feeding box
[0021] Material and structure: usually use transparent plastic or glass box, convenient for observation of experimental objects. The box needs to be ventilated to prevent excessive humidity.
[0022] Size: the size can be a standard feeding box of several tens of centimeters square, or a larger space box according to the experimental scale.
[0023] Ventilation port: the box should have appropriate air holes to avoid excessive humidity or lack of oxygen, and maintain suitable environmental conditions.
[0024] 2. Temperature and humidity control
[0025] Temperature requirement: the suitable temperature range for Diaphorina citri is 22-30℃. In RNA interference experiments, the environmental temperature needs to be kept stable.
[0026] Humidity requirement: wood lice prefer a higher humidity environment (70%-80%). Low humidity can cause dehydration and affect experimental results.
[0027] 3. Feeding substrate
[0028] Plant host: the feeding of Diaphorina citri generally needs to choose suitable host plants, such as citrus plants (such as citrus trees or other related plants) or other plants preferred by wood lice.
[0029] Plant treatment: In RNA interference experiments, RNA interference solutions (such as siRNA, dsRNA, etc.) can be sprayed onto plants, and psyllids will feed on the treated plant sap, thereby interfering with gene expression.
[0030] 4. RNA interference treatment methods
[0031] RNA delivery method:
[0032] Plant treatment: An RNA solution is sprayed onto plant leaves using a sprayer, allowing psyllids to ingest the RNA by feeding on these plants.
[0033] Direct injection: RNA solution can also be injected directly into the psyllid, but this method is more difficult and may affect the health of the psyllid.
[0034] Immersion method: The psyllids are immersed in an RNA solution, or they are allowed to obtain interfering RNA by feeding on plants treated with specific RNA.
[0035] 5. Environmental monitoring
[0036] Light control: Citrus psyllids have certain light requirements, generally needing 12 hours of alternating day and night light to simulate a natural environment. LED lights can be used to control the light intensity and cycle.
[0037] Hygiene and Cleaning: Clean the enclosure regularly to ensure there are no excessive parasites or rotting plant residues to prevent cross-contamination.
[0038] 6. Safety Measures
[0039] Laboratory safety precautions: Personnel should wear appropriate protective clothing and avoid direct contact with RNA solutions or other chemical reagents. After the experiment, all waste should be properly disposed of to ensure that it does not harm the ecological environment or other experimental organisms.
[0040] 7. Other considerations
[0041] Experimental cycle control: RNA interference experiments require control of the psyllid's life cycle to ensure that the RNA interference agent works effectively at each developmental stage. Typically, the post-hatched nymph and adult stages are the most effective for RNA interference.
[0042] Using the above apparatus and methods, you can create a stable experimental environment for RNA interference in citrus psyllids and study the gene function of psyllids using RNA interference technology.
[0043] Please see Figs. 1-3The utility model provides an embodiment: a kind of rearing device for citrus psylla RNA interference experiment, including RNAi pipe, parafilm feeding liquid covering membrane 2 and light-tight aluminium foil tube cover 6, the RNAi pipe includes transparent rearing pipe 4, light-tight pipe plug 5 with vent hole 7 and parafilm sealing film 3, parafilm sealing film 3 and light-tight pipe plug 5 are covered on the upper end and lower end of transparent rearing pipe 4 respectively, parafilm feeding liquid covering membrane 2 is covered on parafilm sealing film 3 and forms the space of the placement feeding liquid main body 1 between parafilm sealing film 3, parafilm sealing film 3 is placed with feeding liquid main body 1, light-tight aluminium foil tube cover 6 is placed outside transparent rearing pipe 4, its height is slightly lower than transparent rearing pipe 4, citrus psylla is placed in transparent rearing pipe 4, the utility model provides a simple, save and fast artificial rearing device, applicable to dsRNA and 20% sucrose mixing, on the basis of not affecting its survival rate, meet the suitable survival environment and nutrient supply of citrus psylla, and combine its biological habit and feeding preference, the observation result under microscope of 20% sucrose / fluorescent dye feeding liquid main body 1 shows that artificial feed can be fed into insect body, the utility model effectively improves the problem that RNA interference operation is complex, time-consuming and high cost for smaller insect body using feeding method, and lays the foundation for further overcoming the insect damage of citrus psylla in the future.
[0044] In the embodiment, the transparent rearing pipe 4 is a acrylic plastic pipe, which has lower manufacturing cost and can reduce the production cost of the device.
[0045] The outer diameter of the transparent rearing pipe 4 is 35 mm, the inner diameter is 33 mm, and the height is 60 mm. In use, the transparent rearing pipe 4 with an outer diameter of 35 mm, an inner diameter of 33 mm and a height of 60 mm can better feed citrus psylla and facilitate observation by experimenters.
[0046] The transparent rearing pipe 4 and the light-tight pipe plug 5 are connected by a detachable buckle 9. In use, the transparent rearing pipe 4 and the light-tight pipe plug 5 connected by the detachable buckle 9 can be easily disassembled by experimenters, so as to facilitate experimenters to add the feeding liquid main body 1.
[0047] In the embodiment, the light-tight pipe plug 5 with the vent hole 7 is a plastic pipe plug, which can further reduce the production cost of the device.
[0048] The parafilm sealing film 3 and the parafilm feeding liquid covering film 2 have a thickness of about 0.3 mm, and in use, the parafilm sealing film 3 and the parafilm feeding liquid covering film 2 with a thickness of 0.3 mm can load the feeding liquid body 1 and prevent the citrus psylla from escaping.
[0049] The air hole 7 of the light-proof tube plug 5 is covered by the gauze 8, and in use, the gauze 8 can maintain the air permeability of the light-proof tube plug 5.
[0050] In use, the transparent feeding tube 4 and the light-proof tube plug 5 are sterilized at high temperature, the transparent feeding tube 4 can be a 35 mm outer diameter and 33 mm inner diameter and 60 mm high acrylic plastic tube with an upper part sealed by the parafilm sealing film 3 to prevent the citrus psylla from escaping.
[0051] Then, the citrus psylla adult is placed in the transparent feeding tube 4, the lower part of the transparent feeding tube 4 is connected with the light-proof tube plug 5 through the buckle 9, the light-proof tube plug 5 can be a light-proof plastic tube plug with an air hole 7 covered by the gauze 8 to allow air to pass through and prevent the citrus psylla from escaping, and the transparent feeding tube 4 is placed on a test tube rack to keep the bottom of the RNAi tube in air circulation.
[0052] Then, the feeding liquid body 1 is added to the parafilm sealing film 3, a layer of parafilm feeding liquid covering film 2 is covered on the upper part of the feeding liquid body 1 and sealed to prevent evaporation and contamination of the feeding liquid body 1, the feeding liquid body 1 is a mixture of 20% sucrose and dsRNA, and the parafilm feeding liquid covering film 2 can prevent the loss and contamination of the feeding liquid body 1.
[0053] Finally, the transparent feeding tube 4 is wrapped with the light-proof aluminum foil tube sleeve 6 for light shielding, the light-proof aluminum foil tube sleeve 6 is slightly lower than the transparent feeding tube 4 in height, the parafilm sealing film 3 and the parafilm feeding liquid covering film 2 are transparent, the main component of the feeding liquid body 1 is 20% sucrose water which is also transparent, and the citrus psylla will climb to the upper part of the transparent feeding tube 4 to pass through the parafilm sealing film 3 by the piercing style mouthparts to suck the feeding liquid body 1 according to the light-seeking characteristics.
[0054] Finally, the whole device is placed in a incubator with a temperature of 28℃ and a humidity of 60% for cultivation. In the initial research of the experiment, it is found that the citrus psylla can maintain normal physiological activity by feeding with 10% sucrose water, and therefore, the feeding device is designed based on 20% sucrose water as one of the components of the feeding liquid body 1.
[0055] In the embodiment, sucrose water with a concentration of 20% is used as artificial feed, and three kinds of feeding liquid main bodies 1 are set for rearing: dsRNA designed and synthesized according to the sequence of the target interference gene and 20% sucrose are mixed in a ratio of 1:1 to serve as the feeding liquid main body 1 for rearing of citrus wood lice, dsRNA designed and synthesized according to the sequence of the target interference gene, fluorescent nanoparticles SPc and 20% sucrose water are mixed in a ratio of 1:1:1 to serve as the feeding liquid main body 1 for rearing of citrus wood lice, and fluorescent dye and 20% sucrose are mixed in a ratio of 1:1 to serve as the feeding liquid main body 1 for rearing of citrus wood lice.
[0056] Through the above steps, the feeding device effectively shortens the interference time and reduces the experimental cost, the interference tube can be reused, a large number of test insects can be disposed of at one time, the experimental error is reduced, the efficient, convenient and accurate effect is achieved in the experimental process, and the problems that the artificial feed of the citrus wood lice in the prior art is usually melia azedarach, and the artificial rearing mode, feed and device are not suitable for dsRNA for RNA interference of the citrus wood lice are solved.
[0057] The embodiments of the utility model are described in detail above in combination with the drawings, but the utility model is not limited to the above-mentioned embodiments, and various changes can be made within the knowledge range possessed by the person skilled in the art without departing from the purpose of the utility model.
Claims
1. A rearing device for citrus psylla RNA interference experiments, characterized by: The RNAi tube comprises a transparent feeding tube (4), a light-proof tube plug (5) with a ventilation hole (7) and a parafilm sealing film (3), the parafilm sealing film (3) and the light-proof tube plug (5) are respectively covered on the upper end and the lower end of the transparent feeding tube (4), the parafilm feeding liquid covering film (2) is covered on the parafilm sealing film (3) and forms a space for placing the feeding liquid main body (1) between the parafilm sealing film (3), the parafilm sealing film (3) is placed with the feeding liquid main body (1), the light-proof aluminum foil tube sleeve (6) is sleeved outside the transparent feeding tube (4) and the height is slightly lower than the transparent feeding tube (4), the citrus psylla is placed in the transparent feeding tube (4).
2. The rearing device for citrus psylla RNA interference experiments according to claim 1, characterized in that: The transparent feeding tube (4) is an acrylic plastic tube.
3. The rearing device for citrus psylla RNA interference experiments according to claim 2, characterized in that: The outer diameter of the transparent feeding tube (4) is 35mm, the inner diameter is 33mm, and the height is 60mm.
4. The rearing device for citrus psylla RNA interference experiments according to claim 3, characterized in that: The transparent feeding tube (4) and the light-proof tube plug (5) are connected through a detachable buckle (9).
5. The rearing device for citrus psylla RNA interference experiments according to claim 4, characterized in that: The light-proof tube plug (5) with the ventilation hole (7) is a plastic tube plug.
6. The rearing device for citrus psylla RNA interference experiments according to claim 5, characterized in that: The thickness of the parafilm sealing film (3) and the parafilm feeding liquid covering film (2) is about 0.3mm.
7. The rearing device for citrus psylla RNA interference experiments according to claim 6, characterized in that: The ventilation hole (7) of the light-proof tube plug (5) is covered by a gauze (8).