CN113717984B - Nucleic acid pesticide for resisting tobacco mosaic virus and synthesis, purification and application thereof - Google Patents
Nucleic acid pesticide for resisting tobacco mosaic virus and synthesis, purification and application thereof Download PDFInfo
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- CN113717984B CN113717984B CN202111032863.3A CN202111032863A CN113717984B CN 113717984 B CN113717984 B CN 113717984B CN 202111032863 A CN202111032863 A CN 202111032863A CN 113717984 B CN113717984 B CN 113717984B
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- mosaic virus
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Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/005—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from viruses
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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
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/60—Isolated nucleic acids
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- 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
- C12N15/63—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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/30—Nucleotides
- C12P19/34—Polynucleotides, e.g. nucleic acids, oligoribonucleotides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/14—Type of nucleic acid interfering N.A.
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
- C12N2770/00011—Details
- C12N2770/14011—Bromoviridae
- C12N2770/14022—New viral proteins or individual genes, new structural or functional aspects of known viral proteins or genes
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- C—CHEMISTRY; METALLURGY
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Abstract
The invention provides a nucleic acid pesticide for resisting tobacco mosaic virus, synthesis, purification and application thereof, wherein both ends of a TMV-cp gene respectively contain a T7 promoter, the sequence is shown as SEQ ID NO.1, a plasmid pT7B-TMV-EcoRI-EcoRI-cp adopts the TMV-cp gene, and EcoRI enzyme cutting sites are introduced at both sides of the T7 promoter to obtain the nucleic acid pesticide; adding an endonuclease EcoRI, digesting to obtain an in vitro transcribed template, adding a nucleoside triphosphate mixture containing inosinic acid, in vitro transcribing and purifying to obtain TMV-cp (I) -dsRNA; the dsRNA modified by inosinic acid is synthesized by a biological method for the first time, and the nucleic acid pesticide has stability under different temperatures and different ultraviolet conditions and has great potential for preventing and treating tobacco mosaic virus; the nucleic acid pesticide has no toxicity and harm to the environment, has good effects in cost control and application, and is expected to become an excellent biological pesticide.
Description
Technical Field
The invention belongs to the technical field of agricultural biology, and particularly relates to a nucleic acid pesticide for resisting tobacco mosaic virus, and synthesis, purification and application thereof.
Background
Tobacco is one of important cash crops in China, and has long planting history. Tobacco virus disease is one of the most common, most extensive and most serious diseases occurring on tobacco in China, and is caused by tobacco mosaic virus (Tobaccomosaicvirus, TMV) infection. TMV is a single-stranded RNA virus, is very stable and has wide hosts, and is very easy to infect tobacco and other solanaceous crops. In recent years, the occurrence of tobacco mosaic virus diseases in China is in an ascending trend year by year, and tobacco production reduction, quality reduction and even harvest failure are caused after viruses are infected, so that the safe production and development of tobacco in China are seriously threatened. Therefore, the prevention and control of tobacco virus diseases and the treatment of easily infected plants are particularly important.
RNA interference (RNAINTERFERENCE, RNAI) is one of the important biological findings in recent years, and two United states scientists that find RNAi mechanisms have therefore gained a Nobel physiology or medical prize in 2006. RNAi is a highly conserved gene silencing phenomenon induced by double-stranded RNA (double-STRANDEDRNA, DSRNA) in the evolution process and degraded by homologous mRNA with high efficiency and specificity, and the technology is widely applied to the fields of agriculture, medical gene therapy of malignant tumors, infectious diseases and the like. RNAi is used in agricultural fields, and is mainly achieved by 3 modes of host-induced gene silencing, virus-induced gene silencing and spray-induced gene silencing. The spray-induced gene silencing mode is similar to that of a traditional pesticide application method, dsRNA is directly sprayed or injected to an acting object, and the effect of silencing a target gene is achieved through pest feeding or pathogen infection, so that the spray-induced gene silencing method is convenient to apply and popularize in agricultural activities.
The dsRNA pesticide has the advantages of being applied to plant pest control: 1) The dsRNA has high specificity and targeting property, so the dsRNA is safe to non-target organisms; 2) RNA belongs to substances naturally existing in the environment or organisms, can be degraded through a natural way, and has no residual risk; 3) Compared with the existing chemical pesticides, the dsRNA is more difficult to generate drug resistance; 4) The production mode is clean production, and the production method does not influence the environment; 5) The production route is a platform technology, and the product can be developed for all kinds of harmful organisms. Therefore, compared with the traditional chemical pesticide control, the nucleic acid pesticide prepared based on the RNA interference technology has potential and good application prospect in controlling plant diseases and insect pests, and the RNA biological pesticide is considered as the third revolution in the pesticide field.
Nucleic acids are affected by a variety of complex factors such as nucleases, light, pH, etc. within and in the environment of the target organism, resulting in poor stability and thus a great deal of disruption in silencing efficiency of the target gene. To improve the stability of nucleic acids, chemical modifications are currently used to enhance the effective delivery of small nucleic acid siRNA drugs comprising 21-22 base pairs, and commonly used chemical modifications mainly include phosphate group modifications, base modifications, ribose modifications, etc., such as 2' -methoxy (2 ' -OMe), 2' -methoxyethoxy (2 ' -MOE), and 2' -F, which are frequently used in ribose modifications. Recent studies have shown that inosinic acid (also known as inosinic acid inosinc acid, abbreviated as I) can be used in siRNA sense strand (SENSE STRAND, ss) modification instead of guanylic acid, and as a result, these modifications have been shown to increase RNAi activity (HARIPRIYAADDEPALLI, 2010). Compared with chemical synthesis and modification of 21 base pair siRNA, the synthesis cost of long-chain dsRNA with hundreds of base pairs is high, chemical synthesis is difficult to realize, mass production is not facilitated, and the research on stability modification of dsRNA is less at present.
Reference is made to:
1.Haripriya Addepalli,Meena,Chang G.Peng,Gang Wang(2010).Modulation of thermal stability can enhance the potency of siRNA,Nucleic Acids Research.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a nucleic acid pesticide for resisting tobacco mosaic virus and synthesis, purification and application thereof. TMV-cp is a gene encoding a capsid protein (cp) of tobacco mosaic virus, and the capsid protein produced by encoding can wrap TMV genome to form virus particles, protect nucleic acid thereof from being destroyed, and the gene plays an important role in viral pathogenicity and infection. In addition, TMV-cp also has the functions of determining the range of virus particles in a host, affecting the onset symptoms of the host, assisting the long-distance transportation of viruses in the host and among leaves, and the like, and is one of key genes of TMV. For the above reasons, the present invention selects this gene as a silencing target gene.
The first object of the present invention is to provide a TMV-cp gene, wherein both ends of the TMV-cp gene respectively contain a T7 promoter, and the corresponding nucleotide sequence is shown as SEQ ID NO. 1.
The second object of the invention is to provide an in vitro transcription template TMV-EcoRI-EcoRI-cp, namely, the in vitro transcription template TMV-EcoRI-EcoRI-cp is obtained by respectively introducing an EcoRI enzyme cutting site at two ends of a TMV-cp gene sequence, and the nucleotide sequence of the in vitro transcription template TMV-EcoRI-EcoRI-cp is shown as SEQ ID NO. 2.
The third object of the present invention is to provide a plasmid pT7B-TMV-EcoRI-EcoRI-cp obtained by introducing EcoRI cleavage sites to the left and right of two T7 promoters, respectively, using the above TMV-cp gene.
Preferably, the primer for introducing EcoRI cleavage site on the left side of the T7 promoter: the forward primer sequence is shown as SEQ ID NO.4, and the reverse primer sequence is shown as SEQ ID NO. 5.
Preferably, the primer for introducing EcoRI cleavage site on the right side of the T7 promoter: the forward primer sequence is shown as SEQ ID NO.6, and the reverse primer sequence is shown as SEQ ID NO. 7.
The fourth object of the present invention is to provide an engineering strain, which converts E.coli DH 5. Alpha. Competent cells by using the plasmid pT7B-TMV-EcoRI-EcoRI-cp to obtain E.coli DH 5. Alpha./pT 7B-TMV-EcoRI-EcoRI-cp strain.
The fifth object of the invention is to provide a tobacco mosaic virus resistant nucleic acid pesticide TMV-cp (I) -dsRNA, the sequence of which is shown as SEQ ID NO. 3.
The sixth object of the present invention is to provide a method for synthesizing TMV-cp (I) -dsRNA as a nucleic acid pesticide against tobacco mosaic virus, wherein an endonuclease EcoRI is added into the plasmid pT7B-TMV-EcoRI-EcoRI-cp, and a Nucleoside Triphosphate (NTP) mixture containing inosinic acid is added for in vitro transcription to obtain TMV-cp (I) -dsRNA.
The seventh object of the present invention is to provide a method for purifying TMV-cp (I) -dsRNA, adding DNaseI into a transcription system, and preserving heat for 0.5-1h at 37-40 ℃ to obtain a TMV-cp (I) -dsRNA crude product; and carrying out anion exchange chromatography on the TMV-cp (I) -dsRNA crude product, and then carrying out tangential flow ultrafiltration concentration to remove ions and unreacted nucleoside triphosphate in the system, thereby obtaining the tobacco mosaic virus resistant nucleic acid pesticide TMV-cp (I) -dsRNA.
The eighth object of the invention is to provide an application of the anti-tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA in preventing and treating tobacco mosaic virus.
By adopting the scheme, the invention has the beneficial effects that:
The invention synthesizes the dsRNA modified by inosinic acid by a biological method for the first time, and the nucleic acid pesticide TMV-cp (I) -dsRNA has stability under different temperatures and different ultraviolet conditions, so that the dsRNA has great potential for preventing and treating tobacco mosaic virus; in addition, the pesticide is nontoxic and harmless to the environment, has good effects in cost control and application, and is expected to be an excellent biological pesticide.
Drawings
FIG. 1 is a schematic diagram showing the insertion of restriction enzyme EcoRI cleavage sites at both ends of the TMV-cp sequence in example 1 of the present invention.
FIG. 2 is an electrophoresis chart of PCR products with EcoRI cleavage sites added to the left side T7 promoter in example 1 of the present invention.
FIG. 3 is a diagram showing EcoRI cleavage of pT7B-TMV-EcoRI-cp plasmid in example 1 of the present invention (M: 5000Marker; lane 1: after cleavage of plasmid pT 7B-TMV-EcoRI-cp; lane 2: before cleavage of plasmid pT 7B-TMV-EcoRI-cp).
FIG. 4 is a diagram showing the EcoRI cleavage of pT7B-TMV-EcoRI-EcoRI-cp plasmid of example 1 of the present invention (M: 5000Marker; lane 1: before cleavage of plasmid pT 7B-TMV-EcoRI-EcoRI-cp; lane 2: after cleavage of plasmid pT 7B-TMV-EcoRI-EcoRI-cp).
FIG. 5 is a graph showing in vitro transcription electrophoresis detection of TMV-cp (I) -dsRNA in example 5 of the present invention.
FIG. 6 is a graph showing comparison of the stability of TMV-cp (I) -dsRNA and TMV-cp-dsRNA at different temperatures in example 7 of the present invention (note: performing analysis of variance using IBMSPSSstatistics19, performing significance analysis using Duncan's new complex polar error method, data in the table are mean.+ -. Standard error, and different lower case letters indicate significant differences at 0.05 level).
FIG. 7 is a graph showing the stability of TMV-cp (I) -dsRNA and TMV-cp-dsRNA of example 8 of the present invention under UV exposure conditions at different times (note: performing analysis of variance using IBMSPSSstatistics, performing significance analysis using Duncan's new complex polar error method, wherein the data in the table are mean.+ -. Standard error, and the difference at the 0.05 level is significant with different lower case letters).
FIG. 8 is a graph showing the stability of TMV-cp (I) -dsRNA of example 9 of the present invention under different temperature conditions.
FIG. 9 is a graph showing the variation of TMV-cp (I) -dsRNA content at various times of UV exposure in example 10 of the present invention (note: analysis of variance using IBMSPSSstatistics, analysis of significance using Duncan's new complex pole difference method, data in the table are mean.+ -. Standard error, and different lower case letters indicate significant difference at 0.05 level).
FIG. 10 is a graph showing changes in the target fragments of TMV-cp (I) -dsRNA at different times of UV exposure in example 10 of the present invention (note: lanes 1-8 represent samples irradiated with UV for 0h, 0.5h, 2h, 4h, 28h, 3d, 5d, 7d, respectively).
FIG. 11 is a graph showing the prevention and treatment of tobacco mosaic virus by TMV-cp (I) -dsRNA and TMV-cp-dsRNA in example 11 of the present invention (note: 1 is TMV-cp (I) -dsRNA treatment group; 2 is TMV treatment group; 3 is TMV-cp-dsRNA treatment group).
Detailed Description
The invention provides a nucleic acid pesticide for resisting tobacco mosaic virus, and synthesis, purification and application thereof.
Since dsRNA molecules are unstable in the environment and are easy to be degraded by microorganisms, illumination, environmental pH, nuclease and other factors, the improvement of the stability of the dsRNA has important significance for improving the gene silencing efficiency. The invention aims to refer to a method for improving the stability of small interfering RNA (SMALL INTERFERING RNA, SIRNA,21-23 nt), and modifies or replaces the base of dsRNA so as to improve the stability of the dsRNA and make the dsRNA more suitable for field spraying. The base modification of nucleic acid is mainly siRNA, but the report of base modification of long-chain dsRNA is less; and at present, the siRNA modification mainly adopts a chemical synthesis method, and for dsRNA, the synthesis cost is high, so that the large-scale production is not facilitated. In the invention, inosinic acid (inosine, abbreviated as I) is introduced into dsRNA in an in vitro transcription mode to replace guanylic acid, and the base modification doping for reducing double-strand pairing stability can improve RNAi activity and finally shows that the silencing efficiency of a target sequence is higher.
The synthesis of dsRNA in the invention adopts a cell-free in vitro transcription technology, and combines purification operation to form a complete integrated technology. The process mainly comprises the self-developed and produced high-activity tool enzymes such as T7RNA polymerase (or mutants thereof), DNaseI, proteinase K and the like. The specific process is as follows: 1) Constructing a plasmid containing a target gene dsDNA, wherein both sides of the gene respectively contain a T7 promoter, and the left side of the left T7 promoter and the right side of the right T7 promoter are respectively introduced with an EcoRI restriction site; 2) High-density fermentation of recombinant strains; 3) Large-scale extraction and purification of plasmids; 4) Linearizing the plasmid to obtain a template for in vitro transcription; 5) Purifying an in vitro transcription template; 6) In vitro transcription is performed by adding substrate NTP, wherein Guanosine Triphosphate (GTP) is replaced with Inosine Triphosphate (ITP): adding T7RNA polymerase (or mutant), NTP, reaction buffer solution and the like into a reaction kettle, and preserving heat for 4-6 hours at 37 ℃ to obtain a dsRNA (I) transcription product containing IMP (inosine monophosphate); 7) Purification of dsRNA: adding DNaseI elimination template, and performing anion exchange chromatography and tangential flow filtration to obtain a large amount of high-purity dsRNA (I).
The invention is further illustrated by the following examples.
Example 1: construction of recombinant engineering Strain E.coli DH 5. Alpha./pT 7B-TMV-EcoRI-EcoRI-cp
1. The construction idea is as follows: the plasmid pT7B-TMV-cp preserved by the company is taken as a template (the cp gene sequence containing the T7 promoter is shown as SEQ ID NO. 1), ecoRI enzyme cutting sites (shown as figure 1) are respectively introduced at two sides of the T7 promoter at two ends of the target gene in a site-directed mutation mode, and sequencing verification is carried out after two mutations, so that the target plasmid pT7B-TMV-EcoRI-EcoRI-cp is obtained. The plasmid is transformed into E.coli DH5 alpha competent cells to obtain recombinant engineering strains E.coli DH5 alpha/pT 7B-TMV-EcoRI-EcoRI-cp. The cp gene sequence of EcoRI inserted into both ends of T7 promoter is shown as SEQ ID NO.2.
SEQ ID NO.1: TMV-cp gene sequence containing T7 promoter (lower case underlined italics T7 promoter)
SEQ ID NO.2: the T7 promoter is inserted with EcoRI TMV-cp gene sequence (uppercase underlined italics EcoRI cleavage site) at both ends
2. Primer design and Synthesis
(1) According to the plasmid pT7B-TMV-cp sequence constructed and deposited by the company, ecoRI recognition sequences (5 '. Cndot.G. AATTC. Cndot.3') were inserted into the left side of the left side T7 promoter (LPT 7) and the right side of the right side T7 promoter (RPT 7), respectively, and the TMV-cp target gene sequence was analyzed on line using NEBriter V2.0 (http:// nc2.Neb. Com/NEButter 2 /).
(2) According to the specification of the Biyundian QuickMutation TM gene site-directed mutagenesis kit, two pairs of primers are respectively designed by utilizing PRIMER PREMIER 5.0.0 software, and the primers are evaluated by utilizing DNAMAN.
(3) The primers designed above were synthesized by Beijing Liuhua macrogene technologies, inc., and the sequences of the primers are shown in Table 1 below:
TABLE 1 primers used to introduce EcoRI cleavage sites on the left and right sides of the T7 promoter
3. Site-directed mutagenesis was inserted into the first EcoRI cleavage site
The pT7B-TMV-cp plasmid was used as a template, and the LPT7-F and LPT7-R primers in Table 1 were used for the first amplification, and the reaction system and the amplification conditions were as shown in Table 2 below:
TABLE 2 PCR amplification System (50 μl)
| Component (A) | Final concentration | Volume of |
| BeyoFusionTMDNA Polymerase | 1/50 | 1μl |
| Primer 1 (10. Mu.M) | 0.4μM | 2μl |
| Primer 2 (10. Mu.M) | 0.4μM | 2μl |
| 10*BeyoFusionTMBuffer(with Mg2+) | 1* | 5μl |
| dNTP mix(2.5mM each) | 0.25mM each | 5μl |
| Plasmid template | 200ng | 2μl |
| ddH2O | —— | 33μl |
| Totalizing | —— | 50μl |
Preparing a PCR reaction mixture according to the table, uniformly mixing, and performing PCR amplification by adopting a two-step method according to the following conditions: pre-denaturation at 95℃for 3min, denaturation at 98℃for 10s, annealing at 68℃for 4min, total 20 cycles, extension at 72℃for 10min, and finally preservation at 8 ℃.
After the PCR was completed, 2. Mu.l of the amplified product was subjected to electrophoresis on a 1.0% agarose gel, and observed by a gel imager to successfully amplify the amplified product to obtain a target fragment, and the result is shown in FIG. 2.
After 1 μl of DpnI endonuclease is added into the obtained PCR product system and mixed uniformly, the template plasmid is digested off by incubation for 15min at 37 ℃, and the product is converted, and the specific operation is as follows:
(1) Dissolving 1 E.coli DH5 alpha competent cell in ice, adding 10 μl of the product into the competent cell, gently mixing, and standing on ice for 30min;
(2) Heat-beating in a water bath at 42 ℃ for 45-60s, and then rapidly standing on ice for 5min;
(3) Adding 900 μl of LB culture medium (without antibody) into the centrifuge tube, mixing, and culturing at 37deg.C and 200rpm for 40-60min;
(4) Centrifuging the cultured bacterial liquid at a low speed for 1min under the condition of 2000rpm, removing 850 μl of culture medium supernatant, and blowing and sucking to mix uniformly;
(5) The bacterial liquid is evenly spread on an Amp-resistant LB solid medium, and is cultured in an inverted way at 37 ℃ for overnight. To increase the efficiency of obtaining positive clones, 2-3 shoots may be simultaneously transformed.
(6) Several monoclonal were selected on the plate, liquid cultured at 37℃and the extracted plasmid was verified by EcoRI digestion, and plasmid extraction was performed with reference to the plasmid miniextraction kit (upgrade version centrifugation column) from Shanghai JieJieR bioengineering Co., ltd., cat No. GK2004.
(7) Through preliminary verification, the plasmids corresponding to the two clones obtained are sent to Beijing Liuhua large gene technology Co., ltd for sequencing. Both positive clones were confirmed by sequencing verification, and thus far an EcoRI endonuclease site was successfully introduced to the left of the left T7 promoter, and the plasmid was named pT7B-TMV-EcoRI-cp. As shown in fig. 3.
4. Site-directed mutagenesis was inserted into the second EcoRI cleavage site
A second amplification was performed using the positive plasmid pT7B-TMV-EcoRI-cp obtained above as a template and the RPT7-F and RPT7-R primers in Table 1, and the same procedure was followed to obtain a double insertion mutant plasmid designated pT7B-TMV-EcoRI-EcoRI-cp as shown in FIG. 4. The corresponding strain was E.coli DH 5. Alpha./pT 7B-TMV-EcoRI-EcoRI-cp.
Example 2: high-density fermentation of engineering strain E.coli DH5 alpha/pT 7B-TMV-EcoRI-EcoRI-cp
1. Strain activation: glycerol tubes stored at-80 ℃ were thawed on ice, streaked onto Amp-resistant plates with an inoculating loop, and incubated in an incubator at 37 ℃ inverted overnight.
2. Seed liquid preparation: one loop of the activated strain was inoculated into LB liquid medium containing Amp antibiotics (Amp final concentration 100. Mu.g/mL), and the seed solution was filled at 200mL/500mL. The culture was carried out at 37℃and 200rpm for 15-16 hours with shaking to give a seed solution OD 600. Apprxeq.3.0.
3. Inoculating a fermentation tank: the fermenter used in this example was 50L, and the components and contents of the fermentation medium are shown in Table 3 below.
TABLE 3 fermentation Medium Components and content
| Name of the name | Proportioning (g/L) |
| KH2PO4 | 10.0 |
| Citric acid monohydrate | 3.55 |
| (NH4)2SO4 | 2.0 |
| MgSO4·7H2O | 2.5 |
| Glucose | 30 |
| Microelement mother liquor | 1mL |
Wherein, the microelement mother liquor formula is 0.1g/L of CoCl 2·6H2 O, 0.1g/L of CuSO 4·5H2 O, 5g/L of FeSO 4·7H2 O, 0.33g/L of MnSO 4·H2 O and 3.8g/L of ZnSO 4·7H2 O, and the microelement mother liquor is filtered, sterilized and stored at 4 ℃ after being prepared, and added each time of use.
The initial liquid loading amount of the fermentation tank is 60%, and the seed liquid is transferred in 10% of inoculation amount. In the fermentation process, the tank pressure is controlled to be 0.1MPa, the air quantity is 1vvm, the pH value is regulated to be 7.0 by ammonia water, and DO (dissolved oxygen) is controlled to be more than 20% by regulating the fermentation rotating speed. And adding sugar in a fed-batch manner after 8 hours of fermentation, gradually increasing the sugar supplementing rate along with the increase of the bacterial concentration (OD 600), and controlling the concentration of residual sugar below 0.3g/L in the whole fermentation process. And fermenting for about 24 hours, and placing the detected thalli OD 600 in a tank with more than 120.
4. And (3) collecting and preserving thalli: at room temperature, the thalli are collected by centrifugation with a Hitachi floor type high-speed centrifuge, the centrifugation condition is 8000rpm and 10min, and the thalli content is 18g/L through calculation. The obtained cells were stored at-20℃in a frozen state.
Example 3: large-scale extraction of plasmid pT7B-TMV-EcoRI-EcoRI-cp (alkaline lysis method)
1. Bacterial heavy suspension: the cells collected in example 2 were taken out and thawed at room temperature, 100L of Tris-HCl buffer (containing 10mmol/L of EDTA, pH=8.0) buffer (50 mmol/L) was added to the stirred tank to resuspend the cells at a rotation speed of 150rpm, and the cells were stirred for about 1 hour until the cells were sufficiently resuspended and the cells were free of caking.
2. Cell lysis: adding NaOH with the same volume of 0.2mol/L and SDS alkali lysate with the concentration of 1.0 percent, and slowly stirring for about 10 minutes to fully crack the thalli.
3. And (3) neutralization: 100L of KAc (pH=5.2) neutralization solution (3 mol/L) was added to the above lysate, and the mixture was gently stirred for 30 minutes.
4. Collecting supernatant: the neutralized product was collected by centrifugation at RT, 10000rpm for 15min, and the pellet was discarded.
5. Further purification of the supernatant: the supernatant obtained in step 4 was filtered through membranes having pore diameters of 10 μm,5 μm and 0.45 μm, respectively, and the filtrate was collected.
6. Concentration of plasmid-containing supernatant: referring to patent CN202110478812.7 (a method and application suitable for large scale extraction of dsRNA) the supernatant volume was concentrated by about 10-fold using tangential flow with a membrane pore size of 500kDa, controlling the flow rate to 60% of full speed.
7. Cellulose adsorption and plasmid solution acquisition: the cellulose filter cake is prepared in advance by referring to patent CN202110478812.7, the plasmid supernatant in the above steps and the incubated cellulose filter cake are fully stirred and mixed uniformly, and the mixture is centrifuged at a low temperature of 9000rpm for 20min at 4 ℃, and the supernatant is discarded to obtain cellulose sediment for adsorbing plasmid DNA. After drying and re-dissolution in buffer, positive pressure filtration was performed to collect plasmid filtrate.
8. Re-concentration of plasmid-containing supernatant: further tangential flow concentration, 300kDa membrane pore size, 80% of full speed flow rate, concentrated the supernatant volume approximately 10-fold.
9. And (3) filtering and sterilizing: the plasmid filtrate was filtered through 0.45 μm and 0.22 μm, respectively, to obtain plasmid pT7B-TMV-EcoRI-EcoRI-cp, the plasmid concentration was controlled at 1500 ng/. Mu.l.
Example 4: linearization of plasmids
According to the total amount m mu g of the plasmid pT7B-TMV-EcoRI-EcoRI-cp, adding m mu l of highly active restriction enzyme EcoRI which is independently developed and produced by company, incubating for 2 hours at 37 ℃, obtaining TMV-cp double-stranded DNA fragments containing T7 promoters at the 5 'and 3' ends, namely an in vitro transcription template.
The in vitro transcription template is taken for tangential flow concentration, the aperture of a filter membrane is 100kDa, the flow speed is 80% of the full speed, and the supernatant volume is concentrated to obtain the template.
Example 5: preparation of TMV-cp (I) -dsRNA by In Vitro Transcription (IVT)
1. In vitro transcription System formulation (1L): 10×T7 Buffer, T7RNA polymerase, NTP (ATP/UTP/CTP/ITP), ddH 2 O and the template obtained in example 4 were added sequentially with reference to the system in Table 4 below. Wherein the final concentration of the in vitro transcription template is controlled to be 50 ng/. Mu.l.
TABLE 4 in vitro transcription System and transcription conditions
| Component (A) | Template/TMV-cp | NTP | T7 RNA polymerase | 10*T7 BTffer | Water and its preparation method |
| Final concentration | 50ng/μl | 3MM each | 0.05～0.1mg/mL | 1*T7 BTffer | / |
| Concentration of | C1 (Unit ng/. Mu.l) | 100mM | C2(mg/mL) | / | / |
| Volume/L | 50/C | 0.03L each | (0.05～0.1)/C2 | 0.1 | upto1L |
| Feeding sequence | 5 | 3 | 2 | 1 | 4 |
2. In vitro transcription of inosinic acid (I) -containing TMV-cp (I) -dsRNA: after the system is prepared, the mixture is stirred uniformly at 100rpm, and the mixture is kept at 37 ℃ for 4.5 hours, so that the dsRNA containing I (SEQ ID NO. 3) is obtained, and a transcription product is named TMV-cp (I) -dsRNA. Samples were taken to detect dsRNA concentration and run the electrophoresis as shown in figure 5.
SEQ ID NO.3: TMV-cp (I) -dsRNA single-stranded sequence (direction 5 '-3'), together with its complementary counterpart, constitutes dsRNA:
UCUUACAIUAUCACUACUCCAUCUCAIUUCIUIUUCUUIUCAUCAICIUIIICCIACCCAAUAIAIUUAAUUAAUUUAUIUACUAAUICCUUAIIAAAUCAIUUUCAAACACAACAAICUCIAACUIUCIUUCAAAIACAIUUCAIUIAIIUIUIIAAACCUUCACCACAAIUAACUIUUAIIUUCCCUIACAIUIACUUUAAIIUIUAUAIIUAUAAUICIIUACUAIAUCCICUAIUCACAICAUUIUUAIIUICAUUUIACACUAIAAAUAIAAUAAUAIAAIUUIAAAAUCAIICIAACCCCACIACUICCIAAACIUUAIACICUACUCIUAIAIUAIACIACICAACIIUIICCAUAAIIAICICUAUAAAUAAUUUAIUAIUAIAAUUIAUCAIAIIAACCIIAUCUUAUAAUCIIAICUCUUUCIAIAICUCUUCUIIUUUIIUUUIIACCUCUIIUCCUICAACUUI.
example 6: purification and lyophilization of TMV-cp (I) -dsRNA
After the transcription of example 5 above was completed, 1U of DNaseI was added to the transcription reaction system per 0.5. Mu.g of template DNA, and an appropriate amount of DNaseI, which was independently developed and produced by the present company, was added, and the template was digested by heat-preserving at 37℃for 1 hour to obtain crude TMV-cp (I) -dsRNA.
The sample TMV-cp (I) -dsRNA was subjected to anion exchange chromatography using a CaptoQImpRes strong anionic filler of Cytiva. And then carrying out tangential flow ultrafiltration concentration, and simultaneously removing ions, unreacted NTP and other substances in the system to obtain the TMV-cp (I) -dsRNA pure product. The obtained pure dsRNA is placed in a clean stainless steel sample tray, the liquid level is not more than 1cm, and the dsRNA is pre-frozen for 1.5 hours at the temperature of minus 80 ℃. And (3) placing the pre-frozen sample into a low-temperature freeze dryer, vacuumizing and performing sectional freeze drying to obtain TMV-cp (I) -dsRNA solid with purity of more than 90%.
Example 7: comparison of stability of TMV-cp (I) -dsRNA and TMV-cp-dsRNA under different temperature conditions
The same process is adopted to prepare I modified TMV-cp (I) -dsRNA and TMV-cp-dsRNA freeze-dried powder before modification, two samples are crushed and packaged into ampere bottles, and 54 ℃ heat storage is respectively carried out for 2 weeks and 35 ℃ heat storage is carried out for 12 weeks in a constant temperature incubator. After the heat storage is finished, 0.1g of a sample is accurately weighed by a ten-thousandth balance, the volume is fixed to 100mL by pure water, the content of nucleic acid is detected by a Nanodrop ultra-micro spectrophotometer (OD 260/OD280 =1.9-2.1), each treatment is carried out for 5 times, each repetition is measured for 3 times in parallel, an average value is taken, and the stability of the two samples at two temperatures is compared. The initial content of TMV-cp (I) -dsRNA and TMV-cp-dsRNA is 64.35% and 63.13% respectively.
FIG. 6 shows that the modified TMV-cp (I) -dsRNA lyophilized powder showed no significant change in nucleic acid content and no significant difference after storage at 54℃for 2 weeks and 35℃for 12 weeks. And the content of the unmodified TMV-cp-dsRNA freeze-dried powder is reduced after the TMV-cp-dsRNA freeze-dried powder is stored at 54 ℃ and 35 ℃, wherein the content of a 54 ℃ treatment group is reduced by 6.4% compared with the initial value, and the content of a 35 ℃ treatment group is reduced by 18.2% compared with the initial value. The preparation and storage of TMV-cp (I) -dsRNA and TMV-cp-dsRNA freeze-dried powder are not carried out in an environment without ribozyme, and the modified TMV-cp (I) -dsRNA freeze-dried powder is stable than the unmodified TMV-cp-dsRNA freeze-dried powder under the experimental condition.
Example 8: stability of TMV-cp (I) -dsRNA under UV exposure conditions at different times
Crushing TMV-cp (I) -dsRNA and TMV-cp-dsRNA freeze-dried powder, spreading the powder in a disposable plastic culture dish with the diameter of 35mm, placing the powder at a position which is 30cm below an ultraviolet lamp in an open mode, respectively irradiating the powder for 0h, 24h and 72h under the ultraviolet lamp, repeating each treatment for 3 times, measuring the nucleic acid content of different treatments by adopting a Nanodrop ultramicro spectrophotometer, and calculating the nucleic acid relative content of different times of ultraviolet irradiation.
As can be seen from fig. 7, the nucleic acid content of the TMV-cp (I) -dsRNA lyophilized powder was not significantly changed after 24h and 72h of uv irradiation, and the difference between the different treatments was not significant. The content of the unmodified TMV-cp-dsRNA freeze-dried powder is reduced after 24 hours and 72 hours of ultraviolet irradiation, and the content of the ultraviolet irradiation 24 hours and 72 hours of treatment groups is respectively reduced by 2.9 percent and 8.1 percent compared with the initial value. Because the preparation and storage of TMV-cp (I) -dsRNA and TMV-cp-dsRNA freeze-dried powder are not performed in a ribozyme-free environment, the modified TMV-cp (I) -dsRNA freeze-dried powder is more stable than the unmodified TMV-cp-dsRNA freeze-dried powder under the experimental condition.
Example 9: determination of stability of TMV-cp (I) -dsRNA under different temperature conditions
Crushing a TMV-cp (I) -dsRNA freeze-dried powder sample, packaging the crushed sample into an ampere bottle, and preserving the sample at constant temperature under different temperature conditions, wherein the specific design is as follows:
1. storing at 54+ -2deg.C for 2 weeks
2. (50+ -2) Deg.C for 4 weeks
3. (45+ -2) Deg.C for 6 weeks
4. Storing at 40+ -2deg.C for 8 weeks
5. Storing at 35+ -2deg.C for 12 weeks
Each treatment was repeated 5 times, 0.1000g of the sample was accurately weighed to 100ml with pure water using a ten-thousandth balance, and the content of nucleic acid was detected using a Nanodrop ultra-micro spectrophotometer. The effective content of TMV-cp (I) -dsRNA sample is 63.81% of the initial mass fraction of TMV-cp (I) -dsRNA. In the embodiment, excel software is adopted for data statistics, IBMSPSSstatistics is adopted for analysis of variance, and Duncan's new complex polar error method is adopted for significance analysis.
As can be seen from FIG. 8, TMV-cp (I) -dsRNA in this example is stable under different temperature conditions, and the content of TMV-cp (I) -dsRNA hardly changes under the conditions of storage at 54 ℃ for 2 weeks, storage at 50 ℃ for 4 weeks, storage at 45 ℃ for 6 weeks, storage at 40 ℃ for 8 weeks and storage at 35 ℃ for 12 weeks, and after statistical analysis, the content of TMV-cp (I) -dsRNA is reduced by 2.5% the highest after storage at 35 ℃ for 84d (12 weeks) without significant difference from the initial content.
Example 10 determination of stability of TMV-cp (I) -dsRNA under UV exposure conditions at various times
The TMV-cp (I) -dsRNA freeze-dried powder is tiled in a disposable plastic culture dish with the diameter of 35mm, the freeze-dried powder is placed at a position of 30cm below an ultraviolet lamp in an opening way, 0h, 0.5h, 2h, 4h, 28h, 3d, 5d and 7d are respectively irradiated under the ultraviolet lamp, each treatment is repeated for 3 times, the change of the nucleic acid content of different treatments is measured by adopting a Nanodrop ultramicro spectrophotometer, the content of the freeze-dried powder TMV-cp (I) -dsRNA before the irradiation of the ultraviolet lamp is 100%, and the change of the content of the TMV-cp (I) -dsRNA after different times of ultraviolet irradiation is calculated. The condition of the target strip is detected by electrophoresis using agarose gel, the sample is diluted 20 times and loaded with 5 mu L, see FIG. 10.
As shown in fig. 9, the effective content of TMV-cp (I) -dsRNA after irradiation of TMV-cp (I) -dsRNA lyophilized powder under uv lamp for 0h, 0.5h, 2h, 4h, 28h, 3d, 5d, 7d was not greatly changed from that before irradiation of uv lamp, and the target bands were all clear as verified by agarose electrophoresis detection (fig. 10). Therefore, under the experimental condition, the TMV-cp (I) -dsRNA freeze-dried powder is stable under the condition of ultraviolet irradiation, and no obvious degradation phenomenon occurs.
Example 11: indoor bioassay effect of tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA
The experiments of this example were carried out in the artificial climate chamber of the company at 10-11 months 2020. Soaking 105 seeds of Yunyan 105 in 65 ℃ warm soup for 15min, soaking in 10% trisodium phosphate solution for 20min, sowing in a matrix mixed by Ping nutrient soil and vermiculite, transplanting two true leaves into a nutrition pot with the same matrix after the two true leaves are unfolded, and selecting seedlings with consistent growth vigor and size to start experimental treatment when the tobacco grows to 4-5 true leaves.
The experimental design is as follows: 1. comparing the clear water with CK; tmv: inoculating only tobacco mosaic virus; 3. tobacco mosaic virus nucleic acid pesticide TMV-cp-dsRNA4.47 mug/mul aqueous solution foliar uniform spraying treatment group; 4. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA4.47 mug/mul aqueous solution foliar uniform spraying treatment group; 5. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA2.24 mug/mul aqueous solution foliar uniform spraying treatment group; 6. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA1.12 mug/mul aqueous solution foliar uniform spraying treatment group; 7. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA0.56 mug/mul aqueous solution foliar uniform spraying treatment group; 8. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA0.28 mug/mul aqueous solution foliar uniform spraying treatment group; 9. tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA0.14 mug/mul aqueous solution foliar uniform spray treatment group.
Other water and fertilizer and agronomic management remained consistent between different treatments, 32 plants per treatment, four replicates. And inoculating TMV virus by adopting an artificial juice friction method after experimental treatment for 24 hours. The clear water control was not inoculated. Each tobacco is inoculated with one leaf.
TMV virus inoculation liquid preparation: tender leaves with significant disease on virus host K326 were cut, 4uTMV disease leaves were placed in a sterilized mortar, and 100mL of 0.01mol/L phosphate buffer (ph=7.0) was added and ground to prepare an inoculum with w=4%.
The requirements of UB23222-2008 'tobacco plant diseases and insect pests grading and investigation method' are adopted to investigate the plant grade by taking the plant as a unit, and the control effect is counted. The specific requirements are as follows:
disease severity classification:
level 0: the whole plant is free from diseases;
stage 1: heart She Maiming or slight flowers and leaves, and the disease plant is not obviously dwarfed;
3 stages: one third of leaves are not deformed, or the sick plant is dwarfed to be more than three fourths of the normal plant height;
5 stages: one third to one half of the leaves of the leaf flowers, or a few leaves deform, or the main vein is blackened, or the diseased plant is dwarfed to be three-thirds of the value of the normal plant height;
7 stages: one half to two thirds of leaf flowers and leaves, or deformation or main vein necrosis, or dwarfing of a diseased plant to one half to two thirds of the normal plant height;
Stage 9: the whole plant leaves and flowers are severely deformed or necrotized, or the diseased plant is dwarfed to be more than one half of the normal plant height.
The calculation method comprises the following steps:
incidence (%) = (number of diseased plants/total number of investigated plants) ×100%
Disease index (%) = [ Σ (number of disease plants or leaves at each stage×the disease grade value)/investigation total number of plants or leaves×the highest grade value) ] ×100% relative prevention effect (%) = [ control disease index-treatment disease index ]/control disease index ]
During growth, growth vigor was observed, the experiment was conducted for 14 days to investigate the disease level, excel software was used for data statistics, IBMSPSSstatistics was used for analysis of variance, and Duncan's new complex polar difference method was used for significance analysis.
In the embodiment, 105 seeds of Yunyan 105 are purchased from Yuxi tobacco seed Limited liability company, pinshi nutrient soil and vermiculite are purchased from Shanghai and Ying gardening Limited company, trisodium phosphate, sodium dihydrogen phosphate and disodium hydrogen phosphate are reagent grade and are purchased from Taitan scientific and technological exploration platform. TMV Virus Strain: purchased from american type culture collection (ATTC), ATCC accession number: PVAS-822 TM. In the experiment, a K326 tobacco mosaic virus system host is adopted for living parasitic seed reproduction.
TABLE 5 tobacco mosaic Virus nucleic acid pesticide TMV-cp-dsRNA indoor bioassay Effect
Note that: the analysis of variance was performed using IBMSPSSstatistics and the significance was performed using Duncan's new complex polar error method, the data in the table were mean.+ -. Standard error, and the difference was significant at the 0.05 level as indicated by the different lower case letters.
From the table above, it can be seen that the tobacco mosaic virus nucleic acid pesticides TMV-cp-dsRNA and TMV-cp (I) -dsRNA both have obvious control effects on TMV. Tobacco is sprayed on leaf surfaces at the same concentration (4.47 mug/mul) by diluting the TMV-cp-dsRNA and the TMV-cp (I) -dsRNA freeze-dried powder, the control effect of the TMV-cp-dsRNA on tobacco mosaic virus is 67.25 percent slightly worse than the control effect 80.39 percent of the TMV-cp (I) -dsRNA treatment group, and the reason for the improved stability of the modified TMV-cp (I) -dsRNA in the environment is possible. In the embodiment, the application concentration of the tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA from 0.14 mu g/mu l to 4.47 mu g/mu l shows obvious control effect on tobacco mosaic virus diseases, and along with the increase of the application concentration, the control effect is enhanced, and the highest relative control effect reaches 80.39%. From the table, the clear water control group has no disease symptoms, which indicates that the tobacco seeds are sterilized and the influence of seed toxicity is eliminated. In addition, experiments show that the tobacco growth vigor of the TMV-cp (I) -dsRNA and TMV-cp-dsRNA foliar spray groups is better than that of the virus-only treatment group (figure 11), and the TMV-cp (I) -dsRNA and TMV-cp-dsRNA can not only interfere TMV, but also improve the tobacco immunity and promote the growth and the cultivation.
From the above results, it is clear that the tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA has great potential for controlling TMV, and in addition, the tobacco mosaic virus nucleic acid pesticide TMV-cp (I) -dsRNA is nontoxic and harmless to the environment, and is expected to be an excellent biopesticide.
Finally, it should be noted that: the above is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that the present invention is described in detail with reference to the foregoing embodiments, and modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
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Claims (1)
1. An application of a tobacco mosaic virus resistant nucleic acid pesticide TMV-cp (I) -dsRNA in preventing and treating tobacco mosaic virus is characterized in that:
the tobacco mosaic virus resistant nucleic acid pesticide TMV-cp (I) -dsRNA is a freeze-drying agent, and the preparation method comprises the following steps:
s1, adding an endoprotease EcoRI into a plasmid containing TMV-EcoRI-EcoRI-cp, adding a nucleoside triphosphate mixture containing inosinic acid for in vitro transcription to obtain TMV-cp (I) -dsRNA, wherein the sequence of the TMV-cp (I) -dsRNA is shown as SEQ ID NO. 3; the sequence of TMV-EcoRI-EcoRI-cp is shown as SEQ ID NO.2
S2, adding DNaseI into a transcription system, and preserving heat for 0.5-1h at 37-40 ℃ to obtain a TMV-cp (I) -dsRNA crude product; anion exchange chromatography is adopted, then tangential flow ultrafiltration concentration is carried out, ions and unreacted nucleoside triphosphate in the system are removed, and purified TMV-cp (I) -dsRNA liquid of the anti-tobacco mosaic virus nucleic acid pesticide is obtained;
s3, pre-freezing the liquid in the S2 at the temperature of minus 80 ℃ and then freeze-drying at low temperature to obtain the TMV-cp (I) -dsRNA freeze-dried agent.
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| CN111513077A (en) * | 2020-05-25 | 2020-08-11 | 中国农业科学院烟草研究所 | RNAi nano preparation, preparation method thereof and application thereof in TMV prevention and treatment |
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| CN111513077A (en) * | 2020-05-25 | 2020-08-11 | 中国农业科学院烟草研究所 | RNAi nano preparation, preparation method thereof and application thereof in TMV prevention and treatment |
| CN113136383A (en) * | 2021-04-29 | 2021-07-20 | 硅羿科技（上海）有限公司 | Method suitable for large-scale extraction of dsRNA and application |
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