CN105746439A - Method and artificial feed for enabling laodelphax striatellus to obtain rice stripe viruses - Google Patents
Method and artificial feed for enabling laodelphax striatellus to obtain rice stripe viruses Download PDFInfo
- Publication number
- CN105746439A CN105746439A CN201610131539.XA CN201610131539A CN105746439A CN 105746439 A CN105746439 A CN 105746439A CN 201610131539 A CN201610131539 A CN 201610131539A CN 105746439 A CN105746439 A CN 105746439A
- Authority
- CN
- China
- Prior art keywords
- rice
- man
- made feeds
- stripe virus
- small brown
- 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
- 241000724205 Rice stripe tenuivirus Species 0.000 title claims abstract description 98
- 238000000034 method Methods 0.000 title claims abstract description 51
- 241001470017 Laodelphax striatella Species 0.000 title abstract description 8
- 241000209094 Oryza Species 0.000 claims abstract description 31
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims abstract description 16
- 229930006000 Sucrose Natural products 0.000 claims abstract description 16
- 239000005720 sucrose Substances 0.000 claims abstract description 14
- 235000021329 brown rice Nutrition 0.000 claims description 83
- 241001498622 Cixius wagneri Species 0.000 claims description 82
- 235000007164 Oryza sativa Nutrition 0.000 claims description 27
- 235000009566 rice Nutrition 0.000 claims description 24
- 239000010408 film Substances 0.000 claims description 15
- 239000011521 glass Substances 0.000 claims description 15
- 239000006228 supernatant Substances 0.000 claims description 12
- 238000007789 sealing Methods 0.000 claims description 11
- 238000000502 dialysis Methods 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 7
- 239000000706 filtrate Substances 0.000 claims description 7
- 239000012528 membrane Substances 0.000 claims description 7
- 239000010409 thin film Substances 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000000463 material Substances 0.000 claims description 6
- 238000005286 illumination Methods 0.000 claims description 5
- 238000002360 preparation method Methods 0.000 claims description 5
- 230000008569 process Effects 0.000 claims description 5
- 230000009514 concussion Effects 0.000 claims description 4
- 230000003203 everyday effect Effects 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 230000001954 sterilising effect Effects 0.000 claims description 3
- 238000004659 sterilization and disinfection Methods 0.000 claims description 3
- 238000005374 membrane filtration Methods 0.000 claims 1
- 231100000252 nontoxic Toxicity 0.000 abstract description 15
- 230000003000 nontoxic effect Effects 0.000 abstract description 15
- 201000010099 disease Diseases 0.000 abstract description 9
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 9
- 241000700605 Viruses Species 0.000 abstract description 7
- 238000011160 research Methods 0.000 abstract description 7
- 241000238631 Hexapoda Species 0.000 abstract description 2
- 230000005540 biological transmission Effects 0.000 abstract description 2
- 238000007796 conventional method Methods 0.000 abstract description 2
- 230000007246 mechanism Effects 0.000 abstract description 2
- 239000000203 mixture Substances 0.000 abstract description 2
- 241001466042 Fulgoromorpha Species 0.000 abstract 1
- 239000007864 aqueous solution Substances 0.000 abstract 1
- 231100000331 toxic Toxicity 0.000 abstract 1
- 230000002588 toxic effect Effects 0.000 abstract 1
- 239000003053 toxin Substances 0.000 abstract 1
- 231100000765 toxin Toxicity 0.000 abstract 1
- 231100000614 poison Toxicity 0.000 description 50
- 239000002574 poison Substances 0.000 description 41
- 239000000243 solution Substances 0.000 description 26
- 229960004793 sucrose Drugs 0.000 description 12
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- 239000003440 toxic substance Substances 0.000 description 9
- 238000011529 RT qPCR Methods 0.000 description 8
- 238000012360 testing method Methods 0.000 description 8
- 235000016709 nutrition Nutrition 0.000 description 7
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 238000010839 reverse transcription Methods 0.000 description 6
- 230000004083 survival effect Effects 0.000 description 6
- 239000008363 phosphate buffer Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 4
- 240000007594 Oryza sativa Species 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 238000001556 precipitation Methods 0.000 description 3
- 230000003612 virological effect Effects 0.000 description 3
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 235000013681 dietary sucrose Nutrition 0.000 description 2
- 239000003085 diluting agent Substances 0.000 description 2
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 description 2
- DGLRDKLJZLEJCY-UHFFFAOYSA-L disodium hydrogenphosphate dodecahydrate Chemical compound O.O.O.O.O.O.O.O.O.O.O.O.[Na+].[Na+].OP([O-])([O-])=O DGLRDKLJZLEJCY-UHFFFAOYSA-L 0.000 description 2
- 229910000397 disodium phosphate Inorganic materials 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 101150012647 pc3 gene Proteins 0.000 description 2
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 2
- 235000011164 potassium chloride Nutrition 0.000 description 2
- 239000001103 potassium chloride Substances 0.000 description 2
- 230000001681 protective effect Effects 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 239000002904 solvent Substances 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- 230000035899 viability Effects 0.000 description 2
- 101710132601 Capsid protein Proteins 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 108020004414 DNA Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 239000007836 KH2PO4 Substances 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 241000522620 Scorpio Species 0.000 description 1
- 241000723848 Tobamovirus Species 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 230000000844 anti-bacterial effect Effects 0.000 description 1
- 238000010805 cDNA synthesis kit Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000006854 communication Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 210000000805 cytoplasm Anatomy 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- DNJIEGIFACGWOD-UHFFFAOYSA-N ethyl mercaptane Natural products CCS DNJIEGIFACGWOD-UHFFFAOYSA-N 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 244000037666 field crops Species 0.000 description 1
- 239000013505 freshwater Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 239000002917 insecticide Substances 0.000 description 1
- 230000036244 malformation Effects 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 238000002161 passivation Methods 0.000 description 1
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 239000004576 sand Substances 0.000 description 1
- 239000009490 scorpio Substances 0.000 description 1
- 230000001932 seasonal effect Effects 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 239000011550 stock solution Substances 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000003934 vacuole Anatomy 0.000 description 1
- 210000002845 virion Anatomy 0.000 description 1
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- 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
- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
-
- 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
- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
- C12N2720/00011—Details
- C12N2720/00051—Methods of production or purification of viral material
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Environmental Sciences (AREA)
- Organic Chemistry (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Immunology (AREA)
- Biochemistry (AREA)
- Virology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Animal Behavior & Ethology (AREA)
- Animal Husbandry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Biomedical Technology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Medicines Containing Plant Substances (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses a method and an artificial feed for enabling laodelphax striatellus to obtain rice stripe viruses. The method for enabling rice stripe viruses to obtain laodelphax striatellus comprises the following step: feeding the laodelphax striatellus with the artificial feed containing rice stripe viruses, thereby enabling the laodelphax striatellus to obtain rice stripe viruses. The composition of the artificial feed is that every 1 muL of the artificial feed contains the rice stripe viruses greater than or equal to 300ng, the balance being a sucrose aqueous solution with concentration being 50g/L. According to the method, the rice stripe virus acquiring rate is higher than 90% which is greatly increased in comparison with that of a conventional method of feeding non-toxic ash-free plant hoppers by toxic rice plants. According to the method, an experimental platform for enabling the laodelphax striatellus to obtain toxin efficiently is established, and the research on the transmission mechanism of rice stripe viruses in the laodelphax striatellus is strongly promoted, and the effective progress of the novel disease-resistant strategy of removing virus in insects is promoted.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of method making small brown rice planthopper obtain rice stripe virus and man-made feeds thereof.
Background technology
Stripe disease is one of main rice disease of China.The normal withered booting of diseased plant or fringe minor malformation are unreal.There is yellow green striped in sword-like leave bottom in jointing sequela, all not withered heart of all types of rice, but heading deformity, solid seldom.Its cause of disease is rice stripe virus (Ricestripevirus, RSV), belongs to very thin Tobamovirus virus.Virion is thread, size 400 × 8nm, is scattered in Cytoplasm, vacuole and core, or becomes the unsetting glomeration such as graininess, sand shape, i.e. Inclusion, seemingly has many filamentouss to tangle and agglomerating.Sick leaf sap dilution point of accumulation 1000～10000 times, passivation temperature be 55 DEG C 3 minutes, subzero 20 DEG C, the longevity in vitro (sick rice) 8 months.
Rice stripe virus propagation between rice plant needs by insecticide amboceptor small brown rice planthopper (Laodelphaxstriatellus).It is research virus levels communication process and the important foundation made mutually of Oryza sativa L.-small brown rice planthopper-RSV three that nontoxic small brown rice planthopper obtains poison experiment.Traditional poison method of raising is to use the rice plants with poison to feed nontoxic without small brown rice planthopper, obtains poison rate and is generally not more than 30%.As Li Shuo et al. adopts fresh in vitro disease leaf to raise poison, the poison rate that obtains of small brown rice planthopper is 11.8% (Li Shuo, Wang Shijuan, slander Jin Yan, Deng. the method that small brown rice planthopper quickly obtains rice stripe virus from vitro sick leaf. Jiangsu's agriculture journal, 2014,30 (2): 449-451.)；For another example the research of Wang Shuan et al. shows, nontoxic small brown rice planthopper is after catching an illness and taking food 2h in rice strain, its band poison rate starts to tend to steadily, substantially remain in about 30% (Wang Shuan, Yang Yizhong, Zhong Chongxiang. strip virus RSV is the research of transfer rate between small brown rice planthopper and rice plant. China's agronomy circular, 2011,27 (05): 328-332.).Visible, traditional method adopting virus-infected plant to feed small brown rice planthopper, its small brown rice planthopper to obtain poison rate relatively low, it addition, the collection of virus-infected plant is subject to the restriction of seasonal factor, constrain the research obtaining poison work.
At present, it is badly in need of a kind of method enabling small brown rice planthopper efficiently to obtain rice stripe virus.
Summary of the invention
First purpose of the present invention is to provide a kind of method making small brown rice planthopper obtain rice stripe virus.
The method making small brown rice planthopper obtain rice stripe virus provided by the present invention, specifically can comprise the steps: to feed the man-made feeds containing rice stripe virus to small brown rice planthopper, so that described small brown rice planthopper obtains rice stripe virus；
Described man-made feeds composed as follows: containing be more than or equal to 300ng (such as 300-1200ng, for another example 600-1200ng) described rice stripe virus in man-made feeds described in every μ L；Surplus is concentration is the aqueous sucrose solution of 50g/L.
In the process, described man-made feeds specifically prepare according to the method comprised the steps: with carry rice stripe virus rice leaf (preferably having infected rice stripe virus and the obvious rice leaf of disease symptom) for material, extract rice stripe virus；The rice stripe virus extracted is joined in the aqueous sucrose solution that concentration is 50g/L, make the final concentration of described rice stripe virus be more than or equal to 300ng/ μ L (such as 300-1200ng/ μ L, for another example 600-1200ng/ μ L), obtain described man-made feeds.Wherein, the rice leaf carrying rice stripe virus described in is preferably the rice leaf carrying rice stripe virus that field gathers.
Wherein, the rice leaf carrying rice stripe virus described in can pass through-80 DEG C of freezen protective.
Further, described " with carry rice stripe virus rice leaf for material, extract rice stripe virus " method, specifically can comprise the steps:
(a1) the described rice leaf liquid nitrogen grinding carrying rice stripe virus is become fine-powdered, add PBS, 4 DEG C of concussion 15min
Wherein, described concussion can be shake instrument concussion with spiral.
The solvent of described PBS is that water, solute and concentration are as follows: potassium dihydrogen phosphate (KH2PO4) 0.2g/L, disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) 2.9g/L, sodium chloride (NaCl) 8.0g/L, potassium chloride (KCl) 0.2g/L；PH is 7.4.
(a2) 4 DEG C, 14000-16000g (concrete such as 15294g) centrifugal 10min, take supernatant.
(a3) filtration sterilization: filter membrane that described supernatant aperture is 0.22 μm is (concrete such as syringe-driven filter that aperture is 0.22 μm, i.e. AcrodiscSyringefilter0.22 μm of supermembranelowproteinbindingNon-pyrogenic) filter, take filtrate.
(a4) described filtrate is carried out dialysis concentration, namely obtains described rice stripe virus.
Wherein, described dialysis concentration is particularly as follows: be transferred in Dialysis tubing by step (a3) gained filtrate the centrifugal 40min of 4 DEG C of 6300g, abandon the solution given bottom described Dialysis tubing, adding concentration in the concentrated solution that described Dialysis tubing top retains is the aqueous sucrose solution of 50g/L, 4 DEG C of centrifugal 40min of 6300g again, until all of described PBS is all replaced by the aqueous sucrose solution that described concentration is 50g/L.
In the process, feed small brown rice planthopper described in normal direction particular by thin film and feed described man-made feeds.
Further, described thin film feeding method specifically can comprise the steps: to be placed in the glass tubing of both ends open by small brown rice planthopper, one end double-layer air-permeable sealed membrane (ventilative parafilm film) of described glass tubing is sealed, the other end first seals with one layer of breathable sealing film, aseptic face outwardly, film adds described man-made feeds, again the aseptic of another layer of breathable sealing film is faced inner envelope feedstuff, being placed by described glass tube horizontal makes the described man-made feeds face in described two-layer breathable sealing film vertical, it is placed in 25 DEG C, periodicity of illumination 16h/8h (16h illumination, 8h is dark) environment in, described small brown rice planthopper in described glass tubing sucks described man-made feeds (free choice feeding) by piercing through sealed membrane, every day changes once described man-made feeds, feed 2 days altogether.
In the present invention, the glass tubing specification of described both ends open is as follows: long 12cm, diameter 2.8cm；Accordingly, the amount of the described man-made feeds put in the middle of described two-layer breathable sealing film is 70 μ L.
In the process, after feeding described man-made feeds to described small brown rice planthopper, may also include and described small brown rice planthopper is transferred to the step raised in healthy water rice seedling (not carrying rice stripe virus) 10 days.
In the process, when feeding described man-made feeds to described small brown rice planthopper, described small brown rice planthopper was two ages.
Second purpose of the present invention is to provide a kind of man-made feeds for making small brown rice planthopper obtain rice stripe virus.
Man-made feeds for making small brown rice planthopper obtain rice stripe virus provided by the present invention, its composition is specific as follows: containing be more than or equal to 300ng (such as 300-1200ng, for another example 600-1200ng) described rice stripe virus in man-made feeds described in every μ L；Surplus is concentration is the aqueous sucrose solution of 50g/L.
3rd purpose of the present invention is to provide a kind of method preparing described man-made feeds.
The method preparing described man-made feeds provided by the present invention is as mentioned before.
4th purpose of the present invention is to provide the application of (I) or (II) as follows:
(I) described man-made feeds or described man-made feeds method of preparing make small brown rice planthopper obtain the application in rice stripe virus；
(II) small brown rice planthopper is made to obtain the method for rice stripe virus or described man-made feeds or described prepare the application in obtaining the small brown rice planthopper carrying rice stripe virus of the man-made feeds method described in.
The present invention gathers band poison rice leaf for material with field, slightly puies forward virus, joins in man-made feeds, by the method that thin film feeds, makes small brown rice planthopper obtain poison, obtains poison rate and reaches more than 90%.The method is with traditional to use susceptible rice strain to feed nontoxic without compared with the method for small brown rice planthopper, obtaining malicious rate and be greatly improved.The present invention establishes the experiment porch making small brown rice planthopper efficiently obtain poison, will promote rice stripe virus research of the mechanism of transmission in small brown rice planthopper body effectively, promotes effective progress of the novel Strategy removing virus in insect bodies.
Accompanying drawing explanation
Fig. 1 makes what small brown rice planthopper obtained rice stripe virus to obtain poison rate and survival rate by thin film feeding method.
Fig. 2 is the solubility curve of the qRT-PCR obtaining poison small brown rice planthopper.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Embodiment 1, man-made feeds containing rice stripe virus preparation
One, the thick extraction of rice stripe virus
1, taking the rice leaf 1g liquid nitrogen grinding carrying rice stripe virus that the land for growing field crops of-80 DEG C of freezen protective gathers and become fine-powdered, (solvent is that water, solute and concentration thereof are as follows: potassium dihydrogen phosphate (KH to add the PBS of 3ml2PO4) 0.2g/L, disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O) 2.9g/L, sodium chloride (NaCl) 8.0g/L, potassium chloride (KCl) 0.2g/L；PH is 7.4), 4 DEG C, shake instrument (rotator-mixer) with spiral and shake 15min.
2,4 DEG C, 12000rpm (is equivalent to 15294g) and is centrifuged 10min, takes supernatant.
3, filtration sterilization: the syringe-driven filter (AcrodiscSyringefilter0.22 μm of supermembranelowproteinbindingNon-pyrogenic) that step 2 gained supernatant aperture is 0.22 μm is filtered, takes filtrate.The purpose of this step is to remove antibacterial.
4, dialysis concentration: step 3 gained filtrate is transferred in Dialysis tubing, 4 DEG C of centrifugal 40min of 7000rpm (being equivalent to 6300g), abandon the solution given bottom Dialysis tubing, the protein concentrated solution that Dialysis tubing top retains adds the sucrose solution (concentration is the aqueous sucrose solution of 50g/L) of 5%, 4 DEG C of centrifugal 40min of 7000rpm (being equivalent to 6300g) again, to the last PBS is all replaced by the sucrose solution of 5%, it is thus achieved that the man-made feeds nutritional solution containing rice stripe virus.
Two, the concentration of rice stripe virus in the man-made feeds nutritional solution of determination step one gained
1, each 1 μ l of rice stripe virus extracting solution (titer) of the man-made feeds nutritional solution (liquid to be measured) of step one gained and the purification of concentration known (100ng/ μ l) is taken respectively, extracting RNA with QIAGENRNeasyMiniKit, concrete operations carry out referring to test kit description.
It is known that the preparation method of the rice stripe virus extracting solution (titer) of the purification of concentration (100ng/ μ l) (the separation purification of virus) specific as follows:
(1) the Oryza sativa L. disease leaf of 300g infection rice stripe virus joins 900mL concentration is 0.01mol/L phosphate buffer (PBS；pH7.2；Containing 0.5% (volumn concentration) mercaptoethanol, 1mmol/LEDTA), low-temperature homogenate, two-layer filtered through gauze.
(2) (volumn concentration) chloroform ice bath stirs 15min to add 20%, stands 10min.
(3) take the centrifugal 15min of supernatant 7500rpm (being equivalent to 7232g), take and reset and add 6% (6g/100ml) PEG6000,1% (1g/100ml) sodium chloride ice bath stirring 40min, stand 4h.
(4) 8000rpm (being equivalent to 8228g) centrifugal 20min, the phosphate buffer (PBS going supernatant, precipitation concentration to be 0.01mol/L；pH7.2；Containing 1mmol/LEDTA) suspend.
(5) the centrifugal 15min of 12000rpm (being equivalent to 12857g), takes the phosphate buffer (PBS that centrifugal 2h, the precipitation 3-5mL concentration of supernatant 30000rpm/min is 0.01mol/L；pH7.2；Containing 1mmol/LEDTA) suspend.
(6) the centrifugal 10min of 10000rpm (being equivalent to 18514g), takes supernatant.
(7) 20%, 25%, 30%, 35%, 40% saccharose gradient (the previous day prepares tubulature 4 DEG C overnight to form gradient).
(8) joining above saccharose gradient by step (6) taken supernatant, 30000rpm (is equivalent to 166716g) and is centrifuged 2h, carefully takes milky band, adds the 0.01mol/L phosphate buffer (PBS of 10 times of volumes；pH7.2；Containing 1mmol/LEDTA) dilution of fully vibrating.
(9) the centrifugal 2h of 30000rpm (being equivalent to 166716g), precipitation is dissolved in the phosphate buffer (PBS that 1-2mL concentration is 0.01mol/L；pH7.2；Containing 1mmol/LEDTA).
(10) the centrifugal 10min of 10000rpm (being equivalent to 18524g), takes supernatant, is the viral solution purified.
The concentration of titer measures: by the concentration of virus in the viral solution of BIO-RADproteinassay test kit detection purification.Result shows, its virus concentration is 100ng/ μ l.
2, reverse transcription:
Use the Reverse Transcription box (iScript of BIORAD companyTMCDNASynthesisKit) RNA that the step 1 extracted is extracted carries out reverse transcription；
Prepare 20 μ l reaction systems: 5 × iScriptreactionmix4 μ l；IScript reverse transcription 1 μ l；The water 11 μ l of nuclease free；RNA template (RNA that namely step 1 is extracted) 4 μ l.
Response procedures: 25 DEG C of 5min；42℃30min；85℃5min.
After reaction terminates, product adds 120 μ l sterilized water dilutions.
3, RealTimePCR:
Using the diluent of the reverse transcription product of the small brown rice planthopper tissue extract of the known RSV concentration (100ng/ μ l) of step 2 acquisition as reference, carry out qRT-PCR with the pair of primers pc3-F/pc3-R of the identical pc3 gene (pc3 is the gene encoding RSV coat protein) being specific to RSV.
Pc3-F:5 '-GATGCGTTGTCTTACCTGACTGC-3 '；
Pc3-R:5 '-CACTATCCCATACCTCGACACCA-3 '.
Prepare 20 μ l reaction systems: SYBRGreenMix10 μ l；The each 0.5 μ l of upstream and downstream primer；DNA profiling (i.e. the diluent of the titer of step 2 acquisition and the reverse transcription product of liquid to be measured) 9 μ l.
The reactant liquor prepared reacts according to qRT-PCR under following condition: preheating (95 DEG C of 3min)；Degeneration (95 DEG C of 15s), annealing (60 DEG C of 15s), chain extension (72 DEG C of 20s) 44 circulation；Generate solubility curve (65 DEG C-95 DEG C).
4, the concentration of rice stripe virus in the man-made feeds nutritional solution of calculation procedure one gained
Test in triplicate, results averaged.
Liquid RSV concentration to be measured/titer RSV concentration=2-(Ct Liquid to be measured -Ct Titer )≈12。
So, the concentration of the RSV in the man-made feeds nutritional solution (liquid to be measured) of step one gained is: 12 × 100ng/ μ l=1200ng/ μ l.
Three, the preparation man-made feeds containing rice stripe virus
(measuring through step 2, wherein the concentration of RSV is 1200ng/ μ l) to the man-made feeds nutritional solution obtained by 5% sucrose solution (concentration is the aqueous sucrose solution of 50g/L) dilution step one, carries out 2 times of dilutions and 4 times of dilutions respectively.Count the man-made feeds nutritional solution stock solution that the step one of not diluted obtains in, obtain the man-made feeds of three kinds of RSV containing variable concentrations altogether.Concrete as shown in table 1.
The concentration of RSV in tri-groups of man-made feeds of table 1A, B, C
| Group | A | B | C |
| Man-made feeds RSV concentration (ng/ μ l) | 1200 | 600 | 300 |
Embodiment 2, made by thin film feeding method small brown rice planthopper obtain rice stripe virus
1, counteracting toxic substances:
nullTake three parts be tens of two age nontoxic small brown rice planthopper，It is individually placed to glass tubing (the long 12cm of 3 identical both ends opens，Diameter 2.8cm) in，One end double-layer air-permeable sealed membrane (ventilative parafilm film) of glass tubing is sealed，The other end first seals with one layer of breathable sealing film，Aseptic face outwardly，Film adds described man-made feeds，Again the aseptic of another layer of breathable sealing film is faced inner envelope feedstuff，The man-made feeds containing RSV of 70 μ l embodiments 1 preparations are put in centre, and (corresponding 3 glass tubings are respectively adopted the A in table 1、B、Tri-groups of man-made feeds of C)，Being placed by described glass tube horizontal makes the described man-made feeds face in described two-layer breathable sealing film vertical，It is placed in 25 DEG C、Periodicity of illumination 16h/8h (16h illumination，8h is dark) environment in，Described small brown rice planthopper in described glass tubing sucks described man-made feeds (free choice feeding) by piercing through sealed membrane，Every day changes once described man-made feeds，Feed 2 days altogether.
2, after step 1 feeds 2 days, the small brown rice planthopper in 3 glass tubings is transferred to respectively in 3 bottles of fresh nontoxic (namely not carrying RSV) rice seedlings, reference numeral A, B, C, raise 10 days.
3, after step 2 raises 10 days, detect whether every small brown rice planthopper in tri-bottles of Oryza sativa L. of A, B, C contains RSV respectively by qRT-PCR method.
Concrete operations are as follows: extract the RNA of every small brown rice planthopper Scorpio by Trizol method, according still further to the operating procedure of 2,3 in embodiment 1 step 2, obtain the qRT-PCR result of every small brown rice planthopper.According to gained amplification curve and solubility curve according to judging whether small brown rice planthopper to be measured obtains poison as follows: first, get rid of the testing sample of non-S shape amplification curve according to amplification curve；Then, in the testing sample of all of S shape amplification curve, determine whether whether small brown rice planthopper to be measured obtains poison according to solubility curve, if gained solubility curve only one of which peak, and solution temperature corresponding to this peak value is 80-85 DEG C, then corresponding small brown rice planthopper preliminary judgement to be measured obtains poison success, otherwise, then corresponding small brown rice planthopper to be measured does not obtain poison success；Finally, be that the q-PCR product obtaining poison sample carries out the agarose gel electrophoresis of 1% (mass percent) by preliminary judgement, run glue detection checking, occur conspicuous object band be defined as obtain poison sample.
4, the testing result according to step 3, add up the small brown rice planthopper fed through tri-groups of man-made feeds containing variable concentrations RSV of A, B, C the poison rate that obtains (obtain poison rate refer to survival small brown rice planthopper obtain poison rate) and survival rate.
In experiment, having carried out parallel twice independent experiment altogether, once obtained poison rate (being provided with three repetitions, result takes average) for calculating, another time is used for calculating survival rate (being provided with twice repetition, result takes average).
5, result
Result shows:
1, after the counteracting toxic substances small brown rice planthopper of two days is transferred to and raises 10 days in nontoxic fresh water rice seedling, RSV comes in small brown rice planthopper body, it is possible to detected by qRT-PCR.Test result indicate that, with the small brown rice planthopper of the man-made feeds counteracting toxic substances that RSV concentration is 1200ng/ μ l obtain poison rate average out to 96%；RSV concentration be the small brown rice planthopper of the man-made feeds counteracting toxic substances of 600ng/ μ l and 300ng/ μ l on average obtain poison rate respectively 71%, 57% (Fig. 1 and Biao 2).The solubility curve (left figure) of the qRT-PCR of the small brown rice planthopper tissue extract (titer) of known rice stripe virus concentration (100ng/ μ l) and part obtain the solubility curve (right figure) of the qRT-PCR of the small brown rice planthopper of poison as shown in Figure 2.
The small brown rice planthopper of the table 2 man-made feeds counteracting toxic substances containing variable concentrations RSV on average obtain poison rate
2, test result indicate that, with the survival rate average out to 70% of the small brown rice planthopper of the man-made feeds counteracting toxic substances that RSV concentration is 1200ng/ μ l；RSV concentration is average viability respectively 75%, 85% (Fig. 1 and the Biao 3) of the small brown rice planthopper of the man-made feeds counteracting toxic substances of 600ng/ μ l and 300ng/ μ l.
The average viability of the small brown rice planthopper of the table 3 man-made feeds counteracting toxic substances containing variable concentrations RSV
Visible, when in man-made feeds, the concentration of RSV, within the scope of 300-1200ng/ μ l, can be obtained by the small brown rice planthopper of counteracting toxic substances and higher obtain poison rate and survival rate simultaneously, especially when RSV concentration is higher than 1200ng/ μ l, the poison rate that obtains of small brown rice planthopper reaches more than 90%.
Comparative example, fed band RSV rice plant method by tradition and make nontoxic to obtain poison without small brown rice planthopper
1, experimental technique
(1) rice plant fallen ill from rice terrace collection, is transplanted to hot-house culture；
(2) nontoxic small brown rice planthopper obtains poison: take 2 age nontoxic small brown rice planthopper, Nature enemy 8 hours, be placed on band poison plant, feed 2 days；
(3) small brown rice planthopper after raising poison is transferred in fresh nontoxic rice seedlings raise；
Start in nontoxic small brown rice planthopper body after (4) 3 days RSV to be detected.The concrete grammar of detection RSV is referring to embodiment.
2, result
Result shows: in laboratory environment, feeds nontoxic small brown rice planthopper with band poison rice plant, and the poison rate that obtains of small brown rice planthopper is on average less than 20%.
It addition, Li Shuo et al. adopts fresh in vitro disease leaf to raise poison, the poison rate that obtains of small brown rice planthopper is 11.8%[1].The research of Wang Shuan et al. shows, nontoxic small brown rice planthopper is after catching an illness and taking food 2h in rice strain, and its band poison rate starts to tend to steadily, substantially remain in about 30%[2].Visible, that traditional employing feeds small brown rice planthopper with viral disease leaf method, its small brown rice planthopper obtain poison rate really relatively low.
[1]. Li Shuo, Wang Shijuan, slander Jin Yan, etc. the method that small brown rice planthopper quickly obtains rice stripe virus from vitro sick leaf. Jiangsu's agriculture journal, 2014,30 (2): 449-451.
[2]. Wang Shuan, Yang Yizhong, Zhong Chongxiang. strip virus RSV is the research of transfer rate between small brown rice planthopper and rice plant. China's agronomy circular, 2011,27 (05): 328-332.
Claims (10)
1. the method making small brown rice planthopper obtain rice stripe virus, comprises the steps: to feed the man-made feeds containing rice stripe virus to small brown rice planthopper, so that described small brown rice planthopper obtains rice stripe virus；
Described man-made feeds composed as follows: containing be more than or equal to rice stripe virus described in 300ng in man-made feeds described in every μ L；Surplus is concentration is the aqueous sucrose solution of 50g/L.
2. method according to claim 1, it is characterised in that: described man-made feeds are to prepare according to the method comprised the steps: with carry rice stripe virus rice leaf for material, extract rice stripe virus；The rice stripe virus extracted is joined in the aqueous sucrose solution that concentration is 50g/L, makes the final concentration of described rice stripe virus be more than or equal to 300ng/ μ L, obtain described man-made feeds.
3. method according to claim 2, it is characterised in that: described " with carry rice stripe virus rice leaf for material, extract rice stripe virus " method comprise the steps:
(a1) the described rice leaf liquid nitrogen grinding carrying rice stripe virus is become fine-powdered, add PBS, 4 DEG C of concussion 15min；
(a2) 4 DEG C, 14000-16000g is centrifuged 10min, takes supernatant；
(a3) filtration sterilization: by the membrane filtration that described supernatant aperture is 0.22 μm, take filtrate；
(a4) described filtrate is carried out dialysis concentration, thus obtaining described rice stripe virus.
4. according to described method arbitrary in claim 1-3, it is characterised in that: in the process, feed small brown rice planthopper described in normal direction by thin film and feed described man-made feeds.
5. method according to claim 4, it is characterized in that: described thin film feeding method comprises the steps: to be placed in the glass tubing of both ends open by small brown rice planthopper, by one end double-layer air-permeable sealed membrane sealing of described glass tubing, the other end first seals with one layer of breathable sealing film, aseptic face outwardly, film adds described man-made feeds, again the aseptic of another layer of breathable sealing film is faced inner envelope feedstuff, being placed by described glass tube horizontal makes the liquid level that the described man-made feeds in described two-layer breathable sealing film are formed vertical, it is placed in 25 DEG C, in the environment of periodicity of illumination 16h/8h, described small brown rice planthopper in described glass tubing sucks described man-made feeds by piercing through sealed membrane, every day changes once described man-made feeds, feed 2 days altogether.
6. according to described method arbitrary in claim 1-5, it is characterised in that: in described method, after feeding described man-made feeds to described small brown rice planthopper, also include described small brown rice planthopper is transferred to the step raised in the rice seedlings not carrying rice stripe virus 10 days.
7. for making small brown rice planthopper obtain the man-made feeds of rice stripe virus, it is composed as follows: containing be more than or equal to rice stripe virus described in 300ng in man-made feeds described in every μ L；Surplus is concentration is the aqueous sucrose solution of 50g/L.
8. the method for man-made feeds described in preparation claim 7, for the method preparing described man-made feeds described in Claims 2 or 3.
9. the man-made feeds described in claim 7 or the method described in claim 8 make small brown rice planthopper obtain the application in rice stripe virus.
10. man-made feeds described in arbitrary described method or claim 7 application in obtaining the small brown rice planthopper carrying rice stripe virus in claim 1-6 and 8.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610131539.XA CN105746439B (en) | 2016-03-09 | 2016-03-09 | Small brown rice planthopper is made to obtain the method and its man-made feeds of rice stripe virus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610131539.XA CN105746439B (en) | 2016-03-09 | 2016-03-09 | Small brown rice planthopper is made to obtain the method and its man-made feeds of rice stripe virus |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105746439A true CN105746439A (en) | 2016-07-13 |
| CN105746439B CN105746439B (en) | 2018-07-06 |
Family
ID=56332860
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610131539.XA Active CN105746439B (en) | 2016-03-09 | 2016-03-09 | Small brown rice planthopper is made to obtain the method and its man-made feeds of rice stripe virus |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105746439B (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106942162A (en) * | 2017-04-25 | 2017-07-14 | 遵义医学院 | A kind of device and its application method suitable for white backed planthopper feeding method RNA interference experiments |
| CN110396503A (en) * | 2019-07-22 | 2019-11-01 | 中国农业大学 | A method of Polerovirus rape yellow virus is obtained using black peach aphid |
| CN114946569A (en) * | 2021-12-30 | 2022-08-30 | 江苏省农业科学院 | Novel method for improving rice stripe virus infection efficiency through leaf inoculation |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101401562A (en) * | 2008-11-13 | 2009-04-08 | 江苏省农业科学院 | Acquiring method for special small brown rice planthopper for inoculating rice black-streaked dwarf virus |
| CN101633908A (en) * | 2009-09-01 | 2010-01-27 | 江苏省农业科学院 | Method for laodelphax striatellus to obtain rice black-streaked dwarf virus (RBSDV) from refrigerated diseased leaves |
| CN102578050A (en) * | 2012-03-02 | 2012-07-18 | 浙江大学 | Method for obtaining brown plant hoppers with rice ragged stunt virus and application of method |
| CN102630640A (en) * | 2012-03-02 | 2012-08-15 | 浙江大学 | Method for obtaining sogatella furcifera carrying SRBSDV (southern rice black-streaked dwarf virus) and application of sogatella furcifera |
| CN102870743A (en) * | 2012-10-12 | 2013-01-16 | 江苏省农业科学院 | Method for preserving rice black-streaked dwarf virus indoor living bodies |
| CN105340837A (en) * | 2015-11-26 | 2016-02-24 | 中国农业科学院植物保护研究所 | Wheat aphid pure artificial feed and artificial mass feeding method |
-
2016
- 2016-03-09 CN CN201610131539.XA patent/CN105746439B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101401562A (en) * | 2008-11-13 | 2009-04-08 | 江苏省农业科学院 | Acquiring method for special small brown rice planthopper for inoculating rice black-streaked dwarf virus |
| CN101633908A (en) * | 2009-09-01 | 2010-01-27 | 江苏省农业科学院 | Method for laodelphax striatellus to obtain rice black-streaked dwarf virus (RBSDV) from refrigerated diseased leaves |
| CN102578050A (en) * | 2012-03-02 | 2012-07-18 | 浙江大学 | Method for obtaining brown plant hoppers with rice ragged stunt virus and application of method |
| CN102630640A (en) * | 2012-03-02 | 2012-08-15 | 浙江大学 | Method for obtaining sogatella furcifera carrying SRBSDV (southern rice black-streaked dwarf virus) and application of sogatella furcifera |
| CN102870743A (en) * | 2012-10-12 | 2013-01-16 | 江苏省农业科学院 | Method for preserving rice black-streaked dwarf virus indoor living bodies |
| CN105340837A (en) * | 2015-11-26 | 2016-02-24 | 中国农业科学院植物保护研究所 | Wheat aphid pure artificial feed and artificial mass feeding method |
Non-Patent Citations (2)
| Title |
|---|
| 冯崇川: "蚜虫薄膜饲毒的简易方法", 《植物保护》 * |
| 李硕 等: "灰飞虱从离体病叶快速获得水稻条纹病毒的方法", 《江苏农业学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106942162A (en) * | 2017-04-25 | 2017-07-14 | 遵义医学院 | A kind of device and its application method suitable for white backed planthopper feeding method RNA interference experiments |
| CN110396503A (en) * | 2019-07-22 | 2019-11-01 | 中国农业大学 | A method of Polerovirus rape yellow virus is obtained using black peach aphid |
| CN114946569A (en) * | 2021-12-30 | 2022-08-30 | 江苏省农业科学院 | Novel method for improving rice stripe virus infection efficiency through leaf inoculation |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105746439B (en) | 2018-07-06 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104521957A (en) | Applications of guaiacol in rice stripe disease | |
| Kawsar et al. | Studies of thrombolytic, antioxidant and cytotoxic properties of two asteraceous plants of Bangladesh | |
| CN105746439A (en) | Method and artificial feed for enabling laodelphax striatellus to obtain rice stripe viruses | |
| CN103599071A (en) | Preparation method of polyinosinic-polycytidylic acid dry powder | |
| CN106668854A (en) | Quadrivalent subunit influenza vaccine and preparation method thereof | |
| CN106727623A (en) | Application of the Tang oligosaccharide in anti-avian leukosis virus preparation is prepared | |
| KR20080084992A (en) | Use of elderberry extract | |
| Talboys | The possible significance of toxic metabolites of Verticillium albo-atrum in the development of hop wilt symptoms | |
| CN115629169A (en) | Extract and preparation containing the same | |
| CN104257745B (en) | A kind of Polysaccharides from Prunella vulgaris L extract and preparation method thereof, preparation and purposes | |
| CN108175743A (en) | A kind of preparation method of Maxingshigan oral liquid | |
| CN103349782B (en) | The preparation technology of Eimeria Tenella Yolk antibody target slow-release preparation | |
| US20040142047A1 (en) | Korean acanthopanax senticosus extract, protein extract, crude protein-polysaccharide which were extracted from korean acanthopanax senticosus, and immunoregulating compositions comprising the same and use thereof | |
| ES2971275T3 (en) | Tobacco leaf extract and use for the treatment of tobacco addiction | |
| CN103932981A (en) | Heparin sodium tube-sealing injection | |
| CN102198263B (en) | Rhizoma Dioscoreae esculentae Sporamin albumen is preparing the application in prevention and therapy tumour medicine and health product | |
| CN107158370A (en) | Racoon dog canine distemper freeze-dried live vaccine and its preparation method and application | |
| CN107056959A (en) | Jerusalem artichoke moderate resistance HSV 1, the composition of RSV, EV 71 and preparation | |
| CN105560302B (en) | Application of geranium water extract in preparation of anti-angiogenesis drugs | |
| Takahashi et al. | Detection of the causal agent associated with hop stunt disease in Japan. | |
| JP2022551319A (en) | American cockroach extract, formulation, preparation method and use thereof | |
| CN107982356A (en) | A kind of oral drugs for treating respiratory tract infection | |
| CN104402995A (en) | Preparation method and application of anti-influenza virus chicken egg-yolk antibody | |
| CN105505867B (en) | For separating the composition and its separating liquid of lymphocyte | |
| CN104231101B (en) | A kind of tree-of-heaven polyoses producing method and the application on control tobacco virus thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |