Preparation method and application of biological herbicide based on RNA (Ribonucleic Acid) interference technology

Abstract

The invention discloses a preparation method and application of a biological herbicide based on an RNA (Ribonucleic Acid) interference technology, which specifically comprises the following steps: designing a specific siRNA (Small Interference Ribonucleic Acid) sequence through a targeted glutamine synthetase gene (GS2), cloning a reverse repeat structure of the specific siRNA sequence to multiple cloning sites of an L4440 vector, and transforming the reverse repeat structure to RNase III defective escherichia coli HT115 for induced fermentation. By optimizing IPTG concentration and induction time, the thalli efficiently express target dsRNA, and a dsRNA aqueous solution is prepared after splitting decomposition and purification. Foliage spraying is adopted during spraying treatment, dsRNA can permeate into plant cells through pores, GS2 mRNA is specifically degraded, ammonium ions in the cells are accumulated, a photosynthetic system collapses, and leaves wither, wither and yellow and finally die within 72 hours. Compared with a traditional chemical herbicide, the herbicide has the advantages of being accurate in targeting, safe, environmentally friendly, free of residues and the like, and an efficient and sustainable biological solution is provided for agricultural weeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological pesticides, and in particular relates to a preparation method of a biological herbicide based on RNA interference technology and application thereof. Background Art

[0002] Chemical herbicides are the most common type of herbicide on the market. They are typically applied by spraying or coating, disrupting the plant's normal physiological metabolism and achieving the desired weed control. While chemical herbicides offer advantages such as rapid effectiveness, wide range of effects, and ease of use, they also present numerous challenges. For example, traditional chemical pesticides have long R&D cycles and high costs, and long-term use can lead to increased weed resistance, reducing their effectiveness. Furthermore, chemical herbicides can easily pollute the environment, affecting soil microbial communities and non-target organisms. Some of their components can also remain in crops and soil, accumulating through the food chain and posing potential risks to human health. Therefore, the development of novel, safe, and highly effective bioherbicides is a pressing need within the pesticide industry.

[0003] RNA interference (RNAi) is a gene silencing technology triggered by endogenous or exogenous double-stranded RNA (dsRNA). It can precisely target and inhibit the expression of key metabolic genes in target plants, thereby achieving efficient and safe weed control. Compared with traditional chemical herbicides, RNAi technology is highly specific, reducing the impact on non-target plants and the ecological environment, while also producing no persistent chemical residues, helping to reduce soil and water pollution. Furthermore, RNAi herbicides can effectively address weed resistance and delay the development of resistant weeds, making them an important direction for the future development of biopesticides. Summary of the Invention

[0004] The purpose of the present invention is to overcome the shortcomings and defects mentioned in the above background technology and provide a biological herbicide based on RNAi technology, by designing specific dsRNA by targeting the tobacco glutamine synthetase gene (GS2), and using a biofermentation method to prepare a highly efficient RNA biopesticide.

[0005] The present invention provides a method for preparing a biological herbicide based on RNA interference technology, and the specific steps are as follows:

[0006] (1) Design target dsRNA sequence

[0007] The tobacco glutamine synthetase gene (GS2) was selected as the target gene, a specific siRNA sequence was designed, and the target dsRNA fragment was obtained by PCR amplification.

[0008] (2) Construction of engineered bacteria

[0009] The designed dsRNA fragment inverted repeat structure was cloned into the multiple cloning site of L4440 vector and transformed into RNaseIII-deficient Escherichia coli HT115 for induced fermentation.

[0010] (3) Induced fermentation to produce dsRNA

[0011] Fermentation was induced in LB and TB liquid media, respectively. By optimizing the isopropyl-β-D-thiogalactopyranoside (IPTG) concentration and induction time, the cells efficiently expressed the target dsRNA. The dsRNA was then lysed and purified to prepare an aqueous solution.

[0012] (4) Indoor spraying experiment

[0013] dsRNA is applied to tobacco leaves through foliar spraying, allowing it to penetrate into plant cells through the stomata and specifically degrade GS2 mRNA, leading to the accumulation of ammonium ions in the cells and the collapse of the photosynthetic system, thereby causing the tobacco leaves to turn yellow, the plants to wilt severely and eventually die, ultimately achieving a weed control effect.

[0014] Furthermore, the nucleotide sequence of the dsRNA is SEQ ID NO. 1.

[0015] Furthermore, the final concentration of IPTG is 0.1 mM.

[0016] Furthermore, the induction time is 6 hours.

[0017] Furthermore, the net content of dsRNA during spraying is 1 μg.

[0018] Furthermore, the composition of the LB liquid medium is: 1% tryptophan, 0.5% yeast extract, and 1% sodium chloride;

[0019] Furthermore, the composition of the TB liquid culture medium is: 1.2% tryptophan, 2.4% yeast extract, 0.72 M K2HPO4, 0.17 M KH2PO4, and 0.4% glycerol.

[0020] Furthermore, among the LB and TB liquid culture media, the TB liquid culture medium is more suitable for the fermentation production of dsRNA.

[0021] Furthermore, the time it takes for tobacco leaves to turn yellow is 3 days, and the time it takes for plants to severely wilt and die is 7 days.

[0022] Beneficial effects of the present invention:

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] (1) In the present invention, by specifically designing dsRNA to target the GS2 gene of tobacco, accidental damage to non-target plants is avoided, the impact on the ecological environment is reduced, and the accuracy and safety of biological control are improved.

[0025] (2) The dsRNA interference technology described in the present invention can effectively break through the resistance barrier and improve the sustainability and stability of weed control by quickly adjusting the target gene or designing multi-gene tandem interference fragments.

[0026] (3) In the present invention, it only takes 3-6 months from the identification of the target gene to the field verification of the dsRNA formulation, which greatly shortens the product development and market launch time compared to the 8-10 year R&D cycle of traditional pesticides.

[0027] (4) The HT115 strain fermentation process described in the present invention can significantly reduce the mass production cost of dsRNA and has higher economic feasibility and commercial potential compared to traditional chemical synthesis methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Gel electrophoresis of PCR amplification products.

[0030] Figure 2 Schematic diagram of the L4440 vector structure.

[0031] Figure 3 Design an electropherogram for an RNA digestion experiment gradient.

[0032] Figure 4 This is a comparison chart of the effects after spraying tobacco seedlings. DETAILED DESCRIPTION

[0033] The present invention will be further described below with reference to specific examples. The following embodiments are merely illustrative and do not limit the present invention.

[0034] Scope of the Invention Unless otherwise specified, the reagents used in the present invention were purchased from biochemical stores; the methods used in the present invention, unless otherwise specified, are conventional methods in the art.

[0035] The composition of the LB solid medium in the present invention is: 1% tryptophan, 0.5% yeast extract, 1% sodium chloride, and 1.5% agarose; the composition of the LB liquid medium is: 1% tryptophan, 0.5% yeast extract, and 1% sodium chloride; the composition of the TB liquid medium is: 1.2% tryptophan, 2.4% yeast extract, 0.72 M K2HPO4, 0.17 M KH2PO4, and 0.4% glycerol.

[0036] Example 1: Design of target dsRNA sequence

[0037] Based on the tobacco glutamine synthetase gene (GS2, Gene ID: 107766022), the present invention selected two target regions (201 bp and 129 bp) and designed specific primers (see Table 1). PCR was used to amplify tobacco genomic DNA, and the quality of the amplified products was tested by TAE gel electrophoresis.

[0038] (1) Design target dsRNA fragment

[0039] dsRNAs were designed based on two target regions of the gene sequence encoding glutamine synthetase in the chloroplasts of Nicotiana tabacum (common tobacco) (Gene ID: 107766022 in the NCBI GenBank). The target regions were 201 bp and 129 bp in length, respectively, and the actual amplified products were approximately 250 bp and 150 bp in length, respectively. Forward and reverse primers of 20 to 24 nucleotides in length were designed based on the target sequence using Primer 3. The primer design results are shown in Table 1, which includes homologous fragments. The actual primers used for PCR amplification ranged from 20 to 24 nucleotides in length. Next, tobacco genome was extracted and PCR amplified using the above primers. The amplification protocol is shown in Table 2. The amplification conditions included preheating at 98°C for 45 seconds, followed by 36 cycles of denaturation at 98°C for 10 seconds, annealing at 56°C for 5 seconds, and extension at 68°C for 5 seconds, followed by a final extension at 68°C for 180 seconds. The reaction was maintained at 4°C with the heated lid set at 105°C. The amplified products were subjected to TAE gel electrophoresis (2% agarose) to verify their quality.

[0040] Table 1 Primer sequence list

[0041] Primer name Primer sequence (5' to 3') Number of bases dsRNA1-F ATGGCTCAGATCTTGGCTCC 20 dsRNA1-R CAGCTGTTCCACTCTGTTCAC 21 dsRNA2-F GGTCCTTACTACTGTGGTGC 20 dsRNA2-R CTGTCCTGGCATAACCTCTC 20

[0042] Table 2 PCR amplification system

[0043] Components Volume usage 4× system Template (extracted tobacco genome) 1 μL 4μL F 1.5 μL 6μL R 1.5 μL 6μL Mix 25 μL 100 μL Deionized water 21 μL 84μL Total volume 50μL 200 μL

[0044] (2) L4440 vector plasmid extraction

[0045] To heat-shock transform the ordered L4440 vector into HT115 (DE3) competent cells, first mix the L4440 vector with the competent cells and heat in a 42°C water bath for 45 seconds to increase cell permeability and facilitate vector entry. Immediately after heat-shock, place the cells on ice for 2 minutes. Then, add the cells to LB liquid medium containing 10 μg / mL ampicillin and incubate at 37°C and 180 rpm for approximately 16 hours. Once the cell suspension becomes turbid and reaches an OD600nm of approximately 8.0, spread the cell suspension evenly on LB solid medium containing 10 μg / mL ampicillin and incubate overnight at 37°C for approximately 16 hours. Then, a single colony was picked on the plate with a toothpick, and a bacterial suspension was made in 10 μL of sterile water. 1 μL of the bacterial suspension was taken for colony PCR in the system as shown in Table 3. The colony PCR conditions were preheating at 98°C for 60 seconds, denaturation at 98°C for 10 seconds, annealing at 56°C for 5 seconds, and extension at 68°C for 5 seconds, repeated 36 times, and finally repaired at 68°C for 180 seconds, kept warm at 4°C, and the hot cover temperature was 105°C.

[0046] After TAE gel electrophoresis (2% agarose concentration) of the colony PCR products, the corresponding successfully transformed bacterial suspension was inoculated into LB liquid culture medium containing 10 μg / mL ampicillin according to the electrophoresis results. The culture was overnight at 37°C and 180 rpm for approximately 16 h. The L4440 vector was extracted using a plasmid recovery kit and the recovery amount was determined. The recovered L4440 plasmid was stored at -20°C.

[0047] Table 3 Colony PCR system

[0048] Components Volume usage 5× system Template (bacterial suspension) 1 μL 5μL F 0.5μL 2.5 μL R 0.5μL 2.5 μL Mix 10 μL 50μL Deionized water 8μL 40 μL Total volume 20 μL 100 μL

[0049] (3) Linearization of L4440 vector

[0050] The L4440 vector was linearized by double enzyme digestion with Sac I and Hind III. The double enzyme digestion system is shown in Table 4. The digestion was carried out at 37°C for 30 min.

[0051] Table 4 Double enzyme digestion system

[0052] Components Volume usage L4440 carrier 40 μL (2 μg) Sac I 2μL Hind III 2μL Buffer 5μL Deionized water 1 μL Total volume 50μL

[0053] (4) One-step homologous recombination cloning

[0054] The linearized vector and two PCR amplification products were added to the system shown in Table 5. The two PCR products were added separately. Under the action of the homologous recombinase, the vector and the target fragment were promoted to combine.

[0055] Table 5 One-step connection

[0056] Components Volume usage Formula for determining dosage Amplified fragment 1 0.1 μL (10 ng) 250bp×0.04ng / bp Amplified fragment 2 0.1 μL (10 ng) 150bp×0.04ng / bp L4440 carrier 2.5 μL (54 ng) 2700bp×0.02ng / bp Buffer 4μL enzymes 2μL Deionized water 11.3μL Total volume 20 μL

[0057] dsRNA design results

[0058] (1) Gene sequence encoding glutamine synthetase in the chloroplasts of [ Nicotiana tabacum (common tobacco) ], SEQ ID NO. 1:

[0059] 1 ATAAACTTTCCATATCCACTCAATCTTGTTACCTTTATAATTAAGACTTCCTCAAAAAAA

[0060] 61 ATTCTTGTTACTCACCATTCTCATATTCTTCTCTCTTCAACACTATTTTTC CAGCATTACC

[0061] 121 AACTTAGGTATGTAACTTATATTCTCTATTATCAATGTCTGTACTTCTTA AGCATTGATG

[0062] 181 ACTAAAAGAAACTTTGTTTTTTTAGAATTGGTTAGTGGGGTTGTTAGTTT CTTGGGGTAT

[0063] 241 TATTGCTGATAACACAATGTGTGTATCATCAATAAGTTTTTCTTGCTGTT TTATTACTGT

[0064] 301 TCTTTTTCAAGATTTCAGTTTTTAGGACCCAAGTTGAACTCAAGTTACTAA GTATCTTTTT

[0065] 361 TTGGTCCTTGTGGTGTTGTGATCTTTGCAGTATGTGCATAAACTGCTATT GAGCTACTGA

[0066] 421 TGTTAGAATCATCATGTTATCCAACTTTAGAACTAAAGCACAGCCAAGTA

[0067] CTGTTTGTA

[0068] 481 GTCTTTGTTTGTTGTTTCTTTTGTCTGCCCTATGAATGTATGCAAACTAT ATCAGATTGT

[0069] 541 AACAAAAGCTAATCTAAGATCTAGACTCAGTGAGTTAGAATGGGTGTGCT TTTCTTATAT

[0070] 601 GTATACATTTAAAATTTTTGAAGTGAGCCTTGACGTAACTGGTAAAGTTG CTGCTATGTG

[0071] 661 ACTAGGAGTTCATGGGTTCGAGCCGTGGAAACAGCGCCTTGCAGAAATGC AGGGTAAGGC

[0072] 721 TGCGTACAACAGACCTTTGTGGTCCGGCCCTTCCCGGACCCTGCACATAG CGGGAACTTA

[0073] 781 GTGCACCGAGCTGCCCTTTATACATTTTAAATTTTCTGCGAGCCAAAAGC TAGGGGCAGA

[0074] 841 AACCGCCCAAGATCGGTCTCTAATTATACAATGGAGAATAACTCTAAAGG TGGCAAAAGC

[0075] 901 ATTTGAATTATGGAAAATACAATATTTTTTCCCTGAAATTATCGCCCATA ACAGAAAACT

[0076] 961 ACAAAGGACAGGTTTTTCTTTTTCCAAAACTAGACTGCATCCTTTACTGA GATTAAAAGT

[0077] 1021 GCAGTGTGATATGATTATGATTACTTATTAATTCTTGATTCTCAGAAGAT ATTAGCATAC

[0078] 1081 TGGCTGAGCTGCATCGTTCATTAGTCATAACTTCATTGAAATTTATAGTT TATCCATGAA

[0079] 1141 TGGCATGATAATAAGACGACTTAAAAAGTAAAATCCTGTTATTTAGTTTA TCTGATGAGG

[0080] 1201 ATATACTTAGTTTGATAGGATTTTAAACTCCTAATTTGGTGCTAAAGTTC CTTGTGTTGT

[0081] 1261 TATTTGAACATTTCAGTTGGTTAGGTGAAGATGGCTCAGATCTTGGCTCC ATCTGCACAA

[0082] 1321 TGGCAGATGAGAATGACAAAGAGCTCAACAGATGCTAATCCCTTGACTTC AAAGATGTGG

[0083] 1381 AGTTCTGTGGTGTTGAAGCAGAATAAAAGACTTGCTGTTAAAAGCTCCGC CAAATTTAGA

[0084] 1441 GTCTTTGCTCTCCAATCTGATAGTGGCACTGTGAACAGAGTGGAACAGCT GCTAAACTTG

[0085] 1501 GATGTAACTCCGTACACTGACAAGATCATTGCTGAATATATTTGGTATAG TATTCTTTGC

[0086] 1561 TATACACATTCTCATACTGTTTATTCAACATTTCACTTGGATGTTTATTA CTCTAAAAAC

[0087] 1621 TCAGAAAACAACATAAAACTCAAAAGTAGGAATTTATAGGTTGTGACTGT TATCTTTTCA

[0088] 1681 CCTTCGTTGTCTACAGCTAGTTCTTTGTGGACAGACTGACCTTATTTCAT TCCATTCAAA

[0089] 1741 ACAGGATTGGAGGGTCTGGAATTGACATGCGCAGTAAATCAAGGGTACA

[0090] TACTGCTGCTT

[0091] 1801 CTCTACTTAACCAAAGAAAATTTGAGAAACTGAATTTGGATTTTGTCACG AATCGTCTCT

[0092] 1861 TAATTGCATGCTTTTGAATTTTCAGACTATTTCAAAGCCAGTCAAGCATG CTTCTGAACT

[0093] 1921 TCCAAAGTGGAACTACGATGGATCAAGTACTGGACAAGCACCTGGAGAA

[0094] GACAGTGAAGT

[0095] 1981 CATTCTATAGTAAGATCAGCAAATGAATTTTTTATTCTTTTTGAACATTT TTCCTTTCTT

[0096] 2041 ACACTCTTCCTTTTGTTTTTTTTTCTTTCAAAAGTTAACCTCATATCATT TTACATCAGC

[0097] 2101 CCTCAGGCAATATTTAAAGATCCTTTCCGCGGTGGCAACAACATCTTGGT GAGTTTGTTA

[0098] 2161 GTTAACAAAACTGATTCATGATGTTTTACTGAATGTTTGATCTCATTGTT TTGAAGTTTG

[0099] 2221 GTGCTATTTGTTATGTAAATTGAAACAGGTTATCTGTGATGCCTACACAC CAGCTGGAGA

[0100] 2281 ACCAATTCCAACAAACAAACGCCATAAAGCTGCTCAGATTTTTAGCGACT CAAAAGTTGT

[0101] 2341 ATCTGAAGTTCCATGGTAACTTTAAATTCATCTGCCTCACTTCACTGGTT TTATGTACCG

[0102] 2401 TTTAATATTTTTTCTTCTGCATATTTTGGGGGCGTCCCTAGAAACCAAGA GAATACCAAC

[0103] 2461 TATTCCCTTTGAACAAACCAACAAAATTATATTGCTTATAGTTCACTAA TTGAGTAAAA

[0104] 2521 CCTCATTCACAAACATCCTAGAAAAATGAATTTACTGAGACATTGTGTTTC CTTGTGCATA

[0105] 2581 AATGCTAAGCAATATTCAAAATCTGCTTCTTAACTCTTTTAACTGTGTCG AAAAGAAAAC

[0106] 2641 TCTTTTAAATGATGGATGGTGAAAATGAAACAATCATTTCTTGATGTTAA TGCAAATCAT

[0107] 2701 TTTTTCTCTCACTGAAAATGGGAATCAAAAGAGTATATGCTGTTATATGGT GTCATGTTGC

[0108] 2761 TTTCCAGTTTTTGAATTTTATCGTGACATTGCTAATGTTGTCTAGCAATT GTTGTTGACT

[0109] 2821 AGGTTTGGAATAGAGCAAGAGTACACCTACTCCAGCAAAATGTAAAGT

[0110] GGCCCTTAGGT

[0111] 2881 TGGCCTGTTGGAGGCTACCCCGGACCTCAGGTAATGGTTGTTTTGCACAA AAGAATGATC

[0112] 2941 AACAGCTCAATAGTCATCCAGCAAAAGTGCAGCCCAGATGTTGCTGTAAA TTAGTTCAAT

[0113] 3001 TTATCTGTCCGATAACCATTTTATCTTCATGGAAACTTTAGGGTCCTTAC TACTGTGGTG

[0114] 3061 CTGGAGCGGATAAATCATTTGGCCGTGATATATCAGATGCTCACTACAAG GCTTGCCTGTGT

[0115] 3121 ATGCTGGAATTAACATTAGTGGTACTAACGGAGAGGTTATGCCAGGACAG GTACCATATT

[0116] 3181 CGTCACATCTCTTGTCTAATACAATAACATCACTGTCACTTATAGTCCTC TAATTGGAGG

[0117] 3241 ATTAACTAAATTTTTTGTGGCAGTGGGAATTTCAAGTAGGTCCTAGTGTG GGAATTGAAG

[0118] 3301 CGGGAGATCACATCTGGTGTGCTAGATACATCCTTGAGGTAATTTTCTTC CTTTGACTGT

[0119] 3361 TGAAATGGTTTATCTCCTACCATTATTAACTGAACTGAGAAATCAGATTT ACCAATTCTG

[0120] 3421 TTTTTTGTTCGTTGATCTAACAGAGAATTACTGAACAAGCAGGAGTTGTT CTCTCACTTG

[0121] 3481 ATCCAACCAATTGAGGTGATCTAAATTTTTTTGATAAT TTTTTTTTTC TTCAAATTAA

[0122] 3541 GTTTAGTTATAAACTCCATATTTTTCTTTACTGGTCTGGTATTTACAGG GTGACTGGAA

[0123] 3601 TGGTGCCGGATGCCACACTAACTACAGGTAGTTCCTTTTTTCAAGAGAGA AACTGGCAAA

[0124] 3661 TCTTGAAAATAAAATCTGGGATATGAATCCTTTAAAATACTGAAATGGGA ATGACATAAG

[0125] 3721 TCACTTGCTCAACATGTAGAAGTCGTTACAGCTACAAATTAGTTGCAAGT GACTTTTACG

[0126] 3781 ACACTCATCATTTTGCTTCATGATTTTTCTTTCCACAAGTTCCTGGTTTC TGTTTAACNC

[0127] 3841 ACTTACTACATTTTCATGGTTTACTATTTGCAGTACACTGAGCATGAGAG AAGAGGGAGG

[0128] 3901 ATTTGAAGTAATAAAGAAAGCGATTCTGAATCTCTCCCTTCGCCACAA

[0129] GGAGCATATAAG

[0130] 3961 TGCTTATGGAGAAGGAAATGAGAGAAGGTTGACTGGAAAGCATGAAAC

[0131] TGCAAGCATTGA

[0132] 4021 CAAATTTTCATGGGTATGCCTGCTGCTGATCTTTCCTTCACTTAGACATA

[0133] AATAGGCCGT

[0134] 4081 ACCAATTGTCCTCATTCCTGTATGATTTTCTAAATTTGCAGGGAGTTGCT

[0135] AACCGTGGTG

[0136] 4141 CCTCAATCCGTGTGGGGCGTGACACTGAGAAGCAAGGCAAAGGTAAG TTTCTGCTTTATT

[0137] 4201 AGAGGAGTAACATTAATATATTCCTTTTGTTTTCTGTGCGAAGAATGGA

[0138] AAATTAGATTA

[0139] 4261 TTTTTGTGGACCACTTTTATCAACATACATCTATCATACAAAGAAAGG

[0140] TAAAATACTGGA

[0141] 4321 TCTCAAAAAAAAAAAAAGATGATTTACTCTATTCTGAACAGCTATGCC

[0142] CGAGCCATGTAT

[0143] 4381 CAATGTTTCTTAGTGCCTTTTAATCAACTGTAAACTATTTAGCTATAC

[0144] GCTTACTTAGGC

[0145] 4441 TGGTATTGATGGTCTTTCTGATGCTGTTTGTTACTCTTTTTTGTTTATGG GATTGGTGGA

[0146] 4501 TGCAGGTTATTTGGAAGACCGCCGCCCAGCTTCAAACATGGACCCCT

[0147] ATGTTGTGACCGG

[0148] 4561 ATTACTTGCCGAAACTACTATACTGTGGGAGCCAACCCTTGAGGCTG

[0149] AAGCTCTTGCTGC

[0150] 4621 TCAAAAGCTCGCATTGAATGTTTAAAGTATTTAAGGGAAAATTGATTC

[0151] ATAATAATCTTC

[0152] 4681 TTAGAGTTCTGAGCTGCTAAGTGAAGAAACTTGTACCTTGTTTAGATT

[0153] CCCTTTTAGGGA

[0154] 4741AAATCTTGTAAAGGAACCACAATTTGTCAGTTACTCCTACAAAAGAGG

[0155] TTCCTTAAGACA

[0156] 4801 ATGAGATTGCTTTGGAGTTGAGGTGTAGTTGTTGGACTA

[0157] Figure 1 The figure shows the separation results of the amplified products by gel electrophoresis. In the figure, M stands for DNA Maker (5000 bp), which serves as a reference for DNA fragment size. Position 1 shows the amplification product of primer dsRNA1, which is approximately 250 bp in length and lacks any observed bands, indicating good purity and amplification. Meanwhile, position 2 shows the amplification product of primer dsRNA2, which is approximately 150 bp in size and, similar to dsRNA1, lacks any additional banding, indicating good amplification quality. These results demonstrate the success of the amplification experiment and the good purity of the products, providing reliable amplification products for subsequent experimental design.

[0158] (2) L4440 vector production effect

[0159] The L4440 vector was successfully recovered at a concentration of 24 ng / μL, as measured by absorbance curve analysis. A 260nm / 230nm ratio of 1.831 indicated the high purity and quality of the recovered material, confirming the effectiveness of the recovery process and providing reliable plasmid support for further engineering bacteria.

[0160] The L4440 vector structure is as follows Figure 2 As shown, the enzyme cutting sites used are indicated in the figure.

[0161] The L4440 vector consists of a double-stranded DNA circular structure with a total length of 2790 base pairs. Its sequence, starting from the first base pair, includes the following components: a T7 promoter, a Sac I restriction site, a Hind III restriction site, an OriF1 promoter (which enables replication), and an AmpR resistance gene (which confers ampicillin resistance). The orderly arrangement of these functional elements provides a fundamental framework for its application in genetic engineering and molecular biology research, and also serves as an important operational foundation for further genetic manipulation and cloning.

[0162] Example 2: Construction of engineered bacteria

[0163] The plasmid, ligated with the target fragment via one-step homologous cloning recombination, was transformed into competent cells (HT115) using the heat shock method for 45 seconds. After cooling, the plasmid was added to LB liquid medium containing 10 μg / mL ampicillin and cultured overnight at 37°C at 180 rpm for approximately 16 hours. The plasmid was then plated onto LB solid medium containing 10 μg / mL ampicillin and cultured overnight at 37°C for 16 hours. A single colony was picked and prepared into a suspension for colony PCR using the system described in Table 3. The colony PCR conditions were: preheating at 98°C for 60 seconds, denaturing at 98°C for 10 seconds, annealing at 56°C for 5 seconds, and extending at 68°C for 5 seconds, repeated 36 times. Finally, the suspension was repaired at 68°C for 180 seconds, followed by a 4°C incubation period with a heated lid at 105°C. The colony PCR products were subjected to TAE gel electrophoresis (2% agarose concentration). According to the electrophoresis results, the successfully constructed bacterial suspension was inoculated into LB liquid medium containing 10 μg / mL ampicillin and cultured overnight at 37°C and 180 rpm for about 16 h.

[0164] Results of engineering bacteria construction: Ten colonies were picked for colony PCR, and the results are shown in Table 6.

[0165] Table 6 Colony PCR results

[0166] Group PCR product determination results Group PCR product determination results A1 94.75 ng / μL B1 121.00 ng / μL A2 90.20 ng / μL B2 71.30 ng / μL A3 73.45 ng / μL B3 138.05 ng / μL A4 43.85 ng / μL B4 71.40 ng / μL A5 83.40 ng / μL B5 120.85 ng / μL A6 - B6 - A7 57.55 ng / μL B7 113.20 ng / μL A8 99.55 ng / μL B8 - A9 72.65 ng / μL B9 - A10 43.80 ng / μL B10 104.20 ng / μL

[0167] In Table 6, Group A represents the results of amplification using primer dsRNA1, while Group B represents the results of amplification using primer dsRNA2. Colony PCR results indicate that the engineered bacteria exhibited promising results. Therefore, colonies corresponding to A1, A8, and B1, B3, and B5 will be selected for expansion and preservation. This screening and further cultivation process will provide a reliable strain base for subsequent fermentation experiments.

[0168] Example 3: Induced fermentation to produce dsRNA

[0169] (1) Small-scale induced expression of dsRNA in strains containing recombinant plasmids

[0170] The engineered bacteria that have been successfully transformed with the L4440 recombinant plasmid were inoculated into LB liquid medium containing 100 μg / mL ampicillin and cultured at 37°C and 180 rpm for about 16 hours to achieve recovery. Then, the recovered bacterial solution was inoculated into LB liquid medium and TB liquid medium containing 100 μg / mL ampicillin at a ratio of 1:100. The culture was carried out at 37°C and 220 rpm for 2 hours so that the OD value of the engineered bacterial solution in the two culture media was 0. 600nmThe OD value of the bacterial solution was about 0.4, indicating that the bacterial solution was in the logarithmic phase of growth. At this time, the RNA expression reached the highest level. Then, IPTG was added at a final concentration of 0.1 mM to induce dsRNA production. The induction was continued at 37°C and 220 rpm for 6 hours. 600nm The total RNA was extracted using the Trizol method.

[0171] (2) Inspection of small-scale induced fermentation products

[0172] First, take 2 mL of the induced bacterial culture (in two portions) and add it to a 1.5 mL centrifuge tube. Centrifuge at 4°C, 12,000 rpm for 1 minute, decant the supernatant, and collect the bacterial pellet. Next, add 1 mL of Trizol reagent to the cells, vortex to mix, and let it sit on ice for 5 minutes. Then, add 200 μL of a mixture of chloroform and isopropanol (24:1 ratio), vortex vigorously to mix, and let it sit on ice for 10 minutes. Centrifuge again at 4°C, 12,000 rpm for 15 minutes. Transfer the supernatant to a new centrifuge tube, mix with an equal volume of isopropanol, and freeze at -80°C for 1 hour. Then, centrifuge at 4°C, 12,000 rpm for 20 minutes, discard the supernatant, and wash the pellet with 1 mL of 75% DEPC ethanol. After washing, centrifuge at 8,000 rpm for 2 minutes, discard the supernatant, and repeat the washing step. After the RNA precipitate is air-dried, it is dissolved in DEPC water. A small amount of the sample is taken and its RNA content is determined by absorbance.

[0173] The total RNA extracted from LB and TB liquid culture media was treated with RNase A using the system described in Table 7, and subjected to TBE gel electrophoresis (3% agarose concentration) to test the quality of the dsRNA.

[0174] Table 7 RNase A digestion system

[0175] Components WT 1 2 3 RNA 9 μg (11 μL) 9 μg (11 μL) 9 μg (11 μL) 9 μg (11 μL) RNase A 20 U (0.57 μL) 8 U (0.23 μL) 8 U (0.23 μL) 5 U (0.15 μL) 1.2M NaCl 7.5 μL (0.3 M) 10 μL (0.4 M) 15 μL (0.6 M) 15 μL (0.6 M) Deionized water 10.93μL 8.77μL 3.77μL 3.85μL Enzyme digestion temperature and duration 37℃, 30min 37℃, 15min 37℃, 15min 37℃, 15min Total volume 30 μL 30μL 30μL 30μL

[0176] (3) Small-scale induced fermentation and product testing results

[0177] Considering that the colony PCR results of group B showed a higher yield, we selected the strain containing only the fragment of the product amplified by the dsRNA2 primer and carried out small-scale induced fermentation in LB and TB liquid media respectively. For these two media, we monitored their OD 600nm The changing trend of the value over time and the relevant data records are shown in Table 8.

[0178] Table 8 OD 600nm Changing trends

[0179] Culture medium type 1 h 1.5 h 2 h LB ≈0 0.20 0.36 TB ≈0 0.24 0.42

[0180] In OD 600nm When the pH approaches 0.4, the bacterial colony is in the logarithmic growth phase, during which RNA expression levels are high. To induce dsRNA expression, IPTG (final concentration 0.1 mM) was added to the culture system to initiate expression of the target fragment in the L4440 vector, thereby promoting the synthesis of the target dsRNA. After 6 hours of induction, total RNA was extracted using the Trizol method and measured using an ultra-micro UV-visible spectrophotometer. RNA was extracted from 2 mL of LB and TB liquid media, yielding approximately 80 μL of sample volume. The RNA concentrations were 593.2 ng / μL (LB liquid media) and 971.12 ng / μL (TB liquid media), respectively. dsRNA yields were further calculated, indicating that TB liquid media produced significantly higher dsRNA yields (38.84 μg / mL) than LB liquid media (23.73 μg / mL), indicating that TB liquid media is more suitable for dsRNA fermentation production.

[0181] like Figure 3 As shown, the DNA marker (5000 bp) is displayed on the far right. The three columns immediately to its left represent bands obtained from RNA extracted from RNase III-deficient Escherichia coli HT115 (DE3) cells that were not transduced with the plasmid and induced with IPTG, followed by RNase A treatment. These bands, representing the experimental group, migrated fastest on the electrophoresis gel and had the smallest molecular weight, presumably indicating partial degradation of the single-stranded RNA by RNase A. Under the WT system, the control group used the same enzyme treatment conditions as the experimental group. Treatment of engineered bacteria induced in TB liquid medium after plasmid transduction revealed faster band migration. This is likely due to the reduced selectivity of RNase A caused by the low salt concentration, coupled with the prolonged RNase A dosage and treatment time, which partially hydrolyzed the dsRNA. In system 1, the salt concentration was also low, but the enzyme dosage and treatment time were moderate, resulting in ideal band positions. The difference between systems 2 and 3 was not significant, suggesting that higher salt concentrations are more suitable for maintaining specificity in enzyme digestion assays.

[0182] Example 4: Indoor spraying experiment

[0183] The experiment involved two treatments: a control group sprayed with deionized water and a control group sprayed with a dsRNA solution. Since the target gene was glutamine synthetase (GS2), which is expressed most abundantly in plant leaves, the dsRNA solution was sprayed directly onto the leaves to ensure effective delivery to the target site. The net concentration of the dsRNA solution was 1 μg, while the control group was sprayed with an equal amount of deionized water. The experimental treatment lasted for one week, during which plant growth was regularly observed.

[0184] like Figure 4 The results showed that tobacco seedlings in the control group (sprayed with deionized water) grew healthily, with leaves maintaining a normal green color and exhibiting no abnormal changes. However, tobacco seedlings sprayed with the dsRNA solution began to yellow their leaves after three days, stunting their growth. After seven days, they were nearing death, with overall plant wilt. This indicates that dsRNA targeting the GS2 gene can effectively inhibit the expression of glutamine synthetase, disrupting nitrogen metabolism in the plant, ultimately causing severe wilting and eventual death of the tobacco plants. This demonstrates the highly effective biological activity of the dsRNA herbicide of this invention and demonstrates its broad potential for application in precision weed control.

Claims

1. A method for preparing a biological herbicide based on RNA interference technology, characterized by the following steps: (1) Designing the target dsRNA sequence: Selecting the glutamine synthetase gene (GS2) of tobacco as the target gene, designing a specific siRNA sequence, and obtaining the target dsRNA fragment by PCR amplification. (2) Constructing engineering bacteria: Cloning the inverted repeat structure of the designed dsRNA fragment into the multiple cloning site of the L4440 vector, and transforming it into the RNase III-deficient Escherichia coli HT115 for induced fermentation. (3) Inducing fermentation to produce dsRNA: Conducting induced fermentation in LB and TB liquid media respectively, and optimizing the concentration of isopropyl-β-D-thiogalactoside (IPTG) and the induction time to enable the efficient expression of the target dsRNA by the bacteria. After lysis and purification, it is prepared into an aqueous solution of dsRNA. (4) Indoor spraying experiment: By means of foliar spraying, applying dsRNA to tobacco leaves, enabling it to penetrate into plant cells through stomata, specifically degrading GS2 mRNA, resulting in the accumulation of ammonium ions in the cells and the collapse of the photosynthetic system, thereby causing the tobacco leaves to wither and turn yellow, and the plants to severely wilt until death, ultimately achieving the herbicidal effect.

2. The preparation method of a biological herbicide based on RNA interference technology according to claim 1, wherein: The nucleotide sequence of the dsRNA is SEQ ID NO.

1.

3. The preparation method of a biological herbicide based on RNA interference technology according to claim 1, characterized in that: The final concentration of IPTG is 0.1 mM.

4. A method for preparing a biological herbicide based on RNA interference technology according to claim 1, characterized in that: The induction time is 6 hours.

5. The preparation method of a biological herbicide based on RNA interference technology according to claim 1, characterized in that: The net content of dsRNA during spraying is 1 μg.

6. The preparation method of a biological herbicide based on RNA interference technology according to claim 1, wherein, Among the LB and TB liquid media, the TB liquid medium is more suitable for the fermentation production of dsRNA.

7. The TB liquid medium according to claim 6, with the composition of 1.2% tryptophan, 2.4% yeast extract, 0.72 M K2HPO4, 0.17 M KH2PO4, 0.4% glycerol.

8. A biological herbicide based on RNA interference technology, characterized in that An aqueous solution of dsRNA prepared by any of the methods according to claims 1-7.

9. The bioherbicide according to claim 8, wherein It does not contain chemical pesticide components and has the advantages of precise targeting, safety, environmental friendliness, and no residue.

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