CN102578050A - Method for obtaining brown plant hoppers with rice ragged stunt virus and application of method - Google Patents
Method for obtaining brown plant hoppers with rice ragged stunt virus and application of method Download PDFInfo
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- CN102578050A CN102578050A CN2012100525829A CN201210052582A CN102578050A CN 102578050 A CN102578050 A CN 102578050A CN 2012100525829 A CN2012100525829 A CN 2012100525829A CN 201210052582 A CN201210052582 A CN 201210052582A CN 102578050 A CN102578050 A CN 102578050A
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Abstract
The invention discloses a method for obtaining brown plant hoppers with rice ragged stunt virus and application of the method. Living rice plants infected with RRSV (rice ragged stunt virus) or frozen rice ill samples are used for feeding the brown plant hoppers, so that the brown plant hoppers are infected with the rice ragged stunt virus, and then the brown plant hoppers are transferred onto healthy rice laminae without the rice ragged stunt virus to be fed with healthy rice laminae without the rice ragged stunt virus until the brown plant hoppers live through the circulation period of the virus and then are infected with the virus. The brown plant hoppers with the virus can be used for screening RRSV-resistant monoclonal antibody with high flexibility and specificity, and accordingly the method provides a material and technical means for early warning the virus and scientifically preventing and controlling the virus. Simultaneously, the brown plant hoppers with the RRSV can be used for rice inoculation experiments to identify resistance of rice in different types and screening out the types of rice capable of resisting diseases, and are served for breeding for disease resistance. In addition, the brown plant hoppers with the RRSV provide a material basis for researching interaction, virus spreading mechanism and the like between the RRSV and brown plant hopper interbody.
Description
Technical field
Field under the present invention is a biological technical field, especially relates to a kind of preparation method and application thereof of carrying the brown planthopper of paddy rice tingia dwarf virus.
Background technology
(Rice ragged stunt virus is a kind of new virus of finding at eighties of last century end of the seventies RRSV) to paddy rice tingia dwarf virus, in Southeast Asia, the local generation in East Asia and some countries of South Asia, Rice Production is caused certain influence.Be popular at present mainly the ground such as Guangxi, Fujian, Hunan, Jiangxi, Guizhou, Yunnan of China.Dark green the stunting of diseased plant performance, the leaf curling indentation is incised, and has elongated arteries and veins swollen at blade back and leaf sheath.Symptoms such as morbidity can not be eared before tillering stage, and the later stage falls ill, and heading is incomplete, grain does not enrich.The general underproduction 10%～20% of morbidity field piece, the serious underproduction can reach 80%～100%, and being influences one of more serious virus disease of rice yield.RRSV is the typical member of Reoviridae Oryzavirus (Oryzavirus), causes the paddy rice tingia disease of stunting.The RRSV genome comprises 10 double-stranded RNA fragments (S1-S10), and the virus that two other genus of the electrophoresis pattern of its morphosis and genome double-stranded RNA and this section-plant reovirus belongs to (Phytoreovirus) and Fijivirus genus (Fifivirus) has bigger difference.RRSV has double capsid, is icosahedral structure of virus, and the diameter of intact virus plastochondria is about 65nm, the about 50nm of core granule.Can be observed under the Electronic Speculum in the viroplast (viro-plasm) that diameter 50～66nm or the virion of 40nm be distributed in susceptible rice leaf bast cell; Also can be observed diameter 40～45nm or two types of crystalline particles of spherical junctions of 50～75nm in the organ or tissue in the band poisonous insect body, assemble or be arranged in dispersedly in the cytoplasmic viroplast.Paddy rice sawtooth leaf dwarf virus is mainly propagated by amboceptor insect brown planthopper, and diseased plant juice, seed and soil all do not pass poison.Its natural host has paddy rice, yard grass, kyllinga brevifolia and barnyard grass grass, and the plant that manual work is infected has kinds more than 10 such as barley, wheat, oat, corn, sugarcane, barnyard grass.The amboceptor insect brown planthopper (Nilaparvatalugens) of RRSV is a kind of migrating property insect, and it is propagated and to be the persistence mode, but transovarian transmission not.The nymph of brown planthopper casts off a skin but does not lose poison, and the distribution of virus in polypide is the highest with content in the salivary gland.It is 3 hours (h) that the amboceptor minimum obtains the poison time, average 9 days (d).The shortest biography poison phase is 1h, and inoculation back 10-36d shows symptom, passes malicious rate and is approximately 40%.Larva maybe be higher than the biography toxic effect rate of adult, female worm and male worm, and long wing type is identical with brachypterism type biography toxic effect rate.Amboceptor passes poison and has the intermittent poison phase that passes usually, can keep 1-4 week or pass poison throughout one's life.The brown planthopper of different geographical population has similar biography poison characteristic.
RRSV is popular in a plurality of provinces of China at present; Had a strong impact on the paddy rice grain-production of China; And disease-resistant variety to use be most economical, the effective method of paddy rice virus disease; This just decision press for the authentication method of setting up a kind of RRSV paddy rice resistant variety, for excavation, the resistant variety of the paddy rice resource of anti-RRSV are identified and breed breeding provides technological means.Because the particularity of RRSV circulation way; Can not inoculate and transovarian transmission not with sick juice frictional inoculation mode; Patent of the present invention is employed in the brown planthopper that the mode of feeding on diseased plant or the frozen sick appearance obtains to carry RRSV, and this provides material and technical support for the screening of the monoclonal antibody specific of the evaluation of paddy rice resistant variety, disease-resistant seed selection, anti-RRSV and mutual work research of brown planthopper and RRSV.And provided by the inventionly make brown planthopper obtain the method for RRSV from freezing blade through the mode of feeding; Solved the problem that ill plant is difficult to long preservation; Widen the time of research, helped screening and the research work such as mutual work of brown planthopper and RRSV of the monoclonal antibody specific of the seed selection of disease-resistant variety, anti-RRSV.
Summary of the invention
The objective of the invention is to overcome the deficiency of prior art, a kind of preparation method and application thereof of carrying the brown planthopper of paddy rice tingia dwarf virus is provided.
The step of preparation method of brown planthopper of carrying paddy rice tingia dwarf virus is following:
1) acquisition of brown planthopper
After the rice field that the area takes place from non-paddy rice tingia dwarf virus is caught brown planthopper, raise at the nontoxic rice shoot of indoor non-insect-proof rice kind, raising temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Treat that brown planthopper adult post-coitum single head worm lays eggs separately; Randomly draw in the population 10% brown planthopper with RT-PCR and dot-ELISA methods analyst band poison situation; Keep not with the offspring of malicious female worm and set up indoor population; In later per generation, randomly drawed in the population 10% brown planthopper and confirms that with RT-PCR, dot-ELISA method brown planthopper is not with paddy rice tingia dwarf virus, promptly obtains the brown planthopper of not carrying paddy rice tingia dwarf virus;
2) brown planthopper obtains poison
Brown planthopper is obtained poison from the susceptible paddy rice diseased plant of freezing preservation
With filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Take out paddy rice diseased plant 1～3 strain of the infection paddy rice tingia dwarf virus of freezing preservation in-10～-80 ℃ of refrigerators; Place and to move in the circular open plastic containers or beaker that seal with nylon gauze after thawing 1～3 hour in greenhouse or the 4 ℃ of refrigerators; Put into then hungry 2～3 hours 20-300 only 1～2 age the brown planthopper nymph; Raise poison and after 1～2 day the brown planthopper of survival is moved to the healthy water rice seedling of supporting worm, raise malicious brown planthopper and on the healthy water rice seedling, follow back after date, detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method through about 10～15 days virus; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus;
Perhaps, brown planthopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber
With filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Carrying circular open plastic containers that paddy rice diseased plant 1～5 strain of RRSV seals with nylon gauze is enclosed in the Temperature and Humidity Control growth chamber and cultivates; Put into the nontoxic brown planthopper nymph in 20～300 1～2 ages after hungry 3 hours in each device; Raise poison and raise 7～12 days until the after date that follows back of tiding over virus on the brown planthopper immigration fresh and healthy paddy rice seedling with survival after 2～10 days; Detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus;
The brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed is identified the resistance of rice varieties, and the disease-resistant variety of Screening of Rice is anti-paddy rice tingia dwarf virus breeding for disease resistance service;
The brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the brown planthopper that will carry paddy rice tingia dwarf virus moves into plantation to be identified in the greenhouse or the 1.5-3 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 5 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole brown planthoppers after 2-7 days; Rice seedling is transplanted to greenhouse or growth chamber; Cultivation temperature is 26-30 ℃, and relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 30-40 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding;
Described brown planthopper of carrying paddy rice tingia dwarf virus is used to screen special, the sensitive monoclonal antibody of anti-paddy rice tingia dwarf virus, is the immunological method of paddy rice tingia dwarf virus in detection paddy rice and the brown planthopper body of core thereby set up with the monoclonal antibody;
The brown planthopper of carrying paddy rice tingia dwarf virus is used to study the mutual work between paddy rice tingia dwarf virus and the amboceptor insect and passes malicious mechanism and provides material base.
The beneficial effect that the present invention compared with prior art has: the mode of feeding of passing through that 1) provides makes brown planthopper obtain the method for paddy rice tingia dwarf virus from freezing blade; Solved the problem that ill plant is difficult to long preservation, the screening of the monoclonal antibody specific that the brown planthopper of band paddy rice tingia dwarf virus is used to carry out disease-resistant variety seed selection, anti-RRSV and the research work such as mutual work of brown planthopper and paddy rice tingia dwarf virus so just can at any time be provided.2) screen the monoclonal antibody that the brown planthopper of carrying RRSV can be used for screening anti-RRSV through serological method; Set up immunological method and can be used for the detection of the RRSV of field crops and brown planthopper effectively; To popular monitoring of generation of this virus disease, for the science prevention and control provide foundation.
Description of drawings
Fig. 1 RT-PCR method is identified the result of the susceptible paddy rice of RRSV;
Fig. 2 dot-ELISA method is identified the result of the susceptible paddy rice of RRSV;
The installation drawing that sick appearance under Fig. 3 artificial condition after the freezing preservation and the susceptible rice plant of living are raised malicious brown planthopper; Sick leaf after a left side-freezing preservation, the susceptible rice plant of the right side-work;
Fig. 4 dot-ELISA methods analyst brown planthopper obtain malicious situation;
Fig. 5 RT-PCR methods analyst brown planthopper obtain malicious situation;
Fig. 6 obtains malicious brown planthopper and screens anti-RRSV monoclonal antibody result;
Fig. 7 RT-PCR method detects and obtains the result that malicious brown planthopper passes malicious ability.
Embodiment
The brown planthopper that paddy rice tingia dwarf virus is carried in utilization of the present invention can be screened the monoclonal antibody of anti-RRSV; Identify that rice varieties is to this viral resistance; For excavation, the resistant variety seed selection of the paddy rice resource of anti-paddy rice tingia dwarf virus provides means and material base, also can be mutual material and the means of providing between further research RRSV and the vector insect.
The acquisition of brown planthopper: after the rice field that the area takes place from non-paddy rice tingia dwarf virus is caught brown planthopper, raise at the nontoxic rice shoot of indoor non-insect-proof rice kind, raising temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Treat that brown planthopper adult post-coitum single head worm lays eggs separately; Randomly draw in the population 10% brown planthopper with RT-PCR and dot-ELISA methods analyst band poison situation; Keep not with the offspring of malicious female worm and set up indoor population; In later per generation, randomly drawed in the population 10% brown planthopper and confirms that with RT-PCR, dot-ELISA method brown planthopper is not with paddy rice tingia dwarf virus, promptly obtains the brown planthopper of not carrying paddy rice tingia dwarf virus;
Brown planthopper is obtained poison from the susceptible paddy rice diseased plant of freezing preservation: with filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Take out paddy rice diseased plant 1～3 strain of the infection paddy rice tingia dwarf virus of freezing preservation in-10～-80 ℃ of refrigerators; Place and to move in the circular open plastic containers or beaker that seal with nylon gauze after thawing 1～3 hour in greenhouse or the 4 ℃ of refrigerators; Put into then hungry 2～3 hours 20-300 only 1～2 age the brown planthopper nymph; Raise poison and after 1～2 day the brown planthopper of survival is moved to the healthy water rice seedling of supporting worm, raise malicious brown planthopper and on the healthy water rice seedling, follow back after date, detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method through about 10～15 days virus; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus;
Brown planthopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber: with filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Carrying circular open plastic containers that paddy rice diseased plant 1～5 strain of RRSV seals with nylon gauze is enclosed in the Temperature and Humidity Control growth chamber and cultivates; Put into the nontoxic brown planthopper nymph in 20～300 1～2 ages after hungry 3 hours in each device; Raise poison and raise 7～12 days until the after date that follows back of tiding over virus on the brown planthopper immigration fresh and healthy paddy rice seedling with survival after 2～10 days; Detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus;
The brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed is identified the resistance of rice varieties, and the disease-resistant variety of Screening of Rice is anti-paddy rice tingia dwarf virus breeding for disease resistance service;
The brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the brown planthopper that will carry paddy rice tingia dwarf virus moves into plantation to be identified in the greenhouse or the 1.5-3 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 5 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole brown planthoppers after 2-7 days; Rice seedling is transplanted to greenhouse or growth chamber; Cultivation temperature is 26-30 ℃, and relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 30-40 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding;
The brown planthopper of carrying paddy rice tingia dwarf virus is used to screen special, the sensitive monoclonal antibody of anti-paddy rice tingia dwarf virus, is the immunological method of paddy rice tingia dwarf virus in detection paddy rice and the brown planthopper body of core thereby set up with the monoclonal antibody;
The brown planthopper of carrying paddy rice tingia dwarf virus is used to study the mutual work between paddy rice tingia dwarf virus and the amboceptor insect and passes malicious mechanism and provides material base.
Below in conjunction with embodiment and accompanying drawing the present invention is described further.
1, infects the acquisition of RRSV paddy rice
1) at RRSV the paddy rice area takes place; According to infecting RRSV paddy rice symptom; Gather to infect the RRSV rice plant, have like the paddy rice tingia dwarf virus generating region collection of in September, 2011 in old town and country, Shidian County, Yunnan dark greenly stunt, the leaf curling indentation is incised, the swollen paddy rice appearance of elongated arteries and veins arranged in that blade back and leaf sheath are sick.
2) the RT-PCR method is identified the sick appearance of paddy rice
Extract total RNA of the sick appearance of paddy rice with reference to Trizol reagent; According to capsid protein gene (CP gene) sequence (number of landing: EU523360) design specific primers, RRSV-CP-F:5 ' CGAATCATCACTGAACAAGTATTTGG 3 ' and RRSV-CP-R:5 ' TCATACACCGGAACCGCTGG 3 ' according to the paddy rice tingia dwarf virus of reporting among the GenBank.Adopt the RT-PCR method to detect and confirm that sick appearance is infected paddy rice sawtooth leaf dwarf virus.The synthetic of the first chain cDNA carries out according to RevertAidTM reverse transcription kit specification, i.e. template ribonucleic acid 2 μ l, downstream primer 1 μ l, RNase Free H
2O 9 μ l.5 min under 70 ℃ take out and put 3-5 min on ice behind the mixing; Add 4 μ l, 5 * RT Buffer, 2 μ l dNTP Mix, 1 μ l Rnasin Inhibitor, 5 min under 37 ℃ behind the mixing; Add 1 μ l Reverse Transcriptase, mix back 42 ℃ of following reaction 1h, 70 ℃ of following 10 min make the revertase inactivation.With cDNA is that template is carried out pcr amplification, and the PCR reaction system is following: preparatory 94 ℃ of 2 min of sex change, and 94 ℃ of 45 sec of sex change, 52 ℃ of 1 min that anneal extends 72 ℃ of 2 min, and cyclic amplification 35 times extends 72 ℃ of 10 min at last.Amplified production carries out electrophoretic analysis in 1% Ago-Gel, and the result is as shown in Figure 1, has identified that the sample of gathering is for infecting the diseased plant of RRSV.
3) the Dot-ELISA method is identified the sick appearance of paddy rice
The liquid nitrogen grinding powdered use in the sick leaf of the paddy rice back of weighing, and (w/v, g/mL) add grinding behind the 0.01 mol/L PBS (pH7.4) press 1:10～30; Centrifugal 3 min of sick juice 5000 rpm; Get on the 3 μ l and check on the NC film, health and susceptible paddy rice leaf juice are set simultaneously respectively as feminine gender and positive control; The dry 10-20 min of room temperature (10 ℃-35 ℃); The NC film is immersed in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim milk powder room temperature and seals 30 min; The NC film is put into the monoclonal antibody incubated at room 30-60 min of the anti-RRSV of appropriateness dilution; Wash film 3～4 times with PBST, each 3 min; The NC film is put into AP enzyme labeling sheep anti-mouse igg two anti-incubated at room 30 ~ 60 min of appropriateness dilution; PBST washes film 4～5 times, each 3 min; 66 μ L NBT and 33 μ L BCIP substrates (Promega) join 10 ml substrate buffer solutions (0.1 mol/L Tris Cl, 0.1 mol/L NaCl, 0.025 mol/L MgCl, pH9.5) mixing, and film is put into substrate solution and reacted, the perusal result.Treat the positive control colour developing obviously, and feminine gender have no when colour developing running water rinsing cessation reaction, Taking Pictures recording result.The result is as shown in Figure 2, has proved that further the sample of being gathered is for infecting the diseased plant of RRSV.The diseased plant of detection back definite band paddy rice tingia dwarf virus (RRSV) is incubated in greenhouse or the material growth or is stored in-10～-80 ℃ of refrigerators.
2, the acquisition of brown planthopper
After the rice field that the area takes place from non-RRSV is caught brown planthopper, raise at the nontoxic rice seedling of indoor non-insect-proof rice kind, raising temperature is 26-30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Treat that brown planthopper adult post-coitum single head worm lays eggs separately; Randomly draw in the colony 10% brown planthopper with RT-PCR and dot-ELISA methods analyst band poison situation; Keep and do not set up indoor population with the offspring of malicious female worm and with it; In later per generation, randomly drawed in the colony 10% brown planthopper and confirms it with RT-PCR and dot-ELISA method and be not with poison really, promptly obtains not the brown planthopper with RRSV;
1) dot-ELISA detects the brown planthopper body and whether is with poison
The single head brown planthopper is put into the centrifuge tube of the eppendorf of 1.5 mL, and add 100 μ L PBS (0.01mol/L, pH7.4); After mashing brown planthopper with toothpick, centrifugal 3 min of 5000 rpm; Get on the 3 μ L and check on the NC film, it is identical that film is dry, monoclonal antibody is hatched, two anti-ly hatch, development step and dot-ELISA detect in the paddy rice RRSV method, and the different just sheep anti-mouse iggs two of two anti-HRP marks for the appropriateness dilution resist; Chromogenic substrate is the chromogenic substrate of HRP, i.e. the TMB chromogenic substrate.The brown planthopper of establishing non-band poison and band poison simultaneously is as feminine gender and positive control.Testing result shows that brown planthopper do not carry RRSV.
2) the RT-PCR method detects brown planthopper and whether is with poison
Extract total RNA of single head brown planthopper with reference to Trizol reagent specification; Auele Specific Primer according to capsid protein gene (CP gene) sequences Design of above-mentioned paddy rice tingia dwarf virus; Adopt the same RT-PCR method of above-mentioned detection paddy rice sample to detect brown planthopper and whether be with poison, testing result proves that further brown planthopper is the insect of non-band poison.
3, brown planthopper is obtained poison from the rice plant that infects RRSV
1) brown planthopper is obtained poison from the susceptible paddy rice diseased plant of freezing preservation
With filter paper preserve moisture down 20-300 only 1 ~ 2 age hungry 2-3 hour of brown planthopper nymph.Take out the sick appearance of the paddy rice 1-3 strain of the infection RRSV of freezing preservation in-10～-80 ℃ of refrigerators; Place in greenhouse (10 ℃-35 ℃) or 4 ℃ of refrigerators and thaw; Move in the circular open plastic containers or beaker that seal with nylon gauze after 1-3 hour; Put into then hungry 2-3 hour 20-300 only 1 ~ 2 age the brown planthopper nymph, so just have preferably air flow property guarantee the brown planthopper survival, can prevent that again brown planthopper from escaping from container simultaneously.Raise after malicious 1-2 days the brown planthopper that survives is moved to the healthy water rice seedling of supporting worm, and the brown planthopper quantity of record survival.Raise malicious brown planthopper and on the healthy water rice seedling, follow back the phase through virus, after about 10-15 days, the brown planthopper quantity of record survival.A part of brown planthopper is carried out above-mentioned RT-PCR and the dot-ELISA method is with malicious rate analysis.The result is like Fig. 4, shown in 5, shows that the survival rate of brown planthopper of the diseased plant of the frozen preservation of feeding is 80%, and being with malicious rate is 30%.
2) brown planthopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber
With filter paper preserve moisture down 20-300 only 1 ~ 2 age hungry 2-3 hour of brown planthopper nymph.Identify the paddy rice diseased plant of carrying RRSV through above-mentioned detection, the circular open plastic containers that seal with nylon gauze are enclosed in the Temperature and Humidity Control growth chamber to be cultivated, and good air flows and guarantees the survival of plant and brown planthopper, avoids the escape of brown planthopper again.Each plastic containers is put 1-5 strain diseased plant plant.Put in each device after hungry 3 hours 20-300 1-2 age nontoxic brown planthopper nymph.Cultivation temperature is 26-30 ℃, and relative moisture is 60 %-75 %, and the photoperiod is 16L:8D.Raise after malicious 2-10 days brown planthopper with survival and move into to raise on the fresh and healthy paddy rice seedling and follow back the phase until what tide over virus, about 7-12 days, and write down the brown planthopper quantity of surviving.And through method detection validation such as above-mentioned RT-PCR, dot-ELISA.130 brown planthoppers that shift out are carried out above-mentioned RT-PCR in the inoculation back and dot-ELISA detects, and it is with malicious rate is 50%.This shows that brown planthopper is higher from the probability that the paddy rice diseased plant of cultivating obtains RRSV.
4, obtain the application of malicious brown planthopper
1) obtains the monoclonal antibody that malicious brown planthopper is used to screen anti-RRSV
Single head band poison brown planthopper is put into the centrifuge tube of the eppendorf of 1.5 mL; And add 100 μ L PBS (0.01mol/L, pH7.4), mash brown planthopper with toothpick after, centrifugal 3 min of 5000 rpm; Get on the 3 μ l and check on the NC, the dry 10-20 min of room temperature (10 ℃-35 ℃); The NC film is immersed in PBST (the 0.01 mol/L PBS that the contains 0.05% Tween-20) confining liquid that contains 5% skim milk powder room temperature and seals 30 min; The NC film is put into different Hybridoma Cell Culture supernatant incubated at room 30-60 min; Wash film 3～4 times with PBST, each 3 min; The NC film is put into sheep anti-mouse igg two anti-incubated at room 30 ~ 60 min of the horseradish peroxidase-labeled of appropriateness dilution; PBST washes film 4～5 times, each 3 min; Develop the color with tmb substrate.If blue spot occurs, then positive, show that this cell line can produce the antibody of anti-RRSV.Testing result is as shown in Figure 5.
2) the biography poison and the paddy rice resistance that obtain malicious brown planthopper are identified
The brown planthopper that above-mentioned diseased plant or the susceptible rice plant of living from freezing preservation obtained poison move into paddy rice seedling (1.5-3 leaf phase) to be identified in the greenhouse or growth chamber raised 2-7 days; Cultivation temperature is 26-30 ℃; Relative moisture is 60 %-75 %, and the photoperiod is 16L:8D.Adopt the rice varieties to be identified of single seedling 5 cephalont methods inoculation 1.5-3 leaf phase, each kind is chosen 100 paddy rice seedlings and is identified, shifts out whole brown planthoppers after 2-7 days, rice seedling is transplanted to greenhouse or growth chamber, grow again.Instituted an inquiry later in 7 days, investigate once every day, continuous observation 40 days.The investigation standard is with reference to the stunt classical symptom of disease of paddy rice tingia: dark greenly stunt, the leaf curling indentation is incised, have elongated arteries and veins swollen at blade back and leaf sheath.Further detect postvaccinal paddy rice with above-mentioned RT-PCR and dot-ELISA method, the paddy rice that the observes classical symptom purpose band that all can increase, the paddy rice of the no disease symptom purpose band that all can not increase, the result is as shown in Figure 6.This shows that brown planthopper obtains can be inoculated on the healthy paddy rice behind the RRSV from freezing preservation or with the disease plant that lives.In like manner; Can connect the viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement rice varieties thereof according to feeding; Promptly set up the authentication method of RRSV paddy rice resistant variety, for excavation, the resistant variety of the paddy rice resource of anti-RRSV are identified and breed breeding provides technological means.
Claims (5)
1. preparation method that carries the brown planthopper of paddy rice tingia dwarf virus is characterized in that its step is following:
1) acquisition of brown planthopper
After the rice field that the area takes place from non-paddy rice tingia dwarf virus is caught brown planthopper, raise at the nontoxic rice shoot of indoor non-insect-proof rice kind, raising temperature is 26～30 ℃, and relative moisture is 60 %～75 %, and the photoperiod is 16L:8D; Treat that brown planthopper adult post-coitum single head worm lays eggs separately; Randomly draw in the population 10% brown planthopper with RT-PCR and dot-ELISA methods analyst band poison situation; Keep not with the offspring of malicious female worm and set up indoor population; In later per generation, randomly drawed in the population 10% brown planthopper and confirms that with RT-PCR, dot-ELISA method brown planthopper is not with paddy rice tingia dwarf virus, promptly obtains the brown planthopper of not carrying paddy rice tingia dwarf virus;
2) brown planthopper obtains poison
Brown planthopper is obtained poison from the susceptible paddy rice diseased plant of freezing preservation
With filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Take out paddy rice diseased plant 1～3 strain of the infection paddy rice tingia dwarf virus of freezing preservation in-10～-80 ℃ of refrigerators; Place and to move in the circular open plastic containers or beaker that seal with nylon gauze after thawing 1～3 hour in greenhouse or the 4 ℃ of refrigerators; Put into then hungry 2～3 hours 20-300 only 1～2 age the brown planthopper nymph; Raise poison and after 1～2 day the brown planthopper of survival is moved to the healthy water rice seedling of supporting worm, raise malicious brown planthopper and on the healthy water rice seedling, follow back after date, detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method through about 10～15 days virus; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus;
Perhaps, brown planthopper is obtained poison from the paddy rice diseased plant of planting in greenhouse or growth chamber
With filter paper preserve moisture down 1～2 age hungry 2～3 hours of brown planthopper nymph; Carrying circular open plastic containers that paddy rice diseased plant 1～5 strain of RRSV seals with nylon gauze is enclosed in the Temperature and Humidity Control growth chamber and cultivates; Put into the nontoxic brown planthopper nymph in 20～300 1～2 ages after hungry 3 hours in each device; Raise poison and raise 7～12 days until the after date that follows back of tiding over virus on the brown planthopper immigration fresh and healthy paddy rice seedling with survival after 2～10 days; Detect the paddy rice tingia dwarf virus in the brown planthopper body with RT-PCR, dot-ELISA method; Analyze it and be with malicious rate, obtain the brown planthopper of carrying paddy rice tingia dwarf virus.
2. application of the brown planthopper of carrying paddy rice tingia dwarf virus of method acquisition according to claim 1; It is characterized in that described brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed identifies the resistance of rice varieties; The disease-resistant variety of Screening of Rice is anti-paddy rice tingia dwarf virus breeding for disease resistance service.
3. a kind of preparation method that carries the brown planthopper of paddy rice tingia dwarf virus as claimed in claim 2; It is characterized in that described brown planthopper of the carrying paddy rice tingia dwarf virus healthy water rice seedling that is used to feed identifies that the method for rice varieties resistance is: the brown planthopper that will carry paddy rice tingia dwarf virus moves into plantation to be identified in the greenhouse or the 1.5-3 leaf phase paddy rice seedling of growth chamber; Adopt single seedling 5 cephalont methods to inoculate rice varieties to be identified; Raise and shift out whole brown planthoppers after 2-7 days, rice seedling is transplanted to greenhouse or growth chamber, cultivation temperature is 26-30 ℃; Relative moisture is 65 %-75 %, and the photoperiod is 16L:8D; Began to observe viral symptom in 7 days later on, continuous observation 30-40 days; Detect paddy rice with RT-PCR and dot-ELISA method; Connect viral symptom of malicious paddy rice and the resistance of RT-PCR and dot-ELISA testing result judgement paddy rice thereof according to feeding.
4. application of the brown planthopper of carrying paddy rice tingia dwarf virus of method acquisition according to claim 1; It is characterized in that described brown planthopper of carrying paddy rice tingia dwarf virus is used to screen special, the sensitive monoclonal antibody of anti-paddy rice tingia dwarf virus, is the immunological method of paddy rice tingia dwarf virus in detection paddy rice and the brown planthopper body of core thereby set up with the monoclonal antibody.
5. application of the brown planthopper of carrying paddy rice tingia dwarf virus that obtains of method according to claim 1 is characterized in that described brown planthopper of carrying paddy rice tingia dwarf virus is used to study the mutual work between paddy rice tingia dwarf virus and the amboceptor insect and passes malicious mechanism to provide material base.
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| CN104521592A (en) * | 2015-01-04 | 2015-04-22 | 连云港市农业科学院 | Large-scale quantitative and strengthened inoculation authenticating method for paddy black-streaked dwarf disease resistance |
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| 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 |
| CN102870743B (en) * | 2012-10-12 | 2014-07-30 | 江苏省农业科学院 | Method for preserving rice black-streaked dwarf virus indoor living bodies |
| CN104521592B (en) * | 2015-01-04 | 2016-09-07 | 连云港市农业科学院 | Inoculation identification method is quantitatively strengthened in the scale of a kind of black streaked dwarf virus of rice resistance |
| CN104521592A (en) * | 2015-01-04 | 2015-04-22 | 连云港市农业科学院 | Large-scale quantitative and strengthened inoculation authenticating method for paddy black-streaked dwarf disease resistance |
| CN104620874A (en) * | 2015-01-04 | 2015-05-20 | 陈丁龙 | Method for inoculating and identifying resistance of soybeans for virus disease of Bemisia tabaci |
| CN104871885B (en) * | 2015-06-18 | 2017-06-13 | 黄山丰乐谷生态农业综合开发有限公司 | A kind of prevention and controls of rice grub brown paddy plant hopper |
| CN104871885A (en) * | 2015-06-18 | 2015-09-02 | 方春华 | Control method for rice pest brown planthopper |
| CN106489564A (en) * | 2015-09-08 | 2017-03-15 | 无锡南理工科技发展有限公司 | A kind of Rice Resistance tingia dwarfing authentication method |
| CN105746439A (en) * | 2016-03-09 | 2016-07-13 | 中国科学院微生物研究所 | Method and artificial feed for enabling laodelphax striatellus to obtain rice stripe viruses |
| CN105746439B (en) * | 2016-03-09 | 2018-07-06 | 中国科学院微生物研究所 | Small brown rice planthopper is made to obtain the method and its man-made feeds of rice stripe virus |
| CN113693032A (en) * | 2021-09-14 | 2021-11-26 | 广西壮族自治区农业科学院 | Method for evaluating rice variety for resisting rice ragged dwarf disease based on artificial inoculation |
| CN113693032B (en) * | 2021-09-14 | 2022-04-12 | 广西壮族自治区农业科学院 | Method for evaluating rice variety for resisting rice ragged dwarf disease based on artificial inoculation |
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