Method for screening bacterial liquid expression of dsRNA vectors

Abstract

The application relates to a method for screening dsRNA carrier expression of bacteria liquid, and relates to a method for screening dsRNA carrier expression of bacteria liquid. In view of the specificity of RNAi and the good application prospect of dsRNA obtained through prokaryotic fermentation, the application provides a method for screening dsRNA carrier expression of bacteria liquid and a method for improving the expression amount of dsRNA of prokaryotic expression. The method provided by the application comprises the following steps: 1, synthesizing a test dsgfp fragment; 2, designing primers; 3, testing dsgfp fragment amplification; 4, recombining the amplification fragment with the selected expression carrier after enzyme cutting, and transferring the plasmid into a competent cell; 5, IPTG induction expression; 6, preparing a dsgfp DNA template prepared in vitro; 7, extracting dsgfp in bacteria liquid of the induced bacteria liquid by using a selected dsRNA extraction method; and 8, determining the most suitable expression carrier and the optimal dsRNA extraction method from the selected expression carrier according to the determination result.

Description

Technical Field

[0001] This invention relates to a method for screening vectors expressing dsRNA. Background Technology

[0002] Studies have shown that dsRNA is extremely unstable in the environment, completely degrading within 3-5 hours under natural conditions; San et al. found that dsRNA completely degrades within 4 hours under ultraviolet irradiation. Furthermore, research on its mechanism of action is not systematic, and factors such as whether plants transmit and amplify dsRNA and the significant differences in the sensitivity of harmful organisms to RNAi have delayed the development of RNAi-based applications. Currently, dsRNA is mainly produced in large quantities through three methods: 1. Synthesizing dsRNA-specific primers containing the T7 promoter and using in vitro synthesis kits to synthesize dsRNA. This method can synthesize dsRNA in large quantities quickly and efficiently, and the synthesized dsRNA can be used directly; however, the cost of in vitro synthesis kits is high, making large-scale application impractical. 2. Prokaryotic fermentation to produce dsRNA, initially used to interfere with *Anoectochilus elegans*, producing strong interference when fed to the nematodes. The constructed dsRNA expression vector is introduced into RNase III-deficient *E. coli* HT115(DE3) competent cells, and dsRNA is produced by induction with isopropyl thio-β-D-galactoside (IPTG). This method can yield large quantities of dsRNA through fermentation at a low cost, making it suitable for large-scale application in practical applications. However, ensuring high bacterial activity and continuous dsRNA production during fermentation and induction still requires further research. Thirdly, expressing dsRNA in transgenic plants can endow them with "lifelong" defenses, effectively inhibiting their growth and development or even causing their death when pests consume them, without human intervention. While this method offers a permanent solution, the safety of transgenic plants and their potential evolution into "super plants" that could disrupt ecosystem balance still require extensive research. Summary of the Invention

[0003] In view of the specificity of RNAi and the promising application prospects of dsRNA obtained by prokaryotic fermentation, this invention provides a method for screening bacterial culture expression vectors for dsRNA and a method for increasing the expression level of prokaryotic dsRNA.

[0004] The present invention provides a method for screening bacterial cultures expressing dsRNA vectors:

[0005] 1. Using the GFP sequence as a template, DSGFP fragments of different lengths were extracted and the bases of the sequence were randomly changed to synthesize test DSGFP fragments with different CG base contents.

[0006] II. Design and synthesize in vitro primers starting with the promoter sequence; design and synthesize dsgfp fragment amplification primers based on double restriction sites selected from candidate expression vectors.

[0007] 3. Using the dsgfp fragment as a template, PCR amplification was performed using dsgfp fragment amplification primers;

[0008] 4. The candidate expression vector is double-digested and then recombined with the dsgfp fragment amplified by PCR in step 3. The recombinant product is then added to Trans-T1 competent cells for culture, and single clones are picked for verification and sequencing. The plasmid that is verified to be correct is transformed into HT115(DE3) competent cells to obtain the initial bacterial culture.

[0009] V. IPTG-induced expression to obtain induced bacterial culture;

[0010] VI. Preparation of in vitro synthesized dsgfp DNA template;

[0011] 7. Extract dsgfp from the induced bacterial culture using the alternative dsRNA extraction method;

[0012] 8. Based on the results of measuring the concentration, purity, and yield of dsgfp in the bacterial culture, determine the most suitable expression vector from the candidate expression vectors and the optimal dsRNA extraction method corresponding to that expression vector.

[0013] The present invention provides a method for increasing the expression level of prokaryotic dsRNA, characterized in that dsRNA with a CG content of 25-35% is selected for expression.

[0014] Furthermore, dsRNA with a CG content of 30% was selected for expression.

[0015] Furthermore, the prokaryotic expression vectors are L4440 and pET-2p.

[0016] The method of the present invention can determine the most suitable expression vector and the optimal dsRNA extraction method corresponding to the expression vector from the candidate expression vectors. Attached Figure Description

[0017] Figure 1 These are test dsgfp fragment sequences of different lengths and CG combinations from Example 1;

[0018] Figure 2 These are 1.5% agarose gel electrophoresis images of dsgfp expression based on pET-2p and L4440 in Example 1: a: dsgfp expression based on pET-2p; b: dsgfp expression based on L4440.

[0019] Figure 3These are grayscale analysis images of 1.5% agarose gel electrophoresis bands of dsgfp expressed by pET-2p and L4440 in Example 1: a: grayscale analysis of the 201bp dsgfp band based on pET-2p and L4440 expression; b: grayscale analysis of the 423bp dsgfp band based on pET-2p and L4440 expression.

[0020] Figure 4 This is a 1.5% agarose gel electrophoresis image of dsgfp in the synthetic bacterial culture in Example 1;

[0021] Figure 5 This is a graph showing the concentration and purity analysis of dsgfp in the five bacterial cultures synthesized in Example 1. a: Detection of dsgfp concentration in the five bacterial cultures diluted to different concentrations. 260 / A 280 b: Five groups of bacterial suspensions were diluted with dsgfp to different concentrations for detection A. 260 / A 230 ;

[0022] Figure 6 This is a purity analysis diagram of pET-2p and L4440 vector expression dsgfp extracted using four methods in Example 1. a: Extracted pET-2p and L4440 vector expression dsgfp, diluted to different concentrations for detection. 260 / A 280 b: Extract pET-2p and L4440 expression dsgfp, dilute to different concentrations, and detect A. 260 / A 230 ;

[0023] Figure 7 These are 1.5% agarose gel electrophoresis images of dsgfp expressed in 50 ml of bacterial culture extracted using the phenol-chloroform method and the Trizol method in Example 1; a: phenol-chloroform method extraction of dsgfp expressed based on pET2p (digested with Dnase I and RNase A, diluted to 500 ng / μl); b: phenol-chloroform method extraction of dsgfp expressed based on L4440 (digested with Dnase I and RNase A, diluted to 500 ng / μl); c: Trizol method extraction of dsgfp expressed based on pET2p (digested with Dnase I and RNase A, diluted to 500 ng / μl); d: Trizol method extraction of dsgfp expressed based on L4440 (digested with Dnase I and RNase A, diluted to 500 ng / μl).

[0024] Figure 8The graph shows the concentration and yield of dsgfp extracted from 1 ml of bacterial culture using the phenol-chloroform method in Example 1. a: Concentration of dsgfp extracted using the phenol-chloroform method based on pET-2p and L4440 expression; b: Yield of dsgfp based on pET-2p and L4440 expression. Detailed Implementation

[0025] The technical solution of the present invention is not limited to the specific embodiments listed below, but also includes any combination of the specific embodiments.

[0026] Specific Implementation Method 1: This implementation method for screening bacterial culture expression vectors for dsRNA is characterized by the following steps:

[0027] 1. Using the GFP sequence as a template, DSGFP fragments of different lengths were extracted and the bases of the sequence were randomly changed to synthesize test DSGFP fragments with different CG base contents.

[0028] II. Design and synthesize in vitro primers starting with the promoter sequence; design and synthesize dsgfp fragment amplification primers based on double restriction sites selected from candidate expression vectors.

[0029] 3. Using the dsgfp fragment as a template, PCR amplification was performed using dsgfp fragment amplification primers;

[0030] 4. The candidate expression vector is double-digested and then recombined with the dsgfp fragment amplified by PCR in step 3. The recombinant product is then added to Trans-T1 competent cells for culture, and single clones are picked for verification and sequencing. The plasmid that is verified to be correct is transformed into HT115(DE3) competent cells to obtain the initial bacterial culture.

[0031] V. IPTG-induced expression to obtain induced bacterial culture;

[0032] VI. Preparation of in vitro synthesized dsgfp DNA template;

[0033] 7. Extract dsgfp from the induced bacterial culture using the alternative dsRNA extraction method;

[0034] 8. Based on the results of measuring the concentration, purity, and yield of dsgfp in the bacterial culture, determine the most suitable expression vector from the candidate expression vectors and the optimal dsRNA extraction method corresponding to that expression vector.

[0035] Specific Implementation Method Two: The difference between this implementation method and Specific Implementation Method One is that a 720bp gfp sequence is used as a template in step one. Other steps and parameters are the same as in Implementation Method One.

[0036] Specific Implementation Method Three: The difference between this implementation method and Specific Implementation Method One or Two is that the lengths of the dsgfp segments selected in step one are 201bp and 423bp, respectively. Other steps and parameters are the same as in Implementation Method One or Two.

[0037] Specific Implementation Method Four: The difference between this implementation method and one of Specific Implementation Methods One to Three is that the content of the base CG in the test dsgfp fragment synthesized in step one is 30%, 50%, and 70%, respectively. Other steps and parameters are the same as in one of Implementation Methods One to Three.

[0038] Specific Implementation Method Five: The difference between this implementation method and one of Specific Implementation Methods One to Four is that in step two, in vitro synthetic primers are designed and synthesized using the T7 promoter as the starting sequence. Other steps and parameters are the same as in one of Implementation Methods One to Four.

[0039] Specific Implementation Method Six: The difference between this implementation method and Specific Implementation Methods One to Five is that the candidate expression vectors mentioned in step two are L4440 and pET-2p. Other steps and parameters are the same as in Specific Implementation Methods One to Five.

[0040] Specific Implementation Method Seven: The difference between this implementation method and Specific Implementation Method Six is ​​that in step two, XhoI and HindIII are selected as restriction enzyme sites based on the candidate expression vector L4440; in step two, KpnI and NotI are selected as restriction enzyme sites based on the candidate expression vector pET-2p. Other steps and parameters are the same as in Implementation Method Six.

[0041] Specific Implementation Method Eight: The difference between this implementation method and Specific Implementation Methods Six or Seven is as follows: In step four, the double digestion system for the L4440 vector is: 6 μl L4440, 2 μl XhoI, 2 μl HindIII, 2 μl 10×Fast Digest Green Buffer, and 8 μl ddH2O, with a total volume of 20 μl; the digestion reaction conditions are 37℃ for 15 min and 80℃ for 10 min; the double digestion system for the pET-2p vector is: 6 μl pET-2p, 2 μl NotI, 2 μl KpnI, 2 μl 10×Fast Digest Green Buffer, and 8 μl ddH2O, with a total volume of 20 μl; the digestion reaction conditions are 37℃ for 15 min and 80℃ for 5 min. Other steps and parameters are the same as in Implementation Methods Six or Seven.

[0042] Specific Implementation Method Nine: The difference between this implementation method and Specific Implementation Methods One to Eight is that the PCR reaction conditions in step three are: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle, 72℃ extension for 30 s, followed by 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; and finally, 72℃ extension for 10 min. Other steps and parameters are the same as in Specific Implementation Methods One to Eight.

[0043] Specific Implementation Method Ten: The difference between this implementation method and Specific Implementation Methods One to Nine is that in step four, the linearized plasmid is recombinated with the dsgfp fragment amplified by PCR in step three using the Clon ExpressII One Step Cloning Kit. Other steps and parameters are the same as in Specific Implementation Methods One to Nine.

[0044] Specific Implementation Method Eleven: The difference between this implementation method and Specific Implementation Methods One through Ten is that in step four, the recombinant product is added to Trans-T1 competent cells, spread on solid culture medium, and cultured upside down overnight. The next day, single clones are picked and cultured at 37°C and 250g for 5 hours. Then, the bacterial culture is verified by PCR using primers for dsgfp fragment amplification. The verified bacterial culture is inoculated into liquid culture medium and cultured at 37°C and 250g for 3.5 hours. After that, the plasmid is extracted, verified by double enzyme digestion, and sequenced. Both the solid and liquid culture media mentioned above are selective media. Other steps and parameters are the same as in Implementation Methods One through Ten.

[0045] Specific Implementation Method Twelve: The difference between this implementation method and Specific Implementation Methods One to Eleven is that: the plasmid verified in step four is transformed into HT115(DE3) competent cells, then plated on solid culture medium and incubated upside down overnight. The next day, single clones are picked and cultured at 37°C and 250g for 5 hours; then, bacterial PCR is performed using dsgfp fragment amplification primers for verification, and the successfully verified bacterial culture is saved as the initial bacterial culture. Other steps and parameters are the same as in Specific Implementation Methods One to Eleven.

[0046] Specific Implementation Method Thirteen: The difference between this implementation method and Specific Implementation Methods One to Twelve is as follows: Step Five: IPTG Induction Expression: The initial bacterial culture is inoculated at a ratio of 1:1000 into selective liquid culture medium and activated overnight at 37°C and 250g centrifugation; then inoculated at a ratio of 1:100 into selective liquid culture medium and cultured at 37°C and 250g centrifugation for 3.5 hours. After that, IPTG solution is added and cultured with shaking for another 5 hours to obtain the induced bacterial culture. Other steps and parameters are the same as in Specific Implementation Methods One to Twelve.

[0047] Total nucleic acid was extracted from 1 ml of the induced bacterial culture, and detected by 1% agarose gel electrophoresis. The electrophoretic bands were analyzed by grayscale analysis using ImageJ software. The successfully induced dsgfp bacterial culture was added to 50% glycerol (1:1) and stored at -80℃.

[0048] Specific Implementation Method Fourteen: The difference between this implementation method and Specific Implementation Methods One to Thirteen is as follows: Step Six: Preparation of the in vitro synthesized dsgfp DNA template: Inoculate the induced bacterial culture at a dilution of 1:1000 into selective liquid culture medium and culture at 37°C and 250g centrifugation for 12–16 h to extract the plasmid; then perform PCR reaction using in vitro synthesized primers, followed by in vitro transcription. The in vitro transcription reaction system is: NTP Buffer Mix 10 μl, Template DNA 8 μl (0.5 μg), T7 RNA polymerase Mix 2 μl, with a total volume of 20 μl; incubate at 37°C for 4–6 h; then anneal the reaction solution in a 70°C water bath for 10 min and cool to room temperature; then add Dnase I and RNase A dilution buffer and react at 37°C for 30 min; thus obtaining the in vitro synthesized dsgfp DNA template. Other steps and parameters are the same as in Implementation Methods One to Thirteen.

[0049] Dissolve 1 μl of the in vitro synthesized dsgfp DNA template in 10 μl of Nuclease-Free water, detect by 1.5% agarose gel electrophoresis, and store the product at -80℃.

[0050] Specific Implementation Method Fifteen: The difference between this implementation method and one of Specific Implementation Methods One to Fourteen is that the alternative dsRNA extraction methods described in step seven include the Trizol method, the phenol-chloroform method, RNA-easy extraction solution, and the 75% alcohol precipitation method. Other steps and parameters are the same as in one of Implementation Methods One to Fourteen.

[0051] Specific Implementation Method Sixteen: This implementation method improves the expression level of prokaryotic dsRNA by selecting dsRNA with a CG content of 25-35% for expression.

[0052] In this embodiment, the prokaryotic expression vector is either L4440 or pET-2p.

[0053] Specific Implementation Method Seventeen: The difference between this implementation method and Specific Implementation Method Sixteen is that dsRNA with a CG content of 30% is used for expression. Other steps and parameters are the same as in Implementation Method Sixteen.

[0054] Example 1

[0055] Screening of bacterial culture vectors for dsRNA expression and methods:

[0056] 1. Using a 720bp gfp sequence as a template, dsgfp fragments of lengths of 201bp and 423bp were extracted and the bases of the sequences were randomly changed to synthesize test dsgfp fragments with CG content of 30%, 50%, and 70%, respectively.

[0057] DSGFP fragments with a length of 201bp and a content of 30% CG (abbreviated as 201-30CG), DSGFP fragments with a length of 201bp and a content of 50% CG (abbreviated as 201-50CG, and so on), 423-30CG, 423-50CG, and 423-70CG; test DSGFP fragment sequences of different lengths and CG combinations in this embodiment are as follows: Figure 1 As shown;

[0058] 2. Design and synthesize in vitro primers using the T7 promoter as the starting sequence (as shown in Table 1); select double restriction sites for the candidate expression vectors (L4440 and pET-2p) to design and synthesize primers for dsgfp fragment amplification (as shown in Table 2).

[0059] Specifically, XhoI and HindIII were selected as restriction enzyme sites based on the candidate expression vector L4440; and KpnI and NotI were selected as restriction enzyme sites based on the candidate expression vector pET-2p.

[0060] Table 1

[0061]

[0062]

[0063] Table 2

[0064]

[0065] 3. Using the dsgfp fragment as a template, PCR amplification was performed using dsgfp fragment amplification primers. The PCR reaction conditions were as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle, 72℃ extension for 30 s, followed by 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; and a final extension at 72℃ for 10 min.

[0066] After PCR amplification, the target band was detected by 1.5% agarose gel electrophoresis, and the band was excised and recovered using a kit.

[0067] IV. The candidate expression vector was double-digested using the Clon ExpressII One Step Cloning Kit and then recombined with the dsgfp fragment amplified by PCR in step III. 10 μl of the recombinant product was then added to 100 μl of Trans-T1 competent cells and plated onto solid LB selective medium (L4440: Ampicillin Amp) + pET-2p: Kanamycin KaNa + Incubate the culture at 37°C upside down overnight. The next day, pick single clones and incubate at 37°C and 250g centrifugation for 5 hours. Then, perform PCR verification using dsgfp fragment amplification primers. The verified bacterial culture is then inoculated 1:100 into selective LB liquid medium (L4440:Amp). + pET-2p:KaNa + The cells were cultured at 37°C and 250g centrifugation for 3.5 hours. Plasmids were then extracted and verified by double enzyme digestion. The remaining bacterial culture was sent to BGI Genomics in Beijing for sequencing. The verified plasmid was then transformed into HT115(DE3) competent cells and plated onto LB selective solid medium (L4440:Amp). + pET-2p:KaNa + Incubate the culture at 37°C inverted overnight. The next day, select single clones and incubate them at 37°C and 250g centrifugation for 5 hours. Perform PCR verification of the bacterial culture using primers for dsgfp fragment amplification. Save the successfully verified bacterial culture as the initial bacterial culture.

[0068] The double digestion system for the L4440 vector consisted of 6 μl L4440, 2 μl XhoI, 2 μl HindIII, 2 μl 10×Fast Digest Green Buffer, and 8 μl ddH2O, for a total volume of 20 μl. The digestion conditions were 37℃ for 15 min and 80℃ for 10 min. The double digestion system for the pET-2p vector consisted of 6 μl pET-2p, 2 μl NotI, 2 μl KpnI, 2 μl 10×Fast Digest Green Buffer, and 8 μl ddH2O, for a total volume of 20 μl. The digestion conditions were 37℃ for 15 min and 80℃ for 5 min. After digestion, the target DNA was recovered by 1% agarose gel electrophoresis.

[0069] V. IPTG-induced expression: The initial bacterial culture was inoculated at a ratio of 1:1000 into LB liquid medium (L4440:Amp). + Tet; pET-2p:KaNa + Tet + Activate overnight at 37°C and 250g centrifugation; then inoculate 1:100 into LB liquid medium (L4440:Amp) + Tet+ pET-2p:KaNa + Tet + The culture was incubated at 37℃ and 250g centrifugation for 3.5h, followed by the addition of IPTG solution and continued incubation with shaking for 5h to obtain the induced bacterial culture. Total nucleic acid was extracted from 1ml of the induced bacterial culture, and the results were detected by 1% agarose gel electrophoresis. ImageJ software was used to analyze the electrophoretic bands using grayscale. The successfully induced dsgfp bacterial culture was stored at -80℃ with 50% glycerol (1:1).

[0070] VI. Preparation of in vitro synthesized dsgfp DNA template: The induced bacterial culture was inoculated 1:1000 into LB liquid medium (L4440:Amp). + Tet + pET-2p:KaNa + Tet + Plasmids were extracted by culturing at 37℃ and 250g centrifugation for 12–16 h. Then, PCR was performed using in vitro synthesized primers (detected by 1.5% agarose gel electrophoresis; the target band was excised and recovered using a kit). In vitro transcription was then performed. The in vitro transcription reaction system consisted of: 10 μl NTP Buffer Mix, 8 μl Template DNA (0.5 μg), and 2 μl T7 RNA polymerase Mix, for a total volume of 20 μl. The mixture was incubated at 37℃ for 4–6 h. The reaction solution was then annealed in a 70℃ water bath for 10 min and cooled to room temperature. Next, 1 / 10 of the reaction solution volume was added with Dnase I and RNase A diluents (diluted 200-fold), and the reaction was carried out at 37℃ for 30 min. This yielded the in vitro synthesized dsgfp DNA template.

[0071] Take 1 μl of the synthesized dsgfp and dissolve it in 10 μl of Nuclease-Free water. Detect the product by 1.5% agarose gel electrophoresis and store it at -80℃.

[0072] 7. Extract dsgfp from the induced bacterial culture using the alternative dsRNA extraction method;

[0073] Alternative dsRNA extraction methods include the Trizol method, the phenol-chloroform method, RNA-easy extraction solution, and the 75% ethanol precipitation method;

[0074] 8. Based on the results of measuring the concentration, purity, and yield of dsgfp in the bacterial culture, determine the most suitable expression vector from the candidate expression vectors and the optimal dsRNA extraction method corresponding to that expression vector.

[0075] Preparation of competent cells in this embodiment:

[0076] The HT115(DE3) second-generation glycerol cryopreservation strain was inoculated at a 1:1000 ratio onto a substrate containing tetracycline (Tetracycline). + In liquid LB medium, culture overnight at 37°C and 250g centrifugation; pick single colonies and culture at 37°C and 250g centrifugation for 5 hours; after activation, dilute the bacterial suspension 1:100 in LB liquid medium (Texas Chromatography-Free). + Incubate at 37℃ and 250g centrifugation for 2–3 h until the OD reaches 0.4–0.6. Place the competent cell culture on ice for 10 min to cool to 0℃, simultaneously pre-cooling with 0.1 mol / L CaCl2 solution on ice, and centrifuge at 4℃ and 4000g for 10 min. Resuspend the precipitate in a 5:1 ratio of pre-cooled 0.1 mol / L CaCl2 solution, place on ice for 15–30 min, and centrifuge at 4℃ and 4000g for 10 min. Add the precipitate to a 25:1 ratio of 0.1 mol / L CaCl2 solution and a 25:1 ratio of 30% glycerol, and place on ice for 10 min. Resuspend the cells, aliquot, and store at -80℃.

[0077] In this embodiment, the concentration and purity of dsgfp synthesized in vitro were determined as follows:

[0078] Two μL of the in vitro synthesized dsgfp DNA template sample was used for detection in a B-500 BIOPHOTO METER. The sample was diluted with nuclease-free water to concentrations of 1000 ng / μl, 500 ng / μl, 250 ng / μl, 125 ng / μl, 62.5 ng / μl, and 31.25 ng / μl, respectively, and A was measured. 260 / A 280 A 260 / A 230 Detect dsgfp diluted to 1.5% agarose gel electrophoresis at 120V for 25 min.

[0079] In this example, the concentration and purity of dsgfp in the bacterial culture were determined as follows:

[0080] Take 2 μL of dsgfp bacterial culture and test it in a B-500 BIOPHOTO METER. Dilute with Nuclease-Free water to concentrations of 1000 ng / μl, 500 ng / μl, 250 ng / μl, 125 ng / μl, 62.5 ng / μl, and 31.25 ng / μl, respectively, and test for A. 260 / A 280 A 260 / A 230 Detect dsgfp diluted to 1.5% agarose gel electrophoresis at 120V for 25 min.

[0081] In this embodiment, five groups of dsgfp synthesized in vitro were diluted to 1000 ng / μl, and 30 μl of the dilution was mixed into 50 ml of un-IPTG-induced bacterial culture. Extraction was performed using the four methods described above, and the concentration was detected by a B-500 BIOPHOTO METER. 260 / A 280 and A 260 / A 280 The optimal extraction method was used to extract 1 ml of bacterial culture for dsgfp and determine the concentration. Based on the loss rate of the extraction method, the dsgfp yield expressed by pET-2P and L4440 was calculated.

[0082] pET-2p and L4440 were induced to express dsRNA via IPTG, and the RNA was detected by 1.5% agarose gel electrophoresis after RNase A digestion (e.g., pET-2p and L4440 were expressed as dsRNA). Figure 2 As shown in the figure, this indicates that the pET-2p-gfp and L4440-gfp expression vectors were successfully constructed.

[0083] This embodiment uses ImageJ software to analyze the grayscale values ​​of dsgfp expressed in the vector. Microsoft Office Excel 2010 and SPSS 25.0 software were used to analyze the in vitro synthesis of dsgfp and the extraction of dsgfpA from bacterial culture. 260 / A 280 A 260 / A 230 Multiple ANOVA and new multiple range test were used for significance analysis. Paired t-tests were used to compare the means between two samples. Using the 250bp and 500bp bands of the molecular weight marker (D2000 Tiangen) as controls (CK), grayscale analysis was performed on five groups of dsgfp with different lengths and CG contents induced by pET-2P and L4440, and the grayscale values ​​of all target dsRNAs were normalized. The results showed that the grayscale values ​​of the five groups of dsgfp with different lengths and CG contents expressed based on the pET-2P vector were significantly higher than those of the five groups of dsgfp with different lengths and CG contents expressed based on the L4440 vector (P < 0.05). Specifically, the grayscale values ​​of pET-2p expression of 201-30CG and 423-30CG were significantly higher than those of L4440 expression of 201-30CG and 423-30CG (e.g., ...). Figure 3 (As shown).

[0084] Dissolve 1 μl of synthesized DSGFP in 10 μl of Nuclease-Free water to prepare a 1.5% agarose gel for electrophoresis detection. Figure 4 The A value of dsgfp in 5 groups of bacterial cultures was detected using a B-500 BIOPHOTO METER. 260 / A 280 A230 / A 260 .Depend on Figure 5 It can be seen that, after serial dilution of the six concentrations, the dsgfp A in each group... 260 / A 280 All values ​​were between 1.9 and 2.2, indicating that the purity of dsgfp was high in the five synthesized bacterial cultures. Figure 5 b shows that the five groups of bacterial suspensions diluted to 31.25 ng / μl with dsgfp dilution, A 260 / A 230 The values ​​were all between 3.2 and 5.0, indicating that the concentration was too low, reaching the minimum detection threshold of the instrument and causing deviation. The dsgfp dilutions in the other five bacterial suspensions were all between 2.2 and 3.0. The results show that the dsgfp in the five combined bacterial suspensions can be used as standards for subsequent experiments.

[0085] Extraction of dsgfp expressed using the pET-2p vector: Using the dsgfp nucleic acid detection results of the target fragment in five combined bacterial cultures as a control (CK), the purity and concentration recovery rates of dsgfp obtained by four extraction methods were compared. Based on the extracted dsgfp nucleic acid detection values, A... 260 / A 280 and A 260 / A 230 Multiple comparison models were constructed using SPSS. The model intercepts (F = 19202.406, P = 0.000) and (F = 922.24, P = 0.000), the extraction method factor (F = 28.408, P = 0.000) and (F = 21.833, P = 0.000) reached highly significant levels, while the concentration factor reached significant (F = 2.868, P = 0.041) and insignificant (F = 0.297, P = 0.909) levels, respectively. The new multiple range test showed no significant difference between different concentrations. Figure 6 and Figure 7 It can be seen that the Trizol method and the phenol-chloroform method (CK is the control) have the highest extraction purity, and the difference between the two is not significant. The next method is the 75% alcohol precipitation method, and the RNA-easy extract has the lowest extraction purity.

[0086] Extraction of dsgfp expressed using the L4440 vector: Using the dsgfp nucleic acid detection results of the target fragment in five combined bacterial cultures as a control (CK), the purity and concentration recovery rates of dsgfp obtained by four extraction methods were compared. Based on the extracted dsgfp nucleic acid detection values, the A... 260 / A 280 and A 260 / A 230Multiple comparison models were constructed using SPSS. The model intercepts (F = 15242.017, P = 0.000) and (F = 734.211, P = 0.000), the extraction method factor (F = 30.300, P = 0.000) and (F = 24.085, P = 0.000) reached highly significant levels, while the concentration factor reached significant (F = 3.453, P = 0.021) and insignificant (F = 0.344, P = 0.880) levels, respectively. The new multiple range test showed no significant difference between different concentrations. Figure 6 and Figure 7 It can be seen that the Trizol method and the phenol-chloroform method (CK is the control) have the highest extraction purity, and the difference between the two is not significant. The next method is the 75% alcohol precipitation method, and the RNA-easy extract has the lowest extraction purity.

[0087] dsgfp from the five bacterial cultures was added to uninduced bacterial cultures, and extraction was performed using four different methods. The concentration and A were then determined. 260 / A 280 A 260 / A 230 Each method was repeated five times. The results showed that there were significant differences in the concentration and purity of dsgfp in the synthesized bacterial solution after extraction compared with that before extraction. SPSS software analysis confirmed these differences to be statistically significant (P < 0.05), as shown in Table 3. The phenol-chloroform method and the Trizol method yielded the highest dsgfp concentration and purity, with the smallest difference from the in vitro bacterial solution synthesis, and calculated extraction loss rates of 21.44% and 23.83%, respectively. The 75% ethanol precipitation method and the RNA-easy extraction solution yielded the lowest dsgfp concentration and purity, with the largest difference from the in vitro bacterial solution synthesis, and calculated extraction loss rates of 32.3% and 37.62%, respectively.

[0088] Table 3

[0089]

[0090] dsgfp was extracted from 1 ml of bacterial culture using the phenol-chloroform method, and the concentration was detected using a B-500 BIOPHOTO METER. The dsgfp yield per ml of bacterial culture was calculated based on the phenol-chloroform extraction loss rate. The results showed that the dsgfp concentrations of the five groups expressing different lengths and CG contents using the pET-2P vector were significantly higher than those expressed using the L4440 vector (P < 0.05). One ml of induced bacterial culture using the pET-2P and L4440 expression vectors could ferment to produce 8.7–24.39 μg of dsgfp and 7.48–22.47 μg of dsgfp respectively. Figure 8(As shown). The Trizol and phenol-chloroform methods were used to efficiently extract complete bacterial total RNA. After digestion with RNase A and DNase I, relatively pure dsgfp was obtained with the lowest loss rate. RNA-easy extraction buffer and 75% ethanol precipitation methods had significantly lower extraction efficiency than the former and were not suitable for rapid extraction of bacterial total RNA and dsgfp. Using the phenol-chloroform method, 1 ml of induced bacterial culture was used to extract dsgfp expressed by pET-2p and L4440, respectively. Nucleic acid concentration was detected, and the expression of 423-30CG by the two vectors was found to be significantly higher than the other four groups, indicating that both pET-2p and L4440 have strong expression capabilities. Concentration analysis was conducted, and the dsgfp yield per milliliter of bacterial culture was calculated based on the extraction loss rate. It was found that the yields of the five groups of dsgfp generated based on pET-2p were all higher than those generated by L4440, reaching a maximum of 24.39 μg / ml. pET-2p can be considered as the relatively optimal expression vector for dsgfp, and the phenol-chloroform method is the best dsRNA extraction method for the pET-2p vector. Furthermore, the expression level of the dsRNA fragment with 30% CG content was about 50% higher than that of the other two dsRNA fragments with CG content.

Claims

1. A method for screening bacterial culture expression vectors for dsRNA, characterized in that... This method is performed in the following steps:

1. Using the GFP sequence as a template, DSGFP fragments of different lengths were extracted and the bases of the sequence were randomly changed to synthesize test DSGFP fragments with different CG base contents. II. Design and synthesize in vitro primers starting with the promoter sequence; design and synthesize dsgfp fragment amplification primers based on double restriction sites selected from candidate expression vectors.

3. Using the dsgfp fragment as a template, PCR amplification was performed using dsgfp fragment amplification primers; 4. The candidate expression vector is double-digested and then recombined with the dsgfp fragment amplified by PCR in step 3. The recombinant product is then added to Trans-T1 competent cells for culture, and single clones are picked for verification and sequencing. The plasmid that is verified to be correct is transformed into HT115 (DE3) competent cells to obtain the initial bacterial culture. V. IPTG-induced expression to obtain induced bacterial culture; VI. Preparation of in vitro synthesized dsgfp DNA template; 7. Extract dsgfp from the induced bacterial culture using the alternative dsRNA extraction method; 8. Based on the results of measuring the concentration, purity, and yield of dsgfp in the bacterial culture, determine the most suitable expression vector and the optimal dsRNA extraction method corresponding to the expression vector from the candidate expression vectors; The lengths of the dsgfp segments selected in step one are 201bp and 423bp, respectively; The CG base contents in the test dsgfp fragments synthesized in step one were 30%, 50%, and 70%, respectively. The candidate expression vectors mentioned in step two are L4440 and pET-2p; XhoI and HindIII are selected as restriction enzyme sites based on candidate expression vector L4440; KpnI and NotI are selected as restriction enzyme sites based on candidate expression vector pET-2p.

2. The method for screening bacterial culture expression dsRNA vectors according to claim 1, characterized in that... The PCR reaction conditions described in step three are as follows: 94℃ pre-denaturation for 3 min; 94℃ denaturation for 30 s, 61℃ annealing for 30 s, 10 cycles, with the annealing temperature decreasing by 0.5℃ per cycle, 72℃ extension for 30 s, followed by 94℃ denaturation for 30 s, 56℃ annealing for 30 s, 72℃ extension for 30 s, 35 cycles; and finally, 72℃ extension for 10 min.

3. The method for screening bacterial culture expression dsRNA vectors according to claim 1, characterized in that... In step four, the recombinant product was added to Trans-T1 competent cells, spread on solid medium, and cultured upside down overnight. The next day, single clones were picked and cultured at 37°C and 250g for 5 hours. Then, the bacterial culture was verified by PCR using primers for dsgfp fragment amplification. The verified bacterial culture was inoculated into liquid medium and cultured at 37°C and 250g for 3.5 hours. After that, the plasmid was extracted, double enzyme digestion was performed for verification, and sequencing was performed. Both the solid and liquid mediums mentioned above are selective media.

4. The method for screening bacterial culture expression dsRNA vectors according to claim 1, characterized in that... In step four, the plasmid that was verified to be correct was transferred into HT115 (DE3) competent cells, then plated on solid culture medium and incubated upside down overnight. The next day, single clones were picked and cultured at 37°C and 250g centrifugation for 5 hours. Then, the bacterial culture was verified by PCR using primers for dsgfp fragment amplification. The successfully verified bacterial culture was saved as the initial bacterial culture.

5. The method for screening bacterial culture expression dsRNA vectors according to claim 1, characterized in that... The alternative dsRNA extraction methods mentioned in step seven include the Trizol method, the phenol-chloroform method, RNA-easy extraction solution, and the 75% ethanol precipitation method.

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