CN112195180A - dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F and application - Google Patents
dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F and application Download PDFInfo
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Abstract
The invention discloses a dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F and application thereof, wherein the dsRNA sequence is shown as SEQ ID NO. 1. Through RNAi interference technology, after dsRNA interference is injected to interfere with a male brown planthopper Selenoprotein F gene, the vas deferens of the male brown planthopper is obviously deformed, the number of sperms in a female insect germ cell mated with the male brown planthopper is obviously reduced, the egg laying amount and the egg hatching rate of the brown planthopper are effectively reduced, the number of brown planthopper populations is controlled, and the function of ecological prevention and control is expected to be fully exerted while the pests are inhibited. The invention can be widely applied to crop breeding, biological pesticide research and development and biological control.
Description
Technical Field
The invention relates to the technical field of molecular biology, in particular to a dsRNA sequence for regulating and controlling a male brown planthopper seminal fluid Selenoprotein F and application thereof.
Background
Nilaparvata lugens (Nilaparvata lugens) for brown plant hopper
Belongs to the hemiptera and the plant hopper family, and is the leading pest on rice in China and many Asian countries. Brown planthopper is a monophagic pest and can only feed and reproduce offspring on rice and common wild rice. When the rice is seriously damaged by the brown planthopper, the rice can be killed in a large area, and the phenomenon of burning of the brown planthopper occurs. Once the brown planthopper is outbreaked and forms a disaster, serious loss can be caused to the rice production. Currently, the prevention and treatment work of brown planthopper is mainly chemical prevention and treatment. However, with the long-term use of a large amount of various chemical agents, the brown planthopper is easy to generate drug resistance, the overuse of the chemical agents is further promoted, serious environmental pollution is caused, the health of human beings is threatened, and the agricultural ecological balance is damaged. Therefore, in the practice of agricultural production, there is an urgent need to find an alternative chemical control method.
RNAi technology is a highly conserved gene regulatory mechanism that prevents the expression of a gene by degrading the mRNA of a target gene or the non-transcribed fragment binding at the 3' end of the mRNA, resulting in the silencing of a specific gene. RNAi has the advantages of target specificity, high efficiency, durability, rapidness and the like, and is widely used for the research of gene functions of various organisms. In the field of pest control, RNAi technology has been accepted by people in recent years as a novel pest control strategy, and is successfully applied to lepidoptera, coleopteran, hemiptera and other pests under laboratory conditions. In RNAi research, the proper target gene and proper introduction method are the key to its success. In the study of entomology, microinjection, feeding, and transgenic plant methods are mainly used.
Selenoprotein F can protect semen in mammals and plays an important role in male fertility. Selenoprotein depletion causes sperm motility to be reduced and sperm life to be shortened.
Disclosure of Invention
The purpose of the invention is as follows: the invention aims to provide a dsRNA sequence for regulating and controlling the seminal fluid Selenoprotein F of a male brown planthopper.
The invention also aims to provide application of the dsRNA sequence in preparing a medicament for preventing and controlling brown planthopper.
The last purpose of the invention is to provide an RNAi interference method for regulating and controlling the semen of the male brown planthopper.
The invention utilizes the transcriptome data of the constituent components of the brown planthopper seminal fluid protein to obtain a Selenoprotein Selenoprotein F gene for regulating and controlling the male brown planthopper seminal fluid, and takes cDNA of the gene as a template and designs a primer to synthesize corresponding dsRNA. The influence of reproductive functions such as sperm transmission of the brown planthopper and the like is adjusted and controlled by dsRNA microinjection.
The technical scheme is as follows: the invention provides a dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F, which is shown as SEQ ID NO. 1.
A primer that amplifies the dsRNA sequence of claim 1 as set forth in SEQ ID NO: 2. SEQ ID NO: 3, respectively.
Use of the dsRNA sequence of claim 1 in preparation of a medicament for controlling brown planthopper.
An RNAi interference method for regulating and controlling the semen of the male brown planthopper, wherein the dsRNA sequence of claim 1 is injected into the newly emerged brown planthopper imago by a microinjection method.
Further, the brown planthopper is a single brown planthopper.
Further, the amount of dsRNA injected by single brown planthopper is 80ng-100 ng.
Has the advantages that: the dsRNA is used for RNAi injection of newly emerged male brown planthoppers, after 1 day, 3 days, 5 days and 7 days after dsRNA injection, the relative expression level of the seleninoprotein F in the male brown planthoppers is obviously reduced, meanwhile, the transmission process of the brown planthoppers sperms is blocked, the egg laying amount and the egg hatchability of the brown planthoppers are reduced, the number of offspring populations is obviously reduced, and the number of the brown planthoppers is effectively controlled.
Drawings
FIG. 1: amplifying an electrophoretogram of the dsRNA, wherein M is Marker, and dsSelenoprotein F is the dsRNA amplified by the invention;
fig. 2: schematic diagram of injecting brown planthopper by using dsRNA of the invention;
FIG. 3: the expression quantity of the Selenoprotein F gene after the male brown planthopper injects dsRNA is shown in a graph, and different lower case letters on the histogram show that the P is more than 0.05 and has significant difference;
FIG. 4: effect of silencing Selenoprotein F Gene by Male Nilaparvata on Male internal genitalia and their mating on female (A: female fertilized sac of dsGFP control group; B: female fertilized sac of dsSelenoprotein F treatment group; C: male vas deferens of dsGFP control group; D: male vas deferens of dsSelenoprotein F treatment group; E, F: male internal genitalia of dsGFP control and dsSelenoprotein F treatment, respectively);
FIG. 5: the influence of the silent Selenoprotein F gene on the reproductive parameters of its mating females, different lower case letters on the histogram indicate significant differences at the level P < 0.05 (A: the amount of eggs laid; B: the pre-egg laying period; C: the egg laying period; D: the number of eggs laid by 15 females during the egg laying period (in days)).
Detailed Description
EXAMPLE 1 Synthesis of dsRNA
(1) Selecting adult male brown planthopper in the insect breeding room, and extracting the total RNA of the brown planthopper male by using an SV total isolation system Kit (Promega, Corporation, Madison, Wis., USA);
(2) removing genomic DNA by using a PrimeScript RT reagent Kit with gDNA Eraser Kit of Takara, and carrying out reverse transcription to synthesize a cDNA template;
(3) downloading a full-length sequence of Selenoprotein F (gene accession number: XM022339255) from NCBI, designing a dsRNA Primer for interfering Selenoprotein F by using software Primer Premier 5.0, wherein the length of a dsRNA template sequence is 226bp, the sequence of the Primer is shown in Table 1 (underlined is a T7 promoter), the designed Primer is synthesized by Shanghai biological technology limited department, and PCR amplification is carried out by Premix TaqTM of Takara (94 ℃, pre-denaturation 5 min; 94 ℃, denaturation 40 s; 55 ℃, annealing 40 s; 72 ℃ extension 1min, 30 cycle reactions; 72 ℃ final extension 10min) to obtain a target fragment;
TABLE 1
(4) After determining the size of the PCR product through Agarose Gel electrophoresis, purifying the PCR product through a MiniBEST Agarose Gel DNA Extraction Kit Ver 4.0 of Takara, connecting the PCR product with a pGEM-T easy vector, and transforming competent cells;
(5) adding LB liquid culture medium without ampicillin (Amp) antibiotics into the transformed competent cells, culturing for 1-2 hours at 37 ℃, smearing the competent cells into LB solid culture medium with Amp (100 mu g/mL) antibiotics, culturing overnight at 37 ℃, and selecting white round and plump monoclonal colonies;
(6) a fresh LB broth containing Amp (50. mu.g/mL) antibiotics was added to the colonies, and plasmids were extracted using the MiniBEST Plasmid Purification Kit Ver 4.0 of Takara, and PCR amplification was performed using the plasmids as templates to synthesize dsRNA having the sequence of SEQ ID No. 1.
EXAMPLE 2 dsRNA microinjection
Subjecting newly emerged male brown planthopper to CO2After anesthesia, injecting the dsRNA synthesized by the invention from the back of the chest of the male brown planthopper (100 ng/head) by using a microinjection instrument, and pulling out the needle after staying for 25-30 seconds; after injection, after 1, 3, 5 and 7 days of eclosion, brown planthopper samples are collected, the change of the expression level of the Selenoprotein F gene is detected, and dsRNA synthesized by taking a green fluorescent protein GFP gene as a template is injected into a control group.
Example 3 silencing of Selenoprotein F Gene by Male Nilaparvata lugens effects on sperm transduction
Taking brown planthopper female adults 2 days after mating, dissecting complete fertilized sacs and various male internal genitals 2 days after eclosion treatment, removing fat bodies and micro-tracheas attached to the internal genitals, cleaning for 3 times by PBS (phosphate buffer solution), adding 4% paraformaldehyde to fix in a wet box for 2h after 10min each time, allowing 1% Triton-100(PBS preparation) to permeate for 40min, and marking cell nuclei by DAPI dye after washing. Finally, the tissue was clamped to a glass slide with micro-forceps, the anti-fluorescence quenching blocking tablet was added dropwise to the sample and carefully covered with a cover slip. Blue nuclei (405nm) were observed under a laser confocal microscope (Zeiss LSM 780, Carl Zeiss Microimaging, Gottingen, Germany) and photographed.
Example 4 quantitative evaluation of application of Male Nilaparvata nilotin F Gene in Nilaparvata nilotica population control
The newly emerged male brown planthopper is subjected to dsRNA microinjection and randomly mated with untreated virgin females at one end, the virgin females are transferred into a glass (the diameter is 10cm, the height is 12cm) containing rice stems in the tillering stage to be continuously raised (the temperature is 26 +/-2 ℃, the humidity is 80 percent, the photoperiod is 16h/8h), and the water depth in the glass is about 1.5 cm. Replacing fresh rice stems in the tillering stage every 24h in the early stage of spawning, and replacing fresh rice stems in the tillering stage every 48h in the second stage of spawning until female insects die. Each treatment is provided with 15 mating groups, and the pre-spawning period (from the emergence of the female insects to the beginning of spawning), the spawning period, the life span and the spawning amount of the female insects and the total spawning amount of the female insects in 15 mating groups in each mating group are recorded.
Two processing groups are set: (1)
as a control group; (2) dsSelenoprotein
As a treatment group. In each treatment, 4 pairs of emerging male and female worms are taken and inoculated to rice in a tillering stage, and the rice is planted in plastic barrels with the diameter of 16cm (4 holes in each barrel and 3 plants in each hole). Counting the number of generations when 3-year nymphs appear on rice stalks (about 25 days), then transferring the 3-year nymphs to plastic bucket-planted rice at a new tillering stage to grow to adults, and counting the sex ratio of male and female filial generations; at the same time dissecting and hatching out nymph of brown planthopperRice shoots were further counted for egg hatchability, and brown planthopper population growth index (PGI ═ N1/No) was calculated for 5 independent biological replicates per treatment. N1-number of progeny Nilaparvata lugens population + eggs of unhatched Nilaparvata lugens, No-number of initial brown planthoppers released
The results show that:
(1) according to example 2, it was found that: after 1 day, 3 days, 5 days and 7 days of emergence of brown planthopper injected with dsRNA, the relative expression level of the Selenoprotein F gene in the treatment group is obviously reduced (figure 3).
(2) According to example 3, it was found that: the vas deferens of brown planthopper males injected with the dsRNA of the invention were misshapen (fig. 4C-4D) compared to the control group, and the misshapen vas deferens affected less sperm to females during male mating. After the male and female worms injected with dsRNA mate, the fertilized sac of the female worm is found to be small in volume, and the number of sperms in the female worm is also remarkably reduced (fig. 4A-4B).
(3) According to example 4 it was found that: after the dsSelenoprotein F treated males mate with the females, the average egg production of the females decreased significantly (fig. 5A), the pre-egg production period of the females was extended (fig. 5B), but the egg production period had no effect (fig. 5C). The egg laying amount of 15 females in the treated group (1832) was significantly lower than the total egg laying amount of 15 females in the control group (4204) (fig. 5D). The egg laying amount of the 15 females in the treated group is obviously lower than that of the control group between 1-3 d and 21-24 d. Within the time interval of 24-27D, the treated group females were ovipositing and were not ovipositing at all (FIG. 5D). The male worms and female worms with silent Selenoprotein F genes mate to result in a significant reduction in the number of offspring (Table 2), and the population growth index is significantly reduced, but the sex ratio of the offspring is not affected.
TABLE 2
Sequence listing
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Claims (6)
1. A dsRNA sequence for regulating and controlling male brown planthopper seminal fluid Selenoprotein F, which is shown as SEQ ID NO. 1.
2. A primer that amplifies the dsRNA sequence of claim 1 as set forth in SEQ ID NO: 2. SEQ ID NO: 3, respectively.
3. Use of the dsRNA sequence of claim 1 in preparation of a medicament for controlling brown planthopper.
4. An RNAi interference method for regulating and controlling the semen of a male brown planthopper is characterized in that: the dsRNA sequence of claim 1 is injected into a newly emerged adult male brown planthopper by microinjection.
5. The method of claim 4, wherein: the brown planthopper is a single brown planthopper.
6. The method of claim 5, wherein: the amount of dsRNA injected by a single brown planthopper is 80ng-100 ng.
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| CN115925856A (en) * | 2022-07-19 | 2023-04-07 | 浙江大学 | Brown planthopper heat shock transcription factor NlHsf1, cDNA and application |
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- 2020-10-19 CN CN202011122641.6A patent/CN112195180A/en active Pending
Non-Patent Citations (1)
| Title |
|---|
| LINQUAN GE等: "Male Selenoprotein F-Like (SPF-L) Influences Female Reproduction and Population Growth in Nilaparvata lugens (Hemiptera: Delphacidae)", 《FRONTIERS IN PHYSIOLOGY》, vol. 10, 25 September 2019 (2019-09-25), pages 2 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115925856A (en) * | 2022-07-19 | 2023-04-07 | 浙江大学 | Brown planthopper heat shock transcription factor NlHsf1, cDNA and application |
| CN115925856B (en) * | 2022-07-19 | 2024-05-28 | 浙江大学 | Brown planthopper heat shock transcription factor NlHsf, cDNA and application |
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