A nucleic acid composition targeting BxGCC gene, nano-preparation and application thereof in prevention and treatment of pine wood nematode disease

Abstract

The present application relates to the technical field of pesticides, in particular to a nucleic acid composition targeting BxGCC gene, a nano preparation and application thereof in prevention and treatment of pine wood nematode disease. The nucleic acid composition comprises hairpin loop, kiss loop, tetra-uridine and siRNA target sequence; the siRNA target sequence is two or more of target 1-target 5; the nucleotide sequences of target 1-target 5 are shown in SEQ ID NO. 1-SEQ ID NO. 5 in turn; any two siRNA target sequences are connected and separated by one or more of hairpin loop, kiss loop and tetra-uridine. The nucleic acid composition and nano preparation provided by the present application have excellent effect in prevention and treatment of pine wood nematode disease, and under optimal conditions, the survival rate of black pine seedlings inoculated with pine wood nematode reaches 96.7% and 93.3% after injection and spraying.

Description

Technical Field

[0001] This invention relates to the field of pesticide technology, and in particular to a nucleic acid composition, nano-formulation targeting the BxGCC gene, and its application in the prevention and control of pine wilt disease. Background Technology

[0002] Pine wilt disease is a major quarantine disease of forest trees worldwide, caused by the pine wilt nematode (Pinus thunbergii). Bursaphelenchus xylophilus It can be spread naturally or by long-distance human intervention via insect vectors such as the pine sawyer beetle. Currently, the effectiveness of its control measures is still not high, and common methods include: injection of abamectin nematicide, quarantine monitoring, etc., with prevention being the primary focus.

[0003] Compared to traditional highly toxic and persistent chemical pesticides, green pesticides offer advantages such as biodegradability, high efficiency, and strong targeting. Nucleic acid pesticides, in particular, hailed as the third generation of pesticides, have a promising future. Nucleic acid pesticides utilize RNA interference (RNAi) technology to achieve targeted control of agricultural and forestry pests through highly specific gene silencing, without affecting non-target organisms. They possess significant advantages such as environmental friendliness, exhibiting low non-target toxicity, controllable degradation, and ecological compatibility. Currently, most developed pesticides are based on dsRNA, but traditional dsRNA is directly derived from the specific long sequences (>500 nt) of the target organism, resulting in relatively high production costs and making them less competitive in the market compared to chemical pesticides. Currently, there are no effective pesticides for controlling pine wilt nematodes, and there is an urgent need for applicable, specific nucleic acid pesticides targeting pine wilt nematodes. Summary of the Invention

[0004] To address the aforementioned problems, this invention provides a nucleic acid composition targeting the BxGCC gene, a nano-formulation, and its application in the prevention and control of pine wilt disease. The nucleic acid composition and nano-formulation provided by this invention exhibit excellent efficacy in preventing and controlling pine wilt disease. Under optimal conditions, trunk injection and spraying resulted in survival rates of 96.7% and 93.3% respectively against pine seedlings inoculated with pine wilt disease.

[0005] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a nucleic acid composition targeting the BxGCC gene, the nucleic acid composition comprising a hairpin loop, a kissing loop, tetrauridine monophosphate, and siRNA target sequences; the siRNA target sequences are two or more of target 1 to target 5; the nucleotide sequences of target 1 to target 5 are as shown in SEQ ID NO.1-SEQ ID NO.5 respectively; any two siRNA target sequences are separated by one or more of the hairpin loop, kissing loop, and tetrauridine monophosphate in tandem.

[0006] Preferably, the nucleotide sequence of the nucleic acid composition is as shown in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.14.

[0007] Preferably, the nucleotide sequence of the gene encoding the nucleic acid composition is as shown in SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 or SEQ ID NO.15.

[0008] This invention provides a nano-formulation comprising a cationic delivery carrier and the nucleic acid composition described in the above-described technical solution.

[0009] Preferably, the mass ratio of the cationic delivery carrier to the nucleic acid composition is 1:0.1-10; the cationic delivery carrier is selected from one or more of cationic polymers, cationic liposomes, natural cationic polysaccharides, and derivatives of natural cationic polysaccharides.

[0010] The present invention provides a method for preparing the nano-formulation described above, comprising: mixing the cationic delivery carrier and the nucleic acid composition, and obtaining the nano-formulation by electrostatic self-assembly.

[0011] Preferably, the mixing method includes a vortex, wherein the vortex rotates at a speed of 1000-3000 rpm for a duration of 1-30 min.

[0012] The present invention provides the application of the nucleic acid composition and / or the nano-formulation described in the above-mentioned technical solutions in the preparation of insecticides, wherein the insecticides target pests including pine wood nematodes.

[0013] This invention provides a method for preventing and controlling pine wilt disease, comprising: The insecticide is applied to the trees or seedlings to be treated by injection and / or spraying; the active ingredient of the insecticide includes the nucleic acid composition and / or the nano-formulation described in the above technical solution.

[0014] Preferably, the concentration of the nucleic acid composition in the insecticide is 100-1000 ng / μL.

[0015] Beneficial effects: This invention designs five siRNA target sequences targeting the BxGCC gene. These siRNA target sequences are then tandemly constructed using hairpin-loop, kissing-loop, and Tetra-U to create multiple long single-stranded RNA sequences resembling dsRNA, capable of forming multiple stem-loop structures. This constitutes the nucleic acid composition targeting the BxGCC gene provided by this invention. This nucleic acid composition exhibits excellent efficacy in controlling pine wilt disease. Under optimal conditions, stem injection and spraying resulted in survival rates of 96.7% and 93.3% for black pine seedlings inoculated with pine wilt disease, respectively, and can be applied to the control of pine wilt disease. Detailed Implementation

[0016] This invention provides a nucleic acid composition targeting the BxGCC gene, the nucleic acid composition comprising a hairpin loop, a kissing loop, tetrauridine monophosphate, and siRNA target sequences; the siRNA target sequences are two or more of target 1 to target 5; the nucleotide sequences of target 1 to target 5 are as shown in SEQ ID NO.1-SEQ ID NO.5 respectively; any two siRNA target sequences are separated by one or more of the hairpin loop, kissing loop, and tetrauridine monophosphate in tandem.

[0017] In one embodiment, the nucleic acid composition comprises five siRNAs with nucleotide sequences as shown in SEQ ID NO. 6, SEQ ID NO. 8, SEQ ID NO. 10, SEQ ID NO. 12, or SEQ ID NO. 14. In another embodiment, the nucleotide sequence of the gene encoding the nucleic acid composition is shown in SEQ ID NO. 7, SEQ ID NO. 9, SEQ ID NO. 11, SEQ ID NO. 13, or SEQ ID NO. 15.

[0018] The hairpin loop of this invention includes a stem region and a loop region, and has the following functions: 1. It enables single-stranded RNA to self-fold and form a stable secondary structure; 2. It reduces the degree of conformational freedom and improves the overall thermodynamic stability; 3. The stem region, with a GC content of approximately 50%, can significantly improve the RNA's resistance to degradation.

[0019] The kissing loops described in this invention are a pair of complementary hairpin loops, which have the following functions: 1. RNA self-assembly and polymerization to form dimers and multimers; 2. Circular RNA splicing.

[0020] The tetrauridine monophosphate (UUUU) described in this invention has the following functions: 1. It acts as a flexible connector; 2. It avoids incorrect pairing.

[0021] This invention designs five siRNA target sequences targeting the BxGCC gene. These siRNA target sequences are then tandemly constructed using hairpin-loop, kissing-loop, and Tetra-U to create multiple long single-stranded RNA sequences resembling dsRNA, capable of forming multiple stem-loop structures. RNA folding predicts that this siRNA possesses a multi-stem-loop dsRNA-like structure (capable of forming square nanostructures). This indicates that the siRNA constructed in this invention is a self-assembling RNA sequence that forms dsRNA-like nanostructures, exhibiting advantages such as high stability and high interference efficiency. The thermodynamic set free energy (ΔG) of the stable assembly containing the five siRNAs is -462.34 kcal / mol (compared to -204.77 kcal / mol for traditional dsRNA), demonstrating greater structural stability. This allows it to significantly resist RNase degradation in plants and maintain biological activity without relying on exogenous vectors. RNA folding predicts a 100% pairing probability at all sites (avoiding single-stranded region degradation).

[0022] Traditional dsRNA requires high-dose application, which is not cost-effective. The nucleic acid composition provided by this invention can significantly reduce the effective concentration and improve delivery efficiency, exhibiting excellent control of pine wilt disease. Under optimal conditions, trunk injection and spraying achieved survival rates of 96.7% and 93.3% respectively against pine wilt-inoculated black pine seedlings. It can be applied to the control of pine wilt disease and has good ecological safety, providing a new paradigm for green control of forest pests and diseases.

[0023] As one embodiment, the method for preparing the nucleic acid composition includes: chemically synthesizing a DNA sequence encoding siRNA and inserting it downstream of the T7 promoter of pET28α to obtain a recombinant plasmid; transforming the recombinant plasmid into *Escherichia coli* (E. coli). E. coli After induction with IPTG (HT115), siRNA was extracted. This preparation method is simple to operate, requires minimal equipment, and produces high RNA concentration, making it applicable to the control of pine wilt disease.

[0024] Based on the above advantages, the present invention provides a nano-formulation comprising a cationic delivery carrier and the nucleic acid composition described in the above-described technical solution. In one embodiment, the cationic delivery carrier is selected from one or more of cationic polymers, cationic liposomes, natural cationic polysaccharides, and derivatives of natural cationic polysaccharides. In another embodiment, the cationic delivery carrier is a polycationic material having a multi-arm structure. In yet another embodiment, the cationic delivery carrier is a polycationic material having a star-shaped structure.

[0025] This invention provides a method for preparing the nanoparticles described above, comprising: mixing the cationic delivery carrier and the nucleic acid composition, and obtaining the nanoparticles through electrostatic self-assembly. In one embodiment, the mass ratio of the cationic delivery carrier to the nucleic acid composition is 1:0.1-10. In another embodiment, the mass ratio of the cationic delivery carrier to the nucleic acid composition is 1:0.8-1.2. In yet another embodiment, the mass ratio of the cationic delivery carrier to the nucleic acid composition is 1:1.

[0026] In one embodiment, the mixing method includes a vortex with a rotational speed of 1000-3000 rpm and a mixing time of 1-30 min. In another embodiment, the vortex has a rotational speed of 2000 rpm and a mixing time of 10 min.

[0027] In this invention, the cationic delivery carrier serves to encapsulate and protect nucleic acids and facilitate their entry into target biological cells. In addition to the specific carriers used in the examples below, those skilled in the art will understand that other carriers with similar charge density and biocompatibility (such as PEI, liposomes, chitosan, etc.) are also suitable for the nucleic acid compositions of this invention.

[0028] When the nucleic acid composition of the present invention is combined with a cationic delivery carrier, it can form nanoparticles with uniform particle size, which significantly enhances the conduction efficiency of siRNA in the wood and phloem of pine trees and greatly extends the shelf life of the drug in the environment.

[0029] Based on the above advantages, the present invention provides the application of the nucleic acid composition and / or the nano-preparation described in the above technical solution in the prevention and control of pine wilt disease.

[0030] Based on the above advantages, the present invention provides the application of the nucleic acid composition and / or the nano-formulation described in the above technical solution in the preparation of insecticides, wherein the insecticides target pests including pine wood nematodes.

[0031] Based on the above advantages, the present invention provides a method for preventing and controlling pine wilt disease, comprising: The insecticide is applied to the trees or seedlings to be treated by injection and / or spraying; the active ingredient of the insecticide includes the nucleic acid composition and / or the nano-formulation described in the above technical solution.

[0032] In one embodiment, the concentration of the nucleic acid composition in the insecticide is 100-1000 ng / μL. In another embodiment, the concentration of the nucleic acid composition in the insecticide is 500 ng / μL. The application concentration of the insecticide can be adjusted according to factors such as the severity of insect infestation and tree age, and it has good insecticidal effect within the range of 100-1000 ng / μL.

[0033] To further illustrate the present invention, the following detailed description, in conjunction with embodiments, provides an example of an siRNA targeting the BxGCC gene, a nano-formulation, and its application, but these should not be construed as limiting the scope of protection of the present invention.

[0034] Example 1 This invention is based on the chloride channel gene BxGCC from the pine wood nematode. The BxGCC gene has the accession number BXY_0592100 in the WormBaseParaSite database, and its nucleotide sequence is shown in SEQ ID NO.22, as follows: ATGGAGAAGGAGAAGAAGAGGAAGAAGAGAGGGGCCAACCAAAACAAAATCAATATTCAGTCGTTGTATGTAGTTGGCGAAGATGGAGGGAGAAGTGGCGGCCCGAGAGGCCGTCAAGACGAGGCCGGCGCCCGCTTCCATGGCCCCCGATTTCGCGACGTAACCCCTTCGGGCCTTACCGAAGCCGGGTTTGCGGGGCTCCCAAAGACGGGAATGCGAGGCGGGGAACGGAGGGGTAA; The amino acid sequence of the protein encoded by the BxGCC gene is shown in SEQ ID NO.23, as follows: MEKEKKRKKRGANQNKINIQSLYVVGEDGGRSGGPRGRQDEAGARFHGPFRRDVTPSGLTEAGVCGAPKDGNARRGTEG.

[0035] Based on a length of 19-21 nt, avoiding four consecutive identical nucleotides (to prevent secondary structure interference), and using design principles such as Ui-Tei rule optimization (e.g., A / U at the 5' end and G / C at the 19th position), five siRNA target sequences were designed and screened, as follows: Target 1: UUCGGCAUUCUGGUCAAAAGG, SEQ ID NO.1; Target 2: AAGGUGAAGUACCUCCCUGGC, SEQ ID NO.2; Target 3: UUCAUCCAGAACGAGACGGUG, SEQ ID NO.3; Target 4: AACCUACAGUUGCUCCGGACA, SEQ ID NO.4; Target 5: UUGAUCCGCCACAAGGAGAAA, SEQ ID NO.5.

[0036] Example 2 Two siRNA target sequences (target 1 and target 2) from Example 1 were tandemly linked using a hairpin loop, a kissing loop, and a tetrauridine motif to construct a single-stranded RNA (SEQ ID NO. 6) containing the tandem siRNA sequence. The coding sequence of this single-stranded RNA (SEQ ID NO. 7) was then chemically synthesized (SEQ ID NO. 24) and inserted downstream of the T7 promoter of pET28a. BamH I and Xho The recombinant plasmid was obtained by transducing the recombinant plasmid between the I restriction sites; the recombinant plasmid was then transformed into Escherichia coli (E. coli). E.coli After inducing HT115 with IPTG (working concentration 1 mM) for 4 h, 1 mL of bacterial culture was taken, centrifuged, and resuspended in 100 μL of extraction buffer. The culture was then lysed with an equal volume of Tris-saturated phenol, centrifuged again, and the supernatant was collected. The target RNA band was detected by electrophoresis, and the RNA concentration was detected by Nanodrop 2000. The extraction buffer consisted of 10 mM Tris (pH 8.0) and 10 mM MgSO4.

[0037] SEQ ID NO.6: GGGCCCGUUGACCGGAAUGACCCUUUUGUUCGGCAUUCUGGUCAAAAGGAGGCUCCGGCCUCACCAAGGAGGUGGUCCAGAACUUUUGUUGUUGGACGUCAUUCAAUCUGGACCGUUGACCGGAAUGACCCUUUUGAAGGUGAAGUACCUCCCUGGCUAACGCAAGUUACCCGUUGACCGGAAUGACCAACUUUUGUCGCUCCAUUGUUUGAUCCCGUUACCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCAGCGUCUCGGCCUCACCGAACAAGGAGGCAG; where the bolded sequences represent target 1 and target 2. SEQ ID NO.7: GGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTA CCTCCCTGGCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAG; SEQ ID NO.24: CGCGGATCCGGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGCTCGAGCGC; where bps 1-3 and bps 309-311 (CGC) are protected bases, bps 4-9 (GGATCC) is the BamHI recognition sequence, and bps 303-308 (CTCGAG) is the BamHI recognition sequence.

[0038] Example 3 siRNA was prepared using a method similar to that in Example 2, the difference being that the target sequence of the single-stranded RNA (SEQ ID NO. 8) was target 1-target 3 as in Example 1, the coding sequence of the single-stranded RNA was shown in SEQ ID NO. 9, and the coding sequence of the chemically synthesized siRNA containing restriction enzyme sites was shown in SEQ ID NO. 25, as detailed below: SEQ ID NO.8: GGGCCCGUUGACCGGAAUGACCCUUUUGUUCGGCAUUCUGGUCAAAAGGAGGCUCCGGCCUCACCAAGGAGGUGGUCCAGAACUUUUGUUGUUGGACGUCAUUCAAUCUGGACCGUUGACCGGAAUGACCCUUUUGAAGGUGAAGUACCUCCCUGGCUAACGCAAGUUACCCGUUGACCGGAAUGACCAACUUUUGUCGCUCCAUUGUUUGAUCCCGUUACCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCAGCGUCUCGGCCUCACCGAACAAGGAGGCAGUUCAUCCAGAACGAGACGGUGCUUCUUUUGGGUCAUUCCGGUCAACGG; Among them, the bold sequences are Target 1 - Target 3; SEQ ID NO.9: GGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGG; SEQ ID NO.25: CGCGGATCCGGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAG TTACCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGG.

[0039] Example 4 siRNA was prepared using a method similar to that in Example 2, except that the target sequences of the single-stranded RNA (SEQ ID NO. 10) were target 1, target 4, and target 5 from Example 1, and the coding sequence of the single-stranded RNA was shown in SEQ ID NO. 11, as follows: SEQ ID NO.10: UAACGCAAGUUACCCGUUGACCGGAAUGACCAACUUUUGUCGCUCCAUUGUUUGAUCCCGUUACCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCAGCGUCUCGGCCUCACCGAACAAGGAGGCAGUUCAUCCAGAACGAGACGGUGCUUCUUUUGGGUCAUUCCGGUCAACGGAACCUACAGUUGCUCCGGACACGACUUUUGGGUCAUUCCGGUCAACGGUUGAUCCGCCACAAGGAGAAACAACUUUUGGGUCAUUCCGGUCAACGGGCCCGUUGUCCUUAUCGGCCGAGACUUUUGGGUCAUUCCGGAAGCCUCCACCGGAAUGACCCUUUUGUCGCAAGACUUUUGUCUCGGCCGAUAAGGACAAC; where the bolded sequences are target point 1, target point 4, and target point 5. SEQ ID NO.11: TAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGGAACCTAC AGTTGCTCCCGGACACGACTTTTGGGTCATTCCGGTCAACGGTTGATCCGCCACAAGGAGAAACAACTTTTGGGTCATTCCGGTCAACGGGCCCGTTGTCCTTATCGGCCGAGACTTTTGGGTCATTCCGGAAGCCTCCACCGGAATGACCCTTTTGTCGCAAGACTTTTGTCTCGGCCGATAAGGACAAC; SEQ ID NO.26: CGCGGATCCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGGAACCTAC AGTTGCCTCCGGACACGACTTTTGGGTCATTCCGGTCAACGGTTGATCCGCCACAAGGAGAAACAACTTTTGGGTCATTCCGGTCAACGGGCCCGTTGTCCTTATCGGCCGAGACTTTTGGGTCATTCCGGAAGCCTCCACCGGAATGACCCTTTTGTCGCAAGACTTTTGTCTCGGCCGATAAGGACAACCTCGAGCGC.

[0040] Example 5 siRNA was prepared using a method similar to that in Example 2, the difference being that the target sequence of the single-stranded RNA (SEQ ID NO. 12) was target 1-target 4 as in Example 1, the coding sequence of the single-stranded RNA was shown in SEQ ID NO. 13, and the coding sequence of the chemically synthesized siRNA containing restriction enzyme sites was shown in SEQ ID NO. 27, as detailed below: SEQ ID NO.12: GGGCCCGUUGACCGGAAUGACCCUUUUGUUCGGCAUUCUGGUCAAAAGGAGGCUCCGGCCUCACCAAGGAGGUGGUCCAGAACUUUUGUUGUUGGACGUCAUUCAAUCUGGACCGUUGACCGGAAUGACCCUUUUGAAGGUGAAGUACCUCCCUGGCUAACGCAAGUUACCCGUUGACCGGAAUGACCAACUUUUGUCGCUCC AUUGUUUGAUCCCGUUACCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCAGCGUCUCGGCCUCACCGAACAAGGAGGCAGUUCAUCCAGAACGAGACGGUGCUUUUUGGGUCAUUCCGGUCAACGGAACCUACAGUUGCUCCGGACACGACUUUUGGGUCAUUCCGGUCAACGG; among them, the bolded sequences are target 1-target 4; GGGCCCGUUGACCGGAAUGACCCUUUUG (SEQ ID NO.16) is a 5' end linker sequence (containing a kissing loop and tetrauridine acid). AGGCUCCGGCCUCACCAAGGAGGUGGUCCAGAACUUUUGUUGUUGGACGUCAUUCAAUCUGGACCGUUGACCGGAAUGACCCUUUUG (SEQ ID NO.17) is the first interval sequence; UAACGCAAGUUACCCGUUGACCGGAAUGACCAACUUUUGUCGCUCCAUUGUUUGAUCCCGUUACCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCAGCGUCUCGGCCUCACCGAACAAGGAGGCAG (SEQ IDNO. 18) is the second spacer sequence; CUUCUUUUGGGUCAUUCCGGUCAACGG (SEQ ID NO.19) is the third spacer sequence (containing tetrauridine acid). CGACUUUUGGGUCAUUCCGGUCAACGG (SEQ ID NO.20) is the fourth spacer sequence (containing tetrauridine acid). SEQ ID NO.13: GGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGGAACCTACAGTTGCTCCGGACACGACTTTTGGGTCATTCCGGTCAACGG; SEQ ID NO.27: CGCGGATCCGGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCACCAAGGAGGTGGTCCAGAACTTTTGTTGTTGGACGTCATTCAATCTGGACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAGTTACCCGTTGACCGGAATGACCAACTTTTGTCGCTCCATTGTTTGATCCCGTTACCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCAGCGTCTCGGCCTCACCGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGGAACCTACAGTTGCTCCGGACACGACTTTTGGGTCATTCCGGTCAACGGCTCGAGCGC。

[0041] Example 6 siRNA was prepared using a method similar to that in Example 2, the difference being that the target sequence of the single-stranded RNA (SEQ ID NO. 14) was target 1-target 5 from Example 1, and the sequence was optimized to a lower ΔG of -462.34 kcal / mol. The coding sequence of the single-stranded RNA is shown in SEQ ID NO. 15, and the coding sequence containing the enzyme cleavage site, synthesized chemically, is shown in SEQ ID NO. 28, as detailed below: SEQ ID NO.14: The bolded sequences represent target points 1 through 5. GGGCCCGUUGACCGGAAUGACCCUUUUG (SEQ ID NO.16) is a 5' end linker sequence (containing a kissing loop and tetrauridine acid). AGGCUCCGGCCUCCUUUUGACCAGAAUGCCGAACUUUUGUUGUUUCUCCUUGUGGCGGAUCAACCGUUGACCGGAAUGACCCUUUUG (SEQ ID NO.29) is the first interval sequence; UAACGCAAGUUAGCCAGGGAGGUACUUCACCUUCUUUUGUCGUGUCCGGAGCAACUGUAGGUUCCGUUGACCGGAAUGACCCUUUUGUCUCCGGACUUUUGAAGCACCGUCUCGUUCUGGAUGAACAAGGAGGCAG (SEQ IDNO. 30) is the second spacer sequence; CUUCUUUUGGGUCAUUCCGGUCAACGG (SEQ ID NO.19) is the third spacer sequence (containing tetrauridine acid). CGACUUUUGGGUCAUUCCGGUCAACGG (SEQ ID NO.20) is the fourth spacer sequence (containing tetrauridine acid). CAACUUUUGGGUCAUUCCGGUCAACGGGCCCGUUGUCCUUAUCGGCCGAGACUUUUGGGUCAUUCCGGAAGCCUCCCACCGGAAUGACCCUUUUGUCGCAAGACUUUUGUCUCGGCCGAUAAGGACAAC (SEQ ID NO. 21) is the 3' end connecting sequence; SEQ ID NO.15: GGGCCCGTTGACCGGAATGACCCTTTTGTTCGGCATTCTGGTCAAAAGGAGGCTCCGGCCTCCTTTTGACCAGAATGCCGAACTTTTGTTGTTTCTCCTTGTGGCGGATCAACCGTTGACCGGAATGACCCTTTTGAAGGTGAAGTACCTCCCTGGCTAACGCAAGTTAGCCAGGGAGGTACTTCACCTTCTTTTGTCGTGTCCGGAGCAACTGTAGGTTCCGTTGACCGGAATGACCCTTTTGTCTCCGGACTTTTGAAGCACCGTCTCGTTCTGGATGAACAAGGAGGCAGTTCATCCAGAACGAGACGGTGCTTCTTTTGGGTCATTCCGGTCAACGGAACCTACAGTTGCTCCGGACACGACTTTTGGGTCATTCCGGTCAACGGTTGATCCGCCACAAGGAGAAACAACTTTTGGGTCATTCCGGTCAACGGGCCCGTTGTCCTTATCGGCCGAGACTTTTGGGTCATTCCGGAAGCCTCCACCGGAATGACCCTTTTGTCGCAAGACTTTTGTCTCGGCCGATAAGGACAAC; SEQ ID NO.28: .

[0042] Example 7 siRNA was prepared using the method described in Example 6. The siRNA (1000 ng / µL) was mixed in equal volumes with a cationic delivery carrier (star-shaped polycation, hereinafter referred to as carrier A) in 20 mM HEPES buffer (pH 7.4). The mixture was vortexed at 2000 rpm for 10 min to form a nano-formulation through electrostatic self-assembly. The encapsulation efficiency of the obtained nano-formulation was >92% (HPLC detection of free siRNA). The final product exhibited >90% stability after 30 days of storage at 4°C protected from light. The cationic delivery carrier was a star-shaped polycation, and the synthesis method was disclosed in the literature [A Facile-Synthesized Star Polycation Constructed as a Highly Efficient Gene Vector in Pest Management. Jianhao Li, Jin Qian, Yuanyuan Xu, Shuo Yan, Jie Shen, and Meizhen Yin

[2019]

[0043] Test Example 1 Botrytis cinerea agar plates: Incubate Botrytis cinerea on potato dextrose agar (PDA) at 25°C for 4-5 days. PDA formulation: 200 g / L potato, 20 g / L glucose, and 15 g / L agar powder.

[0044] Preparation of pine wood nematodes for testing: Pine wood nematodes were reared on Botrytis cinerea agar plates, feeding on mycelia, and reared at 25℃ until they reached adulthood for later use. Nematode assay: 10 μL of siRNA extract or nanoparticles with a concentration of 500 μg / μL from Examples 2-7 were mixed with 90 μL of pine wood nematodes (approximately 50 nematodes, collected by sterile water elution using the Behman funnel method). The mixture was observed for 24 to 48 hours. In addition, 10 μL of sterile PBS was mixed with pine wood nematodes as a control (CK). Each group had 3 wells.

[0045] Method for calculating nematode killing effect: Observe the mortality of pine wood nematodes for 48 hours. After 24 hours, calculate the nematode killing rate based on the number of live nematodes (naturally bent bodies) and dead nematodes (rigid bodies) in a 96-well plate. Correct the cumulative nematode killing rate over the 24-hour observation period, calculate the corrected mortality rate, and perform a significance analysis of the differences.

[0046] The nematicidal effect is calculated according to formulas (1) and (2): Formula (1): Nematode killing rate or control mortality rate (%) = (Initial number of nematodes - Remaining number of surviving nematodes) / Initial number of nematodes × 100; Equation (2): Corrected mortality rate (%) = (nematode killing rate - CK) / (100 - CK) × 100; CK represents the mortality rate of pine wood nematodes in the control holes.

[0047] The results are shown in Table 1. The results showed that all different siRNA combinations and nano-formulations in Examples 2-7 had nematode-killing effects on pine wood nematodes. The combination containing 5 siRNA targets and its formulation showed the best effect, with no significant difference between the two. Dead nematodes began to appear 24 hours after treatment, and the cumulative mortality rate after 48 hours was 44.67%-88.33%, with a corrected mortality rate of 44.67%-88.33%.

[0048] Table 1. Determination of the lethality of siRNA combinations and formulations targeting BxGCC against pine wood nematode.

[0049] Test Example 2 Preparation of pine seedlings and pine wilt nematodes: The pine seedlings used were 1-2 year old susceptible black pine seedlings, which were allowed to acclimate indoors for 2 weeks before inoculation. Pine wilt nematodes were cultured on Botrytis cinerea agar plates. When inoculating the pine seedlings with pine wilt nematodes, the nematodes on the plates were harvested using a funnel method, and 2000 nematodes per seedling were inoculated onto the seedling trunk. The natural mortality rate of the control group (without pesticide application) was observed for calculating the control efficacy. The pine seedlings were cultured in a 25℃, 12-hour light incubation room.

[0050] Preparation of BxGCC-siRNA-nanoform formulation: The siRNA extract or nanoparticles obtained in Examples 2-7 were injected into the stems of pine seedlings inoculated with nematodes at a concentration of 500 ng / μL RNA. A syringe containing 1 mL of the agent was inserted 2-3 mm deep and injected slowly (natural absorption). Ten seedlings were used in each example, and the process was repeated three times. The yellowing of the needles and the survival of the plants were observed every 10 days. The control group was injected with 1 mL of sterile water, and the mortality rate of seedlings infected with pine wilt disease was observed for 30 days. Another group of blank control seedlings were not inoculated with pine wilt nematodes to observe the health of the seedlings themselves and exclude deaths caused by non-pine wilt nematode diseases.

[0051] Method for calculating the control effect: The survival rate of pine seedlings was calculated by observing their survival status on day 30, and a significance analysis of the differences was performed. The results are shown in Table 2.

[0052] The results showed that the different combinations of siRNAs targeting BxGCC and their formulations in Examples 2-7 all had a certain control effect on pine wilt disease. Dead seedlings began to appear from day 10 of observation, and the survival rate on day 30 ranged from 23.3% to 96.7%. Furthermore, the more siRNA targets in the combination, the better the effect, which is consistent with the results of the indoor bioassay experiments. The SPc formulation of the combination of 5 siRNA targets showed the best effect, indicating that formulation facilitates the delivery of siRNA combinations within the plant. Although the synergistic effect of the cationic delivery carrier was masked by the highly active RNA structure (ceiling effect) under high-concentration trunk injection conditions (drug directly enters the xylem), the cationic delivery carrier (Example 7) can impart superior physicochemical properties to the agent. For example, the cationic delivery carrier significantly improved the storage stability (shelf life) of the agent (4℃, 30-day stability >90%), which is crucial for the actual field application and commercialization of pesticides.

[0053] Table 2. Determination of the control efficacy of BxGCC-targeting siRNA combinations and their formulations on inoculated black pine seedlings inoculated with pine wilt nematode.

[0054] Test Example 3 Preparation of pine seedlings and pine wilt nematodes: The pine seedlings used were 1-2 year old susceptible black pine seedlings, which were allowed to acclimate indoors for 2 weeks before inoculation. Pine wilt nematodes were cultured on Botrytis cinerea agar plates. When inoculating the pine seedlings with pine wilt nematodes, the nematodes on the plates were harvested using a funnel method, and 2000 nematodes per seedling were inoculated onto the seedling trunk. The natural mortality rate of the control group (without pesticide application) was observed for calculating the control efficacy. The pine seedlings were cultured in a 25℃, 12-hour light incubation room.

[0055] Preparation of BxGCC-siRNA-nanoform formulation: The siRNA extract or nanoparticles obtained in Examples 2-7 were sprayed onto the entire pine seedlings inoculated with nematodes at a concentration of 500 ng / μL RNA. A second spray was applied every other week (a total of two sprays). Ten seedlings were used for each example, and the process was repeated three times. The yellowing of the pine needles and the survival of the plants were observed every 10 days. A control group was sprayed with sterile water, and the mortality rate of seedlings infected with pine wilt disease was observed for 30 days. Another group of blank control seedlings were not inoculated with pine wilt nematodes to observe the health of the seedlings themselves and exclude deaths caused by non-pine wilt nematode diseases.

[0056] Method for calculating the control effect: The survival rate of pine seedlings was calculated by observing their survival status on day 30, and a significance analysis of the differences was performed. The results are shown in Table 3.

[0057] The results showed that the different combinations of siRNAs targeting BxGCC and their formulations prepared in Examples 2-7 all had a certain control effect on pine wilt disease. The 30-day survival rate was 16.7%-93.3%, which was slightly lower than that of the injection into the trunk. Among them, the combination of 5 siRNA targets was the most effective after being nanoformed, which could increase the number of sprays and improve the control rate.

[0058] Table 3. Determination of the control efficacy of siRNA combinations targeting BxGCC and their formulations on inoculated black pine seedlings inoculated with pine wilt nematode.

[0059] The systemic conduction and synergistic mechanism of the nano-formulation for controlling pine wilt disease described in this invention is as follows: The excellent control efficacy of the nano-formulation provided by this invention in nematode control is attributed to the synergistic effect between the special siRNA secondary structure and the cationic delivery carrier.

[0060] Specifically, the nano-formulation described in this invention enables highly efficient bidirectional transport between the xylem and phloem. Since pine wood nematodes primarily inhabit the resin ducts and vascular tissues of pine trees, the cationic carrier described in this invention compresses siRNA with a multi-stem-loop structure to a nanoscale of approximately 100 nm via electrostatic interaction. The moderate positive charge on the particle surface reduces its non-specific binding to the negatively charged components of plant cell walls, allowing it to be systematically transported throughout the plant via xylem water transport and phloem photosynthetic product distribution, thereby effectively covering all core areas of nematode infection.

[0061] Meanwhile, the nano-formulation described in this invention significantly enhances biomembrane penetration and environmental stability. The unique hairpin and snout loop structures of the siRNA of this invention are thermodynamically more stable than traditional dsRNA. When encapsulated by a cationic carrier and forming a dense core-shell structure, it effectively resists attacks from complex nucleases (RNases) within pine trees, extending the duration of action, shelf life, and in vivo stability of the agent within the tree. Furthermore, the lipophilic or charged properties of the carrier surface enhance its affinity for the nematode body wall and intestinal cell membrane, facilitating efficient entry of siRNA into the nematode body through endocytosis and initiating a highly efficient RNA interference process.

[0062] Furthermore, the nano-formulation described in this invention exhibits multi-target synergistic and cascade amplification effects. After entering nematode cells, the multi-target siRNA, once nanoformulated, can simultaneously inhibit the expression of multiple key chloride ion channel genes. Because the multi-stem-loop structure can generate a higher density of effective siRNA fragments under Dicer enzyme cleavage, it forms a multi-point attack on the physiological system of pine wood nematodes, thereby achieving rapid suppression of nematode populations and significant disease repair even under low-dose conditions.

[0063] In summary, the siRNA combination and nanoformation for killing pine wilt disease provided by this invention have excellent effects in preventing and controlling pine wilt disease. Under optimal conditions, the survival rate of black pine seedlings inoculated with pine wilt disease reached 96.7% and 93.3% when injected into the trunk and sprayed, respectively. Moreover, the preparation method is simple and can be applied to the prevention and control of pine wilt disease.

[0064] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A nucleic acid composition targeting the BxGCC gene, characterized in that, The nucleic acid composition is a nucleic acid whose structure includes a hairpin loop, a kissing loop, a tetrauridine monophosphate, and an siRNA target sequence; the siRNA target sequence is target 1-target 5; the nucleotide sequences of target 1-target 5 are shown in SEQ ID NO.1-SEQ ID NO.5 respectively; the nucleotide sequence of the nucleic acid composition is shown in SEQ ID NO.6, SEQ ID NO.8, SEQ ID NO.10, SEQ ID NO.12 or SEQ ID NO.

14.

2. The nucleic acid composition according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the nucleic acid composition is shown in SEQ ID NO.7, SEQ ID NO.9, SEQ ID NO.11, SEQ ID NO.13 or SEQ ID NO.

15.

3. A nano-formulation, characterized in that, Includes a cationic delivery carrier and the nucleic acid composition of claim 1 or 2.

4. The nano-formulation according to claim 3, characterized in that, The mass ratio of the cationic delivery carrier to the nucleic acid composition is 1:0.1-10; the cationic delivery carrier is selected from cationic polymers and / or cationic liposomes.

5. The nano-formulation according to claim 4, characterized in that, The self-cationic polymer is selected from natural cationic polysaccharides.

6. A method for preparing a nano-formulation, characterized in that, include: The cationic delivery carrier according to any one of claims 3-5 and the nucleic acid composition according to claim 1 or 2 are mixed and then electrostatically self-assembled to obtain the nanoformulation.

7. The preparation method according to claim 6, characterized in that, The mixing method includes a vortex, the vortex rotating at 1000-3000 rpm for 1-30 min.

8. The use of the nucleic acid composition according to claim 1 or 2 and / or the nano-formulation according to any one of claims 3-5 in the preparation of an insecticide, wherein the insecticide targets the pine wood nematode.

9. A method for preventing and controlling pine wilt disease, characterized in that, include: The insecticide is applied to the trees to be treated by injection and / or spraying; the active ingredient of the insecticide includes the nucleic acid composition according to claim 1 or 2 and / or the nano-formulation according to any one of claims 3-5.

10. The method according to claim 9, characterized in that, The concentration of the nucleic acid composition in the insecticide is 100-1000 ng / μL.

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