RNA interference method for parasitic wasps in lepidoptera larvae

Abstract

The invention discloses an RNA (Ribonucleic Acid) interference method for parasitic wasps in lepidoptera larvae, which is characterized in that RNA interference is carried out by using a microinjection method or a nano-material immersion method when the parasitic wasps in the lepidoptera larvae are not separated from hosts or are separated from the hosts to cocoon, so that an RNAi system of the parasitic wasps is successfully constructed, and the defects that the parasitic wasps are small in size in an egg stage, and the parasitic wasps are not damaged in the egg stage can be effectively overcome. And RNAi operation difficulty caused by cocoon wrapping in the pupal stage and short life in the adult stage is avoided. Moreover, the method disclosed by the invention is simple to operate and high in interference efficiency, provides a theoretical basis and a reference effect for further researching gene functions of the parasitic wasps, and has important significance on biological control.

Description

Technical Field

[0001] This invention relates to genetic engineering technology, and more particularly to a method for interfering with RNA in endoparasitic wasps in lepidopteran larvae. Background Technology

[0002] Lepidoptera is the second largest order of insects after Coleoptera, with approximately 200,000 known species worldwide. It is the most concentrated group of agricultural and forestry pests. Both adults and larvae can severely damage plants: adults can pierce fruits, while larvae mainly feed on leaves or bore into stems; some species' larvae even gnaw on roots and stems underground, leading to plant death. Taking rice, a major grain crop in my country, as an example, common lepidopteran pests include nine species such as the rice leaf roller, rice stem borer, and rice bark borer. Among them, the rice stem borer is particularly prominent due to its wide distribution, large population size, severe damage, and strong pesticide resistance. Its larvae bore into rice stems, causing symptoms such as dead heart, dead sheath, and whiteheads, seriously threatening rice yield and food security.

[0003] In the field of biological control, hymenopteran parasitic wasps are important natural enemies of lepidopteran pests. Among them, the rice stem borer parasitic wasp (Cotesia chilonis), as an endoparasitic wasp, is a dominant natural enemy of the rice stem borer larvae. This wasp lays its eggs inside the rice stem borer larvae, and the hatched larvae feed on the host's hemolymph. By the third instar, the larvae gnaw out of the host's body, causing its death. The parasitism rate of this wasp on the rice stem borer in the field is usually 10-30%, and in some areas, the parasitism rate of the overwintering generation can even reach 90%, playing a crucial role in controlling the rice stem borer population.

[0004] RNA interference (RNAi) technology, with its advantages of high efficiency and specificity, has become a powerful tool for pest control and gene function research. However, the application of RNAi technology in endoparasitic wasps faces significant challenges: the main developmental stages are hidden inside the host, limiting conventional introduction methods; the tiny size of the insects increases the difficulty of experimental operations in early stages; and the short lifespan of adults greatly interferes with the efficiency of phenotypic observation and detection. Therefore, although RNAi has a research foundation in some parasitic wasps such as the diamondback moth's braconid wasp, a mature RNAi system for the rice stem borer's braconid wasp is still lacking. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a low-cost and easy-to-operate method for interfering with RNA in endoparasitic wasps of lepidopteran pests.

[0006] Technical solution: The RNA interference method for endoparasitic wasps in lepidopteran larvae of the present invention involves using microinjection or nanomaterial immersion to interfere with RNA during the period when the endoparasitic wasps have not detached from the host or have detached from the host and formed a cocoon. The period when the endoparasitic wasps have not detached from the host is no more than 10 days, and the period when the endoparasitic wasps have detached from the host and formed a cocoon is no more than 7 days.

[0007] Preferably, the step of using microinjection to interfere with RNA during the period when the endoparasitic wasp detaches from the host and spins a cocoon includes: making a hole with a diameter not exceeding 0.5 mm in the abdomen of the endoparasitic wasp cocoon, and injecting dsRNA into the endoparasitic wasp pupa through the hole to complete the RNA interference.

[0008] Preferably, the cocooning period is 1-5 days after cocooning; the step of breaking the cocoon by the endoparasitic wasp includes: opening an initial hole with a diameter not exceeding 0.2 mm in the middle section of the cocoon, observing the position of the pupa, and then enlarging the hole along the abdominal direction of the pupa; the injection site of the endoparasitic wasp pupa is the 1st-3rd abdominal segments; the injection dose of the dsRNA is 5-20 ng.

[0009] Preferably, an injection plate device is used for microinjection. The injection plate device includes a culture dish 1 and a sealing film 2, wherein the sealing film 2 is wrapped around the culture dish 1 around the bottom of the dish. Before the microinjection operation, the endoparasitic cocoon 3 is embedded in the sealing film 2 for fixation, and the injection position is changed by adjusting the endoparasitic cocoon 3.

[0010] Preferably, the step of using nanomaterials for RNA interference during the period when the parasitic wasp in the lepidopteran larva has not yet detached from the host or has detached from the host to form a cocoon includes:

[0011] (1) Mix dsRNA with nanomaterials modified with fluorescent groups at a mass ratio of 1:50-60 and incubate, then add detergent and incubate.

[0012] (2) The nanomaterial / dsRNA complex was dropped onto the surface of the insect body wall;

[0013] (3) After treatment, the insects with the nanomaterial / dsRNA complex were screened to complete the RNA interference.

[0014] Preferably, the fluorescent group in step 1 is a red fluorescent group, and the incubation time is 10-15 min; the screening step in step 3 includes: incubating in the dark for 12-36 h, and screening for fluorescent insects.

[0015] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: 1. It constructs an RNAi system for endoparasitic wasps, which can effectively overcome the difficulties in RNAi operation caused by the small size of endoparasitic wasps in the egg stage, the cocoon-encased pupal stage, and the short lifespan of adult wasps; 2. The method is simple to operate and has high interference efficiency, providing a theoretical basis and reference for further research on the gene function of endoparasitic wasps, and is of great significance for biological control. Attached Figure Description

[0016] Figure 1The diagram shows the pupae of the rice stem borer at different ages, where A is a 1-day-old pupa, B is a 2-day-old pupa, C is a 3-day-old pupa, D is a 4-day-old pupa, E is a 5-day-old male pupa, and F is a 5-day-old female pupa.

[0017] Figure 2 This is a schematic diagram of a homemade injection plate device for injection, where 1 is a culture dish, 2 is a sealing film, and 3 is an internal parasitic bee cocoon;

[0018] Figure 3 A schematic diagram showing the location of RNA interference via microinjection during the cocooning stage of the rice stem borer.

[0019] Figure 4 Statistical results of Caspase-1 expression after injecting dsCaspase-1 and dsGFP into cocoons of the rice stem borer at different ages;

[0020] Figure 5 Microscopic observation of the nanomaterial / dsRNA complex penetrating the host larval body wall and entering the cavity of the Brassicer larvae;

[0021] Figure 6 The figure shows the statistical results of Caspase-1 expression after RNAi via nanomaterial immersion. Detailed Implementation

[0022] The technical solution of the present invention will be further described below.

[0023] Example 1: Preparation of dsRNA

[0024] Caspase-1 was selected as the target gene.

[0025] Based on the cDNA sequence of caspase-1 from the rice stem borer (GenBank: MZ357077.1), the CDS fragment of Cccaspase-1 was cloned, and specific dsRNA primers were designed as shown in Table 1, with dsGFP as the control.

[0026] Table 1 Primers for dsRNA synthesis

[0027]

[0028] Using Cc caspase-1 or GFPCDS fragments as templates, partial sequences of the target gene were amplified by PCR using dsRNA primers. The amplification systems are shown in Table 2, and the reaction conditions are shown in Table 3.

[0029] Table 2 PCR reaction system

[0030] Composition volume 2×Taq master Mix 12.5μL 10μM upstream primer 1μL 10μM downstream primer 1μL template cDNA 100ng ddH2O Add to 25μL

[0031] Table 3 PCR reaction conditions

[0032]

[0033]

[0034] After amplification, electrophoresis was performed on a 1% agarose gel. For dsCaspase-1 amplification products, a 389bp fragment was recovered; for dsGFP amplification products, a 314bp fragment was recovered and purified using the AxyPrep DNA Gel Recovery Kit.

[0035] use The recovered products were cloned using the -T Easy Vector System I to obtain dsCaspase-1 and dsGFP plasmids.

[0036] The cells were transformed into competent DH5α cells and inoculated onto LB plates containing 100 ng / mL ampicillin. The cells were incubated at 37°C for 15 h. Single colonies were picked and sequenced by Shanghai Sangon Biotech Co., Ltd. to screen for dsCaspase-1 / DH5α and dsGFP / DH5α.

[0037] To ensure the accuracy of dsRNA synthesis and obtain a high concentration of DNA template with T7 promoters at both ends, the bacterial culture that was verified by sequencing was expanded and plasmids were extracted. Using the extracted plasmids as templates, the aforementioned dsRNA primers were used for amplification again. Electrophoresis was performed on a 1% agarose gel to detect the amplification. A 389bp dsCaspase-1 DNA and a 314bp dsGFP DNA template with T7 promoters at both ends were recovered.

[0038] Using MEGAscript TM The RNAi kit (catalog number: AM1626) synthesizes dsRNA and purifies it to obtain dsCaspase-1 and dsGFP for subsequent RNA interference.

[0039] Example 2: RNA interference in the cocoons of *Stachys diplophora* using microinjection

[0040] Fix the cocoons of the rice stem borer (Paecilomyces diffusa) at different ages (1-5 days) onto... Figure 2 Microinjection was performed on the homemade injection plate device shown.

[0041] A hole with a diameter of 0.1 mm was made in the abdomen of the endoparasitic wasp cocoon. 1 nL of dsGFP or dsCaspase-1 prepared in Example 1 with a concentration of 10 mg / mL was injected into the endoparasitic wasp pupa through the hole, and the pupa was then placed in an incubator at 27°C for culture.

[0042] Representative images of bee pupae inside cocoons of the rice stem borer at different ages are shown below. Figure 1 As shown, representative images after injection are as follows: Figure 3 As shown.

[0043] 1. Detection of gene expression after RNA interference

[0044] 24 hours after injection, the treated cocoons were dissected and total RNA was extracted. The RNA was then reverse transcribed into cDNA using a kit, and the expression of the target gene in pupae of different ages of *Coprinus spp.* was determined by RT-qPCR. Each treatment contained 5 samples, and each treatment was repeated three times. The amplification system is shown in Table 5, and the reaction conditions are shown in Table 6.

[0045] Table 4 RT-qPCR Primers

[0046]

[0047] Table 5 RT-qPCR reaction system

[0048]

[0049] Table 6 RT-qPCR reaction conditions

[0050]

[0051] The expression of Caspase-1 gene after RNA interference in the cocoons of the rice stem borer at different ages (1-5 days) is as follows: Figure 4 As shown, the transcriptional levels of *C. oryzae* pupae (cocoons) of different ages after microinjection of dsCaspase-1 were significantly reduced by 37.86%, 36.05%, 36.40%, 41.34%, and 35.39% compared to the control (1 day old: t = 3.447, p < 0.05; 2 days old: t = 3.289, p < 0.05; 3 days old: t = 2.842, p < 0.05; 4 days old: t = 2.947, p < 0.05; 5 days old: t = 4.428, p < 0.05).

[0052] 2. Detection of development after RNA interference

[0053] Cocoons were collected in 1.5 mL centrifuge tubes and placed in an incubator at 27°C for continuous culture and observation. Developmental indicators were recorded at regular intervals. Each treatment contained 15 samples, and each treatment was repeated 3 times.

[0054] The developmental indicators of *Papilionophora chinensis* pupae of different ages (1-5 days) after RNA interference are shown in Table 6. After microinjection interference, compared with dsGFP, the total number of offspring of *Papilionophora chinensis* pupae injected with dsCaspase-1 was significantly reduced from 1 to 5 days. Specifically, the total number of 4-day pupae that could emerge as adults decreased from 6.33 in the dsGFP treatment group to 2.00 in the dsCaspase-1 treatment group. The number and emergence rate of 1-4-day pupae were also significantly reduced, with the emergence rate of 4-day pupae decreasing from 42% in the dsGFP treatment group to 13% in the dsCaspase-1 treatment group.

[0055] Table 7. Statistical results of developmental indicators after microinjection interference with endoparasitic wasp cocoons.

[0056]

[0057] Example 3: RNA interference in host larvae using nanomaterial immersion method

[0058] Third-instar larvae of the rice stem borer *Papilionophora braconis*, about to emerge from the host's cocoon (i.e., 9 days after the rice stem borer was parasitized by *Papilionophora braconis*), were selected for interference using a nanomaterial immersion method on the parasitic wasp-host complex. The control group, consisting of third-instar larvae of *Papilionophora braconis* under normal indoor rearing conditions, was not treated with the nanomaterial / dsRNA complex.

[0059] The preparation method for the nanomaterial / dsRNA complex required for each parasitic wasp-host complex is as follows:

[0060] Take 0.25 μL of dsGFP or dsCaspase-1 solution with a concentration of 2000 ng / μL prepared in Example 1 and 13.71 μL of nanomaterials with a concentration of 1 ng / μL;

[0061] The dsRNA solution and nanomaterials were mixed for 15 min, and then phosphate buffered saline (PBS) solution was added and incubated for 15 min to obtain the nanomaterials / dsRNA complex.

[0062] The nanomaterial / dsRNA complex was dropped onto the surface of the body wall of the rice stem borer host, treated in the dark for 15 min, and then incubated in a 27°C incubator in the dark.

[0063] 1. Detection of gene expression after RNA interference

[0064] 24 hours after sample treatment, the parasitized rice stem borers were dissected, and the larvae of *Pachycarpus spp.* inside the rice stem borers were observed and photographed using a fluorescence microscope. Individuals in which nanomaterials were successfully introduced into *Pachycarpus spp.* larvae were collected for RNA extraction. Individuals in which nanomaterials were not successfully introduced could not be used for the next step of the experiment. RNAi efficiency was detected by RT-qPCR. Each treatment was repeated three times, and each treatment contained 5 samples.

[0065] Fluorescence microscopy observation results as follows Figure 5 As shown, the nanomaterial / dsRNA complex successfully entered the larvae of the rice stem borer wasp, and the Caspase-1 gene expression was as follows. Figure 6 As shown, the transcriptional level of *C. dilatatus* immersed in the nanomaterial / dsCaspase-1 complex was significantly lower than that of the dsGFP treatment group (t = 3.891, p < 0.05), indicating that the transcriptional interference was successful.

[0066] 2. Detection of development after RNA interference

[0067] After 24 hours of sample treatment, the samples were incubated in a 27°C incubator in the dark, and developmental indicators were recorded regularly. Each treatment contained 5 samples, and each treatment was repeated 3 times.

[0068] Developmental indicators are shown in Table 7. After the nanomaterial immersion interference, the number of cocoons of rice stem borer larvae immersed in dsCaspase-1 was significantly reduced compared with dsGFP, from 34.33% in the dsGFP treatment group to 21.00% in the dsCaspase-1 treatment group.

[0069] Table 7 Statistical results of developmental indicators after immersion interference with nanomaterials

[0070] deal with dsGFP dsCaspase-1 Control Number of cocoons produced 34.33±4.10 21.00±1.73 36.67±3.38 Number of drones 3.33±2.40 1.33±0.67 5.33±0.33 Number of female bees 9.67±5.36 4.00±1.00 11.00±1.15 Total number (pieces) 13.00±3.51 5.33±1.4 17.00±1.00 Female-to-male ratio 2.90 3.01 2.06 Feathering rate (%) 0.37±0.06 0.25±0.06 0.47±0.44

Claims

1. A method for interfering with RNA from endoparasitic wasps in lepidopteran larvae, characterized in that, The method involves using microinjection or nanomaterial immersion to interfere with RNA during the period when the endoparasitic wasp in a lepidopteran larva is still parasitizing the host or has detached from the host and formed a cocoon. The period when the endoparasitic wasp is still parasitizing the host is no more than 10 days, and the period when the endoparasitic wasp has detached from the host and formed a cocoon is no more than 7 days.

2. The method for interfering with RNA from endoparasitic wasps in lepidopteran larvae according to claim 1, characterized in that, The steps for RNA interference using microinjection during the cocooning period of endoparasitic wasps in lepidopteran larvae include: making a hole with a diameter not exceeding 0.5 mm in the abdomen of the endoparasitic wasp cocoon, and injecting dsRNA into the endoparasitic wasp pupa through the hole to complete the RNA interference.

3. The method for interfering with RNA from endoparasitic wasps in lepidopteran larvae according to claim 2, characterized in that, The cocooning period refers to the cocooning stage from 1 to 5 days after cocooning.

4. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 2, characterized in that, The steps for breaking open the cocoon of the endoparasitic wasp include: making an initial hole with a diameter not exceeding 0.2 mm in the middle section of the cocoon; and after observing the position of the pupa, enlarging the hole along the ventral direction of the pupa.

5. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 2, characterized in that, The injection site for the endoparasitic wasp pupae is the first to third abdominal segments.

6. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 2, characterized in that, The injection dose of the dsRNA is 5-20 ng.

7. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 1, characterized in that, The steps of using nanomaterials for RNA interference during the period when the parasitic wasp in the lepidopteran larvae has not yet detached from the host or has detached from the host to form a cocoon include: (1) Mix dsRNA with nanomaterials modified with fluorescent groups at a mass ratio of 1:50-60 and incubate, then add detergent and incubate. (2) The nanomaterial / dsRNA complex was dropped onto the surface of the insect body wall; (3) After treatment, the insects with the nanomaterial / dsRNA complex were screened to complete the RNA interference.

8. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 7, characterized in that, The fluorescent group mentioned in step 1 is a red fluorescent group.

9. The method for interfering with RNA of endoparasitic wasps in lepidopteran larvae according to claim 7, characterized in that, The incubation time described in step 1 is 10-15 minutes.

10. The method for interfering with RNA from endoparasitic wasps in lepidopteran larvae according to claim 7, characterized in that, Step 3, the screening steps, include: incubating in the dark for 12-36 hours, and screening for fluorescent insects.

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