A fusion protein containing a bt toxin protein, a complex of bt toxin protein RNA and application thereof

Abstract

This invention belongs to the field of biotechnology and relates to a fusion protein containing Bt toxin, a complex of Bt toxin and RNA, and their applications. The recombinant Bt toxin consists of two parts: a Bt toxin protein with toxic effects on hemiptera insects, and a functional domain capable of binding to RNA; the RNA consists of two parts: an aptamer capable of binding to the protein, and double-stranded RNA that interferes with the expression of key genes in the tea leafhopper. The recombinant Bt toxin can be co-expressed with the target double-stranded RNA, forming a protein-RNA complex. Droplets containing the recombinant Bt toxin and RNA complex are ingested by insects, absorbed through the intestines, where the Bt toxin is activated, causing intestinal perforation; while the target double-stranded RNA, once inside the body, can interfere with cysteine ​​proteases (…). Eocvp Gene expression leads to insect death; the two mechanisms work together to achieve the goal of biological control of insects, with significant effects and considerable application value.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a fusion protein containing Bt toxin protein, a complex of Bt toxin protein RNA, and their applications. Background Technology

[0002] Tea is an important economic crop in my country, and the tea green leafhopper is one of the common pests in tea gardens. Due to its rapid adaptation to different climates and environmental conditions, fast reproduction rate, and strong resistance to pesticides, it has become one of the most difficult and significant pests to control in tea gardens. While chemical pesticides have good control effects, long-term use of chemical pesticides easily causes environmental pollution, affects the quality and safety of agricultural products and biodiversity, and does not meet the requirements of sustainable agriculture. Compared with chemical pesticides, biological pesticides have less impact on non-target organisms, are highly selective, safe for humans and animals, have less impact on the ecological environment, induce pest diseases, and are less likely to develop resistance. Therefore, the reduction and efficiency improvement of chemical pesticides and the replacement of chemical pesticides with biological pesticides are imperative.

[0003] The Bt (Bacillus thuringiensis) gene is a gene isolated from Bacillus thuringiensis. Due to the high insecticidal efficacy, safety, and efficiency of its expression product, the Bt toxin protein, it has become the most widely used transgenic insecticidal gene. Because lepidopteran larvae have receptors for the Bt protein in their intestines, it can specifically kill lepidopteran insects without harming other animals. The emergence of transgenic Bt plants has ushered in a new era for modern agriculture.

[0004] Since the discovery of RNA interference (RNAi) mechanisms in 1998, it has been widely applied in gene function, biomedicine, and crop pest and disease control (Fire A et al. Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature, 1998, 391(6669):806-811.). RNAi technology silences the expression of important genes during the growth and development of harmful organisms, leading to growth and developmental disorders or death, thereby reducing the damage of harmful organisms to crops, achieving pest and disease control, and ensuring safe crop production (Gordon KHJ and Waterhouse PM. RNAi for insect-proofplants. Nature, 2007, 25(11), 1231–1232.). RNAi is a promising pest control strategy with great potential for controlling the tea green leafhopper (Empoascapirisuga Matumura) (Zhang, Ruirui, et al. "The Key Roles of Ionotropic Receptor Gene EnouIR25a in Host-Plant Finding by the Tea Green Leafhopper, Empoascaonukii Matsuda." (2023).). RNAi technology has many advantages, including specificity of control targets, ease of target development, convenient and easy application, green and pollution-free operation, no residue, and strong environmental compatibility, providing a good opportunity and huge space for the development of biopesticides. Summary of the Invention

[0005] To address the issues of pesticide residues and insect resistance caused by current chemical pesticides, and the relatively low insecticidal efficiency of broad-spectrum Bt toxin proteins, a method was developed that enhances pest control by co-expressing recombinant Bt toxin proteins with insecticidal double-stranded RNA. This complex degrades into amino acids and nucleic acids after activation, offering advantages such as no environmental pollution, safety for humans and animals, no product residues, and increased yield. It can effectively solve the pesticide residue problem in tea and reduce the impact on biodiversity.

[0006] This invention utilizes rational protein design to obtain two recombinant proteins, both of which can specifically bind to insecticidal double-stranded RNA (dsRNA) to form a recombinant protein-RNA complex for insecticidal use in tea. This complex has the following characteristics: 1) The recombinant protein has two functional domains: a) Bt toxin, a novel, broad-spectrum, multifunctional insecticidal protein with good insecticidal effects against hemiptera insects; b) The recombinant protein contains functional peptide chains at its C- and / or N-terminus, which can specifically bind to insecticidal double-stranded RNA to form a protein-RNA complex. 2) The insecticidal double-stranded RNA consists of two parts: a) an aptamer (TAT) that specifically binds to the functional peptide chains at the C- and / or N-terminus of the recombinant protein; b) the insecticidal double-stranded RNA (this exogenous dsRNA, after being ingested by insects, is degraded into 21-23 nt small interfering RNA (siRNA), triggering RNAi, interfering with the expression of essential insect genes, thus leading to insect death). 3) The recombinant protein and RNA are co-expressed in *E. coli* to form a complex, facilitating large-scale production at low cost.

[0007] The recombinant protein RBP_BPP2_RBP is similar in design to BPP1_RBP, but the difference is that BPP1_RBP has a functional peptide chain at the C-terminus that can bind to the TAT aptamer, while RBP_BPP2_RBP has one such functional peptide chain at both the N-terminus and C-terminus to improve the efficiency of binding RNA.

[0008] The technical solution adopted in this invention is as follows:

[0009] This invention first provides a fusion protein that can be used to control hemipteran insects, comprising, in sequence, a Bt toxic protein and an RNA-binding domain (RBP). The Bt toxic protein is an insecticidal protein with killing activity against hemipteran insects; specifically, the proteins possessing the insecticidal activity of the Bt toxic protein are BPP1 and BPP2 proteins.

[0010] The amino acid sequence of the Bt toxin protein BPP1 is shown in SEQ ID NO:1:

[0011] MGMKFNKLMVSGIAFVCSSTFFTAYIQAAEIEENKNSTTALSESREELFGIVDVDKSFDKMAQYYYFKNLQGKIENGYKFTNYKGSSTNLDKFKTTSDKVSDLQITGLLPEYIGGNTFPNHTEQEQTFSTTSYARTITESVTTTTQSGFSVSGQGGLLNI PIIFPNGTSITASNAGHLSQAKQESDTTRTLTAPSQAVKVPPGKTYRAEVKLVRKSFKANVGFNGTIAPPLSTDIKMNARWEGAGGRPNKTVTKSYATQTLYRDMTPSQQKDLGITFNSTTGQFDVNGKAVIDGIAGSEMEVAIYDITNARTPKLVSKTYY

[0012] The amino acid sequence of the Bt toxin protein BPP2 is shown in SEQ ID NO:2:

[0013] MTNELGSNTQDSNEIDNGIKALDPFNFYRIRTFCGKMLDVVGQSTSDNAMVVQYSINNKPNQNFLVFTLDDGYSIIAAENSGKVLDISEDFFFKGMLIQYHFANSDNQKFLISNNGTIAVKRSGKVFDIPGASTSNDAPVIAYNFNNAANQKFTFERVKTFQVPSPSIGTLPPAPDFKN DINEQLPDKTNPVITHFSTIPYIMANDATFNSHQQIQYSPYYKLVRIQYWEKVTQRILGPRDDYEYNKTKGISKTDQVSMTETVSMSVGADFGFMFKGFSASLSAQITKELSVTKSTSTTEMTEETYKEKYTNPFNYELARAQYMLVNEFYVTRMDGTRITANWTLRDNTQTVTRIFPKS

[0014] Both BPP1 and BPP2 can recognize receptors on the microvilli of midgut epithelial cells in larvae. After interacting with a series of receptor proteins, they form permeability channels on the midgut cell membrane, causing midgut cell damage and detachment, leading to larval cessation of feeding and death. This increases the control efficiency of the protein-RNA complex; the amino acid sequence of the RNA-binding domain RBP, as shown in SEQ ID NO:3: SYGRKKRRQRRRPPQ, can specifically bind to the TAT aptamer to form a protein-RNA complex.

[0015] The fusion protein provided by the present invention encodes a nucleic acid, specifically the nucleotide sequence of which is shown in SEQ ID NO:9 or SEQ ID NO:10.

[0016] This invention provides a protein-RNA complex of a fusion protein and RNA, which is composed of the fusion protein and functional double-stranded RNA.

[0017] Preferably, it is co-expressed in recombinant Escherichia coli and forms a protein-RNA complex.

[0018] More specifically, the functional double-stranded RNA consists of two parts: an RNA aptamer TAT that can specifically bind to recombinant proteins, and a double-stranded RNA with insecticidal function (the positive and negative strands of the double-stranded RNA are connected by the RNA aptamer TAT).

[0019] Specifically, the nucleotide sequence of the RNA aptamer TAT is shown in SEQ ID NO:4: CGAAGCUUGAUCCCGUUUG CCGGUCGAUCGCUUCG.

[0020] The Chain 1 (sense strand) of the functional double-stranded RNA is shown in SEQ ID NO:5: 5'-GGGUCCCAAGAA GUAAAAAUCUCCCCCCAAAGUGUACUUCCCGAUCUAACAAAUUUCACUCUG-3', and the corresponding Chain 2 (paired strand) of the functional double-stranded RNA is shown in SEQ ID NO:13: 5'-CAGAGUGAAAUUUGUUAGAUC GGGAAGUACACUUUGGGGGGAGAUUUUUACUUCUUGGGACCC-3'.

[0021] Alternatively, the Chain 1 (sense chain) of the functional double-stranded RNA is as shown in SEQ ID NO:6: 5'-GGGUCCCAAG AAGUAAAAAUCUCCCCCCAAAGUGUACUUCCCGAUCUAACAAAUUUCACUCUGGUUGGUCAAGAAAUAGUAAACAACUUCAAGUGUGACAAGUGGAGAUCGAUUAUCACACAUGGCGA-3', and the corresponding Chain 2 (paired chain) of the functional double-stranded RNA is as shown in SEQ ID NO:14: 5'-UCGCCAUGUGUGAUAAU CGAUCUCCACUUGUCACACUUGAAGUUGUUUACUAUUUCUUGACCAACCAGAGUGAAAUUUGUU AGAUCGGGAAGUACACUUUGGGGGAGAUUUUUACUUCUUGGGACCC-3'.

[0022] The nucleotides of the functional double-stranded RNA are shown in SEQ ID NO:7 (ShortdsRNA): GGGUCCCAAGAAGUAAAA AUCUCCCCCCAAAGUGUACUUCCCGAUCUAACAAAUUUCACUCUGCGAAGCUUGAUCCCGUUUGC CGGUCGAUCGCUUCGCAGAGUGAAAUUUGUUAGAUCGGGAAGUACACUUUGGGGGAGAUUUUUACUUCUUGGGACCC.

[0023] Or as shown in SEQ ID NO: 8 (LongdsRNA): GGGUCCCAAGAAGUAAAAAUCUCCCCCCAAAGU GUACUUCCCGAUCUAACAAAUUUCACUCUGGUUGGUCAAGAAAUAGUAAACAACUUCAAGUGUGACAAGUGGAGAUCGAUUAUCACACAUGGCGACGAAGCUUGAUCCCGUUUGCCGGUCGAUCGCUUC GUCGCCAUGUGUGAUAAUCGAUCUCCACUUGUCACACUUGAAGUUGUUUACUAUUUCUUGACCAACCAGAGUGAAAUUUGUUAGAUCGGGAAGUACACUUUGGGGGGAGAUUUUUACUUCUUGGGACCC.

[0024] The present invention provides a co-expression recombinant vector comprising an expression cassette of the nucleic acid encoding the fusion protein and the nucleic acid encoding the functional double-stranded RNA.

[0025] Preferably, the nucleic acid encoding the fusion protein and the nucleic acid encoding the functional double-stranded RNA are controlled by different promoters and inserted into different sites of the same recombinant vector.

[0026] Specifically, the nucleotide sequence of the gene encoding the BPP1_RBP recombinant protein is shown in SEQ ID NO:9:

[0027]

[0028] Specifically, the nucleotide sequence of the gene encoding the RBP_BPP2_RBP recombinant protein is shown in SEQ ID NO:10:

[0029]

[0030] The nucleotide sequence of the DNA encoding the functional double-stranded RNA is shown in SEQ ID NO:11:

[0031] GGGTCCCAAGAAGTAAAAATCTCCCCCCAAAGTGTACTTCCCGATCTAACAAATTTCACTCTGCGAAGCTTGATCCCGTTTGCCGGTCGATCGCTTCGCAGAGTGAAATTTGTTAGATCGGGAAGTACACTTTGGGGGGAGATTTTTACTTCTTGGGACCC.

[0032] Or for SEQ ID NO:12:GGGTCCCAAGAAGTAAAAATCTCCCCCCAAAGTGTACTTCCCGATCTAACAAATTTCACTCTGGTTGGTCAAGAAATAGTAAACAACTTCAAGTGTGACAAGTGGAGATCGATTATCACACATGGCGACGAAGCTTGATCCCG TTTGCCGGTCGATCGCTTCGTCGCCATGTGTGATAATCGATCTCCACTTGTCACACTTGAAGTTGTTTACTATTTCTTGACCAACCAGAGTGAAATTTGTTAGATCGGGAAGTACACTTTGGGGGGAGATTTTTACTTCTTGGGACCC.

[0033] The present invention further provides a recombinant bacterium containing the aforementioned co-expression recombinant vector. Preferably, it is a strain deficient in degrading double-stranded RNase, such as *Escherichia coli*.

[0034] The present invention further provides the application of the aforementioned protein-RNA complex in pesticides or pest control for economic crops.

[0035] This invention also provides a method for controlling hemiptera pests, which involves spraying the aforementioned protein-RNA complex as an insecticide onto crops. Preferably, the plant is a tea plant, and the hemiptera pest is the tea leafhopper.

[0036] The advantages of this invention are: the recombinant protein can specifically bind to RNA to form a protein-RNA complex, and the recombinant protein also has insecticidal function, which can enhance the insecticidal effect of the protein-RNA complex. Specifically, droplets containing the recombinant Bt toxin protein-RNA complex are ingested by insects and absorbed through the intestines. The Bt toxin protein is activated, causing intestinal perforation in the insects; while the target double-stranded RNA, after entering the body, can interfere with the expression of the cysteine ​​protease (Eocvp) gene, leading to insect death. The two mechanisms work together to achieve the purpose of insect biological control, with significant effects and great application value. The recombinant Bt toxin protein and the insecticidal double-stranded RNA of this invention are co-expressed and produced in a cell factory, which is convenient for control and operation, easy for large-scale industrial production, and has low production costs. The degradation products of the protein-RNA complex are non-toxic and harmless to humans and environmentally friendly. Attached Figure Description

[0037] Figure 1 Schematic diagram of recombinant protein. A represents the BPP1_RBP recombinant protein; B represents the RBP_BPP2_RBP-double-stranded RNA complex.

[0038] Figure 2 A schematic diagram of functional double-stranded RNA. Where A represents Short dsRNA and B represents Long dsRNA.

[0039] Figure 3 Schematic diagram of co-expression plasmids. A shows the map of plasmid pET-Duet-BPP1_RBP-ShortdsRNA; B shows the map of plasmid pET-Duet-RBP_BPP2_RBP-LongdsRNA.

[0040] Figure 4 Recombinant protein BPP1_RBP and ShortdsRNA were co-expressed. In this diagram, A represents the expression of recombinant protein BPP1_RBP, and B represents the expression of ShortdsRNA.

[0041] Figure 5 Recombinant protein RBP_BPP2_RBP was co-expressed with LongdsRNA. In this diagram, A represents the expression of recombinant protein RBP_BPP2_RBP, and B represents the expression of LongdsRNA.

[0042] Figure 6 Field trial demonstration. A represents the tea garden test site; B represents the application method; and C represents the experimental kit. Detailed Implementation

[0043] The present invention will be further illustrated below with specific embodiments in order to better understand the present invention, but these embodiments do not constitute a limitation thereof.

[0044] Example 1: Construction of co-expression plasmid

[0045] 1. Construction of recombinant proteins

[0046] The recombinant protein (BPP_RBP) has two main functions: one is the Bt toxin protein BPP1, which has insecticidal properties.

[0047] (MGMKFNKLMVSGIAFVCSSTFFTAYIQAAEIEENKNSTTALSESREELFGIVDVDKSFDKMAQYYYFKN LQGKIENGYKFTNYKGSSTNLDKFKTTSDKVSDLQITGLLPEYIGGNTFPNHTEQEQTFSTTSYARTITESVTTTTQSGFSVSGQGGLLNIPIIFPNGTSITASNAGHLSQAKQESDTRTLTAPSQAVK VPPGKTYRAEVKLVRKSFKANVGFNGTIAPPLSTDIKMNARWEGAGGRPNKTVTKSYATQTLYRDMTPSQQKDLGITFNSTTGQFDVNGKAVIDGIAGSEMEVAIYDITNARTPKLVSKTYY) or BPP2

[0048] (MTNELGSNTQDSNEIDNGIKALDPFNFYRIRTFCGKMLDVVGQSTSDNAMVVQYSINNKPNQNFLVFTLDDGYSIIAAENSGKVLDISEDFFFKGML IQYHFANSDNQKFLISNNGTIAVKRSGKVFDIPGASTSNDAPVIAYNFNNAANQKFTFERVKTFQVPSPSIGTLPPAPDFKNDINEQLPDKTNPVITHF STIPYIMANDATFNSHQQIQYSPYYKLVRIQYWEKVTQRILGPRDDYEYNKTKGISKTDQVSMTETVSMSVGADFGFMFKGFSASLSAQITKELSVTKSTSTTEMTEETYKEKYTNPFNYELARAQYMLVNEFYVTRMDGTRITANWTLRDNTQTVTRIFPKS), which can increase the insecticidal effect and improve control efficiency (Banerjee, Rahul, et al. al. "Peptide mediated, enhanced toxicity of abacterial pesticidal protein against southern green stink bug." Microbial Biotechnology 15.7(2022):2071-2082.)(Cao, Beibei, et al. "The crystal structure of Cry78Aa from Bacillus thuringiensis provides insights into its insecticidal activity." Communications Biology 5.1(2022):801.), can be ingested by insects and degraded into toxic active peptides in the alkaline environment of the insect's midgut. These peptides then bind to specific receptors on the striatum cells of the insect's midgut epithelium, causing cell membrane perforation, disrupting cellular osmotic balance, and ultimately leading to the insect ceasing to feed and dying. The other part is the RNA-binding domain (SYGRKKRRQRRRPPQ) (Weeks KM, Ampe C, Schultz SC, et al. Fragments of the HIV-1 Tat Protein Specifically Bind TAR RNA[J]. Science, 1990, 249(4974): 1281-1285.), which can specifically bind to RNA and form a protein-RNA complex.

[0049] 2. Double-stranded RNA design

[0050] Double-stranded RNA mainly consists of two parts: an RNA aptamer that can specifically bind to recombinant proteins (CGAAGCUUGAUCCCGUU UGCCGGUCGAUCGCUUCG)(Yamamoto R, Katahira M, Nishikawa S, et al. A novel RNA motif that binds efficiently and specifically to the Tat protein of HIV and inhibits the transactivation by Tat of transscription in vitro and in vivo[J]. Genes to Cells, 2000, 5(5):371-388.); and functional double-stranded RNA (ShortdsRNA and LongdsRNA, see...). Figure 2 The double-stranded RNA, once absorbed by the tea green leafhopper, is degraded into 21-23 nt interfering RNA (siRNA), which targets and degrades the mRNA encoding cysteine ​​proteinase 1 (GenBank ID: MH036890.1). Cysteine ​​proteinase 1 is an essential gene for the life activities of the tea green leafhopper, expressed throughout its entire life cycle (Qin, Daozheng, et al. "Clarification of the identity of the tea green leafhopper based on morphological comparison between Chinese and Japanese specimens." PLoS One 10.9 (2015): e0139202.). Highly efficient interference with cysteine ​​proteinase 1 gene expression can effectively disrupt its life activities and lead to the death of the tea green leafhopper.

[0051] 3. Construction of co-expression plasmids

[0052] Construction of protein expression plasmids pET-Duet-BPP1_RBP and pET-Duet-RBP_BPP2_RBP:

[0053] The recombinant protein BPP1_RBP gene sequence was inserted into the pET-Duet-1 plasmid, between NdeI and KpnI, to obtain the plasmid pET-Duet-BPP1_RBP for BPP1_RBP protein expression. Similarly, the recombinant protein RBP_BPP2_RBP gene sequence was inserted into the pET-Duet-1 plasmid, between NdeI and KpnI, to obtain the plasmid pET-Duet-RBP_BPP2_RBP for RBP_BPP2_RBP protein expression.

[0054] Construction of co-expression plasmids pET-Duet-BPP1_RBP-ShortdsRNA and pET-Duet-RBP_BPP2_RBP-LongdsRNA:

[0055] The recombinant protein BPP1_RBP gene sequence was inserted into the pET-Duet-1 plasmid between NdeI and KpnI. The double-stranded RNA expression cassette (containing the T7 promoter, template DNA for the double-stranded RNA, and the T7 terminator) gene sequence was inserted between NotI and XhoI in the same co-expression plasmid, resulting in the co-expression plasmid pET-Duet-BPP1_RBP-ShortdsRNA. Figure 3 A). The construction method of pET-Duet-RBP_BPP2_RBP-LongdsRNA is the same ( Figure 3 (B)

[0056] Example 2: Co-expression of recombinant protein BPP1_RBP and ShortdsRNA in Escherichia coli

[0057] 1) The plasmid pET-Duet-BPP1_RBP-ShortdsRNA was transformed into Escherichia coli, and positive transformants were screened and inoculated into LB medium containing 100 mg / L kanamycin. The seed culture was obtained by overnight culture at 37°C.

[0058] 2) Transfer the seed culture to TB medium containing 100 mg / L kanamycin at an inoculation rate of 1%, and incubate at 30°C until the OD600 reaches 0.6-0.8. Add 0.8 mM IPTG to induce the expression of recombinant protein, and collect the cells by centrifugation after incubating at 30°C for 4 hours.

[0059] 3) Take an appropriate amount of cells and extract total RNA using the phenol method, and run RNA PAGE gel to verify RNA expression;

[0060] 4) Take an appropriate amount of cells and resuspend them in lysis buffer (50mM Tris-HCl, 50mM NaCl, 1mM EDTA, pH 7.4). Lyse the cells using an ultrasonicator for 20 min, centrifuge at 12000 rpm for 20 min, and collect the supernatant for later use. Take a small sample for SDS-PAGE analysis to determine protein expression.

[0061] Based on the experimental results, we can conclude that: Figure 4 As shown in Figure A, the protein size obtained after induced expression is approximately 41 kDa, while the recombinant protein (BPP1_RBP) is 38.72 kDa. Therefore, the recombinant protein BPP1_RBP was successfully expressed. Figure 4 As shown in Figure B, compared with the total RNA of strains without the co-expression plasmid, the total RNA extracted after induction containing the co-expression plasmid pET-Duet-BPP1_RBP-Short dsRNA contained overexpressed Short dsRNA (161 nt), as indicated by the arrows in the figure. In conclusion, the co-expression of recombinant protein (BPP1_RBP) and RNA (Short dsRNA) was successful.

[0062] Example 3: Co-expression of recombinant protein RBP_BPP2_RBP and LongdsRNA in Escherichia coli

[0063] 1) The transformation, culture, protein and RNA extraction and analysis of the co-expression plasmid pET-Duet-RBP_BPP2_RBP-Long dsRNA were performed according to the method in Example 2.

[0064] Based on the experimental results, we can conclude that: Figure 5 As shown in Figure A, the size of the protein obtained after induction is between 41 and 53 kDa, and the size of the recombinant protein (RBP_BPP2_RBP) is 47.14 kDa. Therefore, the recombinant protein RBP_BPP2_RBP is successfully expressed.

[0065] Depend on Figure 5 As shown in Figure B, compared with the total RNA of strains without the co-expression plasmid, the total RNA extracted after induction containing the co-expression plasmid pET-Duet-RBP_BPP2_RBP-Long dsRNA contained overexpressed Long dsRNA (291 nt), as indicated by the arrows in the figure. In conclusion, the co-expression of recombinant protein (RBP_BPP2_RBP) and RNA (Long dsRNA) was successful.

[0066] Example 4: Characterization of the control effects of recombinant proteins BPP1_RBP and RBP_BPP2_RBP on tea leafhopper

[0067] 1) Plasmids pET-Duet-BPP1_RBP and pET-Duet-RBP_BPP2_RBP were transformed into Escherichia coli, and positive transformants were screened and inoculated into LB medium containing 100 mg / L kanamycin. The seed culture was obtained by overnight culture at 37°C.

[0068] 2) Transfer the seed culture to TB medium containing 100 mg / L kanamycin at an inoculation rate of 1%, and incubate at 30°C until the OD600 reaches 0.6-0.8. Add 0.8 mM IPTG to induce the expression of recombinant protein, and collect cells after incubating at 30°C for 6 hours.

[0069] 3) Resuspend the cells in lysis buffer (50mM Tris-HCl, 50mM NaCl, 1mM EDTA, pH 7.4), add lysozyme to a final concentration of 0.2mg / ml, incubate at 37℃ for 30min, centrifuge at 12000rpm for 20min and collect the supernatant.

[0070] 4) A certain number of tea leafhoppers in the nymph stage were captured in a square box and sealed with breathable gauze. This not only achieved the effect of ventilation but also prevented the tea leafhoppers from escaping from the test box. At the same time, some fresh tea leaves were placed in the test box for the tea leafhoppers to feed on.

[0071] 5) Spray the lysis buffer (control group), pET-Duet-BPP1_RBP, and pET-Duet-RBP_BPP2_RBP lysis supernatant into the test kits respectively. Figure 6 The pesticide (AC) was placed on the tea trees and covered with a tent to protect them from sunlight and rain. The mortality of the tea green leafhoppers was counted 48 hours after the pesticide was applied.

[0072] Example 5: Characterization of the control effect of protein-RNA complex on tea green leafhopper

[0073] 1) The co-expression plasmids pET-Duet-BPP1_RBP-Short dsRNA and pET-Duet-RBP_BPP2_RBP-Long dsRNA were transformed into E. coli, and cultured according to the method in Example 4.

[0074] 2) The seeds obtained from overnight culture were transferred to TB medium at an inoculation rate of 1% and cultured at 30°C until OD was achieved. 600 Add 1 mM IPTG to the solution at a concentration of 0.6–0.8 to induce the expression of recombinant protein and RNA. After 4 hours, centrifuge to collect the cells.

[0075] 3) Take an appropriate amount of cells and resuspend them in lysis buffer (50mM Tris-HCl, 50mM NaCl, 1mM EDTA, pH 7.4). Add lysozyme to a final concentration of 0.2mg / ml, incubate at 37℃ for 30min, centrifuge at 12000rpm for 20min and collect the supernatant for later use.

[0076] 5) A certain number of tea green leafhoppers at different stages were captured in the experimental box and sealed with breathable gauze to achieve the effect of ventilation. At the same time, some fresh tea leaves were placed in the experimental box for the tea green leafhoppers to feed on.

[0077] 6) Spray the lysate supernatant from the protein-RNA co-expressing bacterial cells evenly into the test kit. Figure 6 The agent (AC) was applied to tea trees and placed on them, then covered with a tent to protect them from sunlight and rain. The results were tallied 48 hours after application. The results are shown in Table 1.

[0078] Table 1. Statistical data on insecticidal effects in field trials

[0079]

[0080] In summary, the experimental results show that, as indicated in Table 1, the mortality rate was 0% in the control group; 14.29% in the group sprayed with BPP1_RBP protein; 18.33% in the group sprayed with RBP_BPP2_RBP protein; 51.67-60% in the group sprayed with BPP1_RBP and ShortdsRNA; and 58.33-68.33% in the group sprayed with RBP_BPP2_RBP and LongdsRNA. Therefore, co-expression of Bt toxin protein and target double-stranded RNA can effectively control the nymphs of the tea green leafhopper.

Claims

1. A protein-RNA complex of a fusion protein and RNA, comprising a fusion protein and a functional double-stranded RNA, characterized in that, The fusion protein includes Bt toxin BPP1 or BPP2, and an RNA-binding domain RBP linked at its N-terminus and / or C-terminus, which are connected by a linker peptide. The amino acid sequence of Bt toxin BPP1 is shown in SEQ ID NO:1, the amino acid sequence of Bt toxin BPP2 is shown in SEQ ID NO:2, and the amino acid sequence of the RNA-binding domain RBP is shown in SEQ ID NO:

3. The functional double-stranded RNA consists of two parts: an RNA aptamer TAT that can specifically bind to recombinant proteins and a double-stranded RNA with insecticidal function. The nucleotide sequence of the RNA aptamer TAT is shown in SEQ ID NO:4, and the positive strand of the nucleotide sequence of the insecticidal double-stranded RNA is shown in SEQ ID NO:5, and the paired strand is shown in SEQ ID NO:13, or the positive strand is shown in SEQ ID NO:6, and the paired strand is shown in SEQ ID NO:14; wherein the positive strand and the paired strand are connected by the nucleotide sequence of the RNA aptamer TAT.

2. The protein-RNA complex of the fusion protein and RNA as described in claim 1, characterized in that, The nucleotides of the functional double-stranded RNA are shown in SEQ ID NO:7 or SEQ ID NO:

8.

3. The protein-RNA complex of the fusion protein and RNA as described in claim 1, characterized in that, It is achieved by co-expressing and forming a protein-RNA complex in recombinant Escherichia coli.

4. A co-expression recombinant vector comprising an expression cassette encoding a fusion protein's nucleic acid and a functional double-stranded RNA's nucleic acid; characterized in that, The fusion protein includes Bt toxin BPP1 or BPP2, and an RNA-binding domain RBP linked at its N-terminus and / or C-terminus, which are connected by a linker peptide. The amino acid sequence of Bt toxin BPP1 is shown in SEQ ID NO:1, the amino acid sequence of Bt toxin BPP2 is shown in SEQ ID NO:2, and the amino acid sequence of the RNA-binding domain RBP is shown in SEQ ID NO:

3. The functional double-stranded RNA consists of two parts: an RNA aptamer TAT that can specifically bind to recombinant proteins and a double-stranded RNA with insecticidal function. The nucleotide sequence of the RNA aptamer TAT is shown in SEQ ID NO:4, and the positive strand of the nucleotide sequence of the insecticidal double-stranded RNA is shown in SEQ ID NO:5, and the paired strand is shown in SEQ ID NO:13, or the positive strand is shown in SEQ ID NO:6, and the paired strand is shown in SEQ ID NO:14; wherein the positive strand and the paired strand are connected by the nucleotide sequence of the RNA aptamer TAT.

5. The co-expression recombinant vector as described in claim 4, characterized in that, The fusion protein's encoding nucleic acid and the functional double-stranded RNA's encoding nucleic acid are controlled by different promoters and are inserted into different sites on the same recombinant vector.

6. The co-expression recombinant vector as described in claim 5, characterized in that, The nucleotide sequence of the coding nucleic acid encoding the functional double-stranded RNA is shown in SEQ ID NO:11 or SEQ ID NO:

12.

7. A recombinant bacterium containing the co-expression recombinant vector as described in claim 6, wherein the recombinant bacterium is a defective strain that degrades double-stranded RNase.

8. The recombinant bacteria as described in claim 7, characterized in that, It is Escherichia coli.

9. The use of the protein-RNA complex of the fusion protein and RNA as described in any one of claims 1 to 3 in the preparation of an insecticide for the tea leafhopper.

10. A method for controlling the tea leafhopper, characterized in that, The method involves spraying the protein-RNA complex of the fusion protein and RNA as described in any one of claims 1 to 3 onto plants as an insecticide.

11. The method as described in claim 10, characterized in that, The plant in question is a tea tree.

Images