Method for expressing dsRNA of escherichia coli without using inducer

Abstract

The invention belongs to the technical field of bioengineering, and particularly relates to a method for expressing dsRNA by escherichia coli without using an inducer. In order to solve the problem that an inducer IPTG (isopropyl-beta-d-thiogalactoside) is still used for expressing dsRNA by escherichia coli, a skeleton vector is modified, an inducible T7 promoter is replaced by a constitutive promoter, and a large amount of dsRNA can be expressed under the condition that no inducer is added. The dsRNA is efficiently produced on a large scale at low cost through escherichia coli, so that it is possible to use the dsRNA for pest control in a field, and finally development of RNAi serving as a core technology for developing nucleic acid biopesticide for targeted control of agricultural pests is promoted.

Description

Technical Field

[0001] The invention belongs to the technical field of bioengineering, and in particular relates to a method for Escherichia coli to express dsRNA without using an inducer. Background Art

[0002] RNA interference (RNAi) refers to the phenomenon that double-stranded RNA (dsRNA), which is widely present in eukaryotes, can degrade specific gene mRNA, resulting in reduced expression of target genes. In recent years, RNAi technology has played a great role in the study of insect gene function, and its application in pest control has also received widespread attention. The development of RNAi technology has accelerated the development of nucleic acid pesticides to a certain extent. Nucleic acid pesticides use the pest's own dsRNA to achieve silencing of target gene transcription levels, thereby inhibiting the growth and development of pests and achieving the purpose of pest control. Compared with traditional pesticides, RNA pesticides have the following characteristics: 1) strong specificity, dsRNA sequence specificity makes it highly specific to target genes and target species; 2) wide range of target selection, transcriptional gene silencing characteristics greatly expand the selection of control targets; 3) green and safe, dsRNA is easily soluble in water and easily degraded in the environment, without residue. Therefore, nucleic acid pesticides have broad application prospects and are expected to lead the "third revolution in the history of pesticide development." The application of RNAi technology in insect pests mainly faces three challenges: screening of target genes, control of dsRNA production costs, and delivery of dsRNA to target pests. After determining the effective target gene, the cost of dsRNA production is an urgent problem to be solved for the large-scale commercialization of nucleic acid pesticides. Therefore, it is urgent to develop a low-cost dsRNA production system to ultimately achieve the widespread application of RNAi technology in practice.

[0003] Escherichia coli is currently the most widely used production strain due to its high transformation efficiency, fast reproduction speed and stable and mature operating system. Scientists have found that RNase III (the enzyme that causes dsRNA degradation)-deficient Escherichia coli HT115 (DE3) can produce dsRNA from an expression vector containing a T7 promoter under the induction of isopropylthiogalactoside (IPTG) to cause degradation of the target gene. Subsequent researchers used single T7 and double T7 promoters to explore the yield of bacterial dsRNA expression and found that the yield of dsRNA expressed by a single T7 promoter in combination with a system with a hairpin structure was higher than that of the target dsRNA expressed by a double T7 promoter. Whether it is a single T7 promoter or a double T7 promoter, it requires an appropriate concentration of IPTG induction to express the corresponding dsRNA, but IPTG is expensive and toxic, and the addition of IPTG in actual industrial fermentation production will increase the risk of pollution, which greatly limits industrial large-scale production. In order to make RNA interference technology widely used in field pest control, it is urgent to build a double-stranded RNA expression system that is inexpensive and can reduce pollution during the production process. In order to solve the problems of using IPTG to express dsRNA in E. coli, we will construct a constitutive vector to express the target dsRNA without any inducer. Summary of the invention

[0004] In order to solve the problem that Escherichia coli still uses the inducer IPTG to express dsRNA, the present invention constructs a constitutive expression vector and also provides a method for expressing dsRNA in Escherichia coli, which expresses the target dsRNA without any inducer, thereby reducing the cost and toxic pollution of expressing dsRNA in industrial production.

[0005] To achieve the above object, the present invention adopts the following technical solutions:

[0006] The present invention provides a constitutive expression vector for expressing dsRNA in Escherichia coli, which is prepared by the following method:

[0007] Step 1, using an endonuclease to digest the backbone vector and remove the T7 promoter already present on the backbone vector;

[0008] Step 2, artificially synthesizing the forward and reverse sequences of the constitutive promoter, and adding corresponding restriction sites and protective bases at both ends, the sequence information is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and synthesizing short double-stranded DNA by slow annealing;

[0009] Step 3, digesting and recovering the double-stranded DNA with restriction sites synthesized in step 2;

[0010] Step 4, connecting the enzyme-digested products obtained in step 1 and step 3;

[0011] Step 5, transforming the ligation product obtained in step 4 into Escherichia coli DH5α, culturing overnight on a resistant medium, picking positive clones, and performing sequencing analysis;

[0012] Step 6, the bacterial solution sequenced correctly in step 5 is preserved and inoculated into a resistance culture medium, and the plasmid is extracted after amplification, that is, the constitutive expression vector.

[0013] Furthermore, the backbone vector is a commercial T444T vector, and the resistance culture medium is a culture medium containing ampicillin resistance.

[0014] The present invention also provides a method for expressing dsRNA in Escherichia coli without using an inducer, characterized in that it comprises the following steps:

[0015] Step 1, transforming the inducible expression vector into a constitutive expression vector (i.e., the above-mentioned constitutive expression vector);

[0016] Step 2, constructing a recombinant expression vector with the constitutive expression vector and the target gene fragment;

[0017] Step 3, the recombinant expression vector is transferred into the host bacteria HT115 (DE3) to express and produce dsRNA.

[0018] Furthermore, the target gene in step 2 is the ATP synthase ATP5A gene of Locust migratoria, the fragment length is 413 bp, and the sequence is shown in SEQ ID NO.9.

[0019] Furthermore, in step 3, the recombinant expression vector is transferred into the host bacteria HT115 (DE3) to express and produce dsRNA, and the specific steps are as follows:

[0020] Step 3.1, transfer the recombinant expression vector into the host bacteria HT115 (DE3), and culture it overnight at 37°C on an LB plate containing ampicillin and tetracycline resistance; the next day, pick a single clone and culture it overnight in 500 μL LB liquid medium;

[0021] Step 3.2, take the bacterial solution from the overnight culture step 3.1 and inoculate it into 50 mL LB liquid culture medium at a volume ratio of 1:100, and continue to expand the culture to allow the E. coli to produce dsRNA; collect the bacterial solution and extract total RNA;

[0022] In step 3.3, take the bacterial solution after overnight culture, centrifuge and discard the supernatant, add ethanol, mix by pipetting and let stand at room temperature, centrifuge again and discard the supernatant, suspend the precipitate with NaCl solution, then let stand at room temperature, centrifuge and obtain the supernatant to obtain the total RNA of Escherichia coli. The dsRNA of the target gene is included in the total RNA.

[0023] Furthermore, the step 3.3 is specifically as follows:

[0024] Take 50mL of bacterial culture after overnight culture, centrifuge at 4℃, 12000rpm for 2min, discard the supernatant, add 2mL of 75% ethanol, mix by pipetting, let stand at room temperature for 5min, then centrifuge at 4℃, 8000rpm for 5min, discard the supernatant, suspend the precipitate with 500μL of 150mmol / L Nacl solution, then let stand at room temperature for 1h, centrifuge at 4℃, 12000rpm for 10min, obtain the supernatant, and obtain the total RNA of Escherichia coli. The dsRNA of the target gene is included in the total RNA.

[0025] Compared with the prior art, the present invention has the following advantages:

[0026] The present invention constructs a constitutive promoter expression vector and uses HT115 (DE3) in combination to express dsRNA in large quantities without using any inducer. The generated dsRNA is functionally verified and found to significantly reduce the expression of the corresponding gene and produce related phenotypes.

[0027] Compared with the traditional T7 promoter for synthesizing dsRNA in Escherichia coli, the method of the present invention for synthesizing dsRNA by using a constitutive promoter in combination with Escherichia coli greatly reduces the use cost and toxicity of the inducer, so that the large-scale use of dsRNA in the field can better meet the needs of the application of RNAi preparations in the field, establishes a low-cost material basis for the research and development of RNAi preparations, and will promote the development of new pest control technologies with RNAi as the core. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of the constitutive expression vector constructed in the embodiment of the present invention;

[0029] Figure 2 The results of expressing LmATP5A-dsRNA using a constitutive expression vector in the examples of the present invention, wherein 1: the expression result of Escherichia coli HT115 (DE3) (the box marked part is the expressed dsRNA); 2: DL1000 Marker;

[0030] Figure 3The expression of LmATP5A-dsRNA in different systems, where M: Marker; 1: E. coli HT115 (DE3) / T444T empty-load expression results induced by IPTG; 2: E. coli HT115 (DE3) / T444T-LmATP5A expression results induced by IPTG (the marked part in the box is the induced expressed dsRNA); 3: E. coli HT115 (DE3) / T444T-Con-LmATP5A expression results (the marked part in the box is the expressed dsRNA);

[0031] Figure 4 This is a graph showing the interference efficiency detection and survival curve of the LmATP5A-dsRNA expressed in the embodiment of the present invention, injected into the migratory locust. DETAILED DESCRIPTION

[0032] The specific implementation of the present invention is further described in detail below in conjunction with the accompanying drawings and examples. The following examples are used to illustrate the present invention, but are not intended to limit the scope of protection of the present invention, which is subject to the claims.

[0033] 1. Raw materials

[0034] T444T was purchased from Beijing Zhongyuan Heju Biotechnology Co., Ltd., and HT115 (DE3) and DH5α (DE3) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0035] 2. Methods

[0036] A method for expressing dsRNA in Escherichia coli without using an inducer in this embodiment comprises the following steps:

[0037] Step 1, transforming the inducible expression vector into a constitutive expression vector, specifically:

[0038] 1) Using the commercial T444T vector as the backbone vector, pciI and BglII endonucleases were used for restriction digestion to remove the T7 promoter at one end of the backbone vector;

[0039] 2) The forward and reverse sequences of the constitutive promoter are artificially synthesized, and the corresponding restriction sites and protective bases are added at both ends. The sequence information is shown in SEQ ID NO.1 and SEQ ID NO.2, and short double-stranded DNA is synthesized by slow annealing;

[0040] 3) digesting the double-stranded DNA with restriction sites obtained in 2) and recovering it;

[0041] 4) connecting the enzyme-digested products obtained in 1) and 3);

[0042] 5) The ligation product was then transformed into E. coli DH5α and cultured overnight at 37°C on an LB plate containing ampicillin resistance. Positive clones were picked and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis;

[0043] 6) The bacterial solution sequenced correctly in 5) was saved and inoculated into 5 ml of LB liquid medium containing ampicillin resistance, and agitated at 200 rpm and 37°C for 12 h to extract the plasmid;

[0044] 7) The plasmid extracted in 6) is then double-digested with KpnI and PsiI endonucleases to remove the T7 promoter at the other end of the backbone vector;

[0045] 8) Artificially synthesizing the forward and reverse sequences of the constitutive promoter, and adding corresponding restriction sites and protective bases at both ends, the sequence information is shown in SEQ ID NO.3 and SEQ ID NO.4, and synthesizing short double-stranded DNA by slow annealing;

[0046] 9) digesting the double-stranded DNA with restriction sites obtained in 8) and recovering it;

[0047] 10) connecting the enzyme-digested products obtained in 7) and 9);

[0048] 11) The ligation product was then transformed into Escherichia coli DH5α and cultured overnight at 37°C on an LB plate containing ampicillin resistance. Positive clones were picked and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing analysis;

[0049] 12) The correctly sequenced bacterial solution in 11) was preserved and inoculated into 5 mL of LB liquid medium containing ampicillin resistance, and the culture was shaken at 200 rpm and 37°C for 12 h to extract the plasmid, i.e., the constitutive expression vector, named T444T-Con, whose structure is as follows: Figure 1 shown.

[0050] Step 2, constructing a recombinant expression vector with the constitutive expression vector and the target gene fragment, specifically:

[0051] 13) using XhoI and SacI endonucleases to perform double digestion on the plasmid extracted in 12), then recovering the digestion products by gel recovery method, and measuring the concentration by Nanodrop spectrophotometer;

[0052] 14) Using SEQ ID NO.5 and SEQ ID NO.6 as primers and locust cDNA as template, amplify the target gene sequence fragment, detect the bands on nucleic acid gel, and confirm that the PCR amplification obtains the correct band size;

[0053] 15) using SEQ ID NO.7 and SEQ ID NO.8 as primers and the product in 14) as a template, PCR amplification was performed to obtain a gene fragment with restriction sites of XhoI and SacI added to both ends of the gene sequence, wherein the target gene is LmATP5A, the gene fragment is 413 bp in length, and the sequence is as shown in SEQ ID NO.9;

[0054] 16) The plasmid and gene fragments digested in 13) and 15) were connected overnight, transformed into competent E. coli DH5α (DE3), and plated for overnight culture;

[0055] 17) Pick a single clone and continue culturing it in liquid culture medium containing ampicillin, identify it by PCR, culture the positive clone in LB overnight, and sequence it;

[0056] 18) Extract the recombinant plasmid, i.e., the recombinant expression vector, from the bacterial solution sequenced correctly in 17).

[0057] Step 3, the recombinant expression vector is transferred into the host bacteria HT115 (DE3) to express and produce dsRNA, specifically:

[0058] 19) The recombinant expression vector prepared in 18) was transferred into HT115 (DE3) and cultured overnight at 37° C. on an LB plate containing ampicillin and tetracycline resistance; the next day, a single clone was picked and cultured overnight in 500 μL of LB liquid culture medium;

[0059] 20) The bacterial solution from the overnight culture 19) was inoculated into 50 mL LB liquid culture medium at a volume ratio of 1:100, and the culture was continued to expand so that the E. coli produced dsRNA; the bacterial solution was collected and the total RNA was extracted;

[0060] 21) Take 50 mL of the bacterial solution after overnight culture in 20), centrifuge at 4°C, 12000 rpm for 2 min, discard the supernatant, add 2 mL of 75% ethanol (prepared in PBS), mix by pipetting and let stand at room temperature for 5 min, then centrifuge at 4°C, 8000 rpm for 5 min, discard the supernatant, suspend the precipitate with 500 μL of 150 mmol / L Nacl solution, then let stand at room temperature for 1 h, centrifuge at 4°C, 12000 rpm for 10 min, obtain the supernatant, and obtain the Escherichia coli total RNA, in which LmATP5A-dsRNA is included.

[0061] 3. Verify the function of expressed dsRNA in locusts

[0062] The total RNA extracted from 21) was subjected to nucleic acid gel detection ( Figure 2), and grayscale scanning quantitative analysis was performed using Image J software. The present invention uses a constitutive promoter to cooperate with Escherichia coli to synthesize dsRNA, and the yield of LmATP5A-dsRNA expressed by 1 ml of bacterial solution is 7.7 μg; while the traditional T7 promoter synthesizes dsRNA in Escherichia coli, and the yield of LmATP5A-dsRNA expressed by 1 ml of bacterial solution is 4.6 μg ( Figure 3 ).

[0063] The dsRNA concentration in 21) was converted and diluted to 30 ng / μL. The LmATP5A-dsRNA expressed by the constitutive promoter in 21) was injected into the third and fourth abdominal segments of the first day of the third instar migratory locust nymphs using a microsyringe. Each locust was injected with 1 μL. The control group was injected with the same dose of dsGFP. Samples were taken at 24 hours to detect the interference efficiency. Compared with the dsGFP group, the expression level of the LmATP5A gene in the dsLmATP5A group decreased by 47.3% ( Figure 4 A), and it was found that the mortality rate of insects in the treated group was as high as 98.3% after 48 hours compared with the control group ( Figure 4 (B).

[0064] In summary, the present invention can express dsRNA in large quantities without any inducer by constructing a constitutive promoter expression vector and using HT115 (DE3). Compared with the traditional T7 promoter synthesizing dsRNA in Escherichia coli, the expression yield of LmATP5A-dsRNA constitutive promoter is higher than that of the traditional T7 promoter. Therefore, the construction of the constitutive promoter expression dsRNA vector greatly reduces the use cost and toxicity of the inducer. In addition, the generated dsRNA is verified by locust body function, and it is found that it can significantly reduce the expression amount of the corresponding gene and produce related phenotypes.

[0065]

[0066]

Claims

1. A constitutive expression vector for expressing dsRNA in Escherichia coli, characterized in that: Prepared by the following method: Step 1, using an endonuclease to digest the backbone vector and remove the T7 promoter already present on the backbone vector; Step 2, artificially synthesizing the forward and reverse sequences of the constitutive promoter, and adding corresponding restriction sites and protective bases at both ends, the sequence information is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and synthesizing short double-stranded DNA by slow annealing; Step 3, digesting and recovering the double-stranded DNA with restriction sites synthesized in step 2; Step 4, connecting the enzyme-digested products obtained in step 1 and step 3; Step 5, transforming the ligation product obtained in step 4 into Escherichia coli DH5α, culturing overnight on a resistant medium, picking positive clones, and performing sequencing analysis; Step 6, the bacterial solution sequenced correctly in step 5 is preserved and inoculated into a resistance culture medium, and the plasmid is extracted after amplification, that is, the constitutive expression vector.

2. A constitutive expression vector for expressing dsRNA in Escherichia coli according to claim 1, characterized in that: The backbone vector is a commercial T444T vector, and the resistance culture medium is a culture medium containing ampicillin resistance.

3. A method for expressing dsRNA in Escherichia coli without the use of an inducer, characterized in that: The following steps are involved: Step 1, transforming the inducible expression vector into a constitutive expression vector; Step 2, constructing a recombinant expression vector with the constitutive expression vector and the target gene fragment; Step 3, the recombinant expression vector is transferred into the host bacteria HT115 (DE3) to express and produce dsRNA.

4. The method for expressing dsRNA in Escherichia coli without using an inducer according to claim 3, characterized in that: In step 1, the inducible expression vector is transformed into a constitutive expression vector, and the specific steps are: Step 1.1, using an endonuclease to digest the backbone vector and remove the existing T7 promoter on the backbone vector; Step 1.2, artificially synthesizing the forward and reverse sequences of the constitutive promoter, and adding corresponding restriction sites and protective bases at both ends, the sequence information is shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO.4, and synthesizing short double-stranded DNA by slow annealing; Step 1.3, digesting and recovering the double-stranded DNA with restriction sites synthesized in step 1.2; Step 1.4, connecting the enzyme-digested products obtained in step 1.1 and step 1.3; Step 1.5, transform the ligation product obtained in step 1.4 into Escherichia coli DH5α, culture overnight on a resistant medium, pick positive clones, and perform sequencing analysis; Step 1.6, the bacterial solution sequenced correctly in step 1.5 is preserved and inoculated into a resistance culture medium, and the plasmid is extracted after amplification, i.e., the constitutive expression vector.

5. The method for expressing dsRNA in Escherichia coli without using an inducer according to claim 4, characterized in that: The backbone vector is a commercial T444T vector, and the resistance culture medium is a culture medium containing ampicillin resistance.

6. The method for expressing dsRNA in Escherichia coli without using an inducer according to claim 3, characterized in that: The target gene in step 2 is the ATP synthase ATP5A gene of Locust migratory locust, the fragment length is 413 bp, and the sequence is shown in SEQ ID NO.

9.

7. The method for expressing dsRNA in Escherichia coli without using an inducer according to claim 3, characterized in that: In step 3, the recombinant expression vector is transferred into the host bacteria HT115 (DE3) to express and produce dsRNA, and the specific steps are as follows: Step 3.1, transfer the recombinant expression vector into the host bacteria HT115 (DE3), and culture it overnight at 37°C on an LB plate containing ampicillin and tetracycline resistance; the next day, pick a single clone and culture it overnight in 500 μL LB liquid medium; Step 3.2, take the overnight cultured bacterial solution in step 3.1 and inoculate it into 50 mL LB liquid culture medium at a volume ratio of 1:100, and continue to expand the culture to allow E. coli to produce dsRNA; collect the bacterial solution and extract total RNA; In step 3.3, take the bacterial solution after overnight culture, centrifuge and discard the supernatant, add ethanol, mix by pipetting and let stand at room temperature, centrifuge again and discard the supernatant, suspend the precipitate with NaCl solution, then let stand at room temperature, centrifuge and obtain the supernatant to obtain the total RNA of Escherichia coli. The dsRNA of the target gene is contained in the total RNA.

8. The method for expressing dsRNA in Escherichia coli without using an inducer according to claim 7, characterized in that: The step 3.3 is specifically as follows: Take 50mL of bacterial culture after overnight culture, centrifuge at 4℃, 12000rpm for 2min, discard the supernatant, add 2mL of 75% ethanol, mix by pipetting, let stand at room temperature for 5min, then centrifuge at 4℃, 8000rpm for 5min, discard the supernatant, suspend the precipitate with 500μL of 150mmol / L NaCl solution, then let stand at room temperature for 1h, centrifuge at 4℃, 12000rpm for 10min, obtain the supernatant, and obtain the total RNA of Escherichia coli. The dsRNA of the target gene is included in the total RNA.

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