CN103060380A - Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus - Google Patents
Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus Download PDFInfo
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Abstract
The invention discloses a method for transforming a plant protoplast by an RNA virus. The method for transforming plant protoplast by the RNA virus in the invention comprises a step of transferring a total RNA extracted from a plant infected by the RNA virus into a plant protoplast, wherein the RNA virus is a multi-component RNA virus, such as a beet necrotic yellow vein virus; and the plant protoplast is a tobacco protoplast, such as a nicotiana benthamiana protoplast. According to the invention, nicotiana benthamiana tobacco leaves are used as raw materials for preparing the protoplast; the plant RNA of an infected leaf infected by the beet necrotic yellow vein virus is inoculated in the nicotiana benthamiana protoplast by a polyethylene glycol mediator, so as to successfully establish infection of the beet necrotic yellow vein virus on the protoplast. The method disclosed by the invention provides an ideal model for researching a pathogenic mechanism between the beet necrotic yellow vein virus and a nicotiana benthamiana host, and has an important theoretical value for discovering interaction between the virus and the host plant and virus evolution.
Description
Technical field
The present invention relates to a kind of method with RNA viruses conversion of plant protoplastis.
Background technology
Sugarbeet Rhizomania is the important disease of harm beet, has become a kind of destructive disease in each beet producing region, the world at present, and is especially very big on Sugarbeet Yield and sugar degree impact.In recent years this disease in the Inner Mongol, there is generation in various degree in the main beet producing regions such as Gansu, Xinjiang and Heilungkiang, have a strong impact on the development of China's beet sugar manufacture industry.The pathogen of this disease is beet necrotic yellow vein virus (Beet necrotic yellow vein virus, BNYVV), this virus is the representative virus that Benyvirus belongs to, propagate (Tamada by Polymyxa betae (Polymyxa beta), T and Baba, T.Beet necrotic yellow vein virus from rhizomania-affected sugar beet in Japan.Ann PhytopatholSoc Japan.1973,39:325-332).BNYVV is just RNA multicomponent virus, and its genome is comprised of 4 ~ 5 RNA, is packaged in respectively in the baculovirus particle of different lengths.Wherein RNA1 and RNA2 the coding " housekeeping gene ", that the various hosts of virus infection are necessary, and RNA3 and RNA4 are not the necessary (Bouzoubaa of virus, S., Niesbach-Klosgen, U., Jupin, I., Guilley, H., Richards, K., and Jonard, G.Shotrened forms of Beet necrotic yellow vein virus RNA-3and-4:internal deletions and a subgenomic RNA.Journal of General Virology, 1991,72:259-226).BNYVV only RNA1 and RNA2 just can this life of systemic infection cigarette, and wherein the P14 of RNA2 coding is that its system motion is necessary, the system motion ability of other small components RNAs and pathogenic having nothing in common with each other.The BNYVV field isolate that the molecule experiment confirm contains RNA4 can produce serious symptoms when inoculating this life cigarette, it is the Newborn Leaves last volume, plant is downgraded (Rahim M.D., Andika I.B., Han C.G H., and Tamada T.RNA4-encoded p31ofBeet necrotic yellow vein virus is involved in efficient vector transmission, symptom severity and silencing suppression in root.Journal of General Virology, 2007,88:1611-1619).Wild type rna 3 is depressed in the selection of this life cigarette and is produced quickly inner absence type RNA3, and the RNA3 of absence type is relevant with the serious symptoms of this life cigarette, Loss of self (Wang Ying then very easily occurs at system's leaf in RNA5, Fan Huiyan, Wang Xian-Bing, Li Min, Han Chenggui, Li Dawei, Yu Jialin.Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3from systemic host Nicotianabenthamiana.Virology Journal, 2011,8:355).Before because the restriction of inoculation system, the correlative study of BNYVV all mainly concentrates on field host's beet or the some other withered spot host (elder brother's promise lamb's-quarters or New Zealand spinach) to be carried out, and the research of itself and viral this life of systemic infection model plant cigarette is still waiting further deeply.
Protoplastis is to remove cell walls, by the exposed cell of plasma membrane parcel.Because protoplastis has lost this barrier of cell walls, not only makes its ideal material that becomes phytology, cytobiology, biotechnology and crop improvement, also become the effective means of plant virus research.Tobacco protoplast be use the earliest in the plant virus research, vegetable material the most widely, it the infecting of plant virus, copy, virus is with the host does mutually, transgenosis is antiviral, have a wide range of applications in aspect the Virus inhibitors etc., for the research of promotion plant virology plays an important role.The inoculation useful virion of protoplastis and viral nucleic acid dual mode.The basic demand of inoculation is: high density, the virus that infectivity is strong (or viral nucleic acid) suspension and freshly prepd high reactivity protoplastis.Transform at present protoplastis and mainly contain polyoxyethylene glycol (PEG) method, electrization and microinjection etc.Wherein the PGE method can promote the fusion of film, assist virus or nucleic acid to infect by broken ring protoplast membrane, owing to can obtain with a small amount of viral nucleic acid the protoplastis of high infection rate, one of the method (Chen Zhanzhou of viral RNA inoculation tobacco protoplast at present commonly used, bad room of Lee. the application of tobacco protoplast in plant virus research. the Agriculture of Anhui science, 2006,34,1119-1122).Utilize at present electric shocking method that BNYVV in-vitro transcription thing is infected beet (Joersbo M, and Brunstedt J.Inoculation of sugar beet protoplasts with beet necrotic yellow vein virus particles by mild sonication.Journal of virological methods.1990,29:63-69) with elder brother's promise lamb's-quarters protoplastis (Gilmer D., Bouzoubaa S., Hehn A., Guilley H., Richards K., Jonard G. Efficient cell-to-cell movement of beet necrotic yellow vein virus requires3 ' proximal genes located on RNA2.Virology, 992,189:40-47) and BY-2 suspension cell device report is arranged successfully, but the system that infects the host Ben Sheng of system cigarette protoplastis is not yet set up.Along with deepening continuously of BNYVV and the research of this life cigarette host interaction mechanism, the foundation that transforms this life cigarette protoplastis system is also day by day important.In addition, because the electric shock conversion method has higher requirement to used instrument in the experiment, thus the popularization of restriction BNYVV transfection protoplasma method.
Summary of the invention
The purpose of this invention is to provide a kind of method with RNA viruses conversion of plant protoplastis.
Method with RNA viruses conversion of plant protoplastis provided by the present invention is that the total RNA that will extract from the plant that infects RNA viruses is transferred in the plant protoplast.
Described method is particularly suitable for the situation that described RNA viruses is many splits RNA viruses.Described many splits RNA viruses refers to that the genome of virus distributes on different nucleic acid chains, be packaged in respectively in the different virions, because genetic information has been separated, an independent plastochondria can not infect, and must be that one group of several infecting simultaneously could all be expressed hereditary property.When the described RNA viruses of band conversion is many splits RNA viruses, adopt aforesaid method, from the plant that has infected corresponding many splits RNA viruses, extract the total RNA of plant, it is transformed the purpose plant protoplast, such method for transformation and traditional method for transformation are (directly with virion or the viral RNA that extracts from virion, or the in-vitro transcription thing that utilizes viral different components infectious clone mixes the rear protoplastis that transforms) compare, more simplify and save cost, and the laboratory without this virus infection sex clone is provided the possibility of carrying out experiment, its reason is as follows: traditional method for transformation, need at first purified virus during conversion, utilize infectious clone in-vitro transcription thing then need to grope the different virus nucleic acid chains between proportioning; For method for transformation provided by the present invention, behind corresponding many splits picornavirus infection plant body, this virus exists with its physiological status in plant materials, not only can dispense the loaded down with trivial details step of a large amount of purified virus particles so use from the plant materials of Influenza Virus the total RNA that directly extracts to transform the purpose plant protoplast, and avoid between the different RNA component proportioning to grope this problem.
In one embodiment of the invention, described many splits RNA viruses is specially beet necrotic yellow vein virus (Beet necrotic yellow vein virus, BNYVV).
In aforesaid method, described plant protoplast can be tobacco protoplast.
In one embodiment of the invention, described tobacco protoplast is specially this life cigarette (Nicotiana benthamiana) protoplastis.
More concrete, described living cigarette protoplastis comes from the young leaves that launches fully of 6-8 leaf this life of phase cigarette, such as first and second expansion leaf.
The young leaves that launches fully of described 6-8 leaf this life of phase cigarette is less than or equal to 0.5mm with leaf area in protoplastis extraction system
2The form of tissue block exist.
Described living cigarette protoplastis specifically prepares according to the method that comprises the steps: with the young leaves that launches fully of described 6-8 leaf this life of phase cigarette (launching leaf such as first and second) shred to leaf area less than or equal to 0.5mm
2, the blade of chopping is placed the enzymolysis solution enzymolysis, thereby obtains described living cigarette protoplastis.
Described first and second refer to from the plant top first young leaves and second young leaves of down counting.
The blade of described chopping and the proportioning of described enzymolysis solution specifically can be the 3g blade: the 5ml enzymolysis solution.
The solvent of described enzymolysis solution is water, and solute and concentration thereof are as follows: cellulase 15g/L, macerozyme 1.5g/L, 2-N-morpholine ethyl sulfonic acid (MES) 20mM, N.F,USP MANNITOL 82g/L, Repone K 20mM, calcium chloride 10mM, bovine serum albumin 1g/L.The pH of described enzymolysis solution can be 5.4-5.5.
Described cellulase can be commercially available various cellulases, especially with cellulase " Onozuka " R-10(Yakult Honsha Co.Ltd) best results, described macerozyme can be commercially available various macerozymes, especially Macerozyme R-10(Yakult Honsha Co.Ltd) best results.
In described living cigarette protoplast preparation method, the time of described enzymolysis can be 3-4h.The temperature of described enzymolysis can be 24 ℃, dark condition.
In the method with RNA viruses conversion of plant protoplastis, described conversion can be polyoxyethylene glycol and transforms.
In the system that described polyoxyethylene glycol transforms, the concentration of described total RNA is more than or equal to 0.5 μ g/ μ l, such as 0.5-3.0 μ g/ μ l, for another example 1.0-3.0 μ g/ μ l; The proportioning of described total RNA, described plant protoplast and described polyoxyethylene glycol can be 2-20 μ g:2 * 10
5Individual: 135mg, such as 20 μ g:2 * 10
5Individual: 135mg.
The present invention is take the material of this life cigarette (Nicotiana benthamiana) tobacco leaf as the preparation protoplastis, the total RNA of plant that will infect the sick leaf of BNYVV through poly-di-alcohol (PEG) mediation is inoculated in this life cigarette protoplastis, successfully sets up BNYVV infecting protoplastis.The present invention provides an ideal model for the mechanism of causing a disease between research beet necrotic yellow vein virus and this lifes cigarette host, does mutually between this virus and host plant and viral evolution has important theory value disclosing.
Description of drawings
Fig. 1 is the electrophorogram of total RNA of extracting from the New Zealand spinach leaf tissue that has infected BNYVV.
Fig. 2 is the microscope picture of this life cigarette protoplastis.
Fig. 3 expects the microscope picture of blue dyeing for this life cigarette protoplastis platform.
Fig. 4 is the Western-blot detected result that the total RNA of incidence of leaf plant of total amount 20 μ g different concns infects this life cigarette protoplastis sampling in 24 hours BNYVV CP expression amount situation.
Fig. 5 is the Western-blot detected result that the different total RNA of incidence of leaf plant of concentration 1.0 μ g/ μ l total amounts infects this life cigarette protoplastis sampling in 24 hours BNYVV CP expression.
Fig. 6 is the Western-blot detected result that the total RNA of concentration 1.0 μ g/ μ l total amount 20 μ g incidence of leaf plants infects this life cigarette protoplastis different time sampling BNYVV CP expression.
Fig. 7 is the detected result that traditional method transforms CP expression amount behind this life cigarette protoplastis.Wherein, CK+ represents the detected result (positive control) of the total protein that extracts from the New Zealand spinach leaf tissue that has infected BNYVV; 1 this traditional method of expression transforms the detected result of CP expression amount behind this life cigarette protoplastis.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
This life cigarette (Nicotiana benthamiana), beet necrotic yellow vein virus (Beet necroticyellow vein virus, BNYVV) and New Zealand spinach (Tetragonia expansa): be recorded in " Wang Ying; Fan Huiyan; Wang xian-B ing; Li Min; Han chenggui; Li Dawei, Yu Jialin (2011) Detection and characterization of spontaneous internal deletion mutants of Beet Necrotic yellow vein virus RNA3 from systemic host Nicotiana benthamiana.Virology Journal2011,8:3351-9 " in the literary composition, the public can obtain from China Agricultural University.
Be used for to extract the enzymolysis solution (during use need with the membrane filtration degerming of 0.2 μ m) of this life cigarette protoplastis: cellulase 15g/L, macerozyme 1.5g/L, 2-N-morpholine ethyl sulfonic acid (MES) 20mM, N.F,USP MANNITOL 82g/L, Repone K 20mM, calcium chloride 10mM, bovine serum albumin (BSA) 1g/L, pH5.4-5.5.More than each concentration be the final concentration of respective components in enzymolysis solution.In the process for preparation of enzymolysis solution, after first other components except calcium chloride and BSA being configured, in 55 ℃ of water-bath 10min, be cooled to room temperature, add again described calcium chloride and BSA.
Wherein, cellulase is cellulase " Onozuka " R-10(Yakult Honsha Co.Ltd), macerozyme is Macerozyme R-10(Yakult Honsha Co.Ltd).
Be used for cultivating the substratum (1L) of this life cigarette protoplastis: take by weighing MS Salt(Sigma M5524) 4.3g, KH
2PO
410mg, inositol 100mg, sucrose 10g, N.F,USP MANNITOL 75g is dissolved in an amount of distilled water and is settled to 1L, high-temperature sterilization, pH5.5.The following solution of every liter of substratum adding filtration sterilization before using:
The VB5 aqueous solution (1000 *) 1mL
2-4-D solution (concentration is 2g/L) 500 μ L
The VB5 aqueous solution (1000 *): the VB5 of 1100mg is added to 100ml ddH
2Among the O.
The 2-4-D of 2-4-D solution: 100mg adds 1ml10M NaOH, adds to 50ml ddH again
2Among the O.
W5 solution: 154mmol/L NaCl, 125mmol/L CaCl
2, 5mmol/L KCl, 2mmol/L MES, solvent are water, pH5.7.More than each concentration be the final concentration of respective components in W5 solution.
MMG solution: 0.4mol/L Mannitol, 15mmol/L MgCl
2, 4mmol/L MES, solvent are water, pH5.7.More than each concentration be the final concentration of respective components in W5 solution.
0.05M PB damping fluid: 9.363g H
3BO
4, 33.230g NaB
4O
710H
2O, 2ml0.5M EDTA, adding distil water is dissolved to 1L, pH8.0.
RNA extracted solution: 100mM Tris-HCl, 0.1mM LiCl, 10mM EDTA, 1.0%(1g/100mL) SDS, solvent is water, pH8.0.More than each concentration be the final concentration of respective components in the RNA extracted solution.
PEG4000 solution: 4g PEG4000(Fluka, cat.No.81240), 2.5mL Mannitol(concentration is 0.8mol/L), 1mL CaCl
2(concentration is 1mol/L), 3mL H
2O.
The extraction of the total RNA of host's blade plant tissue of embodiment 1, virus infection
Present embodiment adopts New Zealand spinach (Tetragonia expansa) as treating infection plant, with many splits RNA viruses beet necrotic yellow vein virus (Beet necrotic yellow vein virus, BNYVV) it is infected, extract total RNA of metainfective New Zealand spinach leaf tissue, to be ready for use on the conversion of protoplastis.Specific as follows:
(1) juice mechanical friction inoculation host New Zealand spinach expands numerous BNYVV: evenly sprinkle a small amount of silicon carbide having infected on the New Zealand spinach blade of BNYVV, get incidence of leaf or isolate scab, grind fully with 0.05M PB damping fluid, the clean emgloves of long hilt broadsword dips a small amount of sick juice friction and smears integrated plate blade, after 5-7 days on blade the circular scab of visible yellow color.
(2) gather the New Zealand spinach blade (the average scab number of every sick leaf is 60-90) of falling ill, about 8-10g vegetable material adds the abundant grind into powder of liquid nitrogen, change in the 50ml centrifuge tube, the 10ml water saturation phenol (pH5.2-6.0) and the isopyknic RNA extracted solution that add rapidly 80 ℃ of preheatings, vibration 20s; Leave standstill the chloroform that adds 10ml behind the 5min, vibration 20s places 5min, the centrifugal 10min of 7000rpm.
(3) get supernatant liquor to new 50ml pipe, add the equal-volume 4M LiCl aqueous solution, place at least 4h for-20 ℃ behind the mixing.The centrifugal 20min of 8000rpm, precipitate resuspended with the DEPC water of 720 μ l, fully draw respectively 360 μ l supernatants after the dissolving and be placed in two 1.5ml centrifuge tubes, add respectively the 3M sodium acetate aqueous solution (pH5.2) of 40 μ l and the dehydrated alcohol of 800 μ l, place at least 2h for-20 ℃ behind the mixing.
Under (4) 4 ℃ of conditions, behind the centrifugal 15min of said mixture 12000rpm, wash one time with 70% ethanol again, dry, add the DEPC aqueous suspension dry powder of 400 μ l, be stored in-20 ℃ after the concentration determination.
The qualification result of the total RNA that extracts as shown in Figure 1, the RNA that extracts has following obvious electrophoretic band, is followed successively by from top to bottom 28S RNA, 18S RNA and 5S RNA, shows to have obtained higher, the more complete total RNA of purity.
Embodiment 2, this life cigarette protoplast preparation
Present embodiment from this life cigarette (Nicotiana benthamiana) for the preparation of the protoplastis that transforms.Specific as follows:
(1) is chosen at first of 6-8 leaf this lifes of the phase cigarette cultivated in greenhouse (24 ℃, 12h illumination, 12h is dark) and second and entirely opens up blade and be used for the protoplastis preparation, it is shredded as far as possible (blade area is less than or equal to 0.5mm
2), the blade with chopping is put in the enzymolysis solution (prescription sees above) rapidly.The proportioning of blade and enzymolysis solution is 3g leaf (being equivalent to about 20 intact leaves) 5ml enzymolysis solution.
(2) leave standstill enzymolysis 3-4h in 24 ℃ of dark places, the large section of microscopically observation of cell is spherical shape and stops enzymolysis, and the centrifugal 1min of 80rpm discharges the cell of enzymolysis.
The enzymolysis solution that (3) will contain protoplastis with 200 purpose nylon leaching films is filled in the 50ml centrifuge tube of placing on ice, removes not enzymolysis cell mass.The accel of the centrifugal 4min(whizzer of 350rpm is transferred to 1, brake will be transferred to 0, makes whizzer keep less acceleration, thereby the physical damnification that protoplastis is subject to is minimized), collect protoplastis (precipitation).
(4) with gently sucking-off of supernatant, the W5 solution (prescription sees above) that adds gently 10-15ml along tube wall, gently protoplastis is suspended (avoiding blowing and beating protoplastis with rifle) as far as possible, 4 ℃, the accel of the centrifugal 4min(whizzer of 350rpm is transferred to 1, brake will be transferred to 0, makes whizzer keep less acceleration, thereby the physical damnification that protoplastis is subject to is minimized), the washing enzymolysis solution.
(5) with the supernatant sucking-off, the operation of repeating step (4) is once namely washed 2 times with W5 solution altogether.
(6) again use W5 solution suspension protoplastis, leave standstill half an hour at least (generally leaving standstill 1-1.5 hour) on ice, make again natural subsidence of protoplastis.
(7) leave standstill in the process, protoplasma cognition sinks to the centrifuge tube bottom gradually, and with gently sucking-off of supernatant, according to an amount of resuspended protoplastis of MMG solution (prescription sees above) of concentration adding of protoplastis, the concentration of general requirement protoplastis is 1 * 10
6Individual/ml(calculates protoplastis concentration with blood cell counting plate), the microscope picture is seen Fig. 2.As shown in Figure 1, the protoplastis number that enzymolysis obtains is more, and cell state is good.High-quality protoplastis generally all is plasma membrane light, opaque, and vacuome is undeveloped, size is even, be the exposed cell of spherical shape.
(8) use platform to expect blue dyeing (working concentration is 0.5g/100ml), draw the protoplastis suspension of 100 μ l steps (7), adding 20 μ l staining fluids is mixed even, room temperature leaves standstill 2min, examine under a microscope after cleaning 2 times with the 0.7M Osmitrol, the protoplastis of living is not colored, and dead protoplastis then is dyed to blueness (platform expects that orchid can not see through normal complete cytolemma).The result as shown in Figure 3.Detected result shows that protoplastis alive wherein accounts for more than 90% of protoplastis sum, and as seen, the protoplastis for preparing by the inventive method has splendid activity.
This life cigarette protoplast transformation of embodiment 3, PEG mediation
Present embodiment will utilize embodiment 1 to obtain the total RNA of plant that extracts from the sick leaf of the New Zealand spinach that has infected BNYVV, method by PEG mediation is transformed in this life cigarette protoplastis that embodiment 2 prepares, be intended to make up this life of the restructuring cigarette protoplastis that changes BNYVV over to, to be ready for use on the mechanism of causing a disease between research BNYVV and this life cigarette host.Specific as follows:
(1) the total RNA of plant that extracts an amount of sick leaf of the New Zealand spinach from being infected by BNYVV that embodiment 1 is obtained is added in the 2ml centrifuge tube (DEPC processings), and (concentration is 1 * 10 to add this life cigarette protoplastis solution that 200 μ l embodiment 2 prepare
6Individual/ml), the total RNA of protoplastis and plant is fully mixed, the PEG4000 solution (prescription sees above) that adds again 220 μ l, abundant mixing (action must softly), room temperature (being generally 23 ℃) was placed after 8-10 minute, the W5 solution (prescription sees above) that adds gently immediately 1ml, gently mixing.
(2) 4 ℃, 400rpm, the accel of 4min(whizzer is transferred to 1, brake will be transferred to 0, makes whizzer keep less acceleration, thereby the physical damnification that protoplastis is subject to is minimized); The sucking-off supernatant adds 1.5ml protoplast culture medium (prescription sees above) at six well culture plates first gently, and the mixed solution that contains this life cigarette protoplastis and the total RNA of plant that step (1) is obtained adds in the substratum gently.Normal temperature (being generally 23 ℃) astigmatism was cultivated 6-48 hour.
(3) according to the needs of experiment purpose, get the restructuring protoplastis of different incubation times, extract total protein, utilize the method (western-blot) of the immune marking to detect the CP expressing quantity of BNYVV virus in the restructuring protoplastis after quantitatively, thereby identify the height of transformation efficiency.Used primary antibodie is Beet necrotic yellow vein virus coating antibody(Agdia, Catalog No:CAB16301), used two anti-are Protein A-Alaline Phosphatase (SIGMA, Catalog No:P7488-500UG).
The present inventor has carried out following optimization to transformation system: this life cigarette protoplastis consumption in embodiment 2 preparations is 2 * 10
5It is individual that (200 μ l, concentration is 1 * 10
6Individual/ml), the PEG4000 consumption is about 135mg(220 μ l, concentration is 4g/6.5ml), the W5 solution usage is 1mL, the protoplast culture medium consumption is under the prerequisite of 1.5mL, and the concentration of the total RNA of plant of embodiment in the transformation system 1 preparation and total amount have been carried out series of optimum (detection time be virus infection protoplastis after 24 hours).See for details following (a) and (b):
(a) total amount of the total RNA of plant of embodiment 1 preparation is 20 μ g in the transformation system, and it is 0.2 μ g/ μ l, 0.5 μ g/ μ l, 1.0 μ g/ μ l, 1.5 μ g/ μ l, 2.0 μ g/ μ l and 3.0 μ g/ μ l that concentration gradient is set.Zero-dose negative control (being denoted as mock) is set simultaneously, and with the total protein that from the New Zealand spinach blade that has infected BNYVV, the extracts positive control (being denoted as CK+) as sample to be tested.3 repetitions are established in experiment.
The result as shown in Figure 4, when the concentration of the total RNA of plant is 0.5 μ g/ μ l and when above, it is 1.0 μ g/ μ l when above that CP protein expression, particularly concentration are all arranged, CP protein expression amount is higher.This shows in the transformation system that when the concentration of the total RNA of plant reached 0.5 μ g/ μ l, BNYVV can infect this life cigarette protoplastis, and concentration is 1.0 μ g/ μ l when above, and it is relatively better to infect effect.In line with the principle of saving cost, selected 1.0 μ g/ μ l are optimum plant total rna concentration.
(b) concentration of the total RNA of plant of embodiment 1 preparation is 1.0 μ g/ μ l in the transformation system, and it is 2 μ g, 5 μ g, 10 μ g, 15 μ g, 20 μ g, 30 μ g and 40 μ g that the total amount gradient is set.Zero-dose negative control (being denoted as mock) is set simultaneously, and with the total protein that from the New Zealand spinach blade that has infected BNYVV, the extracts positive control (being denoted as CK+) as sample to be tested.3 repetitions are established in experiment.
The result as shown in Figure 5, when the total amount of the total RNA of plant is 2 μ g and when above, the CP protein expression is all arranged, and along with the CP expression amount of its same time of increase of total amount also along with increase, the CP expression amount reaches maximum value when RNA total amount 〉=20 μ g, and trends towards gradually steadily.This shows in the transformation system that when the total amount of the total RNA of plant reached 2 μ g, BNYVV can infect this life cigarette protoplastis, and total amount is 20 μ g when above, and it is relatively better to infect effect.In line with the principle of saving cost, selected 20 μ g are the total RNA total amount of optimum plant.
In addition, the present inventor (is in the transformation system to transform this life cigarette protoplastis with the total RNA of plant under optimal conditions also, the concentration of the total RNA of plant is 1.0 μ g/ μ l, and total amount is 20 μ g) after, Western-blot detects the time point of CP expression and measures.Set the detection time point and be followed successively by behind the virus infection protoplastis 6 hours, 12 hours, 18 hours, 24 hours, 36 hours and 48 hours.Zero-dose negative control (being denoted as mock) is set simultaneously, and with the total protein that from the New Zealand spinach blade that has infected BNYVV, the extracts positive control (being denoted as CK+) as sample to be tested.3 repetitions are established in experiment.
The result as shown in Figure 6, BNYVV infects and just can detect viral CP protein expression about protoplastis 12h, reaches maximum value about 24h, and afterwards along with the increase of time, CP protein expression amount has certain reduction, and this may descend relevant with the protoplastis vigor.Be 24-36h behind the virus infection protoplastis detection time that suggestion is best.
Comparative Examples:
Adopt traditional total RNA that from the BNYVV virion, extracts to transform this life cigarette protoplastis, when cultivating 24 hours, detect the CP protein expression amount that transforms BNYVV virus in the rear protoplastis.The method of concrete conversion protoplastis and CP protein expression quantity measuring method carry out with reference to embodiment 3 correlation step.
The result extracts the method (cultivating 24 hours after the protoplast transformation) of the total RNA of plant and compares as shown in Figure 7 among this tradition method for transformation and the embodiment 3, weak effect is few.The CP albumen of expressing in wherein should tradition method for transformation protoplastis is relatively a little less, and this may be relevant with the RNA total concn active or that extract of the virion that extracts.This explanation experimental implementation in the virion leaching process is more loaded down with trivial details, and the risk that exists virus infection power to reduce.
Claims (9)
1. the method with RNA viruses conversion of plant protoplastis is that the total RNA that will extract from the plant that infects RNA viruses is transferred in the plant protoplast.
2. method according to claim 1, it is characterized in that: described RNA viruses is many splits RNA viruses.
3. method according to claim 2, it is characterized in that: described many splits RNA viruses is beet necrotic yellow vein virus.
4. arbitrary described method according to claim 1-3 is characterized in that: described plant protoplast is tobacco protoplast.
5. method according to claim 4, it is characterized in that: described tobacco protoplast is this life cigarette protoplastis.
6. method according to claim 5 is characterized in that: described living cigarette protoplastis comes from the young leaves that launches fully of 6-8 leaf this life of phase cigarette.
7. method according to claim 6 is characterized in that: the young leaves that launches fully of described 6-8 leaf this life of phase cigarette is less than or equal to 0.5mm with leaf area in protoplastis extraction system
2The form of tissue block exist.
8. arbitrary described method according to claim 1-7 is characterized in that: the described polyoxyethylene glycol that is converted into transforms.
9. arbitrary described method according to claim 1-8 is characterized in that: in the system that described polyoxyethylene glycol transforms, the concentration of described total RNA is more than or equal to 0.5 μ g/ μ l; The proportioning of described total RNA, described plant protoplast and described polyoxyethylene glycol is 2-20 μ g:2 * 10
5Individual: 135mg.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609546A (en) * | 2018-12-18 | 2019-04-12 | 中国农业大学 | A kind of development and application of plant multicomponent virus carrier |
| CN114561424A (en) * | 2022-03-24 | 2022-05-31 | 浙江中医药大学 | Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus |
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2013
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Non-Patent Citations (2)
| Title |
|---|
| 李旻: "甜菜坏死黄脉病毒RNA5功能分析及甜菜抗病品种的鉴定", 《中国优秀博硕士学位论文全文数据库 (硕士) 农业科技辑》 * |
| 袁天华 等: "用不同方法把烟草花叶病毒RNA基因导入植物原生质体的研究", 《生物化学与生物物理学报》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109609546A (en) * | 2018-12-18 | 2019-04-12 | 中国农业大学 | A kind of development and application of plant multicomponent virus carrier |
| CN114561424A (en) * | 2022-03-24 | 2022-05-31 | 浙江中医药大学 | Method for seedling injection inoculation of salvia miltiorrhiza and cucumber mosaic virus |
| CN114561424B (en) * | 2022-03-24 | 2024-01-26 | 浙江中医药大学 | Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage |
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