WO2021232536A1 - 一种体外大量获得白蜡虫dsRNA的体系构建方法 - Google Patents
一种体外大量获得白蜡虫dsRNA的体系构建方法 Download PDFInfo
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N57/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds
- A01N57/10—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds
- A01N57/16—Biocides, pest repellants or attractants, or plant growth regulators containing organic phosphorus compounds having phosphorus-to-oxygen bonds or phosphorus-to-sulfur bonds containing heterocyclic radicals
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N2320/00—Applications; Uses
- C12N2320/30—Special therapeutic applications
Definitions
- the invention belongs to the technical field of genetic engineering, and specifically relates to a system construction method for obtaining a large amount of white wax insect dsRNA in vitro.
- White wax worm is a scale insect with great economic value.
- the second-instar male larva of white wax worm lives on the branches of host plants and secretes white waxy covering.
- the wax secreted by this insect becomes white wax (Cerachinensis, also known as white wax). ), that is, the secretion of white wax worm, which is a Chinese specialty.
- White wax is a natural polymer compound with high melting point and chemical stability. It has the characteristics of moisture-proof, lubrication, and light. It is an important chemical raw material and is widely used in the chemical, machinery, food, pharmaceutical, cosmetic, and agricultural industries.
- RNA interference refers to the phenomenon that double-stranded RNA (dsRNA) induces homologous mRNA degradation and silences the expression of target genes in eukaryotes.
- dsRNA transfection mainly includes injection, feeding, soaking, virus infection and gene gun. However, if the research object is too small to move, the infection method of feeding, soaking, and body surface absorption may cause insufficient dsRNA prepared in the kit.
- the insects in this study are white wax worms.
- the insects are small in size and have piercing and sucking mouthparts. They grow and reproduce by absorbing the juice of the host S. sylvestris. They cannot move most of the time, and they infect them by absorption by the skin. Due to body surface absorption and large dosage, dsRNA is unstable and easily degraded in the outside world. It is necessary to spray and absorb the surface skin of white wax insects many times.
- the synthetic dsRNA yield of the kit is low (ul meter), which is not enough for spraying.
- the method of transfection and the synthesis of a large number of kits make the experiment cost expensive. Therefore, in some RNA interference experiments, how to prepare high-yield dsRNA is a problem that needs to be solved by genetic engineering.
- the present invention overcomes the shortcomings in the prior art. , To provide a system construction method for obtaining large amounts of white wax insect dsRNA in vitro, which can provide a reference for how to obtain large amounts of dsRNA at low cost.
- a method for constructing a system for obtaining a large amount of white wax insect dsRNA in vitro including the following steps:
- Step (1) extract the total RNA of white wax worm and reverse transcribed it into cDNA and then carry out PCR amplification.
- the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively, and the product was digested with T4 ligase overnight at 4°C Connect to obtain a recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
- Step (2) Transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply a solid medium to screen a single colony to obtain 302a1-L4440-HT115 bacteria;
- step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
- the total RNA of the white wax worm comprises the total RNA of the white wax worm 302a1 gene.
- the PCR amplification method is used to design primers for the 302a1 gene (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).
- the PCR amplification system is:
- the PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min, and storage at 4°C.
- step (1) the condition system of restriction enzyme digestion is:
- the digestion time is 10 minutes and the temperature is 37°C.
- connection system is:
- the product after ligation is transformed into competent cells DH5 ⁇ , and then coated and cultured on a SOC solid medium containing 100 ⁇ g/mL ampicillin.
- the single colony grown is expanded and multiplied and the plasmid is extracted to obtain 302a1-L4440 Recombinant plasmid.
- the single bacteria grow out later. Use an inoculating loop to pick a single colony into a centrifuge tube containing 100 ⁇ g/mL ampicillin in SOC liquid medium, and culture for 10 hours on a shaker. Extract the plasmid.
- step (2) transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the competent cell strain HT115 (DE3) of Escherichia coli, and coat it with 100 ⁇ g/mL ampicillin And 50 ⁇ g/mL tetracycline SOC solid medium, culture for 12h in the dark, and pick a single colony to obtain 302a1-L4440-HT115 bacteria;
- the invention provides a method for constructing a system for obtaining a large amount of dsRNA in a single-cell cloning manner, which mediates the RNAi of the white wax worm to cause the expression of the target gene to be silenced.
- This method takes the white waxworm RNAi interference as an example.
- the target gene fragment of white waxworm 302a1 is obtained by PCR using the white waxworm cDNA as a template.
- the two ends of the gene fragment are modified by restriction enzymes to form sticky ends.
- Dicer was modified to form a linear L4440 plasmid ligation to form a plasmid 302a1-L4440 recombinant plasmid containing the target gene fragment of the white waxworm.
- the 302a1-L4440 recombinant plasmid was transformed into the competent strain of Escherichia coli HT115 (DE3) to form the 302a1-L4440-HT1115 strain.
- the combined strain is resistant to both ampicillin and tetracycline, cannot synthesize RNA hydrolase, and contains the white wax insect 302a1 gene fragment Therefore, the E. coli can be cultured and propagated in a SOC liquid medium (containing ampicillin and tetracycline) in a single-cell cloning method.
- the propagation process can produce dsRNA after IPTG induction, and a large amount of dsRNA containing the white waxworm 302a1 gene segment can be obtained.
- the recombinant Escherichia coli bacteria liquid is collected, and the bacteria liquid is diluted and sprayed directly on the larval body of the white wax worm.
- the method of the present invention can clone E. coli containing the white wax insect 302a1 target gene by single-cell cloning, and through this Escherichia coli's own secondary metabolism and self-reproduction and replication, obtain a bacterial solution containing a large number of white wax insect gene fragments dsRNA, and combine the white wax insect gene fragments
- the dsRNA bacterial solution is sprayed with white wax worm larvae and enters the body from the surface of the white wax worm, which can effectively interfere with the expression of the target gene.
- RNAi transfection pathway that synthesizes a large amount of dsRNA and enters the body through the body surface provides a reference.
- the present invention has the following beneficial effects:
- the present invention is the first to verify the function of the white waxworm RNA gene by introducing a single-cell cloned microorganism Escherichia coli, so that its genetic material contains the white waxworm 302a1 gene fragment, and the secondary metabolism contains the dsRNA of the 302a1 gene fragment, and there is no RNA hydrolase. During the propagation and self-replication of Escherichia coli, the dsRNA containing the 302a1 gene fragment was enriched.
- the conventional RNAi experiment is synthesized with a kit.
- 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 proficiency of the operator).
- dsRNA can not be stored at ultra-low temperature for more than 1 year; the cost of synthesizing 1ul dsRNA with the kit is between 3 yuan and 5 yuan per ul, that is, the cost of 1ml is between 3000-5000 yuan; taking one gene as an example, an in vitro spray experiment requires 60ml, then The synthesis of the kit is between 180,000 and 300,000 yuan, which is too costly and undesirable; and the cost of each gene experiment with a single-cell cloned microbial coliform is between 800 and 1,500 yuan, cloning the modified 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 resuscitated for repeated use, which greatly reduces the cost of the experiment.
- Figure 1 is a schematic diagram of the construction process of a system for obtaining a large amount of dsRNA in vitro;
- Figure 2 is an electrophoresis diagram of PCR products; among them, a is PCR amplification product, the band is between 250bp and 500bp; M is mark; b ⁇ f different target gene control, because it does not affect the present invention, the present invention does not do this Excessive disclosure
- Figure 3 is a comparison electrophoresis diagram of 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; because the plasmid is in the double enzyme Before cutting, it is circular, so the moving speed of electrophoresis is very slow. After double digestion, the plasmid becomes linear between 2500bp and 2000bp;
- Figure 4 is a picture of 302a1-L4440 recombinant plasmid transformed into E. coli competent cell HT115(DE3) strain on SOC solid medium containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL tetracycline;
- Figure 5 is an electrophoresis diagram before and after induction; where m: DNA molecule Mark; a: after induction; b: before induction.
- a method for obtaining large amounts of white wax insect double-stranded RNA in vitro including the following steps:
- Step (1) extract the total RNA of white wax worm and reverse transcribed into cDNA and then carry out PCR amplification.
- the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively ( Figure 3), and the product after the double digestion was connected with T4 Enzyme ligation overnight at 4°C to obtain recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
- Step (2) Transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply a solid medium to screen a single colony to obtain 302a1-L4440-HT115 bacteria;
- step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
- Step (1) extract the total RNA of white wax worm and reverse transcribed into cDNA and then carry out PCR amplification.
- the amplified product and the L4440 vector plasmid were digested with SacI and SalI respectively ( Figure 3), and the product after the double digestion was connected with T4 Enzyme ligation overnight at 4°C to obtain recombinant plasmid 302a1-L4440 containing the 302a1 gene fragment;
- step (2) the 302a1-L4440 recombinant plasmid obtained in step (1) was transformed into the E. coli competent cell HT115 (DE3) strain, and solid medium was coated to screen a single colony to obtain 302a1-L4440-HT115 strain ( Figure 4);
- step (4) the dsRNA bacterial solution containing the white waxworm 302a1 gene fragment obtained in step (3) is concentrated, and the bacterial solution is used for the RNA interference experiment.
- the white waxworm RNA includes the total RNA of the white waxworm 302a1 gene.
- the primers used in the PCR amplification are ECEGT01-F and ECEGT01-R;
- ECEGT01-F 5'-atgagctccgctctggtataaattcatttggac-3' (SEQ ID NO.6);
- ECEGT01-R 5'-atgtcgacgcctgttgaatcattatcactccta-3' (SEQ ID NO.7);
- the PCR amplification system is:
- the PCR amplification procedure is: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 30 seconds, annealing at 60°C for 30 seconds, extension at 72°C for 1 min, 30 cycles; extension at 72°C for 5 min, and storage at 4°C.
- step (1) the condition system for restriction digestion is:
- the digestion time is 10 minutes and the temperature is 37°C.
- connection system is:
- the product was transformed into competent cells DH5 ⁇ , and then spread and screened on SOC solid medium containing 100 ⁇ g/mL ampicillin. The single colony grown was expanded and multiplied and the plasmid was extracted to obtain the 302a1-L4440 recombinant plasmid.
- step (2) transform the 302a1-L4440 recombinant plasmid obtained in step (1) into the E. coli competent cell HT115 (DE3) strain, and apply it to a strain containing 100 ⁇ g/mL ampicillin and 50 ⁇ g/mL tetracycline. On the SOC solid medium, culture for 12 hours in the dark, and pick a single colony to obtain 302a1-L4440-HT115 bacteria;
- Step (3) The obtained bacterial liquid is further centrifuged at 4°C to concentrate the bacterial cells.
- SOC medium components peptone 20.0g; yeast powder 5.0g; sodium chloride 0.5g; magnesium sulfate heptahydrate 5.0g; D-glucose 3.6g.
- 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 1ml of medium contains 100 ⁇ g of ampicillin.
- SOC solid/liquid medium containing ampicillin and tetracycline 1ml medium contains 100 ⁇ g of ampicillin and 50 ⁇ g of tetracycline.
- 2 ⁇ YT liquid medium containing ampicillin and tetracycline 75 ⁇ g of ampicillin and 12.5 ⁇ g of tetracycline are contained in each milliliter of medium.
- the present invention is a method for obtaining a large amount of dsRNA containing the white wax insect gene fragment by using a single-cell cloned microorganism in the gene function verification of the white wax insect, and is used for the RNAi body surface spray transfection of the white wax insect.
- the purpose of the present invention 1. To explore how to obtain a large amount of dsRNA containing white wax insect gene fragments in vitro; The plasmid containing the white wax insect gene can be stored at low temperature and can be used for downstream RNAi experiments repeatedly. Based on these three purposes, a construction system for obtaining dsRNA in vitro was established
- RNA extraction kit EZ-10 Tatal RNA Minin-presps kit, Shenggong Bioengineering (Shanghai) Co., Ltd.
- kit instructions for the extraction process and use the reverse transcription kit (PrimeScript TM RT reagent Kit with gDNA Eraser, Takara) reverse transcription into cDNA.
- ECEGT01-R 5'-atgtcgacgcctgttgaatcattatcactccta-3' were designed.
- PCR reaction system is shown in Table 1.
- PCR reaction conditions were 94°C pre-denaturation for 5 minutes; 94°C denaturation for 30 seconds, 60°C annealing for 30 seconds, 72°C extension for 1 minute, 30 cycles; 72°C extension for 5 minutes, and 4°C storage.
- Taq PCR Master Mix 25.0 ⁇ l, cDNA 3.0 ⁇ l, ECEGT01-F 10 ⁇ M 2.0 ⁇ l, ECEGT01-R 10 ⁇ M 2.0 ⁇ l, dH 2 O 18.0 ⁇ l, total 50.0 ⁇ l.
- connection system is:
- the 302a1 and L4440 plasmids were digested with double enzymes and then ligated and transformed into 50 ⁇ L DH5 ⁇ competent cells, spread on SOC solid medium containing ampicillin, cultured for 12 hours, and single colonies were picked and cultured in SOC liquid medium.
- Direct PCR amplification with primers ECEGT01-F, ECEGT01-R and bacterial liquid the reaction system is shown in Table 3.
- PCR reaction conditions are 94°C pre-denaturation 5min; 94°C denaturation 30s, 60°C annealing 30s, 72 Extend at °C for 1 min, 30 cycles; extend at 72 °C for 5 min, store at 4 °C. Sent to the company for sequencing, and the result was consistent with the target fragment.
- the 302a1-L4440 recombinant plasmid has been successfully constructed.
- SEQ ID NO.1 to SEQ ID NO.5 is the alignment of the PCR product and the sequence of the 302a1 gene fragment; wherein 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 PCR product sequence was sequenced with primer ECEGT01-R as the template; SEQ ID NO.3 ⁇ SEQ ID NO.5 are the 302a1 gene, and 3 gene fragments from transcriptome data (transcriptome data only exons, no introns) The components are >CL1378.Contig1_All, >CL1378.Contig2_All, >CL1378.Contig3_All.
- SEQ ID NO.3 to SEQ ID NO.5 are 302a1 gene fragments, the underlined part of the 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.
- RNA extracted from the 302a1-L4440-HT115 bacterial solution after induction was one more RNA band than before the induction, while the RNA extracted from the 302a1-L4440-HT115 bacterial solution before the induction did not contain this band.
- the dsRNA of the 302a1 gene fragment was successfully induced and expressed.
- a large amount of bacterial solution containing the dsRNA of the white waxworm 302a1 gene fragment is obtained by transforming the constructed 302a1-L4440 recombinant plasmid into HT115 competent cells, and spreading it on the SOC solid medium containing ampicillin and tetracycline.
- the Escherichia coli 302a1-L4440-HT115 formed into the system was successfully cultured, and dsRNA containing the target 302a1 gene fragment was obtained by induction.
- the culture of the bacterial solution can be expanded and multiplied according to the needs to obtain the required dsRNA bacterial solution.
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Abstract
Description
Taq PCR Master Mix | 25.0μl, |
cDNA | 3.0μl, |
ECEGT01-F 10μM | 2.0μl, |
ECEGT01-R 10μM | 2.0μl, |
dH 2O | 18.0μl, |
总计 | 50.0μl。 |
Sac Ⅰ | 1.0μl, |
Sal Ⅰ | 1.0μl, |
NE Buffer(10x) | 5.0μl, |
扩增产物或L4440 | 4.0μl, |
dH 2O | 39.0μl, |
总计 | 50.0μl。 |
Claims (9)
- 一种体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,包括如下步骤:步骤(1),提取白蜡虫总RNA并反转录为cDNA后进行PCR扩增,扩增产物与L4440载体质粒分别经SacI和SalI双酶切,双酶切后产物利用T4连接酶4℃过夜连接,得到含302a1基因片段的重组质粒302a1-L4440;步骤(2),将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中,涂布固体培养基筛选单菌落,得到302a1-L4440-HT115菌体;步骤(3),将步骤(2)获得的302a1-L4440-HT115菌体液体摇床培养至OD 600=0.38-0.42,加入IPTG诱导,获得含有白蜡虫302a1基因片段的dsRN A菌液;步骤(4),将步骤(3)得到含有白蜡虫302a1基因片段的dsRNA菌液,浓缩菌液,用于RNA干扰实验。
- 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,所述的白蜡虫总RNA包含中白蜡虫302a1基因的总RNA。
- 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,所述的PCR扩增采用的引物为ECEGT01-F和ECEGT01-R;ECEGT01-F:5'-atgagctccgctctggtataaattcatttggac-3'(SEQ ID NO.6);ECEGT01-R:5'-atgtcgacgcctgttgaatcattatcactccta-3'(SEQ ID NO.7)。
- 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,连接后产物转化入感受态细胞DH5ɑ中,在含100μg/mL氨苄青霉素的SOC固培养基涂布筛选培养,长出的单菌落扩大繁殖后提取质粒,即得到302a1-L4440重组质粒。
- 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,步骤(2)的具体方法是:将步骤(1)得到的302a1-L4440重组质粒转化进入大肠杆菌感受态细胞HT115(DE3)菌株中,涂布于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC固体培养基上,避光培养12h,挑取单菌落,得到302a1-L4440-HT115菌体;
- 根据权利要求1所述的体外大量获得白蜡虫dsRNA的体系构建方法,其特征在于,步骤(3)的具体方法是:将302a1-L4440-HT115菌体于含100μg/mL氨苄青霉素和50μg/mL四环素的SOC液体培养基中,37℃避光培养10-12h,然后于含75μg/mL氨苄青霉素和12.5μg/mL四环素的2×YT液体培养基中摇床培养至OD 600=0.38~0.42,加入IPTG诱导培养3~4h,获得含有白蜡虫302a1基因片段的dsRNA菌液。
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CN115011534B (zh) * | 2022-03-23 | 2023-11-03 | 山东农业大学 | 一种茎瘤固氮根瘤菌ors571的突变株、构建方法及应用 |
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