A kind of American white moth nucleic acid pesticide microcapsule and its application

Abstract

The present invention discloses a gypsy moth nucleic acid pesticide microcapsule and its application, belonging to the technical field of biological pesticide preparation. The present invention provides a nucleic acid sequence of a SIFR gene for regulating the growth and development of the gypsy moth, and its full-length cDNA sequence is shown as SEQ ID No. 1, and discloses a dsRNA for targeted silencing of the SIFR gene as described above and the application of a bacterially expressed dsSIFR microcapsule in the control of the gypsy moth. The present invention can effectively silence the SIFR gene of the gypsy moth, resulting in an extension of the development period of the gypsy moth larvae and a significant increase in the mortality rate of the 6th instar larvae. The present invention has the advantages of being pollution-free and reducing the amount of chemical pesticides used.

Description

Technical Field

[0001] The invention belongs to the technical field of biological pesticide preparations, and in particular relates to a gypsy moth nucleic acid pesticide microcapsule and an application thereof. Background Art

[0002] The gypsy moth mainly harms trees, fruit trees, flowers and horticultural crops by feeding on leaves in its larvae, causing significant economic losses to agricultural and forestry production. At present, chemical control is still the main method. Chemical control mainly uses chemical pesticides such as cypermethrin III, avermectin and cyantraniliprole. Although cypermethrin has a good control effect on the gypsy moth, it is harmful to the growth and development of crustaceans such as shrimps and crabs. Therefore, although chemical control is quick and has low control costs, it has disadvantages such as polluting the environment, killing natural enemies, and easily developing drug resistance.

[0003] With the rapid development of biotechnology, the use of RNAi technology to control the harm of pests has become the focus and hotspot of plant protection workers. RNA interference (RNAi) is a technology that introduces endogenous or exogenous double-stranded RNA (dsRNA) into eukaryotic cells, causing specific degradation of host cell mRNA with sequences complementary to dsRNA, and effectively reducing the expression level of target genes. It is currently one of the new technologies most likely to be applied to pest control. Since RNAi technology mainly acts on specific sequences in insects, it has a high degree of target specificity, has little adverse effects on the environment and non-target organisms, and has the advantages of high efficiency and simplicity. It has been widely used in the study of gene function. RNA biopesticides based on RNAi technology have many advantages. Compared with traditional chemical pesticides, they have stronger specificity and are easily degraded in the environment. Therefore, they are green and pollution-free biopesticides. Since dsRNA is extremely easy to degrade, the mass production and stable delivery of dsRNA are crucial for the commercialization of RNA pesticide technology. Summary of the invention

[0004] In view of this, the present invention provides a SIFR gene for regulating the growth and development of the gypsy moth. Inhibiting the expression of this gene can significantly affect the growth and development of the gypsy moth, providing a new and effective way for sustainable prevention and control of the gypsy moth.

[0005] The technical solution adopted by the present invention is as follows: a SIFR gene for regulating the growth and development of the gypsy moth, whose full-length cDNA sequence is shown in SEQ ID No.1 and encodes an amino acid sequence shown in SEQ ID No.2.

[0006] The purpose of the present invention is to provide a dsRNA for inhibiting the growth and development of the gypsy moth, which can target and silence the SIFR gene as described above, wherein the dsRNA positive chain and antisense chain are composed of the nucleic acid sequences shown in SEQ ID No.3 and SEQ ID No.4.

[0007] Another object of the present invention is to provide a recombinant expression vector containing the dsRNA for inhibiting the growth and development of the cunea.

[0008] Furthermore, the construction method of the dsRNA recombinant expression vector of the SIFR gene of the gypsy moth as described above is as follows: extract the total RNA of the gypsy moth, use a reverse transcription kit to synthesize the first chain of cDNA, then use the first chain of cDNA as a template, and design primer sets on both sides of the gene coding region sequence according to the SIFR sequence obtained from the transcriptome data of the gypsy moth larvae: the upstream primer SIFR-F is shown as SEQ ID No.5, and the downstream primer SIFR-R is shown as SEQ ID No.6; then, PCR amplification is performed using the first chain of cDNA as a template to obtain the target fragment HcSIFR, and the dsRNA silencing region is determined according to the conserved structural domain of the cDNA sequence of the fragment HcSIFR, and the primer set of the HcSIFR forward fragment is shown as SEQ ID No.7-8 and the primer set of the HcSIFR reverse fragment is shown as SEQ ID As shown in No.9-10, the fragment HcSIFR is PCR amplified using the primer set of the HcSIFR forward fragment to obtain the target HcSIFR forward fragment, the target HcSIFR forward fragment and the expression vector pET-28a(+) are double-digested with Xho I and Hind III respectively, the desired fragment is recovered, connected and constructed recombinant plasmid pET-28a(+)-dsSIFR(I); then the fragment HcSIFR is PCR amplified using the primer set of the HcSIFR reverse fragment to obtain the target HcSIFR reverse fragment, the target HcSIFR reverse fragment and the recombinant plasmid pET-28a(+)-dsSIFR(I) are double-digested with SacI and BamH I respectively, the desired fragment is recovered, connected and constructed recombinant plasmid pET-28a(+)-dsSIFR, thereby completing the construction of the recombinant expression vector pET-28a(+)-dsSIFR.

[0009] Another object of the present invention is to provide a recombinant bacterium containing a recombinant expression vector of dsRNA for inhibiting the growth and development of the American white moth as described above.

[0010] Furthermore, the method for constructing the recombinant bacteria as described above is as follows: transforming the constructed recombinant expression vector pET-28a(+)-dsSIFR into competent cells of Escherichia coli HT115(DE3), thereby completing the construction of the recombinant bacteria.

[0011] Another object of the present invention is to provide a nucleic acid pesticide microcapsule for controlling the gypsy moth, comprising a capsule core and a capsule wall, wherein the capsule core contains the recombinant bacteria as described above.

[0012] Furthermore, the recombinant bacteria described above are Escherichia coli HT115 (DE3) containing the recombinant expression vector pET-28a (+) -dsSIFR, and the amount of the recombinant bacteria is 2.21-3.07×10 5 cfu / g.

[0013] Another object of the present invention is to provide a nucleic acid pesticide microcapsule for controlling the gypsy moth as described above, which has a water content of 28.29% and a swelling rate of 249.18%.

[0014] Further, the preparation method of the nucleic acid pesticide microcapsule for controlling the American white moth as described above is as follows:

[0015] Step 1, mixing a sodium alginate solution having a concentration of 1% to 3% by mass and a recombinant bacterial suspension in a volume ratio of 2:1 to 1:2 to form a mixed solution;

[0016] Step 2, spraying the mixed solution from the nozzle of the microcapsule granulator into a continuously stirred calcium chloride solution with a concentration of 2% to 4% by mass to form microcapsules;

[0017] Step 3: Allow the microcapsules to stand and solidify in the calcium chloride solution for 15 minutes, filter, wash, and dry to obtain microcapsules.

[0018] Advantages and beneficial effects of the present invention: The present invention provides a nucleic acid sequence of the SIFR gene for regulating the growth and development of the gypsy moth and a sequence encoding an amino acid, and also provides a dsRNA for inhibiting the growth and development of the gypsy moth, which can target and silence the SIFR gene, and the use of bacterially expressed dsSIFR microcapsules in the control of the gypsy moth. The present invention can effectively silence the SIFR gene of the gypsy moth, resulting in an extension of the development period of the gypsy moth larvae and a significant increase in the mortality rate of the 6th instar larvae. The present invention has the advantages of being pollution-free and reducing the use of chemical pesticides. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram for the construction of pET-28a(+)-dsSIFR vector;

[0020] Figure 2 is an agarose gel electrophoresis image of the pET-28a(+)-dsSIFR vector;

[0021] Figure 3 This is a graph for detecting the efficiency of SIFR gene silencing;

[0022] Figure 4 This is the effect of feeding dsSIFR bacterial solution on the weight and food intake of the fourth-instar larvae of the American white moth.

[0023] (a) Effect on the body weight of 4th instar larvae, (b) Effect on the food intake of 4th instar larvae;

[0024] Figure 5 This is a graph showing the effect of feeding dsSIFR bacterial solution on the larval development period of the American white moth;

[0025] Figure 6 This is a graph showing the effect of feeding dsSIFR bacterial solution on larval mortality of the gypsy moth;

[0026] Figure 7 This is the graph of the anti-ultraviolet ability of microcapsules;

[0027] Figure 8 This is the thermal storage stability diagram of microcapsules;

[0028] Fig. 9 This is the storage stability diagram of microcapsules at room temperature and low temperature;

[0029] (a) The stability diagram of microcapsules at room temperature, (b) The stability diagram of microcapsules at low temperature;

[0030] Fig.10 This is a graph showing the effect of feeding dsSIFR microcapsules to the larvae of the American white moth on mortality. DETAILED DESCRIPTION

[0031] In order to better understand the present invention, the technical content of the invention is described in detail with examples below, but the embodiments of the present invention are not limited to these examples. The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0032] Example 1 Full-length cloning of the nymphalidae SIFR gene

[0033] The reading frame (ORF) of the SIFR gene of the gypsy moth is 1425 bp, encoding 474 amino acids, with a molecular weight of 54.7 kDa and a theoretical isoelectric point PI of 9.73, and is a basic protein.

[0034] Extract total RNA from Hyphanthus cunea and use the reverse transcription kit PrimeScript TMThe first strand of cDNA was synthesized by RT reagent Kit (TaKaRa), and then the first strand of cDNA was used as a template. According to the SIFR sequence obtained from the transcriptome data of the nymphalid larvae of the American moth, primer sets were designed on both sides of the gene coding region sequence.

[0035] Forward primer: 5′-ATGGCACCTTTAAGAATATCATCA-3′;

[0036] Reverse primer: 5′-CACTCCCGTGTGGTTGAAG-3′.

[0037] Reaction system:

[0038] 5× PrimeScript buffer 2μL,

[0039] PrimeScript RT Enzyme Mix I 0.5μL,

[0040] Oligo d(T)primer(50μM)0.5μL,

[0041] Random 6mers (100 μM) 0.5 μL,

[0042] Total RNA 0.5μg RNase Free dH2O make up to 10μL.

[0043] The PCR amplification program was as follows: 94°C for 3 min; 94°C for 30 s, 60°C for 30 s, and 72°C for 1 min 30 s, for 35 cycles; and extension at 72°C for 10 min.

[0044] The PCR product was detected by 1% agarose gel electrophoresis, and the target band was recovered by gel recovery kit. The recovered band was connected to the pMD18-T vector overnight, and the ligation product was transformed into DH5α competent cells. The positive clones detected by bacterial liquid PCR were sent to Beijing BGI Biotechnology Co., Ltd. for sequencing to verify the reading frame.

[0045] Example 2 Construction of bacterial expression vector for dsRNA and preparation of bacterial solution

[0046] According to the conserved domain of the cDNA sequence of HcSIFR, the dsRNA silencing region was determined, and the primer sets of HcSIFR forward fragment and HcSIFR reverse fragment were designed respectively (Table 1). The primer sets of HcSIFR forward fragment and reverse fragment were used to perform PCR amplification on the fragment HcSIFR to obtain the target HcSIFR forward fragment and HcSIFR reverse fragment.

[0047] Reaction system:

[0048] 5× PrimeScript buffer 2μL,

[0049] PrimeScript RT Enzyme Mix I 0.5μL,

[0050] Oligo d(T)primer(50μM)0.5μL,

[0051] Random 6mers (100 μM) 0.5 μL,

[0052] Total RNA 0.5μg RNase Free dH2O make up to 10μL.

[0053] The PCR amplification program was as follows: 94°C for 3 min; 94°C for 30 s, 60°C for 30 s, 72°C for 30 s, 35 cycles; and extension at 72°C for 10 min.

[0054] The PCR products were subjected to gel electrophoresis and collected.

[0055] Select Xho I and Hind III as restriction sites to linearize pET-28a(+); prepare the reaction mixture on ice according to the following ingredients.

[0056] Reaction system:

[0057] Green Buffer 5μL,

[0058] Endonuclease-1 2.5μL,

[0059] Endonuclease-2 2.5μL,

[0060] Plasmid 2μL,

[0061] Make up to 50 μL with ddH2O.

[0062] After 3-4 hours in a 37℃ water bath, perform electrophoresis and gel collection.

[0063] According to the instructions of the ClonExpress Ultra One Step Cloning Kit V2, the amount of cloning vector and inserted forward fragment used in the homologous recombination reaction was calculated, and the reaction mixture was prepared on ice according to the calculated results.

[0064] Reaction system:

[0065] 2×CE Mix 5μL,

[0066] Linearized vector 0.03pmol,

[0067] Insert fragment 0.06pmol,

[0068] Make up to 10 μL with ddH2O.

[0069] 50℃ water bath for 5min.

[0070] The homologous recombination reaction product was transformed into Escherichia coli HT115 (DE3), and a single clone was selected for sequencing verification. The plasmid with correct sequencing was named pET-28a (+) -dsSIFR (I);

[0071] Subsequently, Sac I and BamH I were used as restriction sites to linearize the plasmid pET-28a(+)-dsSIFR(I), and homologous recombination was used to insert the reverse fragment. The reaction system was the same as above, and the plasmid with the correct sequencing was named pET-28a(+)-dsSIFR. Figure 1 , 2 It can be seen that the vector construction is complete. The pET-28a(+)-dsSIFR recombinant vector was transformed into Escherichia coli HT115 (DE3) competent cells, and single clones were picked and shaken in LB (containing Kanamycin with a final concentration of 50μg / mL and Tetracycline with a final concentration of 12.5μg / mL), with the empty vector pET-28a(+) as the control; when the OD600 of the bacterial solution was 0.4, IPTG was added to a final concentration of 1mM and continued to be cultured at 37°C and 180rpm for 4h. 50mL of the induced bacterial solution was centrifuged at 5000rpm for 5min, and the supernatant was discarded to collect the bacterial precipitate; 1mL of deionized water was used to resuspend the bacterial body, and then centrifuged at 5000rpm for 2min, and the supernatant was discarded to collect the bacterial precipitate; 800μL of deionized water was added to resuspend the bacterial body.

[0072] Table 1 Primer sequences for constructing the pET-28a(+)-dsSIFR vector of Hyphantria cunea

[0073]

[0074] Example 3 Detection of Silencing Effect of SIFR Gene in Hyphantria cunea

[0075] The bacterial expression dsRNA solution of SIFR gene and GFP gene synthesized in Example 2 was mixed with feed and fed to 4-instar gypsy moth. Active larvae were selected at 48h and 72h, and RNA was extracted using RNeasy Mini Animal Tissue Total RNA Extraction Kit (Qiagen). PrimeScript TMThe first strand of cDNA was synthesized by RT kit (TaKaRa), and the expression of SIFR gene was detected by fluorescence quantitative RT-PCR. Compared with the control GFP gene, the silencing efficiency was 97.58% at 48h and 76.64% at 72h.

[0076] Example 4 Effect of dsRNA of SIFR Gene on Growth and Development of Hyphanthus cunea

[0077] The bacterial solution expressing dsSIFR and dsGFP synthesized in Implementation 2 was mixed evenly with feed and then fed to 4-instar larvae of the cuneiform moth, and the effects on the feeding, weight and development period of the larvae of the cuneiform moth were counted. More specifically, the feed mixed with the bacterial solution expressing dsSIFR and the bacterial solution expressing dsGFP was fed to the cuneiform moth, with 30 larvae in each group, and the larvae were fed normally for 72 hours, and the weight and food intake of a single larva were counted every 24 hours. The molting of each larva was observed and recorded, and the development period and mortality rate were counted.

[0078] Depend on Figure 4 It can be seen that compared with the control dsGFP group, the body weight of the dsSIFR group fed with bacterial solution decreased significantly by 13.33% (P<0.05), and the food intake of the larvae of the dsSIFR group fed with bacterial solution decreased significantly by 21.88% (P<0.05).

[0079] Depend on Figure 5 It can be seen that the dsSIFR group fed with bacterial liquid began to molt to the 6th instar on the 6th day after feeding, and the length of the 5th instar was delayed by an average of 1.1 days compared with the control group.

[0080] Depend on Figure 6 It can be seen that the bacterial solution expressing dsSIFR and dsGFP synthesized in Implementation 2 was mixed evenly with feed and fed to the 6th instar larvae of the American moth. The results showed that the mortality rate of the dsSIFR treatment group increased significantly, and the mortality rate of the larvae in the dsSIFR group fed with bacterial solution was 8 times that of the control group dsGFP, reaching 53.33%.

[0081] Example 5 Preparation of bacterially expressed dsSIFR microcapsules

[0082] The bacterial expression dsSIFR bacterial solution was mixed with a sterile sodium alginate solution with a concentration of 1% to 3% by mass in a volume ratio of 2:1-1:2. After uniform mixing and stirring, granulation was performed using a microencapsulation granulator (B-395Pro, Buchi, Switzerland). Specifically, 50 mL of the liquid inlet was sucked into the liquid inlet, and the mixed solution was dripped into a continuously stirred sterile calcium chloride solution with a concentration of 2% to 4% by mass through a 200 μm nozzle. After stirring and curing with a magnetic stirrer for 15 minutes, the microcapsules were rinsed and filtered to obtain microcapsules, which were dried and stored at room temperature. Microcapsules are a preparation that uses a variety of natural or synthetic polymers to completely encapsulate beneficial microorganisms or other target substances. Encapsulating microorganisms in a physical barrier can protect microorganisms from adverse environmental conditions, improve the stability of the preparation, increase the vitality of the microorganisms, reduce the amount of preparations used, and extend the shelf life of the preparations.

[0083] Example 6 Performance Determination of Bacterial Expression dsSIFR Microcapsules

[0084] The bacterial expression dsSIFR microcapsules prepared according to the method described in Example 5 have an average water content of 28.29%, which can effectively reduce bacterial metabolism and improve the stability of the drug. The average swelling rate is 249.18%, which can effectively delay the release of Escherichia coli and ensure the stability and sustained release of the drug.

[0085] The dsRNA microcapsules prepared in Example 5 were dissolved in a lysis solution (0.2M NaHCO3 and 0.06M Na3C6H5O7·2H2O) after being irradiated with ultraviolet light for 2 hours. The released bacteria were diluted and plated to calculate the survival rate. The results showed that the survival rate of the microcapsules was 29.99% after ultraviolet irradiation, while the survival rate of the unencapsulated bacteria was only 3.09% ( Figure 7 ), indicating that microencapsulation can significantly enhance the UV resistance of Escherichia coli.

[0086] After the E. coli microcapsules prepared in Example 5 were heat treated in a 54°C incubator for 15 days, the survival rate of the microcapsules was 32.23%, and no bacteria were found in the un-microencapsulated bacterial solution ( Figure 8 ), indicating that the microcapsules are effective in protecting bacteria from high temperature damage (P<0.01). The microcapsules were stored in a room temperature incubator and a 4°C refrigerator for 3 months. At each time point, some microcapsules were taken out and placed in lysis solution. The survival rate of the bacteria was calculated after counting by the plate counting method. The results showed that after 90 days of storage at room temperature, the survival rate of the microencapsulated bacteria was 26.97%, which was significantly higher than the survival rate of the bacteria in the bacterial solution of 0.01% (P<0.01), indicating that the microcapsules can significantly delay the inactivation of Escherichia coli cells ( Fig. 9a). After storage at 4℃ for 90 days, the survival rate of the microencapsulated bacteria was 63.98%, which was 0.08% higher than that of the unencapsulated bacteria ( Fig. 9 b). Microencapsulated E. coli can effectively improve the survival rate of bacteria. Storing microcapsules at low temperature (4°C) can more effectively delay the inactivation of bacteria and extend the shelf life of the preparation than storing microcapsules at room temperature.

[0087] Example 7 Bioassay of bacterially expressed dsSIFR microcapsules against the Hyphantria cunea

[0088] The feeds of bacterially expressed dsSIFR microcapsules and bacterially expressed dsGFP microcapsules synthesized in Example 5 were mixed evenly and fed to 6th instar larvae of the cuneiform moth, respectively, and the effect on the mortality of the cuneiform moth larvae was counted. Each group was treated with 10 larvae and 3 biological replicates were performed. Fig.10 It can be seen that when the feeds synthesized in Implementation 5 with the bacterial expression dsSIFR microcapsules and the bacterial expression dsGFP microcapsules were mixed evenly and then fed to the 6th instar larvae of the American white moth, the mortality rate was significantly increased. The mortality rate of the experimental group fed with the bacterial expression dsSIFR microcapsules was as high as 60.00% (P<0.05).

Claims

1. A SIFR gene for regulating the growth and development of the cunea gypsophila, characterized in that: The full-length cDNA sequence is shown as SEQ ID No.1, and encodes the amino acid sequence shown as SEQ ID No.

2.

2. A dsRNA for inhibiting the growth and development of the cunea nymph, characterized in that: The dsRNA can be targeted to silence the SIFR gene as claimed in claim 1, wherein the positive strand and the antisense strand of the dsRNA are composed of the nucleic acid sequences shown in SEQ ID No. 3 and SEQ ID No.

4.

3. A recombinant expression vector containing the dsRNA for inhibiting the growth and development of the cunea as claimed in claim 2.

4. The method for constructing a recombinant expression vector of dsRNA for inhibiting the growth and development of Hyphantria cunea according to claim 3, characterized in that: The construction method is as follows: extract total RNA of the gypsy moth, use a reverse transcription kit to synthesize the first chain of cDNA, then use the first chain of cDNA as a template, and design primer sets on both sides of the gene coding region sequence according to the SIFR sequence obtained from the transcriptome data of the gypsy moth larvae: the upstream primer SIFR-F is shown as SEQ ID No.5, and the downstream primer SIFR-R is shown as SEQ ID No.6; then, PCR amplification is performed using the first chain of cDNA as a template to obtain a target fragment HcSIFR, and a dsRNA silencing region is determined according to the conserved structural domain of the cDNA sequence of the fragment HcSIFR, and a primer set of the HcSIFR forward fragment is shown as SEQ ID No.7-8 and a primer set of the HcSIFR reverse fragment is shown as SEQ ID No.9-10, respectively, and the fragment HcSIFR is amplified by PCR using the primer set of the HcSIFR forward fragment to obtain the target HcSIFR forward fragment, and the target HcSIFR forward fragment and the expression vector pET-28a(+) are respectively transcribed through Xho I and Hind III for double enzyme digestion, recover the required fragments, connect and construct the recombinant plasmid pET-28a(+)-dsSIFR(I); then use the primer set of the HcSIFR reverse fragment to perform PCR amplification on the fragment HcSIFR to obtain the target HcSIFR reverse fragment, and double enzyme digest the target HcSIFR reverse fragment and the recombinant plasmid pET-28a(+)-dsSIFR(I) with Sac I and BamH I respectively, recover the required fragments, connect and construct the recombinant plasmid pET-28a(+)-dsSIFR, thereby completing the construction of the recombinant expression vector pET-28a(+)-dsSIFR.

5. A recombinant bacterium containing the recombinant expression vector of dsRNA for inhibiting the growth and development of the cunea as claimed in claim 3.

6. A method for constructing a recombinant bacterium according to claim 5, characterized in that: The construction method is as follows: the constructed recombinant expression vector pET-28a(+)-dsSIFR is transformed into competent cells of Escherichia coli HT115(DE3), thus completing the construction of the recombinant bacteria.

7. A nucleic acid pesticide microcapsule for controlling the gypsophila cunea, comprising a capsule core and a capsule wall, characterized in that: The capsule core contains the recombinant bacteria as claimed in claim 5.

8. The nucleic acid pesticide microcapsule for controlling the gypsy moth according to claim 7, characterized in that: The recombinant bacteria are Escherichia coli HT115 (DE3) containing the recombinant expression vector pET-28a (+) -dsSIFR; the amount of the recombinant bacteria is 2.21-3.07 × 10 5 cfu / g.

9. A nucleic acid pesticide microcapsule for controlling the American white moth as claimed in claim 8, characterized in that: Its water content is 28.29% and its swelling rate is 249.18%.

10. A method for preparing a nucleic acid pesticide microcapsule for controlling the gypsophila cunea according to any one of claims 7 to 9, characterized in that: Here’s how: Step 1, mixing a sodium alginate solution having a concentration of 1% to 3% by mass and a recombinant bacterial suspension in a volume ratio of 2:1 to 1:2 to form a mixed solution; Step 2, spraying the mixed solution from the nozzle of the microcapsule granulator into a continuously stirred calcium chloride solution with a concentration of 2% to 4% by mass to form microcapsules; Step 3: Allow the microcapsules to stand and solidify in the calcium chloride solution for 15 minutes, filter, wash, and dry to obtain microcapsules.

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