Application of reproductive development-related gene E75 in prevention and control of bactrocera dorsalis

Abstract

The application discloses application of a Bactrocera minax gene E75 , the gene E75 participates in regulating testis development and fecundity of male Bactrocera minax, after designing dsRNA silencing E75 targeting the gene E75 , the male Bactrocera minax appears extremely obvious testis development retardation, and the egg-laying amount and hatching rate of the female Bactrocera minax after mating are obviously lower than those of a control group. After loading ds E75 on two kinds of nanometer materials CS-TPP and SPc, the feeding method RNAi efficiency is obviously improved, the fecundity is obviously decreased, and the number of offspring larvae is obviously reduced, so that the purpose of controlling the population number of Bactrocera minax can be achieved.

Description

TECHNICAL FIELD

[0001] The application belongs to the field of pest green control and genetic engineering, and particularly relates to application of a gene E75 related to reproductive development of Bactrocera dorsalis in control of Bactrocera dorsalis. BACKGROUND

[0002] Bactrocera dorsalis (Hendel) belongs to the order Diptera, the family Tephritidae, and the genus Bactrocera, and causes damage to various fruits and vegetables. Therefore, it is of great significance to develop green control measures for Bactrocera dorsalis for agricultural production.

[0003] The sterile insect technique (SIT) is a green pest control strategy for releasing a large number of sterilized male insects to mate with wild female insects, thereby greatly reducing the number of offspring and reducing the population of the pest or completely eradicating the pest. Because of its target specificity and little impact on the environment, the SIT has been used in many countries to control fruit fly pests that cause significant economic losses. However, traditional radiation sterilization can easily lead to poor ecological fitness and reduced mating competitiveness of sterile males. RNA interference (RNAi)-based SIT can provide an alternative method for traditional radiation sterilization to obtain sterile males without affecting their mating ability and lifespan. Feeding RNAi can cause gene silencing, but the delivery efficiency of directly fed dsRNA is limited, and the use of nanomaterials to encapsulate dsRNA can significantly improve the efficiency of RNAi.

[0004] Male reproductive development is a complex process that is regulated by spermatogenesis-related genes and the endocrine system. Studies have shown that the ecdysone signaling pathway in Drosophila can regulate male courtship behavior. Key genes in the ecdysone pathway, such as EcR-USP, FTZ-F1, E78, and E74, are related to the maintenance of male germ stem cells, the regulation of sperm length, the formation of accessory glands, and the formation of seminal plasma proteins.

[0005] Chitosan-tripolyphosphate (CS-TPP) nanoparticles as a new type of gene carrier have the characteristics of non-toxicity, good biocompatibility and biodegradability, which can improve the efficiency of gene delivery. In Aedes aegypti, CS-TPP nanoparticles improve the silencing effect of target genes, and the mortality rate of Aedes aegypti reaches more than 60%. The new star cationic polymer SPc (Star polycation) has the advantages of low cost, high nucleic acid binding efficiency, excellent nucleic acid delivery performance, high biological safety and high water solubility, which is a good nucleic acid carrier in biological applications. Using SPc to deliver dsRNA can significantly down-regulate the expression of target genes in small tiger moths by about 60%, and inhibit the growth and development of larvae. In addition, SPC has achieved good results in the control of pests such as Chilo suppressalis and aphids. The application of CS-TPP and SPc in these pests shows that they have wide field application and can be used as excellent gene carriers to improve the feasibility of RNAi in pest control. SUMMARY

[0006] In order to solve the above problems, the application selects the key gene E75 of Bactrocera dorsalis male sterility with application value by RNAi technology, and uses the low-cost nanomaterials CS-TPP and SPc to wrap the dsRNA induced by E. coli and feed Bactrocera dorsalis, which can significantly weaken the fertility of male insects and thus significantly reduce the number of offspring.

[0007] In order to achieve the above purpose, the application adopts the following technical solutions:

[0008] Application of Bactrocera dorsalis gene E75: by targeting silencing Bactrocera dorsalis reproductive development related gene E75, the reproductive development of Bactrocera dorsalis male insects is inhibited, and the number of Bactrocera dorsalis population is controlled. Further, the dsRNA for targeting silencing Bactrocera dorsalis gene E75 is composed of nucleotides and nucleotides complementary to the sequence as shown in SEQ ID NO. 1.

[0009] Preferably, the dsRNA for targeting silencing Bactrocera dorsalis gene E75 is fed to Bactrocera dorsalis male insects after being loaded by CS-TPP nanoparticles and SPc nanoparticles.

[0010] The compound for controlling Bactrocera dorsalis includes the dsRNA for targeting silencing Bactrocera dorsalis gene E75. Further, the dsRNA is composed of nucleotides and nucleotides complementary to the sequence as shown in SEQ ID NO. 1. Preferably, the dsRNA is loaded by CS-TPP nanoparticles and SPc nanoparticles.

[0011] Compared with the prior art, the application has the following advantages and beneficial effects:

[0012] The application solves the problems of poor ecological adaptability and decreased mating competition ability of sterile male insects caused by traditional radiation sterility; the dsRNA expressed by E. coli solves the problem of high cost of synthesizing dsRNA by using a kit; and the nano-material mediated feeding method RNAi also solves the problems of complicated conditions of injection method RNAi which is not suitable for field application and low efficiency of feeding method RNAi. The application establishes a high-efficiency and stable dsRNA delivery system and field simulation control technology in B. dorsalis, which not only lays a theoretical foundation for the application of nano-materials in B. dorsalis RNAi, but also provides practical guidance for the green control technology based on SIT technology of RNAi. BRIEF DESCRIPTION OF DRAWINGS

[0013] Figure 1 : Testis phenotype of B. dorsalis after feeding method RNAi of nano-materials loaded with dsE75. Testis morphological dissection diagram of 4-day-old male B. dorsalis after feeding method RNAi, A1, A2 and A3 are testis dissection diagrams of the dsEGFP feeding group, B1, B2 and B3 are testis dissection diagrams of the dsE75 feeding group, C1, C2 and C3 are testis morphological dissection diagrams of the CS-TPP-dsE75 feeding group, and D1, D2 and D3 are testis morphological dissection diagrams of the SPc-dsE75 feeding group. Scale bar: 500 μm.

[0014] Figure 2 : Influence of feeding method RNAi of nano-materials loaded with dsE75 on mating competitiveness. From left to right, they are the dsE75 interference group, the CS-TPP-dsE75 interference group and the SPc-dsE75 interference group.

[0015] Figure 3 : Influence of feeding method RNAi of nano-materials loaded with dsE75 on mating latency. Mating latency statistics are placed in a transparent box with male and female B. dorsalis until the time (min) required for mating begins.

[0016] Figure 4 : Influence of feeding method RNAi of nano-materials loaded with dsE75 on the fecundity of male B. dorsalis. (A) Total amount of eggs laid by a single female, (B) hatching rate, p<0.05.

[0017] Figure 5 : Influence of feeding method RNAi of nano-materials loaded with dsE75 on the sperm activity of B. dorsalis. Green fluorescence represents live sperm, and red fluorescence represents dead sperm. Scale bar: 100 μm.

[0018] Figure 6Effect of dsE75 fed by nanomaterial on the number of sperm and the percentage of dead sperm. The number of live sperm and dead sperm was counted by Image J, and Tukey multiple comparison was used. (A) Total number of sperm, (B) Sperm mortality rate, p<0.05.

[0019] Figure 7 Expression of spermatogenesis-related genes in testis after E75 interference. The relative expression of spermatogenesis-related genes in the testis of Bactrocera dorsalis was detected after the E75 gene was silenced, p<0.05.

[0020] Figure 8 Simulation experiment of nanomaterial loaded with dsRNA. (A) dsEGFP loaded by CS-TPP, namely CS-TPP-dsEGFP; (B) dsE75 loaded by CS-TPP, namely CS-TPP-dsE75; (C) dsEGFP loaded by SPc, namely SPc-dsEGFP; (D) dsE75 loaded by SPc, namely SPc-dsE75.

[0021] Figure 9 Effect of field simulation experiment. A-D are process record pictures in the simulation field control experiment, (A) Bactrocera dorsalis ovipositing; (B) yellow-brown depression appeared on the surface of the honey orange where the eggs were laid; (C, D) larvae feeding. (E) is the analysis of the number of offspring larvae in the field simulation experiment. The number of larvae in 4 batches of honey oranges placed in each cage was counted (counted on the 4th, 8th, 12th and 16th day after placement), and Tukey multiple comparison was used for the total number of 4 batches of larvae, p<0.05. DETAILED DESCRIPTION

[0022] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. The test materials used in the following examples can be obtained by purchase unless otherwise specified.

[0023] Example 1: E. coli expression of Bactrocera dorsalis E75 gene dsRNA

[0024] I. Construction of Bactrocera dorsalis E75 gene vector containing enzyme digestion site

[0025] First, the Bactrocera dorsalis E75 gene containing enzyme digestion site (XM_019990705) was amplified by PCR, and the primer sequences are shown in Table 1.

[0026] Table 1 Primer sequences for cloning fragments of target genes of Bactrocera dorsalis

[0027]

[0028] Bold part is the enzyme digestion site, GAGCTC is the Sac I recognition site; AAGCTT is the Hind III recognition site. (The control group EGFP E. coli expression vector uses the strain previously constructed by the laboratory, which can be constructed by referring to the method of E75 expression vector or can be purchased commercially)

[0029] 2) After agarose gel electrophoresis of the PCR product, the gel was cut and recovered.

[0030] 3) Plasmid L4440 (laboratory preserved, also commercially available) and PCR product double enzyme digestion reaction: the reaction system is shown in the table below, and the enzyme digestion is carried out at 37°C for 3h. The enzyme digestion product was subjected to agarose gel electrophoresis, and the gel was cut and recovered.

[0031]

[0032] 4) Ligation of plasmid L4440 and exogenous gene fragment: the reaction system is shown in the table below, and the ligation is carried out at 16°C overnight.

[0033]

[0034]

[0035] 5) Transformation of the ligation product: take out the Trans 5α competent cells stored in the -80°C refrigerator, thaw on ice, and when it is just thawed, add 50 μL competent cells to 5 μL ligation product, gently centrifuge the tube and mix, ice bath for 30 min (keep still during the process). 42°C heat shock for 45s, immediately placed in ice bath for 2 min. Add 250 μL of antibiotic-free LB liquid medium, and incubate at 37°C on a shaker at 220 r / min for 1h. Take 50 μL of bacterial solution and spread on LB agar containing 60 μg / mL ampicillin resistance, and incubate overnight.

[0036] II. Identification of recombinant vector

[0037] Pick single colony for culture: inoculate white colonies in LB liquid medium containing 60 μg / mL ampicillin resistance and incubate at 37°C at 220 r / min for 4h. Take 1 μL of bacterial solution as PCR reaction template, and use M13F and L4440R as primers to identify the recombinant vector. The colonies with correct fragment size are sent for sequencing verification.

[0038] III. HT115 strain transformation

[0039] The recombinant vector L4440-E75 with correct sequence is transformed into competent E. coli HT115. Refer to the method of constructing orange fruit fly E75 gene vector containing enzyme digestion site. After transformation, the expression vector is identified by referring to the method of identifying the recombinant vector.

[0040] Four: dsRNA expression

[0041] PCR and sequencing to identify the correct HT115 positive bacteria 200 μL in 8 mL LB liquid medium (containing 100 μg / mL ampicillin, 50 μg / mL tetracycline), 37°C, 220r / min shaking culture 8h. The next day, inoculate 2xYT liquid medium (containing 75 μg / mL ampicillin, 12.5 μg / mL tetracycline) in a ratio of 1:100 to expand the culture, 37°C, 220r / min shaking culture to OD 600 0.4-0.6, add IPTG to a final concentration of 0.4mM, continue to shake induction culture 4-5h.

[0042] Five: dsRNA extraction

[0043] 1) Take 40mL of bacteria after induction into a centrifuge tube, 10000r / min, centrifuge for 10min, collect the cell precipitate.

[0044] 2) Add 20mL 1M NH4Ac to dissolve the precipitate, then add 20mL phenol: chloroform: isopropyl alcohol (volume ratio 25:24:1) and mix well, incubate at 65°C for 15min. 10000r / min, centrifuge for 15min, collect the supernatant, precipitate dsRNA with equal volume of isopropanol, ice bath for 30min.

[0045] 3) 12000r / min, 4°C centrifugation for 30min, discard the supernatant. Wash the precipitate with 1mL 75% ethanol.

[0046] 4) Dry the RNA for 5min, dissolve the precipitate in 2mL RNase-free ddH2O.

[0047] 5) Add RNase-free DNase and RNase A solution to the total nucleic acid, 37°C water bath for 30min.

[0048] 6) dsRNA is detected by agarose gel electrophoresis and quality analysis is performed by Nanodrop2000, and the product is stored in a ultra-low temperature freezer at -80°C.

[0049] The dsRNA is named dsE75, which consists of a sense strand and an antisense strand, and the nucleotide sequence of the sense strand is shown in SEQ ID NO. 1, and the nucleotide sequence of the antisense strand is complementary to the sequence shown in SEQ ID NO. 1.

[0050] Example 2: Preparation of nanomaterial delivery dsRNA system

[0051] I. Preparation of CS-TPP-dsRNA nanoparticles

[0052] Reference Dhandapani et al. (Article "Development of CS-TPP-dsRNA nanoparticles to enhance RNAi efficiency in the yellow fever mosquito"), ion cross-linking method was used to prepare CS-TPP-dsRNA nanoparticles with sodium tripolyphosphate (TPP) as cross-linking agent:

[0053] 1) Prepare 100 mM sodium sulfate solution, 0.1 M sodium acetate buffer, 0.02% TPP solution and 0.02% CS solution at room temperature;

[0054] 2) Add 32 μg dsRNA to 100 μL TPP (0.02%) solution;

[0055] 3) Add 500 μL of 0.02% chitosan solution to the solution, heat in a 55°C water bath for 1 min;

[0056] 4) Immediately mix the solution by high-speed vortexing at room temperature for 30 s to facilitate the formation of nanoparticles; centrifuge the mixture at 13000 x g for 10 min at room temperature, and white particles can be seen, which are CS-TPP-dsRNA nanoparticles.

[0057] II. Preparation of SPc-dsRNA nanoparticles

[0058] The preparation method of SPc-dsRNA nanoparticles is as follows: mix 1 mg / mL SPc nano-preparation (obtained from the team of Professor Shen Jie of China Agricultural University, for details, please refer to the article published by the team in 2019: A Facile-Synthesized Star Polycation Constructed as a Highly Efficient Gene Vector in Pest Management.) with dsRNA at a mass ratio greater than or equal to 2:1, high-speed vortex the mixture for 30 s, and incubate at room temperature for 30 min to facilitate the formation of SPc-dsRNA nanoparticles.

[0059] Example 3: Application of dsE75 feeding method RNAi with nano-materials in B. dorsalis

[0060] I. Feeding of dsE75 loaded with nano-materials

[0061] After the E75 dsRNA expressed by E. coli was loaded into nanoparticles, it was coated on artificial feed. The control groups were dsE75, dsEGFP and dsEGFP loaded with nano-materials.

[0062] II. Observation of testis morphology

[0063] Dissect the testes of male adults at 4 days old, which is the 3rd day after feeding with nanomaterial-loaded dsRNA. Observe the testis morphology under bright field using an OPLYMPUSSZX16 stereomicroscope, and take pictures to record the testis development of each male. There are 8 individuals in each treatment. The results show that the anatomical diagrams of the testes of the control group dsEGFP are as shown in A1, A2, A3( Figure 1 ). The color is yellow and the testes are plump; in the dsE75 group of the treatment group, testicular albinism appears as shown in B1, and the development of one-sided testes is smaller in B2; the testicular morphology abnormality of dsE75 wrapped with nanomaterials is more obvious: in CS-TPP-dsE75, the albinism in C1 is more obvious and the individuals are smaller, and the bilateral testes are malformed in C2; in the SPc-dsE75 group, albinism appears in D1 and D3, and the development of one-sided testes is smaller in D2. There are 8 replicates in each of the 4 treatments A, B, C, and D: the testicular morphology of the dsEGFP group is normal; there are 2 pairs of testes with albinism in the dsE, 2 pairs of testes are albino and 1 pair of testes is malformed in CS-TPP-dsE75, and 3 pairs are albino and 1 pair of one-sided testicular morphology is abnormal in the SPc-dsE75 group. Among them, the abnormal ratios of testicular morphology in the dsEGFP group, dsE75 group, CS-TPP-dsE75 group, and SPc-dsE are 0, 25%, 37.5%, and 50% respectively. The testicular morphology of all dsE75 treatment groups is abnormal, indicating that the E75 gene plays a key role in the testicular development of Bactrocera dorsalis. The probability of abnormal testicular development in the CS-TPP-dsE75 group and SPc-dsE75 group wrapped with nanomaterials increases, which also shows that with the increase of interference efficiency, the phenotypic abnormality is more obvious.

[0064] III. Detection of male reproductive ability

[0065] 1. Mating competitiveness and mating latency

[0066] Mark the backs of male insects in the E75 treatment group of the experimental group and male insects in the EGFP group of the control group with different colors of acrylic paint. Put 15 individuals of each into the same transparent box with a size of 14.4 cm × 8.5 cm × 8.5 cm (length × width × height, and the diameter is 6.6 cm). After 2 hours, put 15 virgin female insects as a group. There are 3 replicates, and count the number of mating pairs in the experimental group and the control group. And count the time required from being put in until mating starts (i.e., mating latency). Use the Graph pad Primer7 software to perform a t-test to analyze the difference in mating rate between the treatment group and the control group. The results of mating competitiveness are as Figure 2 . There is no significant difference in all three treatments. The results of mating latency are as Figure 3No significant difference was observed among the four groups of dsEGFP, dsE75, CS-TPP-dsE75 and SPc-dsE75. This indicates that interfering with the E75 gene does not affect the mating latency of male B. dorsalis, and adding CS-TPP and SPc does not affect the mating latency of male B. dorsalis.

[0067] 2. Statistics of egg production and egg hatch rate

[0068] The 13-day-old male B. dorsalis treated with nano-material loaded dsRNA were placed together with the same proportion of 13-day-old female B. dorsalis that were not mated alone. The egg production was observed and counted every other day after mating, and the egg hatch rate was counted. The results of the total egg production of single female are shown in Figure 4 A, the total egg production of single female in the dsEGFP group was 278.1 ± 20.15, in the dsE75 group was 168.9 ± 22.58, in the CS-TPP-dsE75 group was 131.6 ± 26.33, and in the SPc-dsE75 group was 85.1 ± 18.17. The results showed that the total egg production of single female in all dsE75 treated groups was significantly reduced compared with the control group dsEGFP, and there was a significant difference in the total egg production between the SPc-dsE75 group and the dsE75 group, indicating that the dsE75 group wrapped with nano-material SPc can significantly reduce the egg production. The results of the hatch rate are shown in Figure 4 B, the hatch rate of the dsEGFP group was 74.48%, the dsE75 group was 39.20%, the CS-TPP-dsE75 group was 27.50%, and the SPc-dsE75 group was 29.16%. The results showed that the hatch rate of the dsE75 treated with nano-material was significantly reduced compared with the control group dsEGFP; the CS-TPP-dsE75 and SPc-dsE75 groups wrapped with nano-material had a significant difference compared with the dsE75 group without wrapping. This indicates that the dsE75 treated with nano-material wrapping further reduces the reproductive capacity of male B. dorsalis on the basis of improving the interference efficiency.

[0069] Four, sperm quality detection

[0070] 1. Sperm motility detection

[0071] Sperm motility detection kit Sperm Viability Kit (all reagents are provided by the kit) was used to detect 13-day-old male adults. The 13-day-old male adults were dissected and their sperm were stained for motility detection. The specific operation steps are as follows:

[0072] 1) Dissect the testis tissue of B. dorsalis in a culture dish containing HEPES buffer, wash 3 times and then transfer to a clean slide, one pair of testis for each treatment; put the testis in 5 μL HEPES buffer and prick the testis tissue with a pair of tweezers to make the sperm flow into the buffer.

[0073] 2) Transfer all the sperm-containing liquid to an EP tube, centrifuge quickly and then take the supernatant to a new EP tube.

[0074] 3) Prepare the working concentration solution of SYBR-14 in the dark: add 98 μL of HEPES buffer to 2 μL of the storage concentration of SYBR-14. Mix the working concentration solution of SYBR-14 and the supernatant sperm evenly at a ratio of 1:1, incubate at room temperature in the dark for 10 min; add 2 μL of propidium iodide and incubate at room temperature in the dark for 7 min.

[0075] 4) Take pictures in two channels using a Leica laser scanning confocal microscope (Leica TSC SP8), dead sperm are red and live sperm are green.

[0076] 5) Use Image J to count the number of dead and live sperm, at least 5 pictures for each group, and use Graph pad Primer 7 software to analyze the differences between groups by Tukey multiple comparison.

[0077] The number and viability of sperm were detected by double fluorescence staining, live sperm were green and dead sperm were red. By directly comparing the number of green and red sperm in each group, it can be seen that the number of live sperm and the total number of sperm in the dsEGFP treatment group are the most. Figure 5 Further statistical analysis found that there was no significant difference in the total number of sperm between the dsE75 group and the dsEGFP group, and the CS-TPP-dsE75 group and the SPc-dsE75 group were significantly different from the previous two groups, indicating that the two dsE75 treatment groups with added nanomaterials could significantly reduce the total number of sperm ( Figure 6 A). The sperm mortality rate of the three dsE75 treatment groups was significantly higher than that of the dsEGFP group, indicating that the dsE75 treatment inhibited the sperm viability of B. dorsalis ( Figure 6 B). However, it is difficult to clearly analyze the effect of nanomaterials on the number and viability of sperm, because the changes in the groups with and without nanomaterials are not consistent in the A and B figures, but it can be determined that the dsE75 gene can affect the viability of sperm ( Figure 6 ).

[0078] 2, Spermatogenesis-related gene detection

[0079] The expression of spermatogenesis-related genes in the testis after E75 interference was verified by qRT-PCR. The synthesis of related primers was completed by Nanjing Kingsway Biotech Co., Ltd., and the primer sequences are shown in Table 2 below.

[0080] Table 2 Real-time fluorescent quantitative PCR primer sequences

[0081]

[0082]

[0083] The real-time fluorescent quantitative method was performed on a Bio-Rad CFX96 real-time fluorescent quantitative PCR instrument using a 2xT5 Fast qPCR Mix (SYBR Green I) kit. Each sample was set up in three technical replicates, and the corresponding blank control was set up as needed. The total PCR system was 20 μl, as follows. After the end of the fluorescent quantitative PCR, the melting curve was analyzed to ensure specific amplification. The results of real-time fluorescent quantitative PCR were analyzed using 2 -ΔCt Method analysis. The specific steps are as follows:

[0084]

[0085] Real-time fluorescent quantitative PCR reaction program:

[0086] Step 1: pre-denaturation

[0087] 95℃ 3min

[0088] Step 2: PCR reaction

[0089]

[0090] Step 3: melting curve analysis, temperature slowly increased from 55℃ to 94℃ at a rate of 0.5℃ / s, and the fluorescence intensity of the sample was continuously measured to obtain the melting curve, thereby judging the specificity of the amplification reaction.

[0091] After successful interference of E75, the expression of the above-mentioned spermatogenesis-related genes in the testis was detected, as shown in Table 3, the expression of fzo, hop, gudu, rho, and boul genes selected from the spermatogenesis-related genes was significantly down-regulated, suggesting that the E75 gene is involved in the regulation of spermatogenesis. Figure 7

[0092] V. Preliminary application of RNA preparation of B. dorsalis male sterility gene E75

[0093] 1. Simulated field control of B. dorsalis by feeding nanomaterial-loaded dsE75

[0094] ​The simulation control experiment is carried out in a closed greenhouse, and four groups of treatments are set: dsRNA loaded by chitosan cross-linked sodium tripolyphosphate: CS-TPP-dsEGFP and CS-TPP-dsE75, i.e. Figure 8 A and B; dsRNA loaded by star cationic polymer: SPc-dsEGFP and SPc-dsE75, i.e. Figure 8 C and D. Each group has four repetitions, and each repetition corresponds to one gold orange seedling with 8 honey oranges (honey oranges are purchased from a supermarket, and gold orange seedlings are purchased from a fruit farmer. The reason for choosing gold orange seedlings is that the gold orange seedlings are short and have many branches, and can bear fruit), and the honey oranges needed for egg production are replaced every 4 days, and a total of 4 batches of honey oranges are placed. To ensure that each gold orange seedling is an independent small ecological environment, each gold orange seedling is placed in a 40 cm x 40 cm x 60 cm nylon insect cage. Figure 8 Each honey orange-hung gold orange seedling constitutes a small ecological environment Figure 9 A). About 5 days after oviposition, yellow-brown depressions were observed on the surface of the honey oranges where eggs were laid, and a large number of egg particles and hatched first instar larvae were observed when the depressions were peeled off with tweezers Figure 9 B); about 10 days after oviposition, the number of Bactrocera dorsalis larvae was counted by peeling off the honey oranges, and the honey oranges with a large number of larvae had eroded pulp Figure 9 C, D), and most of the larvae were in the second or third instar. It was found in this experiment that the maximum number of larvae in a single fruit was 110 (statistical in this experiment), and it can be seen that Bactrocera dorsalis has strong reproductive capacity and causes serious damage.

[0095] 2. Detection of the control effect of dsE75 loaded by nanomaterials

[0096] The number of offspring larvae was counted after the simulation of control experiments as a standard for detecting the effect of nano-materials loaded with dsE75. Counting the number of offspring larvae can intuitively understand the control effect at the population level. Orange small fruit flies laid eggs on honey oranges for 4 days, then the honey oranges were taken out, and the number of larvae was counted about 7 days later, at which time the larvae were in the 2nd or 3rd instar. The number of offspring larvae (per cage) is the total number of larvae counted in the honey oranges placed at four time points. The experimental data were statistically analyzed and visualized using GraphPad Prism 7.0 and Excel software. For multiple comparisons between multiple samples, Tukey's test in one-way ANOVA was used, and the significance test level was at the p = 0.05 level. The results showed that the number of larvae in all treatment groups with nano-materials was significantly lower than that in the control group, and there was no significant difference between the CS-TPP and SPc treatment groups. The total number of larvae in each treatment was counted, and the number of offspring larvae in the CS-TPP-dsEGFP control group was 1133, and the number of offspring larvae in the CS-TPP-dsE75 treatment group was 456, with a 60% reduction in the number of offspring larvae, with a very significant difference, p < 0.001; the number of offspring larvae in the SPc-dsEGFP control group was 1159, and the number of offspring larvae in the SPc-dsE75 group was 528.3, with a 54% reduction in the number of offspring, with a very significant reduction, p < 0.001; there was no significant difference between the CS-TPP-E75 and SPc-E75 groups Figure 9 ), indicating that nano-materials loaded with dsE75 have a good effect on the control of B. tryoni.

Claims

1. Use of the gene E75 of Bactrocera dorsalis, characterized in that, The dsRNA targeting the B. dorsalis reproductive development related gene E75, consisting of nucleotides as shown in SEQ ID NO. 1 and nucleotides complementary thereto, is fed to male B. dorsalis after being loaded by CSTPP nanoparticles and SPc nanoparticles, thereby promoting abnormal development of testes of the male B. dorsalis.

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