CN114561424A - Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus - Google Patents
Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus Download PDFInfo
- Publication number
- CN114561424A CN114561424A CN202210300333.0A CN202210300333A CN114561424A CN 114561424 A CN114561424 A CN 114561424A CN 202210300333 A CN202210300333 A CN 202210300333A CN 114561424 A CN114561424 A CN 114561424A
- Authority
- CN
- China
- Prior art keywords
- inoculation
- salvia miltiorrhiza
- leaves
- cucumber mosaic
- virus
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
- 241000304195 Salvia miltiorrhiza Species 0.000 title claims abstract description 52
- 238000011081 inoculation Methods 0.000 title claims abstract description 51
- 235000011135 Salvia miltiorrhiza Nutrition 0.000 title claims abstract description 44
- 238000000034 method Methods 0.000 title claims abstract description 35
- 241000724252 Cucumber mosaic virus Species 0.000 title claims abstract description 32
- 239000007924 injection Substances 0.000 title claims abstract description 23
- 238000002347 injection Methods 0.000 title claims abstract description 23
- 241000196324 Embryophyta Species 0.000 claims abstract description 32
- 241000700605 Viruses Species 0.000 claims abstract description 22
- 235000002637 Nicotiana tabacum Nutrition 0.000 claims abstract description 19
- 244000061176 Nicotiana tabacum Species 0.000 claims abstract description 5
- 241000208125 Nicotiana Species 0.000 claims description 15
- 239000007788 liquid Substances 0.000 claims description 5
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 238000000227 grinding Methods 0.000 claims description 4
- 240000008067 Cucumis sativus Species 0.000 claims description 3
- 235000010799 Cucumis sativus var sativus Nutrition 0.000 claims description 3
- 239000007853 buffer solution Substances 0.000 claims description 3
- 241000207746 Nicotiana benthamiana Species 0.000 claims description 2
- 230000000644 propagated effect Effects 0.000 claims description 2
- 230000001902 propagating effect Effects 0.000 claims 1
- 238000002255 vaccination Methods 0.000 claims 1
- 201000010099 disease Diseases 0.000 abstract description 20
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 20
- 208000024891 symptom Diseases 0.000 abstract description 9
- 239000000463 material Substances 0.000 abstract description 8
- 208000015181 infectious disease Diseases 0.000 abstract description 6
- 230000003950 pathogenic mechanism Effects 0.000 abstract description 4
- 239000005723 virus inoculator Substances 0.000 abstract description 4
- 230000003993 interaction Effects 0.000 abstract description 2
- 239000000284 extract Substances 0.000 abstract 1
- 239000012528 membrane Substances 0.000 description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 7
- 239000000243 solution Substances 0.000 description 6
- 239000000020 Nitrocellulose Substances 0.000 description 5
- 239000006180 TBST buffer Substances 0.000 description 5
- 229920001220 nitrocellulos Polymers 0.000 description 5
- 244000052769 pathogen Species 0.000 description 5
- 230000001717 pathogenic effect Effects 0.000 description 5
- 239000000843 powder Substances 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 229910010271 silicon carbide Inorganic materials 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 238000001035 drying Methods 0.000 description 4
- 230000009385 viral infection Effects 0.000 description 4
- 230000003612 virological effect Effects 0.000 description 4
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 239000013256 coordination polymer Substances 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000011159 matrix material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012216 screening Methods 0.000 description 3
- 239000008223 sterile water Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- HYXITZLLTYIPOF-UHFFFAOYSA-N 1,6,6-trimethyl-8,9-dihydro-7H-naphtho[1,2-g]benzofuran-10,11-dione Chemical compound O=C1C(=O)C2=C3CCCC(C)(C)C3=CC=C2C2=C1C(C)=CO2 HYXITZLLTYIPOF-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000017276 Salvia Nutrition 0.000 description 2
- 240000007164 Salvia officinalis Species 0.000 description 2
- 241000612118 Samolus valerandi Species 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 230000000903 blocking effect Effects 0.000 description 2
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000002458 infectious effect Effects 0.000 description 2
- KWGKDLIKAYFUFQ-UHFFFAOYSA-M lithium chloride Chemical compound [Li+].[Cl-] KWGKDLIKAYFUFQ-UHFFFAOYSA-M 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 230000007480 spreading Effects 0.000 description 2
- 230000009885 systemic effect Effects 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- QRXMUCSWCMTJGU-UHFFFAOYSA-N 5-bromo-4-chloro-3-indolyl phosphate Chemical compound C1=C(Br)C(Cl)=C2C(OP(O)(=O)O)=CNC2=C1 QRXMUCSWCMTJGU-UHFFFAOYSA-N 0.000 description 1
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 1
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 1
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 244000062175 Fittonia argyroneura Species 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 240000001140 Mimosa pudica Species 0.000 description 1
- 235000016462 Mimosa pudica Nutrition 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 241000767350 Salvia virus Species 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- WCHSQACWYIQTKL-XPWFQUROSA-N [[(2r,3s,4r,5r)-5-(6-aminopurin-9-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2r,3r,4r,5r)-2-(6-aminopurin-9-yl)-4-hydroxy-5-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Chemical compound C1=NC2=C(N)N=CN=C2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1OP(O)(=O)OP(O)(=O)OC[C@H]([C@@H](O)[C@H]1O)O[C@H]1N1C(N=CN=C2N)=C2N=C1 WCHSQACWYIQTKL-XPWFQUROSA-N 0.000 description 1
- 230000000840 anti-viral effect Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 208000026106 cerebrovascular disease Diseases 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 230000002526 effect on cardiovascular system Effects 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 239000012160 loading buffer Substances 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 244000000010 microbial pathogen Species 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000008239 natural water Substances 0.000 description 1
- 230000007918 pathogenicity Effects 0.000 description 1
- 239000003415 peat Substances 0.000 description 1
- 239000010451 perlite Substances 0.000 description 1
- 235000019362 perlite Nutrition 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 239000002574 poison Substances 0.000 description 1
- 231100000614 poison Toxicity 0.000 description 1
- 235000020183 skimmed milk Nutrition 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 238000009331 sowing Methods 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000010455 vermiculite Substances 0.000 description 1
- 235000019354 vermiculite Nutrition 0.000 description 1
- 229910052902 vermiculite Inorganic materials 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Images
Classifications
-
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8206—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated
- C12N15/8207—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by physical or chemical, i.e. non-biological, means, e.g. electroporation, PEG mediated by mechanical means, e.g. microinjection, particle bombardment, silicon whiskers
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/18—Testing for antimicrobial activity of a material
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Analytical Chemistry (AREA)
- Immunology (AREA)
- Toxicology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention discloses a seedling inoculation method for salvia miltiorrhiza and cucumber mosaic virus, belonging to the technical field of agricultural biology. The method extracts total RNA from tobacco leaves infected with cucumber mosaic virus, adopts a needle tube injection mode to carry out injection inoculation when the salvia miltiorrhiza plants are cultured to 6-8 leaf stages, and successfully establishes an infection system of the cucumber mosaic virus on the salvia miltiorrhiza plants. Provides an ideal experimental model for researching the pathogenic mechanism between the cucumber mosaic virus and the salvia miltiorrhiza host, and has important theoretical value for revealing the interaction between the virus and the host plant and the virus evolution. Compared with the friction inoculation method, the method has the advantages that the inoculation efficiency is greatly improved, and the salvia miltiorrhiza seedlings can be subjected to symptom observation two weeks after inoculation. The invention can complete high-efficiency inoculation with a small amount of disease-sensitive materials, the virus inoculation amount is controllable, the total RNA amount of the injection leaves is small, the damage to the leaves is small, and the field disease condition is closer.
Description
Technical Field
The invention relates to the field of plant virus inoculation, in particular to a seedling stage injection inoculation method for salvia miltiorrhiza and cucumber mosaic virus.
Background
Salvia miltiorrhiza is a large traditional Chinese medicinal material commonly used in China, and is widely applied to treating cardiovascular and cerebrovascular diseases. In recent years, the demand of salvia miltiorrhiza is increasing day by day, and the planting area is also expanding. However, the salvia miltiorrhiza mainly carries out asexual propagation in production, so that the problem of virus infection is serious, and the development of the salvia miltiorrhiza industry is limited. Research shows that the main pathogen of the salvia virus disease is Cucumber Mosaic Virus (CMV), can cause the salvia to degenerate, has higher disease incidence in fields, has the disease incidence rate of 60-80 percent in some fields, shows typical symptoms of mosaic, mottle, leaf curl, yellowing, dwarfing and the like, has tiny root system of a disease-sensitive plant, greatly reduces the yield, and sharply reduces the content of medicinal components such as tanshinone II and the like, thereby seriously affecting the quality of the salvia.
The establishment of artificial inoculation method of plant virus to relevant host plant is the precondition and basis for researching pathogenicity and harmfulness of virus and screening resistant variety of host plant. The mechanical friction inoculation method is a commonly used plant virus inoculation method at present, but the mechanical friction inoculation needs more susceptible materials, has certain requirements on host range of the susceptible materials, and some plants can be successfully infected only by the friction of juice of plants of the same genus or the same susceptible plants. In addition, mechanical attrition inoculation requires spraying carborundum onto the leaves in advance, causing micro-wounds to provide conditions for viral infection. But the dosage of the carborundum is not well controlled, and certain technical difficulty is caused by time and labor waste. At present, there is no report on successful infection of salvia miltiorrhiza by plant viruses through mechanical friction inoculation. The research on the salvia miltiorrhiza viral disease mainly adopts a field natural disease occurrence mode, the method obtains disease-sensitive materials by creating and utilizing natural disease occurrence conditions, however, factors influencing field disease occurrence such as temperature, humidity, the number of pathogenic microorganisms and the like are many, a disease nursery needs to be established, the identification period is long, the labor cost is high, the result is unstable, and the repeatability of experimental batches is poor. In addition, the salvia miltiorrhiza viral disease is caused by various plant viruses, the influence on the inoculation result is complex, and the consistency, objectivity and reliability of the result are influenced if the field natural attack cannot clearly play the role of the pathogen with the main effect. The establishment of a single pathogen inoculation method is the key for researching the pathogenic mechanism of the salvia miltiorrhiza viral disease.
Disclosure of Invention
The invention aims to provide a more efficient and highly operable salvia miltiorrhiza seedling inoculation method aiming at the current situation that the artificial inoculation system of cucumber mosaic virus on salvia miltiorrhiza, which is the main pathogen of salvia miltiorrhiza viral disease, is lacked at present and aiming at the defects of the traditional mechanical friction method. The method not only realizes the rapid identification of the virus resistance of the salvia miltiorrhiza material in the seedling stage, but also provides an ideal model for researching the pathogenic mechanism between the cucumber mosaic virus and the salvia miltiorrhiza, and has important theoretical value for disclosing the interaction between the modified virus and host plants and the virus evolution.
The technical scheme adopted by the invention is as follows: taking tobacco leaves infected with cucumber mosaic virus as a material of a poison source, extracting plant total RNA in the leaves, inoculating the plant total RNA into the salvia miltiorrhiza seedlings in an injection inoculation mode, and successfully establishing the infection of the cucumber mosaic virus on salvia miltiorrhiza plants.
The technical scheme adopted by the invention is as follows:
a seedling stage inoculation method for salvia miltiorrhiza and cucumber mosaic virus is characterized in that total RNA with the concentration of more than or equal to 0.5 mu g/mu l is extracted from tobacco leaves infected with cucumber mosaic virus and used as an inoculation liquid, and injection inoculation is carried out on the back of leaves cultured to 6-8 leaf stages of salvia miltiorrhiza plants in a needle tube injection mode.
Further, the cucumber mosaic virus-infected tobacco leaves were propagated by the following method:
grinding tobacco leaves infected by cucumber mosaic virus with 0.05M PB buffer solution, and inoculating virus juice onto healthy tobacco leaves by mechanical friction method to expand the virus source.
Further, the tobacco is Nicotiana benthamiana or Nicotiana tabacum.
Further, the injection inoculation is specifically as follows:
culturing Saviae Miltiorrhizae radix plant to 6-8 leaves stage, spreading 3-5 pinholes on the back of the third and fourth leaves with needle of injector, and slowly injecting total RNA from the back of the leaves to complete inoculation.
Further, the total RNA amount of the inoculated plant per leaf injection at the time of the injection inoculation is 200. mu.l or more.
Further, after inoculation, the salvia miltiorrhiza seedlings are placed in an artificial greenhouse for culture management, the temperature is kept between 20 ℃ and 22 ℃ day and night, and the temperature is adjusted to 25 ℃ day and night after 3 days.
Compared with the field natural morbidity method adopted in the current production, the seedling-stage artificial inoculation method for the salvia miltiorrhiza and cucumber mosaic virus has the advantages of pure inoculation pathogen, high inoculation efficiency, good repeatability and the like. The method can be carried out indoors, the conditions are controllable, the method is not influenced by seasons and external environment conditions, the virus infection condition can be identified only in about two weeks, and the method is suitable for screening disease-resistant breeding of the salvia miltiorrhiza. Meanwhile, the plant total RNA extracted from the diseased leaves is injected to inoculate the plant leaves, but the conventional virus RNA extracted from virions or agrobacterium tumefaciens infected by the virus is not injected, so that the experimental method is greatly simplified, the experimental cost is saved, and the possibility of researching the virus is provided for laboratories without the infected virus clone. The traditional mechanical friction inoculation method damages the cell wall by spraying carborundum, so that viruses enter cytoplasm through an exposed membrane, and the technology can not quantitatively or synchronously infect, so that the experimental treatment is difficult to reliably determine. The injection inoculation method can control the virus inoculation amount, and the locking of the leaves is smaller and is closer to the disease occurrence condition in the field.
Drawings
The invention is further described below with reference to the drawings and examples;
FIG. 1 is a diagram of plant symptoms after 2 weeks of inoculation of Salvia miltiorrhiza seedlings with total RNA of leaf plants infected with cucumber mosaic virus.
FIG. 2 is a diagram showing the results of detecting the expression of coat protein of cucumber mosaic virus in diseased leaves of the Salvia miltiorrhiza Bunge system.
Detailed Description
Example 1 extraction of Total RNA from Virus-infected host leaf plant tissue
1. Cucumber mosaic virus source propagation: uniformly spreading a small amount of 800-mesh carborundum on healthy Bunge tobacco leaves, taking 1-2 leaves of the Bunge tobacco infected by cucumber mosaic virus, adding 0.05M boric acid buffer solution into the carborundum for full grinding, taking clean PE gloves with two hands, dipping a small amount of disease juice, rubbing and smearing the whole leaves, and observing the leaves with obvious disease symptoms after 2 weeks.
2. Collecting the leaf of the Bunsen tobaccos (flower leaves, shrunken and curly) which are typical symptoms of cucumber mosaic virus disease, adding liquid nitrogen, fully grinding into powder, quickly taking 700 ul of plant powder by using an Eppendorf tube, adding 350 ul of 80 ℃ preheated water saturated phenol (pH5.2-6.0) and equal volume of RNA extraction buffer (100mM Tris-HCl (pH8.0), 0.1mM LICL, 10mM EDTA, 1.0% SDS), and oscillating for 15 sec; standing for 5 min. 350ml of chloroform was added thereto, shaken for 15sec, left for 5min and centrifuged at 12000rpm for 15 min.
3. Taking supernatant into a new RNase-free Eppendorf tube, adding equal volume of 4M LiCl solution, mixing, and standing at-20 deg.C for more than 5 hr. Then centrifuged at 12000rpm for 15min at 4 ℃. The supernatant was transferred to a new centrifuge tube. And (5) drying the precipitate at room temperature or drying the precipitate by an ultra-clean bench.
(4) Washing the precipitate once with 70 vol% ethanol, washing the precipitate again with anhydrous ethanol, and drying at room temperature or ultra-clean bench drying. Storing the dry powder at-20 deg.C, adding appropriate amount of DEPC ddH before inoculation and injection2Dissolving the precipitate with O to obtain RNA solution with total RNA concentration not lower than 0.5. mu.g/. mu.l, and placing the total RNA solution in ice bath for later use.
Example 2 cucumber mosaic Virus injection inoculation of Salvia miltiorrhiza seedling plants
1. The salvia miltiorrhiza seeds are sterilized in a sodium hypochlorite solution with the concentration of 3 wt% for 15min, washed with sterile water for 3 times and dried. The method comprises the steps of mixing northeast peat soil, vermiculite and perlite in a ratio of 1:1:1 to form a planting matrix, placing the planting matrix into a seedling pot, carrying out natural water washing on the matrix, then carrying out sowing on salvia miltiorrhiza seeds, covering a small amount of soil on the seeds, covering a seedling pot cover, carrying out transplanting and seedling division after a second true leaf is produced, wherein each pot is divided into one plant, and the salvia miltiorrhiza seedlings can be inoculated when the salvia miltiorrhiza seedlings grow to 6-8 leaves.
2. Taking 1ml of plant total RNA by using a 1ml sterile injector, pricking 3-5 holes in the back surfaces of the 3 rd and 4 th leaves of the salvia miltiorrhiza by using a needle head on the injector without piercing the leaves, slowly injecting the plant total RNA into the back surfaces of the salvia miltiorrhiza leaves after removing the needle head, infiltrating the whole leaves as much as possible, wherein the amount of the plant total RNA injected into each leaf is about 200 mu l. Keeping the day and night temperature at 20-22 deg.C after inoculation, adjusting the temperature to 25 deg.C after 3 days, and managing the normal growth of seedling.
3. Symptom observation is carried out on the salvia miltiorrhiza seedlings after 2 weeks of inoculation, and the result is shown in figure 1, because the inoculation wound surface is small, the salvia miltiorrhiza seedlings after injection inoculation grow well, the salvia miltiorrhiza seedlings controlled by the injection inoculation sterile water have no symptom, and the systemic leaves of the salvia miltiorrhiza seedlings inoculated with the total RNA of the cucumber mosaic plant show the symptoms of leaf and leaf downward curling.
Example 3 Virus Western blot detection of diseased Salvia miltiorrhiza plants
1. Salvia miltiorrhiza seedling leaves with obvious cucumber mosaic virus infection symptoms obtained in example 2 are used for Western blot detection, powder of plant materials ground in liquid nitrogen with a volume of 100 ul is taken by an Eppendorf tube, 100 ul of 1 Xprotein loading buffer (100mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.2% bromophenol blue and 5% beta-mercaptoethanol) is added, the mixture is oscillated and boiled in a boiling water bath for 10min, the mixture is immediately placed on ice for 2min, centrifuged at 12000rpm for 10min, and the supernatant is taken and placed in a new centrifuge tube for standby.
2. Preparing PAGE gel (4.5% concentrated gel and 12.5% separation gel), loading 20 μ l of sample to PAGE gel for electrophoresis, firstly performing 80V electrophoresis, increasing the voltage to 120V after the sample enters the separation gel, stopping electrophoresis until bromophenol blue just comes out, and transferring protein to nitrocellulose membrane by an electric transfer method (200mA, 80 min). The transferred nitrocellulose membrane was rinsed once in TBST buffer and transferred to 10ml of blocking solution (TBST + 5% skim milk powder). Blocking at 37 ℃ for at least 2hr or at 4 ℃ overnight.
3. Directly adding a certain volume of cucumber mosaic virus CP specific antiserum into the sealing solution after sealing, and reacting at 37 deg.C for at least 1 hr. The nitrocellulose membrane was rinsed 3 times for 10min each in TBST buffer. The nitrocellulose membrane was added to an AP-A secondary antibody diluted (1:5000) with TBST and reacted at 37 ℃ for 30 to 60 min. The membrane was washed again with TBST 3 times for 10min each. The nitrocellulose membrane was placed in alkaline phosphatase buffer (AP) containing NBT at 330. mu.g/ml and BCIP at 165. mu.g/ml under dark conditions to develop a clear band. As shown in FIG. 2, CP expression of cucumber mosaic virus was detected in 6 samples of total RNA from plants injected with diseased leaves, but not in the Salvia miltiorrhiza plants injected with sterile water control. Two weeks after injection inoculation, the cucumber mosaic virus can infect the salvia miltiorrhiza to the systemic leaves and begin to express the CP protein. Repeated inoculation experiment results show that the inoculation success rate of the method is as high as 70-80%.
The method can realize the cucumber mosaic virus and the artificial inoculation of the salvia miltiorrhiza plant, and can carry out the system infection after 2 weeks. The injection inoculation liquid is plant total RNA extracted from diseased leaves of tobacco (the tobacco or the common tobacco) instead of conventional virus infectious clone, thereby greatly simplifying the experimental method and providing possibility for researching the virus for laboratories without the virus infectious clone. In addition, the injection inoculation can also control the inoculation amount of a single plant, has less damage to leaves and is closer to the field morbidity, and is suitable for developing the research on pathogenic mechanisms between cucumber mosaic virus and salvia miltiorrhiza hosts and the germplasm resource screening of antiviral salvia miltiorrhiza.
The above embodiments are intended to illustrate the present invention, not to limit the present invention, and any modifications and changes made within the spirit of the present invention and the scope of the claims fall within the scope of the present invention.
Claims (6)
1. A seedling stage inoculation method for salvia miltiorrhiza and cucumber mosaic virus is characterized in that total RNA with the concentration of more than or equal to 0.5 mu g/mu l is extracted from tobacco leaves infected with cucumber mosaic virus and used as inoculation liquid, and injection inoculation is carried out on the back of leaves cultured to 6-8 leaf stages of salvia miltiorrhiza plants by adopting a needle tube injection mode.
2. The method of claim 1, wherein the cucumber mosaic virus-infected tobacco leaves are propagated by:
grinding tobacco leaves infected by cucumber mosaic virus with 0.05M PB buffer solution, inoculating virus juice to healthy tobacco leaves, and propagating the virus source.
3. The method according to claim 1, wherein the tobacco is a Bunsen tobacco Nicotiana benthamiana or a common tobacco Nicotiana tabacum.
4. The method according to claim 1, characterized in that the injection vaccination is in particular: 3-5 pinholes are poked on the back of the third and fourth unfolded leaves of the salvia miltiorrhiza cultured to the 6-8 stage by using a needle of an injector and the leaves cannot be poked through, so that a micro wound is caused, and the total RNA is injected from the back of the leaves to finish the inoculation.
5. The method of claim 1, wherein: the total RNA amount of each leaf of the inoculated plant is more than 200 mul.
6. The method of claim 1, wherein: after inoculation, the salvia miltiorrhiza seedlings are placed in an artificial greenhouse for culture management, the temperature is kept between 20 and 22 ℃ day and night, and the temperature is adjusted to 25 ℃ day and night after 3 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210300333.0A CN114561424B (en) | 2022-03-24 | 2022-03-24 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210300333.0A CN114561424B (en) | 2022-03-24 | 2022-03-24 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114561424A true CN114561424A (en) | 2022-05-31 |
| CN114561424B CN114561424B (en) | 2024-01-26 |
Family
ID=81720572
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210300333.0A Active CN114561424B (en) | 2022-03-24 | 2022-03-24 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114561424B (en) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101321455A (en) * | 2004-02-23 | 2008-12-10 | 以色列国农业部 | Engrafted plants resistant to viral diseases and methods of producing same |
| CN103060380A (en) * | 2013-01-25 | 2013-04-24 | 中国农业大学 | Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus |
| CN108048601A (en) * | 2017-12-20 | 2018-05-18 | 江苏省农业科学院 | A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application |
-
2022
- 2022-03-24 CN CN202210300333.0A patent/CN114561424B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101321455A (en) * | 2004-02-23 | 2008-12-10 | 以色列国农业部 | Engrafted plants resistant to viral diseases and methods of producing same |
| CN103060380A (en) * | 2013-01-25 | 2013-04-24 | 中国农业大学 | Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus |
| CN108048601A (en) * | 2017-12-20 | 2018-05-18 | 江苏省农业科学院 | A kind of capsicum Cucumber Mosaic Virus Seedling Inoculation method and its application |
Non-Patent Citations (7)
| Title |
|---|
| DAVID E.SCHLEGEL: "Highly Infectious Phenol Extracts from Tobacco Leaves Infected with Cucumber Mosaic Virus", VIROLOGY, vol. 11, pages 330 * |
| G.E.HOLCOMB 等: "Natural Infection of Salvia uliginosa with Cucumber Mosaic Cucumovirus", HORTSCIENCE, vol. 33, no. 7, pages 1215 - 1216 * |
| SOUMYA SINHA 等: "Characterization and evolutionary analysis of Cucumber mosaic virus isolate infecting Salvia sclarea in India", 3 BIOTECH, vol. 11, no. 11, pages 1 - 7 * |
| VERMA, V, S.: "Salvia yellow vein mosaic virus", GARTENBAUWISSENSCHAFT, vol. 39, no. 5, pages 565 - 566 * |
| 丁远杰 等: "丹参病毒病病原鉴定研究", 中草药, vol. 34, no. 12 * |
| 吴志明, 温春秀, 彭卫欣, 吴智红, 王云逸, 谢晓亮: "黄瓜花叶病毒丹参株系外壳蛋白基因的克隆和序列分析", 河北农业科学, no. 02, pages 24 - 30 * |
| 杨立;缪作清;杨光;邵爱娟;黄璐琦;申业;王雪;陈美兰;: "丹参枯萎病及其病原菌的研究", 中国中药杂志, no. 23, pages 59 - 62 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114561424B (en) | 2024-01-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| van Spronsen et al. | Cell biological changes of outer cortical root cells in early determinate nodulation | |
| CN101803569A (en) | Method for inducing strawberry stolons and eliminating viruses in test tube by high temperature processing in combination with shoot tip culture | |
| CN102181473B (en) | Construction method for plant root related functional gene research model | |
| CN110172474B (en) | Induction and rapid propagation method for hairy roots of purple ginseng | |
| CN101213938B (en) | Method for cultivating detoxification tissue culture bulb of hyacinth | |
| CN106244625B (en) | A kind of tobacco seed genetic transforming method of mediated by agriculture bacillus rapidly and efficiently | |
| Sood et al. | Development of a low cost micropropagation technology for an endangered medicinal herb (Picrorhiza kurroa) of North-Western Himalayas | |
| CN109511553A (en) | A kind of coptis hairy highly effective revulsion induction method | |
| CN103695458A (en) | Methods for efficient induction production of Glycyrrihiza uralensisi Fisch hairy roots and production of licorice root secondary metabolites by using Glycyrrihiza uralensisi Fisch hairy roots | |
| CN114561424A (en) | Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus | |
| CN107501399A (en) | Vitis davidii Foex transcription factor VdWRKY70 and its application in plant resistance to environment stress kind is cultivated | |
| CN104845929B (en) | Tuniclike psammosilene root plant cell suspension cultures and its method for building up | |
| Chen | Mycoplasmalike organisms in sieve tube elements of plants infected with blueberry stunt and cranberry false blossom | |
| CN108795981A (en) | The method for creating Resistance Strain of Cotton black striped plant bug new germ plasm using the RNAi technology of mediated plant | |
| CN106718942A (en) | The tissue-culturing rapid propagation and domestication culture techniques of North China's dendrobium candidum | |
| CN106613970B (en) | The quick breeding by group culture method of sealwort leaf elegant jessamine | |
| CN103026966B (en) | Method for identifying tomato yellow leaf curl virus resistance by utilizing detached leaf | |
| Danial et al. | Seed histology of recalcitrant Eurycoma longifolia plants during germination and its beneficial attribute for hairy roots production | |
| CN104839022B (en) | The abductive approach of tuniclike psammosilene root feather shaped root system | |
| CN103931476A (en) | Method for cultivating cruciferae plants for observing plasmodiophoromycetes infection | |
| CN104630261B (en) | A kind of method improving flower genetic transformation transient expression efficiency | |
| Ma et al. | First report of Fusarium oxysporum causing basal stem rot on Panax ginseng in China | |
| CN103060380B (en) | Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus | |
| CN102640642B (en) | Method for pierce-inoculating geminivirus infectious clone-containing solid colony to plant, and application thereof | |
| Cohen et al. | A noncirculative whitefly-borne virus affecting tomatoes in Israel |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |