CN109456947A - A kind of method that efficient indoor winter saves propagation southern rice black-streaked dwarf virus - Google Patents
A kind of method that efficient indoor winter saves propagation southern rice black-streaked dwarf virus Download PDFInfo
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- 241000756822 Southern rice black-streaked dwarf virus Species 0.000 title claims abstract description 87
- 238000000034 method Methods 0.000 title claims abstract description 33
- 241000209094 Oryza Species 0.000 claims abstract description 65
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 65
- 235000009566 rice Nutrition 0.000 claims abstract description 65
- 241000176086 Sogatella furcifera Species 0.000 claims abstract description 62
- 239000002574 poison Substances 0.000 claims abstract description 18
- 231100000614 poison Toxicity 0.000 claims abstract description 18
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract description 10
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 8
- 210000002845 virion Anatomy 0.000 claims abstract description 7
- 238000000605 extraction Methods 0.000 claims abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 6
- 239000000284 extract Substances 0.000 claims abstract description 5
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract description 5
- 230000001681 protective effect Effects 0.000 claims abstract description 5
- 238000002347 injection Methods 0.000 claims description 14
- 239000007924 injection Substances 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 14
- 238000007710 freezing Methods 0.000 claims description 12
- 230000008014 freezing Effects 0.000 claims description 12
- 230000001902 propagating effect Effects 0.000 claims description 7
- 210000002751 lymph Anatomy 0.000 claims description 5
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 claims description 4
- 239000006228 supernatant Substances 0.000 claims description 4
- 238000005119 centrifugation Methods 0.000 claims description 3
- 229910000147 aluminium phosphate Inorganic materials 0.000 claims description 2
- 239000000872 buffer Substances 0.000 claims description 2
- 239000000203 mixture Substances 0.000 claims description 2
- 238000003860 storage Methods 0.000 claims description 2
- 241000700605 Viruses Species 0.000 abstract description 16
- 208000015181 infectious disease Diseases 0.000 abstract description 9
- 238000004321 preservation Methods 0.000 abstract description 7
- 239000000843 powder Substances 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 2
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- 238000001514 detection method Methods 0.000 description 12
- 238000003757 reverse transcription PCR Methods 0.000 description 11
- 238000001962 electrophoresis Methods 0.000 description 5
- 238000000520 microinjection Methods 0.000 description 5
- 241001674048 Phthiraptera Species 0.000 description 3
- 230000003321 amplification Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 201000010099 disease Diseases 0.000 description 3
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 231100000252 nontoxic Toxicity 0.000 description 3
- 230000003000 nontoxic effect Effects 0.000 description 3
- 238000003199 nucleic acid amplification method Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 241001498622 Cixius wagneri Species 0.000 description 2
- 241000196324 Embryophyta Species 0.000 description 2
- 241000702658 Fijivirus Species 0.000 description 2
- 241000702247 Reoviridae Species 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 108090000565 Capsid Proteins Proteins 0.000 description 1
- 101710132601 Capsid protein Proteins 0.000 description 1
- 102100023321 Ceruloplasmin Human genes 0.000 description 1
- 101710094648 Coat protein Proteins 0.000 description 1
- 102100021181 Golgi phosphoprotein 3 Human genes 0.000 description 1
- 101710125418 Major capsid protein Proteins 0.000 description 1
- 101710141454 Nucleoprotein Proteins 0.000 description 1
- 241000819999 Nymphes Species 0.000 description 1
- 241001085205 Prenanthella exigua Species 0.000 description 1
- 101710083689 Probable capsid protein Proteins 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 238000009644 cyrogenic grinding Methods 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 230000006806 disease prevention Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 210000002919 epithelial cell Anatomy 0.000 description 1
- 210000001035 gastrointestinal tract Anatomy 0.000 description 1
- 238000000227 grinding Methods 0.000 description 1
- 238000000703 high-speed centrifugation Methods 0.000 description 1
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- 102000039446 nucleic acids Human genes 0.000 description 1
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- 102220182402 rs80133178 Human genes 0.000 description 1
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- 238000010257 thawing Methods 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
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- C12N7/00—Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
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- C12N2720/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsRNA viruses
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Abstract
A kind of method that efficient indoor winter saves propagation southern rice black-streaked dwarf virus, it is the rice freezen protective that will infect southern rice black-streaked dwarf virus (SRBSDV), liquid nitrogen processing is lower will to freeze rice grind into powder, SRBSDV virion is extracted with phosphate buffer (pH 7.4) again, and the viral extract of extraction is injected into the internal of white backed planthopper, it can be obtained the white backed planthopper of the carrying southern rice black-streaked dwarf virus for passing poison.The white backed planthopper that infection southern rice black-streaked dwarf virus is obtained using this method, can solve the bottleneck that rice of catching an illness is difficult to long-term preservation (especially rice cannot survive under winter cryogenic conditions).The present invention can be also used for interaction of the research SRBSDV virus between the intracorporal infection mechanism of white backed planthopper, and virus and mediator white backed planthopper.
Description
Technical field
The invention belongs to field of biotechnology, specifically, being a kind of efficient indoor winter preservation propagation south rice
The method of black-streaked dwarf virus.
Background technique
Southern rice black-streaked dwarf virus (southern rice black-streaked dwarf virus, SRBSDV)
Be the 2nd group of Reoviridae (Reoviridae) Fijivirus category (Fijivirus) a novel species (Zhou Guohui etc.,
2008).White backed planthopper (Sogatella furcifera) is the unique propagation mediator of the virus, with persistently, walk around to proliferous type side
Formula is propagated.SRBSDV is since, due to passing the activity of migrating of virus mediator white backed planthopper, fast propagation, to south China rice
The Rice Production in area causes great loss.The virus also results in the Rice Production of Vietnam, Japan and South Korea serious
It influences.2009 and 2010, the area that China's rice is infected by SRBSDV was up to 33.33 ten thousand hm2With 66.67 ten thousand hm2.Hereafter, by
In actively taking disease prevention and control measure, the occurring area of the virus is controlled in 250,000 hm2Left and right.But according to Liu Wancai etc.
(2016) it reports, from 2016, which had the trend that comes back again.
Since virus report, the researcher with regard to there are many is active in research disease-white backed planthopper-rice interaction pass
System, effective prevention and control measure etc..According to the occurrence regularity of SRBSDV, " controlling worm diseases prevention " is to control effectively arranging for disease prevalence
It applies.But by the white backed planthopper service life is short and rice of catching an illness be difficult to long-term preservation (especially under the cryogenic conditions in winter, rice
It is easily withered) limitation, researchers generally require 4 annual~May is coastal in south China and the rice regions such as Hainan Province
Field acquisition catch an illness rice, taking back laboratory qualification and expanding numerous rear can be used for further scientific research.Acquisition and identification dye
This process of sick rice is complicated, time-consuming and laborious and expends financial resources, it is often more important that significantly limits laboratory research
The progress of the virus.Some effort have also been made in terms of saving the virus indoors in researcher.CN102630640A discloses one kind
Preparation method and its application for carrying the white backed planthopper of southern rice black-streaked dwarf virus, describe white backed planthopper in freeze-thaw
The rice caught an illness on feeding, white backed planthopper can carry the virus.In fact, this method and unstable, Liu et al. (2016) card
Bright white backed planthopper can obtain poison from the rice of defrosting, but virus can not cause this white in the internal duplication of white backed planthopper
Backward flight lice will not propagate SRBSDV.And pass through electron microscope observation, Liu et al. discovery, the virion of SRBSDV after freezing
Coat protein rupture, so that virion enteral in white backed planthopper loses protection and digested, or because of virus capsid protein
Rupture cannot cannot break through the middle Gut barrie r of white backed planthopper by the Receptor recognition on midgut epithelial cell without can enter middle intestines.
So far, the method that indoor winter saves SRBSDV is still study the virus during urgent need to resolve ask
Topic.
Summary of the invention
The technical problems to be solved by the present invention are: being directed to the deficiency of above-mentioned existing SRBSDV Techniques of preserving, one kind is provided
Efficient interior winter saves the method for propagating southern rice black-streaked dwarf virus.
In order to solve the above technical problems, the technical scheme adopted by the invention is that: a kind of efficient indoor winter preservation biography
The method for broadcasting southern rice black-streaked dwarf virus, the method steps are as follows:
A. the paddy rice low freezen protective of southern rice black-streaked dwarf virus will be infected；
B. the phosphate buffer for being 7.4 with pH value extracts to extract SRBSDV virion freezing rice, obtains
SRBSDV extracting solution；
C. SRBSDV extracting solution is injected into the lymph not with the white backed planthopper of SRBSDV, obtains that taking for poison can be passed
White backed planthopper with SRBSDV.
Cryogenic freezing storage temperature in the step A is -60~-100 DEG C.
SRBSDV extracting solution is after the rice liquid feeding nitrogen of freezing is pulverized in the step B, and it is 7.4 that pH value, which is added,
Phosphate buffer, every gram of freezing rice add 800~2000 μ L phosphate buffers, mix, and in 4 DEG C of 0.5~3h of extraction, centrifugation is taken
Supernatant obtains.
In the step C, the injection volume of SRBSDV extracting solution is that every white backed planthopper injects 20~150nL；For injecting
White backed planthopper be 3~5 age nymphs and sprout wings 1~2d adult, injection position be chest of the white backed planthopper in addition to foot and wing.
Specifically, steps are as follows for operation of the present invention:
A. it the acquisition and preservation of contamination rice: from the rice sample of field acquisition suspected infection SRBSDV, is examined through RT-PCR
After surveying identification, taking qualification result is the rice of SRBSDV infection, is placed in -60~-100 DEG C of freezen protectives；
B. the extraction to SRBSDV virion: using liquid nitrogen flash freezer grinding rice at powder, then the phosphoric acid for being 7.4 with pH value
Buffer is in 4 DEG C of extraction 0.5~3h of Rice Powder to extract SRBSDV virion, and after high speed centrifugation, supernatant is
SRBSDV extracting solution；
C. SRBSDV extracting solution is injected into the lymph not with the white backed planthopper of SRBSDV with microinjection instrument；
D. the white backed planthopper after injection SRBSDV is fed with the rice seedling of health, after 3~5d, is carried on the back with RT-PCR method dialogue
The malicious situation of the band of plant hopper is checked that SRBSDV is expanded in white backed planthopper body；
E. the white backed planthopper of the injection of healthy rice seedlings feeding 5d SRBSDV is transferred on the rice of early tillering stage, into
Row man power single stem rice passes poison test.The detection of rice band poison situation is carried out to rice leaf with RT-PCR method, white backed planthopper smoothly will
SRBSDV is passed on rice；
F. nontoxic white backed planthopper is connected to detection in step E and detects the band of white backed planthopper after 7d on virulent rice
Malicious situation, white backed planthopper smoothly can obtain poison from this rice.
The method that the present invention uses microinjection, by PBS (phosphate buffer, pH=7.4) from the infection SRBSDV of freezing
The viral extract extracted in rice is injected directly into the lymph not with the white backed planthopper of SRBSDV, as a result SRBSDV energy
Amplification is replicated in white backed planthopper body, and white backed planthopper can pass to the virus on healthy rice.Method provided by the invention
Can the SRBSDV of freezing be gone in white backed planthopper body and be replicated, and white backed planthopper can pass to SRBSDV on rice, solve
SRBSDV material subculture problem in winter room, researcher no longer need annual to acquire the rice of infection SRBSDV and white from field
Backward flight lice.
In this way, virus is extracted the present invention provides the SRBSDV material of freezen protective is ground, then through PBS, finally with aobvious
The method of microinjection by virus injection to the white backed planthopper body not with SRBSDV in, successfully make white backed planthopper with poison method.
This method successfully solves the technical bottleneck that SRBSDV and subculture cannot be effectively saved in winter room, goes on smoothly section for researcher
It learns research and provides the guarantee of experimental material.
Detailed description of the invention
Fig. 1 is the electrophoretogram of SRBSDV in the rice sample of RT-PCR detection field acquisition.
Wherein: M maker；1~4 is rice sample.
Fig. 2 is electrophoretogram of the white backed planthopper with malicious situation after RT-PCR detection injection SRBSDV extracting solution 5d.
Wherein: M maker；1~30 is the white backed planthopper sample that injection has SRBSDV virus.
Fig. 3 is that RT-PCR detection injection obtains the electrophoretogram that malicious white backed planthopper passes the rice band poison situation of poison.
Wherein: M maker；1~30 rice sample to be passed poison.
Fig. 4 is that RT-PCR white backed planthopper of the detection without SRBSDV is obtained from the malicious successfully rice of the malicious white backed planthopper biography of injection band
The electrophoretogram of poison.
Wherein: M maker；1~30 is the white backed planthopper sample for obtaining poison.
Specific embodiment
Below in conjunction with specific embodiment, the invention will be further described.
1. acquisition, identification and the preservation of field SRBSDV infection rice
From the rice sample of field acquisition suspected infection SRBSDV, using all pretty equal (2010, Zhou Qian, Zhu Junzi, Liang Jin
Just, southern rice black-streaked dwarf virus is waited quickly to detect [J] genomics and applied biology, 2010,29 (5): 1009-
1012.) the SRBSDV in RT-PCR method detection rice.Specifically: referring to precious bioengineering (Dalian) Co., Ltd
(TaKaRa) nucleic acid extraction kit specification (Code No.9767) extracts rice sample total serum IgE, then with the first of TaKaRa
Chain cDNA synthetic agent box (Code No.6210) synthesizes cDNA.PCR reaction system: Ex Taq Version 2.0 (TaKaRa)
10 μ L, each 1 μ L of upstream and downstream primer (S10F/S10R), 1 μ L of cDNA template add ddH2O complements to reaction system final volume
20 μ L, PCR response procedures are as follows: 94 DEG C of 2min of initial denaturation are denaturalized 94 DEG C of 30s, and anneal 55 DEG C of 30s, 72 DEG C of extension 1min, circulation
Amplification 30 times, last 72 DEG C of extensions 10min.It is expected that amplification purpose band is 477bp, 1.5% Ago-Gel of amplified production
Electrophoresis is analyzed, as a result as shown in Figure 1, rice sample is SRBSDV infection.The rice for being accredited as the SRBSDV positive is encapsulated in
Valve bag is placed in -80 DEG C of preservations.
2. the acquisition and raising of nontoxic white backed planthopper
Never the rice field that SRBSDV occurs collects white backed planthopper nymph, and the indoor non-nontoxic seedling of insect-proof rice is raised,
Temperature is 25~30 DEG C, relative humidity 70% or so, photoperiod 14L:10D.It is more than indoor raising three generations.And survey sample,
Ensure white backed planthopper without SRBSDV virus.
The preparation of 3.SRBSDV extracting solution
The rice 0.5g for taking cryogenic freezing adds liquid nitrogen grinding, and powder is transferred to the sterile centrifugation tube of 1.5mL, is added 800 μ L's
PBS (phosphate buffer, pH=7.4) oscillation mixes, 1.5h (therebetween, mixing is rocked in not timing) under the conditions of centrifuge tube is placed in 4 DEG C,
12000rpm/min is centrifuged 10min, and supernatant is SRBSDV extracting solution.
4. SRBSDV is injected into white backed planthopper body by microinjection
Collect 4 age white backed planthopper nymphs of aforementioned indoor raising, CO21~2min is anaesthetized, microinjection instrument is used
SRBSDV extracting solution is injected into white backed planthopper from the mesopodium and metapedes base portion of white backed planthopper by (Drummond Scientific)
In lymph, volume injected is the every worm of 100nL/.White backed planthopper after injection is raised with the rice seedling for being uninfected by SRBSDV virus
It supports.After 5d, RT-PCR method single head detects the malicious situation of white backed planthopper band.Detection method is the same as the field rice band poison in abovementioned steps 1
As a result detection is shown in that Fig. 2, display SRBSDV are expanded in white backed planthopper body.
5. white backed planthopper passes poison and obtains malicious test
The white backed planthopper with SRBSDV virus that injection is obtained, is transferred on the rice of early tillering stage with 10 heads/plant, is adopted
Malicious method is connect with single plant, is repeated 30 times.White backed planthopper removes after feeding 3d on rice.Rice is placed in solarium's culture of no worm
After 30d, the detection of rice band poison situation is carried out to rice leaf with RT-PCR method (the same), testing result is shown in Fig. 3, shows white backward flight
Lice smoothly passes to SRBSDV on rice.The 3 age white backed planthoppers not with SRBSDV virus are transferred to above-mentioned detection band poison
Rice on, after 7d using RT-PCR method (the same) single head detection white backed planthopper band poison situation, detect 30 cephalonts altogether, as a result see
Fig. 4, white backed planthopper can smoothly obtain SRBSDV virus from this rice.
Claims (6)
1. a kind of efficient indoor winter saves the method for propagating southern rice black-streaked dwarf virus, which is characterized in that this method
Steps are as follows:
A. the paddy rice low freezen protective of southern rice black-streaked dwarf virus will be infected；
B. the phosphate buffer for being 7.4 with pH value extracts to extract SRBSDV virion freezing rice, obtains SRBSDV
Extracting solution；
C. SRBSDV extracting solution is injected into the lymph not with the white backed planthopper of SRBSDV, obtains the carrying that can pass poison
The white backed planthopper of SRBSDV.
2. a kind of efficient indoor winter as described in claim 1 saves the method for propagating southern rice black-streaked dwarf virus,
It is characterized in that, the cryogenic freezing storage temperature in the step A is -60~-100 DEG C.
3. a kind of efficient indoor winter as described in claim 1 saves the method for propagating southern rice black-streaked dwarf virus,
It is characterized in that, SRBSDV extracting solution is that phosphoric acid buffer is added after the rice liquid feeding nitrogen of freezing is pulverized in the step B
Liquid, 1g freezing rice add 800~2000 μ L phosphate buffers, mix, and in 4 DEG C of 0.5~3h of extraction, centrifugation takes supernatant, obtains
It arrives.
4. a kind of efficient indoor winter as described in claim 1 saves the method for propagating southern rice black-streaked dwarf virus,
It is characterized in that, the injection volume of SRBSDV extracting solution is the every worm of 20~150nL/ in the step C.
5. a kind of efficient indoor winter as described in claim 1 saves the method for propagating southern rice black-streaked dwarf virus,
It is characterized in that, the white backed planthopper in the step C for injection is the adult of 3~5 age nymphs and the 1~2d that sprouts wings.
6. a kind of efficient indoor winter as described in claim 1 saves the method for propagating southern rice black-streaked dwarf virus,
The injection position chosen when it is characterized in that, injecting white backed planthopper in the step C is chest of the white backed planthopper in addition to foot and wing.
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| CN113693033A (en) * | 2021-09-14 | 2021-11-26 | 广西壮族自治区农业科学院 | Method for evaluating resistance of rice variety to southern rice black-streaked dwarf disease based on artificial inoculation |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113693033A (en) * | 2021-09-14 | 2021-11-26 | 广西壮族自治区农业科学院 | Method for evaluating resistance of rice variety to southern rice black-streaked dwarf disease based on artificial inoculation |
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