CN111607589A - A system construction method for obtaining a large amount of dsRNA in vitro - Google Patents

Abstract

The invention relates to a method for constructing a system for obtaining a large amount of dsRNA from ashworm in vitro. First, the total RNA of ashworm is extracted and reverse transcribed into cDNA, followed by PCR amplification. After the product is connected, the obtained recombinant plasmid is transformed into Escherichia coli competent cell HT115 (DE3) strain, a solid medium is coated to screen a single colony, and then the single colony is cultured on a liquid shaker to OD ₆₀₀ =0.38‑0.42, and IPTG is added to induce The bacterial solution was then concentrated and used for RNA interference experiments. In the invention, single-cell cloning of Escherichia coli contains the target gene of the ashworm, and through the self-metabolism of the Escherichia coli and a large amount of self-reproduction and replication, a large amount of bacteria liquid containing the dsRNA gene of the ashworm is obtained, and the bacteria liquid containing the dsRNA is sprayed on the larvae of the ashworm , enter the body from the surface of the worm body, and effectively interfere with the expression of the target gene.

Description

A system construction method for obtaining a large amount of dsRNA in vitro

technical field

The invention belongs to the technical field of genetic engineering, and in particular relates to a system construction method for obtaining a large amount of dsRNA in vitro.

Background technique

White wax worm, Ericerus pela (Chavannes), commonly known as wax worm, belongs to the genus Ericerus (Chavannes, 1819) in the taxonomy of the Hemipetera family Coccoidea. Cerachinensis is a kind of scale insect with great economic value. The 2nd instar male larvae of Cerachinensis live on the branches of host plants and secrete a white waxy covering. The wax secreted by this insect becomes Cerachinensis, also known as Cerachinensis. ), that is, the secretion of the waxworm, which is a special product of China. White wax is a natural polymer compound with high melting point and chemical stability. It has the characteristics of moisture-proof, lubricating and glossing. It is an important chemical raw material and is widely used in chemical industry, machinery, food, medicine, cosmetics, agriculture and other industries.

RNA interference (RNAi) refers to the phenomenon that in eukaryotes, homologous mRNA is degraded by double-stranded RNA (dsRNA) and the expression of target genes is silenced. The main methods of dsRNA transfection include injection, feeding, soaking, virus infection and gene gun. However, if the research subject is too small to move, the infection methods of feeding, immersion, and surface absorption may result in insufficient dsRNA prepared by the kit.

The insects in this study are P. chinensis, which are small in size and have piercing-sucking mouthparts. They grow and reproduce by absorbing the sap of the host, and they cannot move most of the time. They will be infected by skin absorption. Due to body surface absorption and large dosage, dsRNA is unstable and easily degraded in the outside world. It needs to be sprayed and absorbed on the body surface skin of white wax insects for many times. Therefore, in some RNA interference experiments, how to prepare high-yield dsRNA is a problem that needs to be solved in genetic engineering.

SUMMARY OF THE INVENTION

In the process of RNAi transfection, due to objective reasons, the research objects cannot use micro-dose transfection methods such as gene guns, but when in vitro transfection is adopted, large doses of dsRNA are required. Therefore, the present invention overcomes the deficiencies in the prior art. , to provide a system construction method for obtaining a large amount of dsRNA in vitro, which can provide a reference for how to obtain a large amount of dsRNA at low cost.

For achieving the above object, the technical scheme adopted in the present invention is as follows:

A method for constructing a system for obtaining a large amount of dsRNA in vitro, comprising the following steps:

In step (1), the total RNA of the worm was extracted and reverse transcribed into cDNA, followed by PCR amplification. The amplified product and the L4440 vector plasmid were double digested with SacI and SalI respectively, and the double digested product was digested with T4 ligase overnight at 4°C connected to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;

In step (2), the 302a1-L4440 recombinant plasmid obtained in step (1) is transformed into the Escherichia coli competent cell HT115 (DE3) strain, and the solid medium is coated to screen a single colony to obtain the 302a1-L4440-HT115 bacterial cell;

step (3), culturing the 302a1-L4440-HT115 bacterial cell liquid obtained in step (2) on a shaker to OD ₆₀₀ =0.38-0.42, adding IPTG for induction, and obtaining a dsRNA bacterial liquid containing the 302a1 gene fragment of the waxworm;

In step (4), the dsRNA bacterial solution obtained in step (3) containing the 302a1 gene fragment of the white wax worm is concentrated, and the bacterial solution is used for the RNA interference experiment.

Further, it is preferred that the total RNA of the white wax worm comprises the total RNA of the white wax worm 302a1 gene.

Further, preferably, in step (1), the PCR amplification method is used to design a 302a1 gene primer (the primer name is ECEGT01) containing SacI and SalI double restriction sites, wherein

ECEGT01-F: 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO. 6);

ECEGT01-R: 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO. 7).

Further, preferably, in step (1), the PCR amplification system is:

The PCR amplification program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 5 min, and storage at 4 °C.

Further, it is preferred that, in step (1), the conditional system of enzyme cleavage is:

The digestion time was 10 min and the temperature was 37°C.

Further, preferably, the connection system is:

Further, it is preferred that the connected product is transformed into the competent cell DH5ɑ, and the SOC solid medium containing 100 μg/mL ampicillin is coated and screened for culture, and the single colony that grows is expanded and propagated to extract the plasmid, i.e. to obtain 302a1-L4440. recombinant plasmids. Wherein, there is no specific limitation on the method of expanding reproduction, preferably after the single colony that grows out, pick a single colony with an inoculation loop and put it in a centrifuge tube containing 100 μg/mL ampicillin SOC liquid medium, and cultivate it on a shaking table for 10 hours. Extract the plasmid.

Further, preferably, the specific method of step (2) is: transform the 302a1-L4440 recombinant plasmid obtained in step (1) into Escherichia coli competent cell HT115 (DE3) strain, and coat it on the HT115 (DE3) strain containing 100 μg/mL ampicillin and 50 μg/mL tetracycline on the SOC solid medium, cultured in the dark for 12 h, and single colonies were picked to obtain 302a1-L4440-HT115 cells;

Further, preferably, the specific method of step (3) is: 302a1-L4440-HT115 bacterial cells are cultured in the SOC liquid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline for 10-12 h at 37° C. , and then shaken in 2×YT liquid medium containing 75 μg/mL ampicillin and 12.5 μg/mL tetracycline to OD ₆₀₀ =0.38-0.42, add IPTG to induce culture for 3-4 h, and obtain a dsRNA bacterial solution.

The invention provides a method for constructing a system for obtaining dsRNA in large quantities by single-cell cloning, which mediates the RNAi of the white wax worm to cause the silencing of target gene expression. In this method, the RNAi interference of P. elegans was taken as an example, and the target gene fragment of P. cinerea 302a1 was obtained by PCR using P. cinerea cDNA as a template, and the two ends of the gene fragment were modified by restriction endonucleases to form sticky ends. Dicer modified to form a linear L4440 plasmid ligation to form a plasmid 302a1-L4440 recombinant plasmid containing the target gene fragment of ashworm. The 302a1-L4440 recombinant plasmid was transformed into the E. coli competent HT115 (DE3) strain to form the 302a1-L4440-HT1115 strain, which was resistant to both ampicillin and tetracycline, could not synthesize RNA hydrolase and contained the 302a1 gene fragment of A. , so the Escherichia coli can be cultured and propagated by shaking the SOC liquid medium (containing ampicillin and tetracycline) by single-cell cloning, and its propagation process can be induced by IPTG to produce dsRNA, and a large amount of dsRNA containing the 302a1 gene fragment of the white wax worm can be obtained, The recombinant Escherichia coli bacterial liquid was collected, and the bacterial liquid was diluted and sprayed directly on the larvae of the white wax worm. According to the method of the invention, single-cell cloning of Escherichia coli containing the target gene of Escherichia coli 302a1, and through the secondary metabolism and self-reproduction and replication of the Escherichia coli, a bacterial liquid containing a large amount of dsRNA gene fragments of Escherichia coli can be obtained, The dsRNA bacterial solution is sprayed on the larvae of the white wax worm, and enters the body from the surface of the worm body, which effectively interferes with the expression of the target gene. It is an insect type that cannot be micro-injected during the RNAi transfection process due to the small size of the insect and no chewing mouthparts. , synthesizing a large amount of dsRNA and providing a reference for the RNAi transfection pathway into the body through the body surface.

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

The present invention is the first to verify the RNA gene function of P. elegans by introducing single-cell cloned microorganism Escherichia coli, so that its genetic material contains P. elegans 302a1 gene fragment, and the secondary metabolism contains dsRNA of the 302a1 gene fragment, and there is no RNA hydrolase. The dsRNA containing the 302a1 gene fragment was enriched during the propagation and self-replication of E. coli. When the research object is too small, such as the worms in this study are small in size and cannot move, when the RNAi transfection process requires dsRNA spraying on the body surface or transfection methods that require a large amount of dsRNA such as soaking and feeding, this method can provide A large number of dsRNAs are easy to popularize and apply.

Taking the spraying experiment of ashworm as an example, conventional RNAi experiments are synthesized by kits, the amount of dsRNA synthesized in a single time is 20ul, and the concentration is 0 to 1000ug/ml, (the concentration is related to the operator's proficiency), the synthetic dsRNA can not be stored at ultra-low temperature for more than 1 year; the cost of synthesizing 1ul of dsRNA with the kit is 3 yuan to 5 yuan per ul, that is, the cost of 1ml is between 3000-5000 yuan; taking a gene as an example, an in vitro spraying experiment requires 60ml, then The synthesis of the kit is between 180,000 and 300,000 yuan, and the cost is too high. The cost of each gene experiment of single-cell cloned microorganism E. coli is between 800 and 1,500 yuan. The cloned E. coli strain, It can be stored in ultra-low temperature for 2-3 years, and can also be expanded according to the requirements of the experiment. In the later stage of ultra-low temperature storage, it can still be activated and recovered for repeated use, which greatly reduces the experimental cost.

Description of drawings

Figure 1 is a schematic diagram of the construction process of the system for obtaining a large amount of dsRNA in vitro;

Fig. 2 is the electrophoresis diagram of PCR product; wherein, a is PCR amplification product, and the band is between 250bp-500bp; M is mark; excessive disclosure;

Figure 3 shows the electrophoresis images of the L4440 plasmid before and after double digestion; among them, a1: control, circular L4440 plasmid (before double digestion); b1: linear L4440 plasmid (after double digestion); M: mark; It is circular before cutting, so the moving speed of electrophoresis is very slow, and the plasmid is linear between 2500bp-2000bp after double enzyme digestion;

Figure 4 is a picture of 302a1-L4440 recombinant plasmid transformed into E. coli competent cell HT115 (DE3) strain and cultured on SOC solid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline;

Figure 5 is the electrophoresis diagram before and after induction; wherein, m: DNA molecule Mark; a: after induction; b: before induction.

Detailed ways

The present invention will be further described in detail below in conjunction with the embodiments.

Those skilled in the art will understand that the following examples are only for illustrating the present invention and should not be construed as limiting the scope of the present invention. If no specific technology or condition is indicated in the examples, the technology or condition described in the literature in the field or the product specification is used. If the materials or equipment used are not marked with the manufacturer, they are all conventional products that can be obtained through purchase.

Example 1

A method for obtaining a large amount of double-stranded RNA ash worms of ash worms in vitro, comprising the following steps:

In step (1), the total RNA of the worm was extracted and reverse transcribed into cDNA, followed by PCR amplification. The amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively (Figure 3), and the product after double digestion was connected by T4. The enzyme was ligated overnight at 4°C to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;

In step (2), the 302a1-L4440 recombinant plasmid obtained in step (1) is transformed into the Escherichia coli competent cell HT115 (DE3) strain, and the solid medium is coated to screen a single colony to obtain the 302a1-L4440-HT115 bacterial cell;

Step (3), culturing the 302a1-L4440-HT115 bacterial cell liquid obtained in step (2) on a shaker to OD ₆₀₀ =0.38, adding IPTG for induction, and obtaining a dsRNA bacterial liquid containing the 302a1 gene fragment of the waxworm;

In step (4), the dsRNA bacterial solution obtained in step (3) containing the 302a1 gene fragment of the white wax worm is concentrated, and the bacterial solution is used for the RNA interference experiment.

Example 2

In step (1), the total RNA of the worm was extracted and reverse transcribed into cDNA, followed by PCR amplification. The amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively (Figure 3), and the product after double digestion was connected by T4. The enzyme was ligated overnight at 4°C to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;

In step (2), the 302a1-L4440 recombinant plasmid obtained in step (1) was transformed into the E. coli competent cell HT115 (DE3) strain, and the solid medium was coated to screen a single colony to obtain 302a1-L4440-HT115 bacterial cells (Fig. 4);

step (3), culturing the 302a1-L4440-HT115 bacterial cell liquid obtained in step (2) on a shaker to OD ₆₀₀ =0.42, adding IPTG for induction for 3.5 h, and obtaining a dsRNA bacterial liquid containing the 302a1 gene fragment of the white waxworm;

In step (4), the dsRNA bacterial solution obtained in step (3) containing the 302a1 gene fragment of the white wax worm is concentrated, and the bacterial solution is used for the RNA interference experiment.

In step (1), the P. elegans RNA comprises the total RNA of P. elegans 302a1 gene.

The primers that described PCR amplification adopts are ECEGT01-F and ECEGT01-R;

ECEGT01-F: 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO. 6);

ECEGT01-R: 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO. 7);

Described PCR amplification system is:

The PCR amplification program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 5 min, and storage at 4 °C.

In step (1), the condition system of enzyme cleavage is:

The digestion time was 10 min and the temperature was 37°C.

The connection system is:

After the ligation, the product was transformed into competent cells DH5ɑ, and the SOC solid medium containing 100 μg/mL ampicillin was coated and screened for culture. The single colony that grew was expanded and propagated and the plasmid was extracted to obtain the 302a1-L4440 recombinant plasmid.

The specific method of the step (2) is: transform the 302a1-L4440 recombinant plasmid obtained in the step (1) into the Escherichia coli competent cell HT115 (DE3) strain, and coat it on a liquid containing 100 μg/mL ampicillin and 50 μg/mL tetracycline. On SOC solid medium, culture in the dark for 12h, pick a single colony to obtain 302a1-L4440-HT115 cells;

The specific method of step (3) is as follows: 302a1-L4440-HT115 cells are cultured in SOC liquid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline at 37° C. for 10-12 h in the dark, and then cultured in 75 μg/mL SOC liquid medium containing 75 μg/mL tetracycline. mL ampicillin and 12.5 μg/mL tetracycline in 2×YT liquid medium were shaken and cultured to OD ₆₀₀ =0.42, and IPTG was added to induce the culture for 3 h to obtain a dsRNA bacterial solution containing the 302a1 gene fragment of the white waxworm.

In step (3), the obtained bacterial liquid was further centrifuged at 4°C to concentrate the bacterial cells.

Applications

All media involved in the experiment were purchased from Sangon Bioengineering (Shanghai) Co., Ltd., including:

SOC medium components: peptone 20.0 g; yeast powder 5.0 g; sodium chloride 0.5 g; magnesium sulfate heptahydrate 5.0 g; D-glucose 3.6 g.

SOC solid medium components: SOC medium: 34g, agar powder: 12g, distilled water: 1L

SOC liquid medium components: SOC medium: 34g, distilled water: 1L

SOC solid/liquid medium containing ampicillin: 1 ml of medium contains 100 μg of ampicillin.

SOC solid/liquid medium containing ampicillin and tetracycline: 1 ml of medium contains 100 μg of ampicillin and 50 μg of tetracycline.

2×YT medium components: yeast powder 10.0g; peptone 16.0g; sodium chloride 5.0g.

In 2×YT liquid medium containing ampicillin and tetracycline: 75 μg of ampicillin and 12.5 μg of tetracycline per ml of medium.

Note: "/" in the text means "or". The 302a1 gene can be replaced with any desired gene for interference experiments.

The invention is a method for obtaining a large amount of dsRNA containing the gene fragments of the ashworm by using single-cell cloned microorganisms in the verification of the gene function of the ashworm, which is used for the spraying and transfection of the body surface of the ashworm. Objectives of the present invention 1. To explore how to obtain a system construction method for obtaining a large amount of dsRNA containing ash worm gene fragments in vitro; 2. To reduce the cost of synthesizing dsRNA with conventional kits in RNAi experiments; 3. Once the preparation is successful, the modified and Plasmids containing the white wax worm gene are cryopreserved, and downstream RNAi experiments can be repeated. Based on these three purposes, a construction system for obtaining dsRNA in vitro was established

1 Construction of recombinant vector

The total RNA was extracted by RNA extraction kit (EZ-10Tatal RNA Minin-presps kit, Sangon Bioengineering (Shanghai) Co., Ltd.), the extraction process was referring to the kit instructions, and reverse transcription kit (PrimeScript ^™ RT) was used to extract total RNA. reagent Kit with gDNA Eraser, Takara) was reverse transcribed into cDNA. Primers ECEGT01-F: 5'-atgagctccg ctctggtataaattcatttggac-3'; ECEGT01-R: 5'-atgtcgacgcctgttgaatcattatcactccta-3' were designed according to the sequence of the 302a1 gene fragment. cDNA was used as a template for PCR amplification [Taq PCRMaster Mix (2X, with Red Dye), Sangon Bioengineering (Shanghai) Co., Ltd.], and the PCR reaction system is shown in Table 1. PCR reaction conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min, and storage at 4°C.

Table 1

Taq PCR Master Mix 25.0 μl, cDNA 3.0 μl, ECEGT01-F 10μM 2.0 μl, ECEGT01-R 10μM 2.0 μl, dH₂O 18.0 μl, total 50.0 μl.

The electrophoresis detection results of the amplified fragments were between 250-500 bp (Figure 2), as expected. According to the process of vector construction in Figure 1, the 302a1 gene fragment gel recovery product and L4440 vector were digested with SacI and SalI (NEB (Beijing) Co., Ltd.) in a water bath at 37°C for 10 min. The reaction system is shown in Table 2. The cleavage product was recovered and purified, and the cleavage product was detected by agarose gel electrophoresis (Fig. 3). Then, T4 ligase was used for ligation at 4°C overnight to obtain the 302a1-L4440 recombinant plasmid.

Table 2

SacⅠ 1.0 μl, SalⅠ 1.0 μl, NE Buffer(10x) 5.0 μl, Amplification product or L4440 4.0 μl, dH₂O 39.0 μl, total 50.0 μl.

The connection system is:

The 302a1 and L4440 plasmids were double digested and transformed into 50 μL DH5α competent cells, spread on the SOC solid medium containing ampicillin, cultured for 12 h, and a single colony was picked and cultivated in the SOC liquid medium. The primers ECEGT01-F, ECEGT01-R and bacterial solution were used for direct PCR amplification. The reaction system was shown in Table 3. PCR reaction conditions: PCR reaction conditions were pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30s, annealing at 60°C for 30s, and 72°C Extension at ℃ for 1 min, 30 cycles; extension at 72 ℃ for 5 min, and storage at 4 ℃. Sent to the company for sequencing, the results were consistent with the target fragment, and the 302a1-L4440 recombinant plasmid has been successfully constructed.

Sequence Listing SEQ ID NO.1 to SEQ ID NO.5 are the alignment of the PCR product and the 302a1 gene fragment sequence; SEQ ID NO.1 is the sequence of the PCR product sequenced with the primer ECEGT01-F as the template; SEQ ID NO.2 The sequence of the PCR product was sequenced with the primer ECEGT01-R as the template; SEQ ID NO.3~SEQ ID NO.5 is the 302a1 gene, and the transcriptome data (transcriptome data only has exons, no introns) 3 gene fragments The compositions are >CL1378.Contig1_All, >CL1378.Contig2_All, >CL1378.Contig3_All, respectively. Among them, SEQ ID NO.3 to SEQ ID NO.5 are 302a1 gene fragments, the underlined partial sequence overlaps with SEQ ID NO.1 and SEQ ID NO.2, and the PCR product belongs to the 302a1 gene fragment, that is, the expansion band is the desired target gene.

Inducible expression of 2dsRNA

The 302a1-L4440 recombinant plasmid obtained in step (1) was transformed into the Escherichia coli competent cell HT115 (DE3) strain, spread on the SOC solid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline, and cultivated in the dark 12h, pick a single colony to obtain 302a1-L4440-HT115 cells; 302a1-L4440-HT115 cells were cultured in SOC liquid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline at 37°C in the dark for 10- After 12h, the cells were shaken in 2×YT liquid medium containing 75μg/mL ampicillin and 12.5μg/mL tetracycline to OD ₆₀₀ =0.4, induced by IPTG for 4h, and the cells were collected. After induction, the bacterial liquid was extracted for total RNA detection, and the results were shown in Figure 5. The results of gel electrophoresis showed that the RNA extracted from 302a1-L4440-HT115 bacterial solution after induction had one more RNA band than that before induction, while the RNA extracted from 302a1-L4440-HT115 bacterial solution before induction did not contain this band. The dsRNA-induced expression of the 302a1 gene fragment was successful.

Among them, the bacterial solution to obtain a large amount of dsRNA containing the 302a1 gene fragment of the white wax worm is: transform the constructed 302a1-L4440 recombinant plasmid into HT115 competent cells, and spread it on the SOC solid medium containing ampicillin and tetracycline to avoid Incubate in light for 12 hours, pick single bacteria into SOC liquid medium (containing 100 μg/mL ampicillin and 50 μg/mL tetracycline), cultivate at 37°C for 10-12 hours in the dark, and then place in 2×YT liquid medium (containing 75 μg/mL ampicillin). Penicillin and 12.5 μg/mL tetracycline) were cultured to OD ₆₀₀ =0.38-0.42.

result:

The constructed Escherichia coli 302a1-L4440-HT115 was successfully cultured. After induction, dsRNA containing the target 302a1 gene fragment was obtained. The culture of the bacterial liquid can be expanded and propagated according to the needs to obtain the required dsRNA bacterial liquid.

The foregoing has shown and described the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited by the above-mentioned embodiments, and the descriptions in the above-mentioned embodiments and the description are only to illustrate the principle of the present invention. Without departing from the spirit and scope of the present invention, the present invention will have Various changes and modifications fall within the scope of the claimed invention. The claimed scope of the present invention is defined by the appended claims and their equivalents.

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acagtattag atgatggcca cagttttcaa tgcctctgat tacctatact ctgttcatat 2040

gcctgtaaat atacacttgc acttgcatgt ggagaaaata ttttgagcaa attaatacgt 2100

caaatagtat tataatatta cattttcgtt aacactcact cacagtccta caatggcagt 2160

agggtagaga tgctgattga ggaatcagta atgccgggct cgaatcctac caagggcaac 2220

gttttataaa cattttttttt gttttaagat aaatttttttt ttttaaaatg gttattagat 2280

tctctgtttt tt 2292

<210> 4

<211> 395

<212> DNA

<213> artificial sequence()

<400> 4

attcatcgaa acagactatt cctatcaatt ccaaaaaaag tcttgatagt tctggcaaaa 60

aatcttaaaa ttgagtatta gctcgaagat ccaaaaactt tttaataact tcgtcggtac 120

gcggcagata caatcttaca ttcggaattt tactaatacc tttttgaaat attttccgga 180

tacgacccca ctctgaacca ttcctcggta ttagacaccc agaatttcat aattcccgtc 240

tttcaaggct atatttttct aaaataagat gacttcttct ctgcggatat cgaccttcgc 300

atcgatacac agcttctatg tttttgggtt tgaaaaccta gacaacagta acacctggta 360

caatttcttc ctttaataac tgtctcgaat tgctg 395

<210> 5

<211> 4904

<212> DNA

<213> artificial sequence()

<400> 5

gttgaatttt taatctacgt tcgaaatttt attcagcgtg ttagtttttg cccgaagtat 60

tattggatgg ccgaaagatt ggttgatcga ttgcataatg aattaataaa actgcggttg 120

ttatgcggga cggtagataa cttaccggat tatatagtga attactcggc cagtagtgct 180

gggaatcgta atacgcagcc gtcatatcat ttatttcatt gtcacctcga tataaagtgg 240

ttacacatta cgcttttgta tcaattaatt gtaataaaac agagacttgg aactgaaaaa 300

tcttccgaag aatatgaaac ttctttgaat tttcgtgcat tagataactt cgttgagctc 360

ctgatgtcag atttaattat cattactctc aaaagatttc aacagatttc catcgacgat 420

cttgctgtta aatcgttatt tacgtgtact tgcgtacgag aaatgtggaa gctgctgcag 480

ctaagtgttg accatcttca taggcgcggt ttcatatcga atttctggca ttttgtgaac 540

cgaatttgtg ataaaattgc attaaacagt gttattcaaa cagactacaa tgattgttca 600

atgttaaaaa tgccgcaaac ctactcttct tcccatccat ctatgtttgt ggtttggttg 660

ttgaaacact taataaaatt gtggagctct ggtgaaaaac tggaaattga atccaattat 720

ttgcttttag aaaatgcaat aaaaatatgc gtaaattcgg aaccagctga atcctgtttg 780

cgtgttttcc tattttttgt ggaaaatatt ttgaattctg attgggaaat gcgaactgag 840

ccacttgtac ttctttggga gtattttaac aaaaagctta attccttgtt ttacattcct 900

ggatcgtcga ttagtggaat tgctgtcatg agcaaaagtg catcaggctt attgacgcag 960

ttgaacaata aattagaaca atttgtttta gacaaaaata tggacagttt tcatttgttt 1020

cttcgattac tgggaagaaa cttgaaaaaa tggcgtgaca attcgcgctt ttggaaccaa 1080

ttaaaaggtc gtatatactc caagttttca gcttccaagt tatcatccct gaatgagatc 1140

ggattgtatc attttgtatc gttattttta actttggctc ttaccaccga tctcaatgaa 1200

gtgagcaaga agttgcaatc cttcctcaac ttacttattg cagaaaatct tgatgaagct 1260

cgattaaaat tagtctggaa agctcatata gctttgattt tattgtttgc cgagaacaac 1320

ctagatataa gcattgtgtc gaatccatta atggatatgg tagaaacagc cgtacagcta 1380

ccaaattgct tgagtttaat gagggtattt ttggaggggt ttcaagattt gatagattgc 1440

acaaataatt tggaactggg gcagtacatt tttatcggaa actggatttc ttcctatctt 1500

tcgcaatgca atgaatcgga ttcgaatagg tttctggaga ctatatcggt attaatcaat 1560

aaactgaatt ctctaatgtc agctcagtta ccacttggtc aagacaatca attgatcctt 1620

atgtataatg cattatggga acacttggta ccgtatttga aaaaaataat gagctccgcg 1680

tcgaacaatt cttaccaggt accggatatt gctgccacac ttaccacatt atcattcaaa 1740

tcttgttttc ctctcagtac tagaaatcgg gctgtagact tactaacagt tttcgttagt 1800

cacgattcaa ttgatgtcag gttgttatgt cgttatctta gccaagtatc gcgttccatg 1860

cattcgcatt tattaaataa ttttagcatt actttgatca agggatggtt aagatgcctt 1920

attcttctat cgagcgatga agagttagaa gaggatctcg ctagatttac cacatttatc 1980

accacgttac ccgaaataca aaaactattt tcagaaacac aaatcaccct gaatctgcat 2040

tcagattatt ttgagaattt tctcatggca ttgcagttga aatactcatc acttcaagat 2100

ttccattcaa aaaataagtt tagtagccaa gtatggtcgt atttcgctaa tattgagcga 2160

tggctgttgc atttagtaaa aattcctaaa tcaataaatt tggccatgaa tatttaccga 2220

aacgttggac agatgttttt taagattccg gaaatgctgt acactaagtc tcgtccaaat 2280

agtttgctgc agtcttttttt ggatttatta cttttaccga tacaaattcg aaatcccgag 2340

tttaaactta attgtcattt aagcgatgcg ataaaaaaat actttctact tgttcatttg 2400

tggtcttttc aaattaggtt ggtgaacagt tttctctgtt caagtgtttt gaattctcgg 2460

aacgctttga attaactagt aaatcaatta ttgaaatact atgctgcgca tttttaagaa 2520

agcgagccaa aacacctcac caatacgctt ttcaaacatt aattttcctc aataatctca 2580

tcagattaca caaaaataac gacgctgttg taaaatgttt tataaaaaat gctctggaga 2640

ggatatgtga tgtcgtaatg ttctgtgaag atattagtcc ctgtaaaaaa gaggcaaaag 2700

atatattgca gtttaccttg gatttaaata ttcttcgaag tgaatcagat gttggaaatt 2760

ttttaatcga agtgttcggt aaactctgtc aagagcatct agctttcaat tcgggaaact 2820

tattcgattt gttcgaggaa attatttccg tagctccgga ttcagtggct cgatttttac 2880

cgcagtttag agaaaaaatc aaagaagttg aacataaacg aggtgttgga tttgatcgtt 2940

cgttaaggaa aggacttgaa cgaatcgagg aagcactcgg tagtatacgg aataatcaaa 3000

gaacaaatag tttctgttaa ttcagatttt caaatatgta aattatgtaa acttatgtgt 3060

cgtatatgtg agtgaaaaaa tatatgtttt taatttaagg agcgtcgttt taatttgttt 3120

tcatttttac aattcccaat acgtagttca atgctttcta tatttgtttt tgaattgttt 3180

tttctattct gttttggatt gtcatttaaa actcacgcat atttttacgt atttttttt 3240

cggatgcttt attgacaata attttcaatg atgaattttt ttcgaatttt atgatagtaa 3300

aaataatgca tgtgaagaaa ttatattgtt ggtttcctag actgaatttc tgtaccttca 3360

ccagcgagat taagccattt gatgcgatac ctggtcctaa atcactttcc ttcattagga 3420

cccttgagat ttacttgcca gtgattggca attatggatt taaaaaatta catcacgatg 3480

gattccaaaa attacagcaa ttcgggacaa tcattaaaga agaaattgta ccaggtgtta 3540

cagtcgtctt ggttttcaaa cccgaagaca tagaagctgt gtatcggtgt gaaggtcgat 3600

atccgcagag aagaagtcat cttattttag aaaaatatcg ccgtgaaaga ccggaattgt 3660

ataattctgg aggtctcata ccgacgaatg gtccagagtg gggtcgtatc cggaagatat 3720

ttcaaaaaga tattagtaaa attcagaatg tgagattgta tctgcctcgt accgacggag 3780

ttattaaaaa gtttttggac cttcgaacca ataatcagtc ttctgatttt ctgccggagc 3840

tatcaagact ctttctcgaa ttgataggat tagtctgttt cgatgaatcg ctcaactgtt 3900

tttcggaatt tgaattggaa ccaaactcgc aaacatcaaa tttgattgag gcggctgcgt 3960

gcatgaatag tgtgatttca cgtacagacg gtgggttaca actttggaaa cacttcaata 4020

ctcctgcata tcgcaaattt tccaaagctc atagatatat agaaatagtt gctcaaaaat 4080

ttttgcataa aaaattaaaa aacttggacg accggcctgt tgaatcatta tcactcctag 4140

atcaattcct taaaaatgaa aatttagatg aaaaagatgt cttaggcgcg atagttgatt 4200

ttctcctcgc tggtattgat acgatggctt atacatcgtc ttttgctctt tatcacttag 4260

ctaaaaatga agaaaagcaa gagaaaatgt ttctagaact gaataaatta atgacagcgg 4320

acgaatcaat tactgaagaa atgttacaaa aatcggttta cacgaaagct gttatgaggg 4380

aaattcatcg tatgaaccct gtatctattg gcattggtag aattttatca gaagatgcta 4440

ttctatcgaa ttatcatgtt ccagcgggga ccatggttat tacacaaaat caagtatctt 4500

gtcgactgcc agaatatttt gccagtccaa atgaatttat accagagcgt tggttacgag 4560

aatcaggaag cgaacaaata catccgtact tggttttacc tttcggacac gggccccgga 4620

cttgtattgc tcgtcgattg gcagaacaga acttacaact tttgttgatg aacattgttc 4680

acaaattcaa actgaagtgg ctgggtaacg atttggattg tgtatccgtg ctgattaata 4740

aaccggaaaa acctgttcag attcattttc agccaagaac gtaaattatc gattcaatga 4800

gtacttaacc tttttttcat ttgtactgtg gcagtgtgtc atttctttgg accaacaaat 4860

tctacactgt tgaaattttt gatgaaatct catttatagc caat 4904

<210> 6

<211> 33

<212> DNA

<213> artificial sequence()

<400> 6

atgagctccg ctctggtata aattcatttg gac 33

<210> 7

<211> 33

<212> DNA

<213> artificial sequence()

<400> 7

atgtcgacgc ctgttgaatc attatcactc cta 33

Claims (9)

1. a system construction method that obtains a large amount of ash worm dsRNA in vitro, is characterized in that, comprises the steps: Step (1), extract the total RNA of the white wax worm and reverse-transcribe it into cDNA, and then carry out PCR amplification. The amplified product and the L4440 vector plasmid were double digested with SacI and SalI respectively, and the double digested product was digested with T4 ligase overnight at 4°C connected to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment; In step (2), the 302a1-L4440 recombinant plasmid obtained in step (1) is transformed into the E. coli competent cell HT115 (DE3) strain, and the solid medium is coated to screen a single colony to obtain the 302a1-L4440-HT115 cell; step (3), culturing the 302a1-L4440-HT115 bacterial cell liquid obtained in step (2) on a shaker to OD ₆₀₀ =0.38-0.42, adding IPTG for induction, and obtaining a dsRNA bacterial liquid containing the 302a1 gene fragment of P. chinensis; In step (4), the dsRNA bacterial solution obtained in step (3) containing the 302a1 gene fragment of the white wax worm is concentrated, and the bacterial solution is used for the RNA interference experiment. 2. The system construction method for obtaining a large amount of dsRNA in vitro according to claim 1, characterized in that, the total RNA of described ash worm comprises the total RNA of the 302a1 gene of ash worm. 3. the system construction method that obtains in vitro a large amount of white wax worm dsRNA according to claim 1, is characterized in that, the primer that described PCR amplification adopts is ECEGT01-F and ECEGT01-R; ECEGT01-F: 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO. 6); ECEGT01-R: 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO. 7). 4. The system construction method for obtaining a large amount of dsRNA in vitro according to claim 1, characterized in that, in step (1), the PCR amplification system is: Taq PCR Master Mix (2X, with Red Dye) 25.0μl, cDNA 3.0 μl, ECEGT01-F 10 μM 2.0 μl, ECEGT01-R 10 μM 2.0 μl, _ddH2O 18.0 μl, Total 50.0μl; The PCR amplification program was as follows: pre-denaturation at 94 °C for 5 min; denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s, extension at 72 °C for 1 min, 30 cycles; extension at 72 °C for 5 min, and storage at 4 °C. 5. The system construction method for obtaining a large amount of dsRNA in vitro according to claim 1, characterized in that, in step (1), the conditional system for enzyme cleavage is: SacⅠ 1.0 μl, SalⅠ 1.0 μl, NE Buffer (10x) 5.0 μl, Amplification product or L4440 4.0 μl, dH ₂ O 39.0 μl, 50.0 μl total; The digestion time was 10 min and the temperature was 37°C. 6. the system construction method that obtains in vitro a large amount of ash worm dsRNA according to claim 1, it is characterised in that the connection system is: 4.0ul of amplified product after digestion, L4440 1.0ul after digestion, T4 enzyme 1.0ul, T4 Buffer 2.5ul, _H2O 11.5ul, 20.0ul in total. 7. the system construction method that obtains in vitro a large amount of white wax worm dsRNA in vitro according to claim 1, it is characterized in that, product is transformed into competent cell DH5ɑ after ligation, in the SOC solid medium coating screening containing 100 μg/mL ampicillin After culture, the single colony that grows is expanded and propagated, and the plasmid is extracted to obtain the 302a1-L4440 recombinant plasmid. 8. The system construction method for obtaining a large amount of dsRNA in vitro according to claim 1, wherein the specific method of step (2) is: transform the 302a1-L4440 recombinant plasmid obtained in step (1) into Escherichia coli to sense In the HT115 (DE3) strain of morphological cells, it was spread on SOC solid medium containing 100 μg/mL ampicillin and 50 μg/mL tetracycline, and cultured in the dark for 12 h, and a single colony was picked to obtain 302a1-L4440-HT115 cells. 9. The system construction method for obtaining a large amount of dsRNA in vitro according to claim 1, characterized in that, the specific method of step (3) is: placing 302a1-L4440-HT115 thalline in a solution containing 100 μg/mL ampicillin and 50 μg /mL tetracycline in SOC liquid medium, cultured at 37°C in the dark for 10-12h, and then shaken in 2×YT liquid medium containing 75μg/mL ampicillin and 12.5μg/mL tetracycline to OD ₆₀₀ =0.38~ 0.42, add IPTG to induce culture for 3-4 hours, and obtain a dsRNA bacterial liquid containing the 302a1 gene fragment of A. chinensis.

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