A RNA interference method for Bemisia tabaci eggs

Abstract

The invention discloses a method for RNA interference of whitefly eggs, comprising the following steps: dsRNA, star-shaped polymer nanomaterials and surfactants are mixed to obtain an interference reagent; clean cotton seedlings are placed in a greenhouse insect cage to receive eggs for 12h-48h, cotton seedlings are taken out and the number of eggs on the cotton seedlings is recorded; the interference reagent is drawn with a pipette and dripped on the egg body, the dripping process keeps the pipette from touching the egg body, and the droplet is absorbed by the egg body, and the cotton seedlings with eggs are placed back in the greenhouse insect cage to continue to be raised; after the preset time of raising, samples of some eggs are taken, RNA is extracted, and RNA interference efficiency is identified, and the remaining eggs are normally raised in the greenhouse insect cage until the adult emerges, the number of emerging adults is recorded, and the phenotypic changes of adults are observed. The RNA interference method of whitefly eggs of the present invention can eliminate the damage of microinjection to eggs, and while ensuring the hatching rate of eggs, interference with target genes is achieved, breaking through the bottleneck of the interference technology of whitefly eggs.

Description

Technical Field

[0001] The invention relates to the technical field of insect RNA interference methods, and in particular to an RNA interference method for whitefly eggs. Background Art

[0002] Bemisia tabaci belongs to the order Hemiptera, family Aleyrodidae. As an important agricultural pest worldwide, it is widely distributed in China, Japan, Malaysia, India, the United States and other countries. Bemisia tabaci is a rapidly evolving composite species. B whitefly was first introduced into my country in 1949 and broke out in the 1990s, causing huge economic losses to my country's agricultural production. Q whitefly was first discovered in Yunnan Province, my country in 2003. At present, Q whitefly has replaced B whitefly as the main harmful species in my country. Bemisia tabaci is a polyphagous pest with a wide host spectrum. Its host plants involve 74 families and 420 species, mainly including Cruciferae, Cucurbitaceae, Leguminosae, Solanaceae, etc. It harms host plants by directly feeding on plant sap, causing plant weakness; secreting honeydew, inducing sooty mold; acting as a plant virus carrier, spreading plant viral diseases. In view of the severity of the harm caused by Bemisia tabaci, it is urgent to analyze its functional genes or study its genetic control.

[0003] RNA interference (RNAi) is a highly conserved gene expression regulation mechanism in the process of biological evolution. It is induced by double-stranded RNA (dsRNA) homologous to the target gene sequence, and is a specific gene expression silencing phenomenon. At present, the operation technology of RNAi in insects is relatively mature. There are three main methods for introducing dsRNA or siRNA into insects, namely injection, feeding and tissue culture. Injection and feeding are currently the most common methods for introducing dsRNA molecules into cells. The injection method inevitably causes mechanical trauma and postoperative infection to the insect body, and has high requirements for injection instruments and operation techniques. It is mostly used in laboratory research on medium and large insects. For insects with small size and more body fluids, such as whiteflies, the operation is difficult and the experimental effect is not good. Compared with the injection method, the feeding method has the advantages of simple operation, less trauma and less time consumption, and has a good application in RNAi experiments on adult whiteflies. However, due to the difficulty in mastering the culture conditions of the tissue culture method, it is currently only used in fruit flies, Aedes albopictus and Aedes aegypti. In addition, there are technologies that show that dsRNA can be introduced by body wall penetration, which can penetrate the insect body wall into the target cells to correctly target the target gene and regulate gene expression. This method has been tried to be applied to the interference of whitefly nymphs and has achieved initial results.

[0004] After years of research, the RNAi technology for adults of Bemisia whitefly has become relatively mature. There are also reports on the RNAi operation technology for nymphs of Bemisia whitefly. However, there is no successful experience of RNAi for Bemisia whitefly eggs at home and abroad. This is mainly because there are the following difficulties in RNAi operation on Bemisia whitefly eggs: 1. The eggs of Bemisia whitefly are only 0.2mm long and almost invisible to the naked eye. All experimental operations must be completed under a microscope, which requires extremely high operating skills of the experimenter; 2. The eggs of Bemisia whitefly have an egg stalk at the end, which is inserted into the cracks on the leaf surface through the ovipositor, and it is impossible to achieve in vitro operation of the eggs while ensuring the hatching rate of the eggs; 3. Compared with nymphs and adults of Bemisia whitefly, it is difficult to introduce dsRNA or siRNA into insects with Bemisia whitefly eggs. In addition, the special physiological stage of the eggs also makes it impossible to use feeding methods to introduce dsRNA. In addition, the structure of the egg shell is complex, and there is a hydrophobic wax layer secreted by egg cells in the egg shell, which can prevent the evaporation of water in the egg and the invasion of water-soluble substances. The egg shell is covered with a sticky layer secreted by the female insect's accessory gland, which can also prevent the invasion of foreign objects. This makes it difficult to copy the body wall penetration system of the nymph to interfere with the egg. Based on the above reasons, the technical bottleneck of the interference of whitefly eggs has not been solved by those skilled in the art. Summary of the invention

[0005] The technical problem to be solved by the present invention is to provide an RNA interference method for whitefly eggs, which can avoid the damage to eggs caused by microinjection and achieve interference with target genes while ensuring the hatching rate of eggs.

[0006] In order to solve the above technical problems, the present invention provides an RNA interference method for whitefly eggs, comprising the following steps:

[0007] The dsRNA, the star-shaped polymer nanomaterial and the surfactant are mixed to obtain an interference reagent;

[0008] The clean cotton seedlings were placed in a greenhouse insect cage to incubate eggs for 12 hours to 48 hours, and the cotton seedlings were taken out and the number of eggs on the cotton seedlings was recorded;

[0009] The interfering reagent is sucked with a pipette and dripped onto the egg body, and the pipette is kept from touching the egg body during the dripping process. After the droplet is absorbed by the egg body, the cotton seedlings with eggs are placed back into the insect cage in the greenhouse for further breeding;

[0010] After the preset feeding time, samples of some eggs were taken to extract RNA and measure the RNA interference efficiency. The remaining eggs were raised normally in greenhouse insect cages until adults emerged, the number of emerged adults was recorded, and the phenotypic changes of adults were observed.

[0011] In one embodiment, the dsRNA is synthesized by dsTra-F2 primer and dsTra-R3 primer, the nucleotide sequence of the dsTra-F2 primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the dsTra-R3 primer is shown in SEQ ID NO: 2.

[0012] In one embodiment, in the interfering agent, the added mass ratio of the dsRNA: the star-shaped polymer nanomaterial: the surfactant is 1:1:1.

[0013] In one embodiment, the concentration of the dsRNA is 0.5 μg / μl-1 μg / μl.

[0014] In one embodiment, the surfactant is an alkyl glycoside.

[0015] In one embodiment, the interfering reagent is dripped onto the egg body in an amount of 0.2 μl / egg to 0.4 μl / egg.

[0016] The implementation of the present invention has the following beneficial effects:

[0017] The present invention provides the RNA interference method for whitefly eggs, which can avoid the damage to eggs caused by microinjection, achieve interference with target genes while ensuring the hatching rate of eggs, and break through the bottleneck of the interference technology for whitefly eggs. In addition, the amount of dsRNA used in the present invention is very small, as low as 0.2 μl / egg, which greatly reduces the experimental cost while ensuring the interference efficiency, and has a good application prospect. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 The efficiency of Bttra RNA interference in Bemisia tabaci adults and eggs. DETAILED DESCRIPTION

[0019] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is described in further detail below.

[0020] The present invention provides an RNA interference method for whitefly eggs, comprising the following steps:

[0021] The dsRNA, the star-shaped polymer nanomaterial and the surfactant are mixed to obtain an interference reagent;

[0022] The clean cotton seedlings were placed in a greenhouse insect cage to incubate eggs for 12 hours to 48 hours, and the cotton seedlings were taken out and the number of eggs on the cotton seedlings was recorded;

[0023] The interfering reagent is sucked with a pipette and dripped on the egg body, and the pipette is kept from touching the egg body during the dripping process. After the droplet is absorbed by the egg body, the cotton seedlings with eggs are placed back into the insect cage in the greenhouse for further breeding;

[0024] After the preset feeding time, samples of some eggs were taken to extract RNA and test the RNA interference efficiency. The remaining eggs were raised normally in greenhouse insect cages until adults emerged, the number of emerged adults was recorded, and the phenotypic changes of adults were observed.

[0025] In one embodiment, the dsRNA is synthesized by dsTra-F2 primer and dsTra-R3 primer, the nucleotide sequence of the dsTra-F2 primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the dsTra-R3 primer is shown in SEQ ID NO: 2. It should be noted that the Transformer gene is an ancient and conservative key gene in insect sex determination. It participates in sex regulation by regulating the sex-specific alternative splicing of the downstream doublesex and fruitless genes. The Transformer gene plays an important role in female development. Interference with pre-embryonic female insects will cause male splicing of doublesex, and incomplete development or varying degrees of masculinization of female offspring. Exploring the role of transformer in the sex determination cascade of whiteflies is helpful to understand the sex determination mechanism of whiteflies, lay the foundation for exploring the sex differentiation of whiteflies, and provide a theoretical basis for genetic manipulation of sex of whiteflies. Using the interference method for whitefly eggs of the present invention to carry out functional verification of the transformer gene in the pre-embryonic stage is conducive to a better understanding of the sex determination mechanism of whiteflies. In addition, the inventors found that most sex-determining genes are highly expressed in whitefly eggs, and are expressed at lower levels or even absent in nymphs and adults. Therefore, to verify the function of transformer genes, it is necessary to interfere with whitefly eggs.

[0026] However, since the eggs of whiteflies are small and thin, the mortality rate is extremely high when RNA interference is performed by microinjection. The interference efficiency is not high when the body wall of nymphs is penetrated. Currently, no suitable method has been found to perform RNA interference on the eggs of whiteflies.

[0027] The present invention uses an interfering agent with a specific component, drips the interfering agent onto the egg body, and the droplets can be absorbed by the egg body, thereby breaking through the bottleneck of the interference technology of the whitefly egg. Among them, the star-shaped polymer nanomaterial has a strong permeability, combined with the affinity of the surfactant, the dsRNA is successfully infiltrated into the egg of the whitefly, eliminating the damage to the egg by microinjection, and while ensuring the hatching rate of the egg, the target gene is interfered, solving the problem of interference with the whitefly egg.

[0028] Furthermore, due to the special physiological stage of the eggs, it is impossible to use the feeding method to achieve the introduction of dsRNA. In addition, the structure of the egg shell of Bemisia tabaci is complex. There is a hydrophobic wax layer inside the egg shell to prevent the evaporation of water in the egg and the invasion of water-soluble substances. There is also a viscose layer outside the egg shell to prevent the invasion of foreign objects, which makes it difficult to copy the body wall penetration system of the nymph for the interference of the egg. Therefore, in terms of the composition of the interference reagent, the Bemisia tabaci eggs cannot imitate the interference reagent system of the adult and nymph of Bemisia tabaci, but need to use a specific interference reagent system to achieve the stable introduction of dsRNA molecules while ensuring the survival rate of the Bemisia tabaci eggs.

[0029] In one embodiment, the surfactant is an alkyl glycoside, and the alkyl glycoside can be selected from APG0810. It should be noted that the above-mentioned surfactant listed in the present invention is a new type of non-ionic surfactant with relatively comprehensive performance, which has the characteristics of ordinary non-ionic and anionic surfactants, has high surface activity, good ecological safety and compatibility, and is internationally recognized as the preferred "green" functional surfactant. In this experiment, the addition of APG can increase the permeability of star-shaped polymer nanomaterials and promote the stable introduction of dsRNA molecules. Other surfactants will destroy the protein stability of the eggshell, resulting in a decrease in the survival rate of whitefly eggs.

[0030] In one embodiment, the star-shaped polymer nanomaterial includes a star-shaped polymer P1 and / or a star-shaped polymer P2. The star-shaped polymer P1 or the star-shaped polymer P2 can self-assemble in an aqueous solution to form nano-microspheres. The chemical structure of the star-shaped polymer P1 is shown in formula (A):

[0031]

[0032] Formula (A)

[0033] The chemical structure of the star polymer P2 is shown in formula (B):

[0034]

[0035] In one embodiment, the concentration of the dsRNA is 0.5 μg / μl-1 μg / μl. In the interference reagent, the mass ratio of the dsRNA: the star-shaped polymer nanomaterial: the surfactant is 1:1:1. Among them, too much or too little addition of the dsRNA will affect the interference efficiency; too much addition of the star-shaped polymer nanomaterial will affect the survival rate of whiteflies; too much addition of the surfactant will affect the normal hatching of eggs.

[0036] In summary, the present invention provides the RNA interference method for whitefly eggs, which can avoid the damage to the eggs caused by microinjection, achieve interference with the target gene while ensuring the hatching rate of the eggs, and break through the bottleneck of the interference technology for whitefly eggs. In addition, the amount of dsRNA used in the present invention is very small. In one embodiment, the amount of the interference reagent dripped on the egg body is 0.2μl / egg-0.4μl / egg. While ensuring the interference efficiency, the experimental cost is greatly reduced, and it has a good application prospect.

[0037] The present invention is further described below with specific embodiments:

[0038] Example 1

[0039] RNA interference method for Bemisia tabaci eggs

[0040] Experimental Materials and Methods

[0041] Test insects

[0042] Q whiteflies were first collected from poinsettias (Euphorbia pulcherrima Willd.) sold at Beijing Zhongshu Dasenlin Flower Market in June 2009, and then subcultured on cotton (Gossypium herbaceum L.cv. Zhongmian 49) in a glass greenhouse. To maintain the purity of the population, mitochondrial cytochrome oxidase I (mtCOI) was used for monitoring every 2-3 generations.

[0043] Test reagent

[0044] RNA extraction reagent Trizol (Invitrogen, Carlsbad, CA, USA), cDNA reverse transcription kit RT reagent Kit (Takara Biotech), dsRNA synthesis kit T7RiboMAX TM Express RNAi System, fluorescence quantitative kit SuperReal PreMix Plus (SYBR Green) (TIANGEN, Beijing, China) and gel recovery kit (Promega), star polymer nanomaterials (90 nm microspheres self-assembled in aqueous solution by star polymer P2 in Example 2 of Chinese patent application 201810350562.7 (invention name: a star polymer and its preparation method and application)), alkyl glycoside (Wanqi, APG0810), DNA marker (TIANGEN, Beijing, China), agarose (Beijing Qingke), ES-Taq Mix DNA polymerase. Other commonly used chemical reagents in laboratories (analytical grade) were commercially available.

[0045] Main instruments

[0046] Fluorescence quantitative PCR instrument (ABI QuantStudio 3), PCR instrument (Bio-Rad S1000), Nanodrop (ThermoScientific Nanodrop 2000), centrifuge (Eppendorf 5417R), water bath (Beijing Liuyi Manufacturing Plant), electrophoresis instrument and gel imaging system (Bio-Rad), constant temperature box and high pressure sterilizer (SanYo), tissue grinder (Shanghai Jingxin Technology), pipette (Eppendorf), ultrapure water instrument (ZMQ55VOTI Mini Q), clean bench (Suzhou Purification Technology Co., Ltd.).

[0047] RNA extraction and cDNA synthesis

[0048] The total RNA of Bemisia tabaci was extracted using Trizol reagent. The specific steps are as follows:

[0049] 1) Use a dissecting needle to pick about 300 eggs of Bemisia tabaci and place them in a 1.5 ml centrifuge tube and freeze them in liquid nitrogen to prevent RNA degradation.

[0050] 2) Add 1 ml of Trizol and 1 grinding bead to the whitefly sample, grind in a pre-cooled tissue grinder for 2-3 minutes to make it fully homogenized, and let it stand on ice for 5 minutes.

[0051] 3) Add 300 μl of chloroform, vortex vigorously for 30 seconds to mix thoroughly, let stand on ice for 5 minutes, and centrifuge at 12000 rpm at 4°C for 15 minutes.

[0052] 4) Gently pipette 400 μl of the supernatant into a clean centrifuge tube.

[0053] 5) Add an equal volume of pre-cooled isopropanol, slowly invert to mix, place on ice for 10 min to precipitate RNA, and centrifuge at 12000 rpm at 4°C for 10 min.

[0054] 6) Pour off the supernatant, add 1 ml of pre-chilled 75% anhydrous ethanol for rinsing, and centrifuge at 8000 rpm at 4°C for 5 min.

[0055] 7) Pour off the ethanol and carefully remove the remaining liquid, dry at room temperature, add 10 μl pre-cooled DEPC water to dissolve, and use Nanotrop to measure the RNA concentration and OD 260 / OD 280 The integrity of RNA was checked by 1% agarose gel electrophoresis.

[0056] According to the instructions of the TaKaRa company's reverse transcription kit cDNA was synthesized using RT reagent Kit and the product was stored at -20°C for later use.

[0057] Fluorescence quantitative PCR analysis

[0058] The Transformer gene is an ancient and conserved key gene in insect sex determination. It participates in sex regulation by regulating the sex-specific alternative splicing of downstream doublesex and fruitless. At the same time, it is reported to play an important role in female development. Interference with pre-embryonic female insects will lead to male splicing of doublesex, and incomplete development or varying degrees of masculinization of female offspring. Exploring the role of transformer in the sex determination cascade of whiteflies will help us understand the sex determination mechanism of whiteflies, lay the foundation for exploring the sex differentiation of whiteflies, and provide a theoretical basis for genetic manipulation of sex in whiteflies. Therefore, we used the newly invented interference method for whitefly eggs to verify the function of the transformer gene in the pre-embryonic stage.

[0059] 1. Screening of fluorescence quantitative primers: Use Primer Premier 5.0 software to design specific primers that meet the requirements of fluorescence quantitative test (annealing temperature ≈ 60°C, product size between 100-200bp, avoid interfering fragments), then use Bemisia tabaci egg cDNA as a template, use the designed primers, first perform ordinary PCR, and detect PCR products by electrophoresis to determine that the product size is correct and is a single bright band, and send it to the company for sequencing to verify the specificity of the primers.

[0060] Table 1 Primer list

[0061]

[0062] Table 2 PCR reaction system

[0063]

[0064] RT-qPCR was performed using an ABI QuantStudio 3 real-time fluorescence quantitative PCR instrument. The template concentration was gradient diluted and the amplification efficiency of the primers was tested according to the system and procedure recommended by the fluorescence quantitative kit. The reaction system is shown in Table 3 and the reaction procedure is shown in Table 4.

[0065] Table 3

[0066]

[0067] Table 4

[0068]

[0069] The melting curve was obtained during the extension at 72°C, and finally the appropriate fluorescence quantitative primers were screened (Table 1).

[0070] 2. Selection of internal reference gene: SDHA was selected as the internal reference gene of the present invention.

[0071] 3. Data analysis: RT-qPCR data were processed using ABI, and the relative expression of each gene was calculated based on 2-ΔΔCt, where the amplification efficiency (E) of the target primer was calculated using the formula E=[10(-1 / slope)-1]×100%.

[0072] dsRNA synthesis

[0073] The full-length sequence of the verified transformer gene was input into the website of siRNA binding sites in Drosophila genes http: / / www.flyrnai.org / cgi-bin / RNAi_find_primers.pl to design the corresponding dsRNA primers. The primers were used to perform PCR with the cDNA of whitefly eggs as template, and the fragments with the same size as the target band were recovered and sequenced for verification. After verification, the dsRNA synthesis kit T7 RiboMAX was used according to the instructions of the manual. TM The dsRNA was synthesized using the ExpressRNAi System and the product was stored at -20°C until use.

[0074] RNA interference of whitefly eggs

[0075] 1) Preparation of interference reagent: Synthesize dsRNA using the screened dsRNA primers. Dilute the newly synthesized dsRNA to 1 μg / μl, and mix the star polymer nanomaterial, alkyl glycoside and dsRNA in a volume ratio of 1:1:1 for later use.

[0076] 2) Cultivation of cotton seedlings: Sow soaked cotton seeds into seedling pots, and when the cotton seedlings grow one true leaf with a diameter of about 2 cm, pull up the cotton seedlings with their roots and set aside.

[0077] 3) Inoculation of cotton seedlings with eggs: Check the cotton seedlings under a microscope to ensure that there are no other whitefly nymphs and eggs on the seedlings. Place the clean cotton seedlings in the insect cage in the greenhouse for 24 hours to inoculate eggs. Take out the cotton seedlings and record the number of eggs under a microscope.

[0078] 4) Interference of eggs: Use a 10μl pipette to absorb the prepared interference reagent and carefully drop it on the eggs under a microscope, taking care not to let the tip of the pipette touch the egg body. After the droplet is absorbed by the egg body, place the cotton seedlings with eggs in a small insect cage and continue to raise them.

[0079] 5) Phenotypic observation: After 2 days of interference, samples of some eggs were taken to extract RNA and measure the RNA interference efficiency. The remaining eggs were raised normally in small insect cages until the adults emerged, the number of emerged adults was recorded, and the phenotypic changes of the adults were observed under a microscope.

[0080] Comparison of two RNA interference methods in Bemisia tabaci

[0081] RNA interference experiments were conducted on Bttra using the adult feeding method and the egg interference method of Bemisia tabaci, respectively. The exogenous gene EGFP was used as a control, and three biological replicates were set up for each experiment. Two days after the interference, RT-qPCR was used to detect the interference efficiency of Bttra. The experimental results are shown in the figure. Figure 1 As shown in FIG. 1 , wherein Egg represents the egg of Bemisia tabaci, and Adult represents the adult of Bemisia tabaci. Figure 1 It can be seen that both methods can effectively reduce the expression level of Bttra.

[0082] In summary, the present invention provides the RNA interference method for whitefly eggs, which can avoid the damage to the eggs caused by microinjection, achieve interference with the target gene while ensuring the hatching rate of the eggs, and break through the bottleneck of the interference technology for whitefly eggs. In addition, the amount of dsRNA used in the present invention is small, which greatly reduces the experimental cost while ensuring the interference efficiency, and has a good application prospect.

[0083] The above is a preferred embodiment of the invention. It should be pointed out that a person skilled in the art can make several improvements and modifications without departing from the principle of the invention. These improvements and modifications are also considered to be within the scope of protection of the invention.

Claims

1. A method for RNA interference of whitefly eggs, characterized in that: The following steps are involved: RNA from Bemisia tabaci was extracted and reverse transcribed into cDNA; The verified full-length sequence of the transformer gene was used to design dsRNA primers, and PCR was performed using the cDNA of Bemisia tabaci as a template, followed by sequencing verification; After verification, the dsRNA synthesis kit RiboMAX was used TM The dsRNA is synthesized by the Express RNAi System, wherein the dsRNA primers include a dsTra-F2 primer and a dsTra-R3 primer; the nucleotide sequence of the dsTra-F2 primer is shown in SEQ ID NO: 1, and the nucleotide sequence of the dsTra-R3 primer is shown in SEQ ID NO: 2; The dsRNA, the star-shaped polymer nanomaterial and the surfactant are mixed in a mass ratio of 1:1:1 to obtain an interference reagent; the surfactant is an alkyl glycoside; The clean cotton seedlings were placed in a greenhouse insect cage to incubate eggs for 12 hours to 48 hours, and the cotton seedlings were taken out and the number of eggs on the cotton seedlings was recorded; The interfering reagent is sucked with a pipette and dripped on the egg body, and the pipette is kept from touching the egg body during the dripping process. After the droplet is absorbed by the egg body, the cotton seedlings with eggs are placed back into the insect cage in the greenhouse for further breeding; After the preset feeding time, samples of some eggs were taken to extract RNA and measure the RNA interference efficiency. The remaining eggs were raised normally in greenhouse insect cages until adults emerged, the number of emerged adults was recorded, and the phenotypic changes of adults were observed.

2. The RNA interference method for whitefly eggs according to claim 1, characterized in that: The concentration of the dsRNA is 0.5 μg / μl-1 μg / μl.

3. The RNA interference method for whitefly eggs according to claim 1, characterized in that: The amount of the interfering reagent dripped onto the egg body is 0.2 μl / egg-0.4 μl / egg.

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