Low-cost, large-scale production process of a dsRNA nanopesticide for citrus psyllids
Patent Information
- Application Number
- CN202610971301.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2026-07-01
- Publication Date
- 2026-10-27
AI Technical Summary
[0040]与现有技术相比，本发明具有以下有益效果：
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Figure CN122979296A_ABST
Abstract
Description
Technical Field
[0001] This invention belongs to the field of agricultural biotechnology, specifically relating to a low-cost, large-scale production process for a double-stranded RNA (dsRNA) nanopesticide targeting citrus psyllids. Background Technology
[0002] Huanglongbing (HLB) is a devastating disease of citrus caused by *Candidatus Liberibacter asiaticus* (CLAs), and there is currently no effective treatment. The Asian citrus psyllid (*Diaphorina citri Kuwayama*) is the sole vector for HLB, therefore controlling the psyllid is a crucial measure for HLB control.
[0003] Traditional chemical pesticides for controlling citrus psyllids present problems such as pesticide residues, environmental pollution, and pest resistance. RNA interference (RNAi) technology, based on the specific degradation of homologous mRNA by exogenous double-stranded RNA, inhibits the expression of key target genes in pests, thereby interfering with their growth and reproduction. It has advantages such as high specificity, high environmental safety, and low likelihood of developing pesticide resistance, and has become a new direction for agricultural pest control.
[0004] However, the commercial application of dsRNA pesticides faces two major bottlenecks: first, the production cost of dsRNA is high, and the cost of chemical synthesis and in vitro transcription kits is expensive, making it difficult to meet the needs of field applications; second, naked dsRNA is easily degraded by ultraviolet light and nucleases in the field, and it is difficult to penetrate the midgut cell membrane of insects, resulting in low delivery efficiency.
[0005] Current research has employed microbial fermentation to produce dsRNA, but these methods generally suffer from low yields, complex purification processes, and high costs. Meanwhile, existing nanocarriers, such as layered bimetallic hydroxides and metal-organic frameworks, have complex preparation processes and high costs, making them unsuitable for large-scale production.
[0006] Therefore, developing a low-cost, high-efficiency, and industrially viable process for producing dsRNA nanopesticides for citrus psyllids has significant practical importance and application value. (Invention Content)
[0007] (I) Technical Problem to be Solved The purpose of this invention is to overcome the shortcomings of existing technologies and provide a low-cost, large-scale production process for dsRNA nanopesticides for citrus psyllids, solving problems such as high dsRNA production costs, complex nanocarrier preparation, and low delivery efficiency in existing technologies. (II) Technical Solution
[0008] To achieve the above objectives, the present invention adopts the following technical solution:
[0009] A low-cost, large-scale production process for a dsRNA nanopesticide for citrus psyllids includes the following steps:
[0010] Step 1: Construction of recombinant expression vector
[0011] Key genes of the citrus psyllid were selected as targets, namely the DcSnf7 gene and / or the DcIap5 gene;
[0012] Design specific primers to amplify the target gene fragment using citrus psyllid cDNA as a template;
[0013] The amplified target gene fragment was inserted in reverse repeat into the L4440 vector carrying a bidirectional T7 promoter to obtain a recombinant expression vector.
[0014] Step 2: Construction and activation of engineered strains
[0015] The recombinant expression vector was transformed into RNase III-deficient Escherichia coli HT115 (DE3) competent cells to obtain engineered strains;
[0016] The engineered strain was inoculated onto LB solid medium containing ampicillin and cultured at 37°C for 12-16 hours. Single colonies were then picked for PCR verification.
[0017] The verified single colony was inoculated into LB liquid medium containing ampicillin and cultured at 37°C with shaking at 200 rpm for 12-16 h to serve as the seed culture.
[0018] Step 3: High-density fermentation to produce dsRNA
[0019] Inoculate the seed culture into TB fermentation medium at an inoculation rate of 5%-10% and culture at 37℃ and 200 rpm with shaking.
[0020] When the OD600 reaches 0.6-0.8, add lactose to a final concentration of 4-8 g / L for induction, and continue culturing for 12-16 h;
[0021] After fermentation, collect the cells by centrifugation at 4°C and 8000 rpm for 10 minutes.
[0022] Step 4: Low-cost extraction and purification of dsRNA
[0023] The collected bacterial cells were resuspended in STE buffer, and lysozyme was added to a final concentration of 1 mg / mL. The cells were then incubated at 37°C for 30 min.
[0024] Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture, shake vigorously for 10 min, and centrifuge at 4℃ and 12000 rpm for 15 min;
[0025] Take the supernatant, add 0.1 volume of 3M sodium acetate (pH 5.2) and 2 volumes of anhydrous ethanol, and place at -20℃ for 30 min;
[0026] Centrifuge at 12000 rpm for 15 min at 4℃, discard the supernatant, wash the precipitate twice with 75% ethanol, dry at room temperature, and dissolve in RNase-free water;
[0027] Add DNase I and RNase A, incubate at 37°C for 30 min to remove DNA and single-stranded RNA, and obtain purified dsRNA.
[0028] Step 5: Low-cost preparation of chitosan nanocarriers
[0029] Chitosan (molecular weight 40-100kDa, degree of deacetylation 70-85%) was dissolved in 1% acetic acid solution and stirred until completely dissolved to prepare a chitosan solution of 1-2 mg / mL.
[0030] The chitosan nanocarrier mother liquor was obtained by filtration and sterilization using a 0.22 μm filter membrane.
[0031] Step 6: Preparation of dsRNA-chitosan nanocomposite
[0032] The purified dsRNA solution was mixed with the chitosan nanocarrier stock solution at a mass ratio of 1:(2-5), and gently stirred at room temperature for 30 min.
[0033] Adjust the pH to 5.5-6.0 with 0.1M NaOH solution and continue stirring for 10 minutes;
[0034] Add an appropriate amount of sucrose as a freeze-drying protectant, and freeze-dry to obtain the finished product of citrus psyllid dsRNA nanopesticide.
[0035] Further, the nucleotide sequence of the DcSnf7 gene in step 1 is shown in SEQ ID NO:1, and the nucleotide sequence of the DcIap5 gene is shown in SEQ ID NO:2.
[0036] Further, the formulation of the TB fermentation medium in step 3 is as follows: 12 g / L tryptone, 24 g / L yeast extract, 4 mL / L glycerol, 2.31 g / L KH2PO4, and 12.54 g / L K2HPO4.
[0037] Furthermore, the fermentation process described in step 3 adopts a fed-batch fermentation method. When the OD600 reaches 2.0, glucose solution is added at a rate of 1-2 g / (L·h) until the fermentation is completed.
[0038] Furthermore, the preferred mass ratio of dsRNA to chitosan in step 6 is 1:3.
[0039] Furthermore, the final concentration of sucrose in step 6 is 5%-10% (w / v). (III) Beneficial Effects
[0040] Compared with the prior art, the present invention has the following beneficial effects:
[0041] Low production cost: This invention uses high-density fermentation of Escherichia coli HT115 (DE3) to produce dsRNA, uses inexpensive lactose as an inducer to replace expensive IPTG, and uses TB medium to replace LB medium. The dsRNA yield can reach more than 80 μg / mL, and the production cost is reduced to less than 0.3 USD / g, which is far lower than the existing technology level.
[0042] Simple and easily scalable process: This invention uses a one-step method to prepare chitosan nanocarriers and dsRNA-chitosan nanocomposites, which does not require complex chemical reactions and expensive equipment, has a short production cycle, and is suitable for large-scale industrial production.
[0043] Good stability and high delivery efficiency: The dsRNA-chitosan nanocomposite prepared by this invention has an average particle size of 200-300nm and a Zeta potential of +20-+30mV. It can effectively protect dsRNA from ultraviolet light and nuclease degradation. It can intelligently release dsRNA in the alkaline environment of the citrus psyllid midgut, significantly improving delivery efficiency.
[0044] With good control effect and high safety: The dsRNA nano-pesticide for citrus psyllid produced by this invention has a mortality rate of over 85% against citrus psyllid nymphs and adults, and is safe for non-target organisms such as bees and ladybugs, with no pesticide residues, which meets the requirements of green agricultural development. Attached Figure Description
[0045] To provide a more detailed description of the technical solutions of the embodiments of this invention, the accompanying drawings used in the following description of the embodiments are briefly introduced. Obviously, the drawings in the following description are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on such drawings without creative effort.
[0046] Figure 1 is a flowchart of the overall production process of the dsRNA nanopesticide for citrus psyllids of the present invention.
[0047] Figure 2 is a schematic diagram of the structure of the recombinant expression vector L4440-DcSnf7.
[0048] Figure 3 shows the particle size distribution and zeta potential of the dsRNA-chitosan nanocomposite.
[0049] Figure 4 is a schematic diagram of the transmission electron microscopy (TEM) of the dsRNA-chitosan nanocomposite.
[0050] Figure 5 is a bar chart comparing the mortality rates of 3rd instar nymphs of citrus psyllids at 7 days in different treatment groups. Detailed Implementation
[0051] The present invention will be further described in detail below with reference to specific embodiments.
[0052] Example 1
[0053] Based on the DcSnf7 gene sequence of the citrus psyllid published in GenBank (accession number: XM_008482345.1), specific primers were designed:
[0054] Upstream primer: 5'-GCGGATCCATGAGTAAGAAGCTGAAGAAG-3'(BamHI)
[0055] Downstream primer: 5'-GCAAGCTTTTACTTCTTCTTCTTCTTCTT-3'(Hind III)
[0056] Total RNA was extracted from citrus psyllids and reverse transcribed into cDNA. The cDNA was then used as a template for PCR amplification. The PCR reaction system consisted of: 25 μL of 2×Taq PCR MasterMix, 1 μL each of forward and reverse primers, 2 μL of cDNA template, and 21 μL of RNase-free water. The PCR conditions were: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; and a final extension at 72℃ for 10 min.
[0057] The PCR amplification products were double-digested with BamHI and HindIII, and the L4440 vector was also double-digested with the same restriction endonucleases. The digested products were separated by agarose gel electrophoresis, and the target fragment and vector fragment were recovered using a gel extraction kit.
[0058] Mix the target fragment and the vector fragment at a molar ratio of 3:1, add T4 DNA ligase, and ligate overnight at 16°C.
[0059] The ligation product was transformed into Escherichia coli DH5α competent cells, plated on LB solid medium containing ampicillin, and incubated at 37°C for 12-16 h.
[0060] Single colonies were selected for PCR and sequencing verification to obtain the recombinant expression vector L4440-DcSnf7.
[0061] Example 2
[0062] The recombinant expression vector L4440-DcSnf7 was transformed into Escherichia coli HT115 (DE3) competent cells, plated on LB solid medium containing ampicillin (100 μg / mL) and tetracycline (12.5 μg / mL), and cultured at 37°C for 12-16 h.
[0063] A single colony was picked and inoculated into 5 mL of LB liquid medium containing the same antibiotic, and cultured at 37°C and 200 rpm for 12 h with shaking to obtain the primary seed culture.
[0064] The primary seed culture was inoculated at a rate of 5% into 100 mL of TB liquid medium containing the same antibiotic, and cultured at 37°C and 200 rpm with shaking until the OD600 reached 0.6-0.8, which was then used as the secondary seed culture.
[0065] The secondary seed culture was inoculated into a 5L fermenter at an inoculation rate of 10%. The fermentation medium was TB medium, and the fermentation conditions were: temperature 37℃, stirring speed 300rpm, aeration rate 1vvm, and pH controlled at 7.0±0.2.
[0066] When the OD600 reaches 0.6-0.8, lactose with a final concentration of 6 g / L is added for induction, and at the same time, 50% glucose solution is added at a rate of 1.5 g / (L·h), and the culture is continued for 14 h.
[0067] After fermentation, the cells were collected by centrifugation at 4℃ and 8000rpm for 10 minutes, and weighed. The yield of wet cells could reach more than 25g / L.
[0068] Example 3
[0069] Take 10g of wet bacterial cells and resuspend them in 100mL of STE buffer (10mM Tris-HCl pH8.0, 100mM NaCl, 1mM EDTA).
[0070] Add lysozyme to a final concentration of 1 mg / mL, incubate at 37°C for 30 minutes, gently shaking several times during the incubation period.
[0071] Add an equal volume of phenol-chloroform-isoamyl alcohol (25:24:1) mixture, shake vigorously for 10 min, and centrifuge at 4℃ and 12000 rpm for 15 min.
[0072] Carefully aspirate the supernatant and transfer it to a new centrifuge tube. Repeat step 3 once.
[0073] Take the supernatant, add 0.1 volume of 3M sodium acetate (pH 5.2) and 2 volume of anhydrous ethanol, mix gently, and incubate at -20°C for 30 min.
[0074] Centrifuge at 12,000 rpm for 15 min at 4℃, discard the supernatant, wash the precipitate twice with 75% ethanol, and dry at room temperature for 5-10 min.
[0075] Dissolve the precipitate in 10 mL of RNase-free water, add DNase I to a final concentration of 10 μg / mL and RNase A to a final concentration of 20 μg / mL, and incubate at 37 °C for 30 min.
[0076] Repeat steps 3-6 once, and finally dissolve the precipitate with 2 mL of RNase-free water to obtain purified dsRNA.
[0077] The concentration and purity of dsRNA were determined using a UV spectrophotometer. The OD260 / OD280 ratio was between 1.8 and 2.0, and the dsRNA yield could reach over 80 μg / mL of fermentation broth.
[0078] Example 4
[0079] Weigh 1g of chitosan (molecular weight 60kDa, degree of deacetylation 80%) and slowly add it to 100mL of 1% acetic acid solution. Stir magnetically at room temperature until completely dissolved.
[0080] Adjust the pH to 5.0 with 0.1M NaOH solution and continue stirring for 30 minutes.
[0081] The mixture was sterilized by filtration through a 0.22 μm microporous membrane to obtain a 10 mg / mL chitosan nanocarrier stock solution, which was stored at 4 °C for later use.
[0082] Example 5
[0083] Take 1 mL of dsRNA solution with a concentration of 1 mg / mL and slowly add it dropwise to 3 mL of chitosan solution with a concentration of 1 mg / mL while stirring (the mass ratio of dsRNA to chitosan is 1:3).
[0084] Stir gently at room temperature for 30 minutes, adjust the pH to 5.8 with 0.1M NaOH solution, and continue stirring for 10 minutes.
[0085] Add sucrose to a final concentration of 8% (w / v) as a freeze-drying protectant, dissolve it completely, and then dispense it into vials.
[0086] Pre-freeze to -40℃, then freeze-dry to obtain a white powdery citrus psyllid dsRNA nanopesticide product.
[0087] Example 6
[0088] The dsRNA nanopesticide for citrus psyllids was diluted with distilled water to different concentrations (50 μg / mL, 100 μg / mL, 200 μg / mL, 400 μg / mL).
[0089] Take fresh citrus shoots, soak them in a diluted solution for 10 seconds, remove them, let them dry, and then put them into an insect cage.
[0090] Thirty healthy 3rd instar nymphs of citrus psyllids were introduced, with each concentration repeated three times, and water treatment served as a control.
[0091] The psyllids were raised under conditions of 25±1℃, 70±5% relative humidity, and 16L:8D photoperiod. The mortality of the psyllids was observed and recorded daily for 7 consecutive days.
[0092] The results showed that when the dsRNA concentration was 200 μg / mL, the mortality rate of citrus psyllids after 7 days reached 86.7%, which was significantly higher than that of the naked dsRNA treatment group (43.3%).
Claims
1. Claims A low-cost, large-scale production process for dsRNA nanopesticides for citrus psyllids, characterized by: Includes the following steps: Step 1: Construct a recombinant expression vector carrying key genes of the citrus psyllid; Step 2: Transform the recombinant expression vector into Escherichia coli HT115 (DE3) to obtain the engineered strain; Step 3: Producing dsRNA using a high-density fermentation process; Step 4: Extract and purify dsRNA; Step 5: Prepare chitosan nanocarriers; Step 6: Mix dsRNA with chitosan nanocarrier to prepare dsRNA-chitosan nanocomposite.
2. The production process according to claim 1, characterized in that, The key genes for the citrus psyllid mentioned in step 1 are the DcSnf7 gene and / or the DcIap5 gene.
3. The production process according to claim 1, characterized in that, The recombinant expression vector mentioned in step 1 is the L4440 vector carrying a bidirectional T7 promoter.
4. The production process according to claim 1, characterized in that, The fermentation medium mentioned in step 3 is TB medium, the inducer is lactose, and the final concentration is 4-8 g / L.
5. The production process according to claim 1, characterized in that, The fermentation process described in step 3 adopts a fed-batch fermentation method, with glucose solution added at a rate of 1-2 g / (L·h).
6. The production process according to claim 1, characterized in that, The dsRNA extracted in step 4 was extracted using the phenol-chloroform extraction method, and purified using the DNase I and RNase A enzymatic digestion method.
7. The production process according to claim 1, characterized in that, The chitosan mentioned in step 5 has a molecular weight of 40-100 kDa and a degree of deacetylation of 70-85%.
8. The production process according to claim 1, characterized in that, The mass ratio of dsRNA to chitosan in step 6 is 1:(2-5).
9. The production process according to claim 1, characterized in that, In step 6, sucrose is added as a freeze-drying protectant, with a final concentration of 5%-10% (w / v).
10. The application of the dsRNA nanopesticide for citrus psyllid prepared by the production process according to any one of claims 1-9 in the control of citrus psyllid.