CN109022483A - The method of TRV carrier mediated Gene Silencing systemic vaccination tree peony floral organ - Google Patents
The method of TRV carrier mediated Gene Silencing systemic vaccination tree peony floral organ Download PDFInfo
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Abstract
The invention discloses a kind of methods of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated: TRV viral vectors being converted Agrobacterium, infects floral organ in a manner of injection inoculation after cultivation to the positive colony containing TRV.Through the inoculated tree peony floral organ of the method for the present invention, the presence of TRV viral vectors can detecte in the tissue such as petal, stamen, gynoecium, sepal, and the symptom that TRV infects occur.The present invention be application of the Gene Silencing technology in the research of tree peony development of floral organs process related gene provide it is a kind of quick, efficiently infect method.
Description
Technical field
The invention belongs to field of plant genetic project technology, it is related to TRV carrier in Gene Silencing and infects floral organ
The method of official.
Background technique
Tree peony (Paeonia suffruticosa) belongs to Paeoniaceae Paeonia sect. Moutan, is important ornamental flower and medicine
With plant, in recent years, it has been found that ornamental character of the tree peony in addition to its large flower and brilliant color, and a kind of excellent oil crops,
Seed oil has the characteristics that oil production is high, oily quality is good.However, tree peony is as xylophyta, genetic conversion system still not at
It is ripe, and tree peony growth year is long, from seedling to 3~4 years time of needs of blooming, studies tree peony flower development and fruit development
There are great bottlenecks for relevant gene.
Virus induced gene silencing (virus-induced gene silencing, VIGS) is the base after a kind of transcription
Because of silent technology, various plants have been widely used in as a kind of effective reverse Genetics Technique.The work of VIGS
It is that plant can generate specific endonucleases Dicer during resisting virus infection with principle, by virus replication
The double-stranded RNA of middle generation is cut into the small molecules interference RNA (siRNA) of 21~24 nucleotide.SiRNA in plant cell into
After the amplification of one step, with the combination such as single stranded form and Agronaute 1 (AGO1) albumen, the silencing complex of RNA induction is formed
(RNA-induced silencing complex,RISC).RISC can specifically cause same with siRNA in plant cytoplasm
The degradation of the mRNA in source, so as to cause the silencing of target gene.
VIGS technology mainly realizes the silencing to endogenous gene by viral vectors.Tobacco rattle virus (Tobacco
Rattle virus, TRV) it is a kind of using more extensive viral vectors, with host range is wide, silence efficiency is high, maintains
The advantages that time is long.VIGS tests the double base table of RNA1 and RNA2cDNA that common silent carrier is Tobacco rattle virus (TRV)
Up to carrier TRV1 and TRV2 (Wang S B, Tian S L, Shah S N, et al.Cloning and
characterization of the CarbcL gene related to chlorophyll in pepper(Capsicum
annuum L.)under fruit shade stress.Frontiers in Plant Science,2015,6(6):
850.).TRV1 includes the gene of the RNA polymerase of coding RNA dependence, motor protein and 16kD protein, is the auxiliary of VIGS system
Help viral vectors.TRV2 includes capsid protein (Cp) gene, multiple cloning sites (MCS) etc., for being inserted into target gene.
But tree peony, as xylophyta, mutant is not easy to obtain, and brings inconvenience to the molecular breeding of this kind of plant.
There is no relevant reports for application in relation to VIGS in peony growth course at present.
Summary of the invention
The purpose of the present invention is to provide a kind of Gene Silencing systemic vaccination tree peony floral organs that TRV is carrier mediated
The method of official, this method simple, efficient, low cost can make TRV viral vectors disseminate developmental tree peony floral organ.
In order to achieve the above objectives, the invention adopts the following technical scheme:
1) by viral vectors TRV1 and TRV2 or by viral vectors TRV1 and inserted with the recombination of target gene
TRV2 converts Agrobacterium respectively, the positive Agrobacterium containing TRV1 that conversion is obtained and the positive containing TRV2 or recombination TRV2
Agrobacterium mixes in infecting in buffer after cultivation, obtains mixed bacteria liquid；
2) after step 1), mixed bacteria liquid is drawn with the syringe with syringe needle, then by syringe needle in developmental tree peony
Injection inoculation is inserted into inside peony plant and carried out at floral organ, until the peony organ surface has liquid to ooze out (water stain shape)
When stop injection；
3) use the following conditions to cultivate peony plant after injecting: temperature for 21~25 DEG C, humidity for 60~
70%, intensity of illumination is 1800~2000Lx, and 16h illumination/8h is dark.
Preferably, in the step 3), phenotype observation is carried out to tree peony floral organ in peony plant incubation, and adopt
Sample, the accumulation for extracting the TRV viral vectors for using the method for sxemiquantitative to detect each institutional framework of floral organ after RNA.
Preferably, the conversion uses freeze-thaw method.
Preferably, the buffer that infects includes following components: 10mM 2-morpholine ethane sulfonic acid (MES), 100 μM of acetyl fourths
Ketone musk (AS) and 10mM MgCl2。
Preferably, the positive Agrobacterium containing TRV1 and another positive Agrobacterium (such as containing TRV2) are cultivated respectively
After being 0.6~0.8 to OD600 value, then it is 1:1 according to volume ratio that being infected buffer and being resuspended and adjust OD600, which is 1~1.5,
It is mixed.
Preferably, stage of development of the floral organ in inoculation is in aobvious flower bud phase between the circle peach phase.
Preferably, the insertion position of the syringe needle is specially sepal base portion, and with bennet junction, cell tissue is thicker.
The beneficial effects of the present invention are embodied in:
The present invention carries out the inoculation of TRV viral vectors to the floral organ in growth course using with needle applicator, does not need
Macrocyclic stable expression and sterile working can reach and infect purpose, can be used in a short time verifying purpose gene in flower
Function in the different structure of organ, and the influence to flower development and fruit development, the present invention is at low cost, easy to operate, fits
The function of verifying gene in the ontologies plant such as tree peony is closed, is male with colored and fruit development related gene especially in tree peony
The research of gene function provides new foundation and direction in pellet.
Further, it is injected from sepal base position, can be convenient, is efficiently that infecting comprising viral vectors is slow
Fliud flushing is transported in each institutional framework of floral organ, and entire petal is made apparent water stain shape occur, and effect is infected in raising.
Detailed description of the invention
Fig. 1 is TRV1, TRV2 carrier (pTRV1, pTRV2) structure chart.
Fig. 2 is inoculation method schematic diagram；Wherein: (a) Inoculating date；(b) inoculation method；(c) inoculation is completed.
Fig. 3 is the floral organ phenotype after being inoculated with 12 days；Wherein: lower view is longitudinal partly cut-away of top view.
Fig. 4 is the internal anatomy of floral organ different structure after being inoculated with 12 days.
Fig. 5 is the accumulation of the viral vectors of half-quantitative detection floral organ different structure.
Specific embodiment
Invention is further described in detail with reference to the accompanying drawings and examples, and its object is to better understand this hair
The protection scope that bright content is not intended to limit the present invention.
The present invention is based on VIGS systems, and proposing one kind can the TRV used in research peony development related gene
The method of carrier mediated Gene Silencing systemic vaccination tree peony floral organ.
(1) raw materials and reagents source
1) peony plant: Varieties of Peony is that Feng Dan (buy from the limited public affairs of Shaanxi Zhong Zi state industry tree peony industry development by seed
Department), it plants under identical environment (planting site: Xibei Univ. of Agricultural & Forest Science & Technology's resource garden).
2) VIGS tests silent carrier TRV1 and TRV2 (referring to Fig. 1) used, is presented by Tsinghua University professor Liu Yule
(in October, 2011), but can voluntarily be constructed according to existing literature.
3) Agrobacterium GV3101 is purchased from marine growth.
4) LB liquid medium formula: 10g/L tryptone, 5g/L yeast extract, 10g/LNaCl；
LB solid plate culture medium: production plate then adds 15g/L agar powder in LB liquid medium.
5) YEP Liquid Culture based formulas: 10g/L tryptone, 10g/L yeast extract, 5g/L NaCl；
YEP solid plate culture medium: production plate then adds 15g/L agar powder in YEP fluid nutrient medium.
(2) inoculation method
1, the conversion and culture of Agrobacterium
1.1) TRV2, TRV1 plasmid are transformed into Agrobacterium GV3101 competent cell with frozen-thawed method respectively, liquid
Nitrogen freeze-thaw method converts Agrobacterium method particularly includes: takes 1~2 μ L viral vectors plasmid to be added in 1 pipe competence GV3101, ice
Upper placement 30min；Quick-frozen 5min in liquid nitrogen is placed into, taking-up is placed in 5min in 28 DEG C of water-bath, later ice bath 2min.It is added
900 μ L YEP fluid nutrient mediums, 180rpm, 2~4h of jog.4000rpm is centrifuged 1min at room temperature, abandons 800 μ L supernatants, then gently
Suction, which is beaten, makes cell suspend, and obtains conversion fluid.It draws 200 μ L of conversion fluid and is spread evenly across YEP solid plate culture medium (containing 50 μ g/mL
Rif+50 μ g/mL Gen+50 μ g/mL Kan) on, it is inverted for 28 DEG C and is protected from light culture 2 days.It picks from the plate single bacterium and falls within 300 μ L
In YEP fluid nutrient medium (containing 50 μ g/mL Rif+50 μ g/mL Gen+50 μ g/mL Kan), cultivate 5h (28 DEG C, 250rpm).So
Template is done with bacterium solution afterwards, PCR detects positive colony.It is spare that obtained positive strain is protected into bacterium.
1.2) the GV3101 Agrobacterium containing TRV2, TRV1 viral vectors is inoculated in 5mL YEP fluid nutrient medium respectively
In (containing 50 μ g/mL Kan+50 μ g/mL Rif+50 μ g/mL Gen), stayed overnight respectively at 28 DEG C of shaken cultivations.
1.3) 500 μ L bacterium solutions is taken within second day to be inoculated in 25mL YEP fluid nutrient medium (containing 50 μ g/mL Kan+50 μ g/mL
Rif+50 μ g/mL Gen) in, 28 DEG C of shaken cultivations are stayed overnight, until OD600 value is 0.6~0.8, (OD600 value and culture infect
Activity is related), 4000r/min is centrifuged 20min, outwells supernatant, retains bacterium precipitating.
1.4) buffer (10mM MES+100 μM AS (Acetosyringone)+10mM MgCl is infected with 20mL2, molten
Agent is water) thallus is resuspended, and be 1.2~1.5 with infecting buffer to adjust OD600 value.
1.5) after 1.4), the GV3101 bacterium solution containing TRV1 and the GV3101 bacterium solution containing TRV2 are pressed to the ratio of 1:1
Example (volume ratio) mixes, and 28 DEG C, place 3h (mixing well) under 90r/min, obtains mixed bacteria liquid.
2, it infects
In the tree peony circle peach phase, the petal that growth and development is not infected by germ normally, is chosen, (in March, 2018) is inoculated with.
Mixed bacteria liquid is drawn with the syringe with syringe needle, for tree peony sepal base portion, carries out injection inoculation.Angle is (circumferential from 2 to 3
On) injected, until petal surface is full of water stain.After inoculation, potted plant is moved on in phjytotron, cultivates item
Part are as follows: temperature is 23 ± 2 DEG C, and humidity is 60~70%, and intensity of illumination is 1800~2000Lx, the dark (training of 16h illumination/8h
Feeding condition is conducive to the breeding of TRV viral vectors, infects effect to reach good).
3, it detects
12 days after inoculation, phenotype observation is carried out to the floral organ after inoculation, and is grouped and knits sampling extraction RNA, uses semidefinite
The accumulation of the method detection viral vectors of amount.
3.1) extraction for the RNA that tree peony floral organ each group is knitted
1. taking 2mL centrifuge tube, it is added 700 μ L lysates (665 μ L SL and 35 μ L beta -mercaptoethanols)；
2. 100mg is taken to organize, tissue abrasion mixes at fine powder, concussion in liquid nitrogen, puts after shaking when sample is more on ice
It sets.12000rpm, 2min (4 DEG C)；
3. Aspirate supernatant, and be transferred in CS Filter column, 12000rpm, 2min (4 DEG C), gentle aspiration supernatant, and
It is transferred in the centrifuge tube of new RNase-Free.
4. dehydrated alcohol (for 0.4 times of supernatant volume) gently is added, after mixing, all liq is transferred to CR3
In adsorption column, 12000rpm, 15sec (4 DEG C) abandon waste liquid, CR3 adsorption column are placed back in collecting pipe.
5. the protein liquid removal RW1,12000rpm, 15sec (4 DEG C) of 350 μ L is added in CR3 adsorption column, waste liquid is abandoned, it will
CR3 adsorption column places back in collecting pipe.
6. plus the DNase I (preparing in advance) of 80 μ L is stood 15min (room temperature) in CR3 adsorption column centre.
7. the protein liquid removal RW1 of 350 μ L is added in CR3 adsorption column, 1min, 12000rpm, 15sec are stood, abandons waste liquid,
CR3 adsorption column is placed back in into collecting pipe.
8. 500 μ L rinsing liquid RW (with preceding plus ethyl alcohol) is added into CR3 adsorption column, 12000rpm, 15sec abandon waste liquid.
9. it is primary to repeat 8. step.
10. CR3 adsorption column is put into new centrifuge tube by 12000rpm after, 2min (4 DEG C), is added dropwise in the center of adsorbed film
30~50 μ L ddH2O is stood 3min (room temperature), 12000rpm, 1min, as RNA extracting solution.
3.2) synthesis of cDNA
By electrophoresis detection RNA mass, nucleic acid-protein quantitative instrument measures rna content.The synthesis reference of the first chain of cDNART reagent Kit with gDNA Eraser (TAKARA), is expanded later with 18S, for measuring
The quality of cDNA.
3.3) half-quantitative detection virus accumulation
Taking the cDNA of the 2 above-mentioned synthesis of μ L is template, is expanded with TRV2-U/TRV2-L primer PCR, detects TRV2, reactant
It is as follows:
TRV2-U:5 '-TTACGACGAACCAAGGGAGTACTAC-3 '
TRV2-L:5 '-AGTCACAATTAGCCCTATTTAGATGT-3 '
PCR response procedures: 94 DEG C of 2min；94 DEG C of 30s, 54 DEG C of 30s, 72 DEG C of 30s, 28 circulations；72℃5min.
3.4) result and analysis
It is inoculated with as shown in Fig. 2, showing flower bud phase in tree peony, until bud is full of water stain shape outside bud.After inoculation, peony flowers
Apparent difference (Fig. 3, Fig. 4) is presented with compareing and (not being inoculated with).12 days after inoculation, there is significant distortion symptom (virus in petal
Infection causes), and compared to control, there is discolouration phenomena in the peony petal of inoculated viral vectors.12 days after inoculation, stamen top
End blacks, and not loose powder, the blackening of stamen whole, shrivelled 20 days after inoculation.There is phenomenon of losing water in sepal top.Extract floral organ
The RNA of different structure carries out viral vectors detection, can detect the accumulation (Fig. 5) of viral vectors.
In short, inoculation method disclosed by the invention, easy, quickly TRV viral vectors can be inoculated into developmental
In tree peony floral organ, tree peony floral organ is made to can detecte virus load in the institutional frameworks such as petal, stamen, gynoecium, sepal
The presence of body.The present invention provides for application of the Gene Silencing technology in the research of peony growth course related gene
It is a kind of it is quick, efficiently infect method.New think of is provided for research tree peony flower development and fruit development related gene
Road.
<110>Xibei Univ. of Agricultural & Forest Science & Technology
<120>method of TRV carrier mediated Gene Silencing systemic vaccination tree peony floral organ
<160> 2
<210> 1
<211> 25
<212> DNA
<213>artificial sequence
<400> 1
ttacgacgaa ccaagggagt actac 25
<210> 2
<211> 26
<212> DNA
<213>artificial sequence
<400> 2
agtcacaatt agccctattt agatgt 26
Claims (7)
1. a kind of method for the Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated, it is characterised in that: should
Be inoculated with tree peony floral organ method the following steps are included:
1) by viral vectors TRV1 and TRV2 or by viral vectors TRV1 and recombination TRV2 inserted with target gene points
It Zhuan Hua not Agrobacterium, the positive Agrobacterium containing TRV1 that conversion is obtained and the positive Agrobacterium containing TRV2 or recombination TRV2
It mixes after cultivation in infecting in buffer, obtains mixed bacteria liquid；
2) after step 1), mixed bacteria liquid is drawn with the syringe with syringe needle, then by syringe needle in developmental tree peony floral organ
Injection inoculation is inserted into inside peony plant and carried out at official, until the peony organ surface stops injection when having liquid exudation；
3) use the following conditions to cultivate peony plant after injecting: for temperature for 21~25 DEG C, humidity is 60~70%,
Intensity of illumination is 1800~2000Lx.
2. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: the method for the inoculation tree peony floral organ is further comprising the steps of: right in peony plant incubation
Tree peony floral organ carries out phenotype observation, and samples, extracts the TRV disease for using the method for sxemiquantitative to detect each structure of floral organ after RNA
The accumulation of poisonous carrier.
3. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: the conversion uses freeze-thaw method.
4. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: the buffer that infects includes following components: 10mM 2-morpholine ethane sulfonic acid, 100 μM of acetosyringones
And 10mM MgCl2。
5. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: will corresponding positive Agrobacterium cultivate be 0.6~0.8 to OD600 value after, infected buffer and be resuspended simultaneously
Adjusting OD600 is 1~1.5, is then that 1:1 is mixed according to volume ratio.
6. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: stage of development of the floral organ in inoculation is in aobvious flower bud phase between the circle peach phase.
7. a kind of Gene Silencing systemic vaccination tree peony floral organ that TRV is carrier mediated according to claim 1
Method, it is characterised in that: the insertion position of the syringe needle is specially sepal base portion.
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Cited By (1)
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| CN114990155A (en) * | 2022-04-06 | 2022-09-02 | 东北林业大学 | Method for establishing virus-induced gene silencing system at rabdosia lophanthide flower part |
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