Escherichia coli recombinant bacteria for high-efficiency expression of dsRNA based on double-plasmid system and application thereof

Abstract

The application discloses a recombinant Escherichia coli for efficiently expressing dsRNA, which is a TG1 strain with gene defects and into which the following plasmids are introduced: a first plasmid comprising a T7 RNA polymerase expression module; and a second plasmid comprising a dsRNA expression module. rnc The application also discloses a method for producing dsRNA, and application of the recombinant Escherichia coli in preparing nucleic acid pesticides. rnc The application provides a new recombinant Escherichia coli with gene defects for producing dsRNA in the field of nucleic acid pesticides. rnc The knockout has no significant influence on the growth rate of the recombinant Escherichia coli, and the recombinant Escherichia coli has great industrial fermentation potential. The recombinant Escherichia coli adopts a double-plasmid expression system, and the dsRNA expression module structure with double T7 terminators is used to improve the expression amount and purity of dsRNA synthesis.

Description

Technical Field

[0001] This invention belongs to the field of nucleic acid pesticide technology, specifically relating to a recombinant Escherichia coli strain that efficiently expresses dsRNA based on a dual plasmid system and its application. Background Technology

[0002] RNA interference (RNAi) is a natural immune mechanism in eukaryotes, and nucleic acid pesticides developed based on this technology belong to the category of novel biological pesticides. The basic principle involves delivering artificially synthesized double-stranded RNA (dsRNA) or short interfering RNA (siRNA) into the target pest's body. The siRNA is then cleaved by the Dicer enzyme and loaded onto a RISC complex, thereby achieving specific degradation or translational inhibition of the mRNA, ultimately blocking the function of key genes, leading to developmental arrest or death of the pest.

[0003] Compared with traditional pesticides, nucleic acid pesticides have advantages such as high specificity, environmental friendliness, and rapid development, and have received widespread attention from scholars in the field of plant protection in recent years. Currently, there are several industrialized cases of nucleic acid pesticides based on RNAi technology, such as BioDirect, a sprayable nucleic acid pesticide developed by Bayer for controlling Varroa destructor in bees, and Ledprona, a sprayable nucleic acid pesticide developed by Greenlight Biosciences for controlling potato beetles.

[0004] The synthesis and preparation technology of double-stranded RNA (dsRNA) is crucial for the practical application of RNAi (RNAi) nucleic acid pesticides in the field. Currently, the production of dsRNA mainly relies on two strategies: in vitro transcription and microbial fermentation. The preparation of dsRNA using *E. coli* fermentation is the most economical method reported to date. Generally, *E. coli* strains with the *rnc* gene deficiency are used, along with pET or L4440 series vectors, to synthesize dsRNA with the assistance of the lysogen DE3 system. Among these, the *rnc* gene knockout strain HT115(DE3) has been frequently reported in the literature, and when combined with pET series vectors, its dsRNA yield can reach 4.23 mg / L. In addition, other *E. coli* strains have also been modified for dsRNA expression. For example, CN 110229839 A discloses the BL21(DE3) strain with rnc gene knockout, whose dsRNA yield is more than 5 times higher than the commercial HT115(DE3)–L4440 system; CN 118440881 A also discloses the JM109(DE3) strain with knockout of rnc and byproduct pathway-related genes, whose dsRNA expression level reaches 5.419 mg / L. However, compared with other chassis cells, the dsRNA yield of *E. coli* is still relatively low. For example, *Corynebacterium glutamicum* can achieve a dsRNA yield of 1 g / L in 0.3 L fed-batch fermentation, which is the highest value reported in currently published bacterial systems; *Saccharomyces cerevisiae* has also been reported by companies to have a pilot-scale yield of 1–1.5 g / L. Therefore, increasing the expression level of dsRNA in Escherichia coli and reducing the cost of large-scale industrial production are of great significance for promoting the widespread application of nucleic acid pesticides in agricultural production.

[0005] E. coli fermentation offers advantages such as low cost, convenient genetic manipulation, clear genetic background, and high regulatory acceptance, making HT115(DE3) the mainstream strain for dsRNA production via fermentation. CN 115786199 A discloses a method for producing dsRNA using this strain, but this strain still suffers from low yield, low dsRNA purity, extraction difficulties, and cell stability issues in the later stages of fermentation. Therefore, the development of the nucleic acid pesticide industry still requires further screening for more efficient and stable dsRNA production chassis cells. Summary of the Invention

[0006] To address the problems of low yield, low purity, and poor cell stability in existing technologies for producing dsRNA using E. coli fermentation, this invention aims to provide a novel genetically engineered bacterial system. This system can achieve efficient and high-purity expression of dsRNA, meeting the yield and cost requirements of industrial-scale nucleic acid pesticide production.

[0007] To achieve the above objectives, a first aspect of the present invention provides a recombinant Escherichia coli strain that efficiently expresses dsRNA, wherein the recombinant Escherichia coli strain is an rnc gene-deficient TG1 strain and is transformed with the following plasmid:

[0008] The first plasmid contains a T7 RNA polymerase expression module; and

[0009] The second plasmid contains a dsRNA expression module.

[0010] Preferably, the first plasmid is a pACYC-derived plasmid.

[0011] Furthermore, the T7 RNA polymerase expression module is driven by a heat-inducible promoter.

[0012] Furthermore, the thermally induced promoter is the pHsh promoter.

[0013] Preferably, the second plasmid is a pET28-derived plasmid.

[0014] Furthermore, the dsRNA expression module includes a dual T7 promoter.

[0015] Furthermore, the dsRNA expression module also includes a dual T7 terminator.

[0016] Preferably, the second plasmid is a single-copy dsRNA expression vector containing the sequence shown in SEQ ID NO.14; the second plasmid may also be a double-copy dsRNA expression vector containing the sequence shown in SEQ ID NO.15.

[0017] A second aspect of the present invention provides a method for producing dsRNA, the method comprising:

[0018] S1. Fermentation culture of recombinant Escherichia coli as described above;

[0019] S2. The expression of T7 RNA polymerase is activated by heat induction, thereby expressing dsRNA;

[0020] S3. Recover dsRNA from bacterial cells.

[0021] Furthermore, the thermal induction involves adjusting the culture temperature to 42°C.

[0022] A third aspect of the present invention provides the application of the above-mentioned recombinant Escherichia coli in the preparation of nucleic acid pesticides.

[0023] The present invention has the following beneficial effects:

[0024] 1. This invention provides a novel recombinant Escherichia coli strain with an rnc gene deficiency for the production of dsRNA in the field of nucleic acid pesticides; the rnc knockout of this strain has no significant effect on its growth rate and has great potential for industrial fermentation.

[0025] 2. This recombinant bacterium uses a dual plasmid expression system, which increases the expression level of T7 RNA polymerase in the dsRNA production module; at the same time, the structure of the dsRNA expression module with dual T7 terminators is optimized to improve the quality and purity of dsRNA synthesis.

[0026] 3. The dsRNA expression level of this recombinant bacterium is the highest reported to date. Its dsRNA expression level is 13.87 times higher than that of the commonly used strain HT115(DE3) / pT7B-TMV, and the purity of the dsRNA produced is also significantly improved. Attached Figure Description

[0027] Figure 1 This is a nucleic acid gel electrophoresis image of Escherichia coli JM109(DE3) transformant colonies validated by PCR amplification.

[0028] Figure 2 A bar chart comparing the growth status of different Escherichia coli RNC-deleted strains.

[0029] Figure 3 SDS-PAGE images comparing the expression levels of T7 RNA polymerase in different E. coli lysates.

[0030] Lane M: Protein Marker (RealBand four-color pre-stained protein marker, 10~180 kDa, purchased from Sangon Biotech); Lane 1: E. coli HT115(DE3) without IPTG induction; Lane 2: E. coli HT115(DE3) with IPTG induction; Lane 3: E. coli W3110Δrnc; Lane 4: E. coli W3110Δrnc / pACYC-pHsh-T7RNAp; Lane 5: E. coli TG1Δrnc; Lane 6: E. coli TG1Δrnc / pACYC-pHsh-T7RNAp.

[0031] Figure 4 This is a schematic diagram of the construction of the single-copy dsRNA expression vector pET28-ter-TMV-cp.

[0032] Figure 5 This is a construction map of the double-copy dsRNA expression vector pET28-ter-TMV-cpX2. Detailed Implementation

[0033] To illustrate the construction and application of the recombinant Escherichia coli strain that efficiently expresses dsRNA based on a dual-plasmid system, specific embodiments are provided below. These embodiments are merely illustrative of the technical solutions of this invention and do not constitute a limitation on the scope of protection of this invention. Furthermore, it should be understood that after reading the teachings of this invention, all those skilled in the art and capable of performing various modifications or alterations to this invention, and these equivalent forms also fall within the scope of protection defined by the appended claims.

[0034] Plasmids pKD46, pKD13, and pCP20 are commonly used plasmids in the Red homologous recombination system. Unless otherwise specified, ligation in all molecular cloning steps was performed using T4 DNA ligase at 16°C overnight.

[0035] Unless otherwise specified, the experimental materials, operating methods, etc. used in the following embodiments can be obtained through conventional commercial channels, are known in the technical field, or are performed in accordance with the product instructions.

[0036] Example 1: Knockout of the rnc gene in Escherichia coli

[0037] In this embodiment, the rnc gene of Escherichia coli was knocked out using the Red homologous recombination system, and the growth status of several commonly used Escherichia coli strains (JM109(DE3), W3110 and TG1) after the rnc gene was knocked out was compared. Strains that were not affected by polarity effects were selected as chassis cells.

[0038] The specific steps are as follows:

[0039] 1.1 Preparation of the knockout box rnc::kan

[0040] Using primers rnc-inact-F (sequence shown in SEQ ID NO.1) and rnc-inact-R (sequence shown in SEQ ID NO.2) containing upstream and downstream homologous arms of the target gene rnc and part of the kanamycin (kan) encoding gene sequence (see Table 1), the knockout cassette fragment rnc::kan was obtained by PCR amplification using pKD13 plasmid as a template and PrimeSTAR® Max DNA Polymerase (Takara Biotech, R045Q).

[0041] The PCR amplification system is shown in Table 2. The amplification program was: denaturation at 98℃ for 5 seconds; annealing at 58℃ for 5 seconds; extension at 72℃ for 15 seconds; repeated for 35 cycles.

[0042] Table 1 Knockout cassette amplification primers

[0043]

[0044] Note: Homologous arm sequences are indicated by underscores.

[0045] Table 2. PCR amplification system for knockout cassette fragments (100 μL)

[0046]

[0047] 1.2 Knockout and Validation of the Escherichia coli RNC Gene

[0048] (1) Plasmid pKD46 was transformed into competent cells of Escherichia coli JM109 (DE3), W3110 and TG1 strains (the competent cells of the above strains were purchased from Shanghai Weidi Biotechnology Co., Ltd.), and spread on LB solid plates containing 100 µg / mL ampicillin (Amp) resistance, and cultured overnight at 30°C.

[0049] (2) Pick a single colony that has grown on the above plate and put it into 20 mL of LB liquid medium containing 100 µg / mL Amp and 4 mM L-arabinose, and incubate at 30 °C until the OD of the bacterial culture reaches the specified value. 600 Once the saturation reaches 0.6-0.7, remove the cells and place them in an ice bath at 4°C for 20 min, then centrifuge at 6000 rpm for 1 min to collect the cells. Wash the collected cells twice each with sterile ddH2O and 10% glycerol to prepare electrocompetent cells.

[0050] (3) Take 180µL of electrocompetent cells and 1µg of knockout cassette fragment rnc::kan that has been placed on ice for 10 min and pre-cooled, mix well, electroporate once at 180V 200Ω 5ms, add 1mL of LB medium, revive and incubate at 30℃ for 3h, then spread on LB solid plates containing 50µg / mL Kan and 100µg / mL Amp, and incubate overnight at 30℃ to obtain single colonies of transformant E.coli rnc::kan / pKD46.

[0051] (4) The plasmid pCP20 was transformed into E. coli rnc::kan / pKD46 gene knockout bacteria to eliminate the kan resistance gene. The transformed bacteria were then plated on LB agar plates containing 50 µg / mL chloramphenicol. Transformants were screened using LB agar plates containing 50 µg / mL kan and LB agar plates without antibiotics. Transformants that did not grow on kan plates or on antibiotic-free plates were selected. Colony PCR amplification was performed using the primers shown in Table 3 with 2xRapid Taq Plus Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd., P223-01) for verification. Figure 1 This is a nucleic acid gel electrophoresis image of Escherichia coli JM109(DE3) transformant colonies validated by PCR amplification. Figure 1In the above, lane 2 shows the DL5000 DNA Marker (purchased from Yisheng Biotechnology Co., Ltd., 10504ES); lane 1 shows the wild-type strain control amplified with primers K1-F (sequence shown in SEQ ID NO.3) and K2-R, showing a band of 1000bp; lane 3 shows no band after amplification of the transformant with primers K1-F and K1-R (sequence shown in SEQ ID NO.4); lane 4 shows no band after amplification of the transformant with primers K2-F (sequence shown in SEQ ID NO.5) and K2-R (sequence shown in SEQ ID NO.6), indicating that the kan resistance gene has been lost; lane 5 shows a band of 498bp amplified with primers K1-F and K2-R, indicating that the obtained transformant is a mutant strain E. coli JM109(DE3)Δrnc with the rnc gene knocked out. The rnc gene knockout transformants of strains W3110 and TG1 were also verified using the same primers and PCR conditions. The results were consistent with those of JM109(DE3), indicating that the corresponding rnc gene knockout mutant strains were successfully obtained.

[0052] The colony PCR amplification system is shown in Table 4. The amplification program was as follows: 95℃ pre-denaturation for 1 min; 95℃ denaturation for 10 s; 52℃ annealing for 30 s; 72℃ extension for 1 min; repeated for 35 cycles.

[0053] Table 3. Primers for gene knockout validation

[0054]

[0055] Table 4. Colony PCR amplification system (20 μL)

[0056]

[0057] 1.3 Comparison of growth status of different Escherichia coli RNC-deleted strains

[0058] Single colonies of *E. coli* strains JM109(DE3)Δrnc, W3110Δrnc, and TG1Δrnc (with the rnc gene knocked out) were inoculated into LB broth, with strain HT115(DE3) as a control. The cultures were incubated overnight at 37°C using a shaker at 200 rpm. The OD of the fermentation broth for each strain was measured using a spectrophotometer. 600 The values ​​were compared to assess the growth intensity of different RNC-deficient bacteria.

[0059] Figure 2 A bar chart comparing the growth status of different E. coli RNC-deleted strains, such as... Figure 2 As shown, the growth OD of strains W3110Δrnc and TG1Δrnc is... 600The values ​​were all higher than those of strain HT115(DE3), and compared with the wild type, the knockout of the rnc gene had less impact on strains W3110Δrnc and TG1Δrnc, which can be used as potential chassis cells for dsRNA fermentation production.

[0060] Example 2: Construction and expression of T7 RNA polymerase expression vector

[0061] 2.1 Construction of T7 RNA polymerase expression vector

[0062] Expression elements containing the constitutive promoter pHsh and T7 RNA polymerase sequence with hot-start characteristics were obtained using gene synthesis methods. The specific sequences are shown in SEQ ID NO.7.

[0063] After obtaining the cloning vector pUC57-T7RNAP containing the expression element from Sangon Biotech (Shanghai) Co., Ltd., primers T7-F (sequence shown in SEQ ID NO. 8) and T7-R (sequence shown in SEQ ID NO. 9) were designed. The T7 RNAP sequence was obtained by PCR amplification using this plasmid as a template. The expression vector backbone was then obtained by PCR amplification using primers pAC-F (sequence shown in SEQ ID NO. 10) and pAC-R (sequence shown in SEQ ID NO. 11) with the commercial plasmid pACYCDuet-1 (purchased from Novagen) using the rapid PCR polymerase PrimeSTAR® Max DNA Polymerase (Takara Biotech, R045Q). The PCR amplification system is shown in Table 6. The amplification program was: 98℃ denaturation for 5 s; 55℃ annealing for 5 s; 72℃ extension for 35 s; repeated for 35 cycles.

[0064] The T7 RNA polymerase expression vector pACYC-T7RNAp was obtained by homologous recombination of the above T7RNAP and pACYC vector backbone using a recombinant cloning kit (purchased from Novizan Biotechnology Co., Ltd., C116).

[0065] Table 5 Primers for constructing the T7 RNA polymerase expression vector

[0066]

[0067] Table 6. PCR amplification system (100 μL) for constructing the T7 RNA polymerase expression vector

[0068]

[0069] 2.2 Expression of T7 RNA polymerase

[0070] S1. The T7 RNA polymerase expression vector pACYC-T7 RNAp was transformed into E. coli TG1Δrnc and E. coli W3110Δrnc to obtain expression strains E. coli TG1Δrnc / pACYC-T7 RNAp and E. coli W3110Δrnc / pACYC-pHsh-T7RNAp, respectively. These expression strains were inoculated into 50 mL LB medium containing 50 μg / mL chloramphenicol and cultured at 37℃ and 200 rpm in a shaker until the OD600 reached 0.5-1.0 (approximately 2 h). To induce the Hsh promoter, the culture temperature was adjusted to 42℃ and cultured for another 18 h before centrifugation to collect the bacterial cells.

[0071] E. coli HT115(DE3), E. coli W3110Δrnc, and E. coli TG1Δrnc were used as control strains. E. coli HT115(DE3) was inoculated into 50 mL LB medium containing 25 μg / mL tetracycline and cultured at 37℃ and 200 rpm for 2 h. Then, 0.5 mM IPTG was added for induction, and the culture was continued for 18 h before centrifugation to collect the bacterial cells. E. coli TG1Δrnc and E. coli W3110Δrnc were inoculated into 50 mL LB medium without antibiotics and cultured at 37℃ and 200 rpm for 16–18 h before centrifugation to collect the bacterial cells.

[0072] S2. Add 20 mL of 20 mM phosphate buffer to the collected bacterial cells, shake to mix, place on ice and use an ultrasonic disruptor to disrupt the bacterial cells. The disruption parameters are 200W power, 1 s disruption, 3 s pause, and a total disruption time of 20 min. Centrifuge the obtained disruption solution at 12000 r / min for 20 min, and take the supernatant for SDS-PAGE electrophoresis.

[0073] Figure 3 To compare the expression levels of T7 RNA polymerase in different E. coli lysates, based on Figure 3 The results showed that, compared with the control strain E.coli TG1Δrnc, the expression strain E.coli TG1Δrnc / pACYC-pHsh-T7RNAp had a significant overexpression band at around 100kDa, which is consistent with the theoretical value of molecular design (99kDa). This indicates that the expression vector can achieve significant overexpression of T7 RNA polymerase in E.coli TG1Δrnc.

[0074] Under the same loading conditions, the overexpression bands of DE3-expressing strain E.coli HT115 (DE3) with lysogen and E.coli W3110Δrnc / pACYC-phsh-T7RNAp were not significant, indicating that the expression level of T7 RNA polymerase was lower than that of strain E.coli TG1Δrnc / pACYC-phsh-T7RNAp.

[0075] Example 3: Construction of a single-copy dsRNA expression vector

[0076] This embodiment uses the TMV-cp sequence as the target (TMV-cp sequence information can be found in CN 113717984 A). The target gene with a double T7 promoter was obtained by digestion with EcoRI, and then ligated with pET28a to construct a dsRNA expression vector. Simultaneously, terminator modules were added to both ends using an enzyme digestion and ligation method. A schematic diagram of the construction is shown below. Figure 4 As shown.

[0077] The specific steps are as follows:

[0078] S1. Digest plasmid pT7B-TMV-EcoRI-EcoRI-cp with EcoRI (refer to CN 113717984 A), recover the TMV-cp fragment using a gel extraction kit (purchased from Tiangen Biotech Co., Ltd.), and ligate it with pET28a, which was also digested with EcoRI, to obtain pET28-TMV-cp plasmid.

[0079] S2. Using pET28a plasmid as a template, and ter-F (sequence shown in SEQ ID NO.12) and ter-R (sequence shown in SEQ ID NO.13) as primers (primer sequences are shown in Table 7), PCR amplification was performed using PrimeSTAR® Max DNA Polymerase (Takara Biotech, R045Q) to obtain a TMV-cp fragment with a T7 terminator at one end. The PCR amplification system is shown in Table 8. The amplification program was: 98℃ denaturation for 5 s; 55℃ annealing for 5 s; 72℃ extension for 15 s; repeated for 35 cycles.

[0080] S3. The fragment is double-digested with SphI and SalI respectively, and then ligated to the pET28-TMV-cp plasmid backbone which has also been double-digested, to obtain the pET28-ter-TMV-cp plasmid, which is the single-copy dsRNA expression vector obtained in this embodiment, and its sequence is shown in SEQ ID NO.14.

[0081] Table 7 Primer sequences for dsRNA expression vector construction

[0082]

[0083] Note: The underlined bases are T7 terminator sequences.

[0084] Table 8 PCR amplification system (100 μL)

[0085]

[0086] Example 4: Construction of a double-copy dsRNA expression vector

[0087] The TMV-cp sequence gene with T7 terminator and T7 promoter at both ends, as described in Example 3, was synthesized using gene synthesis methods. This gene was then blunt-end ligated to the EcoRV-digested pET28-ter-TMV-cp plasmid to obtain a double-copy dsRNA expression vector (pET28-ter-TMV-cpX2). The construction map is shown below. Figure 5 As shown, its sequence is shown in SEQ ID NO.15.

[0088] Example 5: Determination of dsRNA expression levels in different hosts

[0089] In this embodiment, single-copy and double-copy dsRNA expression vectors were transformed into *E. coli* TG1Δrnc / pACYC-T7 RNAp to obtain the final recombinant strains *E. coli* TG1-ATS and *E. coli* TG1-ATD. *E. coli* HT115(DE3) / pT7B-TMV was used as a control (strain construction method referred to CN 115786199 A), and *E. coli* TG1Δrnc without plasmid was used as a blank control. The dsRNA expression levels of the recombinant strains were compared. The specific methods are as follows:

[0090] 5.1 Fermentation of recombinant strains E. coli TG1-ATS and E. coli TG1-ATD

[0091] Recombinant E. coli TG1-ATS and E. coli TG1-ATD, preserved in 10 µL glycerol tubes, were inoculated into 20 mL of LB medium containing 50 µg / mL chloramphenicol and 50 µg / mL kanamycin, respectively, and cultured overnight at 37°C and 200 rpm. The next day, 1 mL of the bacterial culture was transferred to 50 mL of LB liquid medium containing 50 µg / mL chloramphenicol and 50 µg / mL kanamycin, and cultured at 37°C and 200 rpm until OD (digesterone) was reached. 600 The concentration was set to 0.5~1.0. The temperature was adjusted to 42℃ and cultured for another 18 hours before fermentation was stopped.

[0092] 5.2 Fermentation of recombinant E. coli HT115(DE3) / pT7B-TMV

[0093] The recombinant E. coli HT115(DE3) / pT7B-TMV, preserved in 10 µL glycerol tubes, was inoculated into 20 mL of LB medium containing 25 µg / mL tetracycline and 100 µg / mL ampicillin, and cultured overnight at 37 °C and 200 rpm in a shaker. The next day, 1 mL of the bacterial culture was transferred to 50 mL of LB liquid medium containing 25 µg / mL tetracycline and 100 µg / mL ampicillin, and cultured at 37 °C and 200 rpm in a shaker for 2 h. Then, 0.5 mM IPTG was added for induction, and fermentation was stopped after 18 h of further culture.

[0094] 5.3 Extraction of dsRNA

[0095] Take 1-3 mL of the bacterial culture and extract total RNA using the total RNA extraction reagent (Trizol) (purchased from Sangon Biotech, B511311). Refer to the reagent instructions for specific methods.

[0096] Total RNA was dissolved in 50 µL of nuclease-free water, and 2 µL of DNase I (100 U / µL) and 2 µL of RNase T1 (50 U / µL) were added. The mixture was reacted in a metal bath at 37 °C for 1 h. After purification using an RNA magnetic bead purification kit, the RNA was dissolved in 50 µL of nuclease-free water to obtain a dsRNA solution.

[0097] 5.4 Calculation of dsRNA Expression Level and Purity Detection

[0098] The nucleic acid concentration of the obtained dsRNA solution was determined using a NanoDrop microspectrophotometer, and the dsRNA content per unit volume of fermentation broth was calculated, which is the final dsRNA expression level (mg / L). Simultaneously, the purity (%) of the obtained dsRNA solution was determined by high-performance liquid chromatography. The results are shown in Table 9.

[0099] Table 9 Comparison of dsRNA expression levels in different Escherichia coli strains

[0100]

[0101] It can be seen that compared with the control strain E. coli HT115(DE3) / pT7B-TMV, the recombinant E. coli TG1Δrnc with the dual plasmid system showed a significant increase in dsRNA expression levels, increasing by 4.72-fold and 13.87-fold, respectively. Furthermore, the strain E. coli TG1Δrnc-ATD with the dual-copy dsRNA expression module showed a 2.94-fold increase in dsRNA expression levels compared with the single-copy strain E. coli TG1Δrnc-ATS.

[0102] Based on the liquid chromatography purity test results, compared with the dual promoter design of pT7B-TMV in CN 115786199 A, the dual promoter and dual terminator design in this embodiment can improve the purity of dsRNA by more than 20%. Furthermore, the expression of dsRNA in recombinant E. coli TG1Δrnc-ATD has little impact on its final cell density; Table 9 shows its OD... 600 Slightly lower than the blank control, indicating that it can be used as a production strain to meet the needs of industrial dsRNA production.

[0103] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A recombinant Escherichia coli bacterium for efficiently expressing dsRNA, characterized in that, The recombinant E. coli bacteria are rnc a TG1 strain with genetic defects and into which the following plasmids have been introduced: a first plasmid comprising a T7 RNA polymerase expression module; and a second plasmid comprising a dsRNA expression module; the T7 RNA polymerase expression module comprises a sequence as shown in SEQ ID NO. 7; the dsRNA expression module comprises a double T7 promoter; the dsRNA expression module further comprises a double T7 terminator; the second plasmid comprises a single copy dsRNA expression vector sequence as shown in SEQ ID NO. 14 or a double copy dsRNA expression vector sequence as shown in SEQ ID NO.

15.

2. The recombinant E. coli bacterium of claim 1, wherein, the vector backbone of the first plasmid is pACYC.

3. A method of producing dsRNA, characterized by, the method comprises: S1, fermenting and culturing the E. coli recombinant bacteria according to any one of claims 1-2; S2, activating the expression of T7 RNA polymerase by heat induction, so as to express dsRNA; S3, recovering dsRNA from the bacterial cells.

4. The method of claim 3, wherein, the heat induction is adjusting the culture temperature to 42℃.

5. Use of the E. coli recombinant bacteria according to any one of claims 1-2 in the preparation of nucleic acid pesticides.

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