CN103820490A - Method for culturing male sterile plant through virus-induced gene silencing system - Google Patents

Abstract

The invention provides application of a virus-induced gene silencing (VIGS) carrier system in culturing a dicot male sterile plant. A VIGS-technology-based efficient practical plant sterile strain creation technical system is created, the sterile rate is more than 96% and can be up to 98%, the system has the characteristic of unstable inheritance, the infertility is not inherited in the later generations, and the gene contamination or the problem about biosafety caused by uncontrollable biotechnology diffusion is avoided. Besides, the method is simple to operate, the consumed time is short, the workload in the field is low, the cost is greatly saved, and the breeding efficiency is improved.

Description

A kind of method that uses the gene silencing system of virus induction to cultivate male sterile plants

Technical field

The carrier system and the method that the present invention relates to cultivate with animal nutrition male sterile plants, relate in particular to dicotyledons.

Background technology

It is stable that cultivation has sterility, and thoroughly, the good male sterile strain of each side proterties is to realize the key of plant hybridization breeding objective.At present, the main path of cultivating male sterile strain in production has cytoplasmic sterility system, the line with genic sterile chemical emasculation system etc. of unifying.

Cytoplasmic male sterility is most widely used general and one of the most effective approach in current rape heterosis utilization, but the sterility of this system sterile line not thoroughly, be subject to the impact of envrionment temperature.Under initial bloom stage cold condition, be prone to trace-pollen, the generation of micro mist will force maternal self-fertility, and F1 generation seed restoring degree is reduced, and on producing, application exists certain risk.For example, for rape, cytoplasmic sterility system micro mist problem is the technical barrier that breeding circle both at home and abroad is not yet thoroughly captured at present, is also the large hidden danger of rape three line for hybrid seed production ubiquitous.Nucleus sterile line sterility is thoroughly stable, is not subject to environmental influence, and restorer is many, easily obtains strong advantage combination.The weak point of this system is to exist in its sterile line 50% fertile plant, 50% fertile plant want artificial removal's sterile line in parent propagation and cross-fertilize seed production in, cause production cost increase, once and remove not in time, in cross-fertilize seed, there will be a certain proportion of sterile strain.Through improving, people have been bred as recessiveness and dominant genic male sterile three is, solved a difficult problem that needs to pull out 50% fertile plant in the breeding of core sterile parent and cross breeding seed process, but this system exist the problem that maintenance line is less, parent's seed selection is more difficult.Chemical emasculation refers to before male plant Organ Differentiation or in growth course, sprays interior absorption medicament, through a series of physiological and biochemical procedures, and to stop the formation of pollen or the normal development of inhibition pollen, and the male sterile causing.Utilizing male sterilant to cultivate kind is a kind of novel rapeseed breeding method, at home and abroad application is comparatively extensive, but this systematic cross kind is owing to being subject to ambient conditions impact, field complex operation, and due to problems such as pesticide residue, on producing, large-area applications also exists certain limitation.

In recent molecular biology research, utilizing animal nutrition to cultivate male sterile plants is an important research direction.Compared with traditional sterile line breeding, the cultivation of biotechnology sterile line can not be subject to the restriction of genetic resources, can change from molecular level the means realization of plant fertility by transgenosis etc.But the stable sterile line of utilizing genetically modified organism technology to cultivate is not only faced with the safety problem of transgenosis diffusion, and there is equally the shortcoming of above-mentioned nucleus sterile line.

The gene silencing (virus induced gene silencing, VIGS) of virus induction is also a kind of biotechnology that can be used for regulating plant native gene expression level.The gene silencing that VIGS causes is a kind of PTGS (post-transcriptional gene silencing, PTGS) phenomenon, is ubiquitous inherited immunity mechanism in plant materials.VIGS technology by inserting goal gene fragment in virus vector, and infect host plant, utilize naturally occurring inherited immunity mechanism in plant materials, by endogenous goal gene mRNA degraded, make plant occur the phenotype that goal gene afunction or expression level decline.Compared with plant transgenic technology, VIGS is without cultivating transfer-gen plant, and have easy and simple to handle, obtain the advantages such as phenotype is quick, be widely used in the research with plant disease-resistant, environment stress, cell signalling and the genes involved function such as grow at present.But, utilize the reticent plant endogenous gene of VIGS technology to have the problem of a silence efficiency always, one acquisition has the ratio of phenotype material lower than 30%, has limited this technology application.

Summary of the invention

The object of the invention is to utilize biotechnology foundation not to be subject to breeding resource limit, can to utilize the existing high-quality germ plasm resource of educating, quickly breeding sterility is the stable and good male sterile line of proterties thoroughly, and has the pollination Controlling System that field is simple to operate, breeding cost is low.

The object of the invention is to realize by following measures:

VIGS carrier system is in the application of cultivating in dicotyledonous male sterile plants.

Preferably, above-mentioned VIGS carrier system derives from Tobacco rattle virus (TRV virus), comprises helper viral vector pTRV1 (containing the RNA1 fragment of TRV virus) and expressing viral vector pTRV2 (containing the RNA2 fragment of TRV virus).

Above-mentioned pTRV1 carrier comprises following structure: be followed successively by the RNA polymerase (RdRp) that left margin (LB), 2 × 35S promoter, 2 × 35S promoter drive the Tobacco rattle virus RNA expressing to rely on, rich halfcystine 16K albumen, motion albumen (MP), independently shear ribozyme (Rz), transcription terminator (NOS _t), right margin (RB); Above-mentioned pTRV2 carrier comprises following structure: be followed successively by left margin (LB), 2 × 35S promoter, 2 × 35S promoter and drive the Tobacco rattle virus coat protein (CP) expressed, multiple clone site (MCS), independently shear ribozyme (Rz), transcription terminator (NOS _t), right margin (RB).As shown in Figure 9.In the time utilizing the reticent plant gene of VIGS method, utilize in the pTRV2 carrier that the multiple clone site (MSC) between pTRV2 support C P and Rz is structured in target fragment.PTRV1 and pTRV2 carrier all have the border, left and right of T-DNA, for object fragment in carrier being imported to vegetable cell infecting plant process, to start copying of Tobacco rattle virus.PTRV1 is containing the RNAl cDNA clone of TRV virus, by virus replication is assembled necessary; PTRV2 is containing TRV viral RNA 2cDNA clone and multiple clone site (MCS).

Above-mentioned VIGS carrier system is with plant fertility regulatory gene, be mainly recessive male sterility gene, be preferably COI1 gene (Coronatine-insensitivel), MSP1 gene (MultipleSporocyte), TDR gene (tapetum degeneration retardation) and UDT1 gene (undeveloped Tapetuml) etc., can affect after gene silencing that microsporocyte growth, tapetal development, pollen sac and extine are grown and the gene that causes male-sterile character.

Above-mentioned dicotyledons is preferably cress.

The sequence of rape C OI1 gene is SEQ ID NO.1, and wherein the sequence fragment of Nucleotide (1181) to (1694) can be for the whole rape C OI1 of silence gene.The sequence of tobacco COI1 gene is SEQ ID NO.2, and wherein Nucleotide (444) to (952) can be for the whole tobacco COI1 of silence gene.SEQ ID NO.3 is the Tobacco rattle virus RNA1 sequence fragment in pTRV1 carrier, and SEQ ID NO.4 is pTRV2 carrier sequence.

Use above-mentioned VIGS carrier system to cultivate the method for dicotyledonous male sterile plants, comprise the following steps:

(1) clone plant fertility regulatory gene, builds the restructuring pTRV2 virus vector that carries plant fertility regulatory gene;

(2) will carry restructuring pTRV2 virus vector, the pTRV1 carrier of plant fertility regulatory gene import Agrobacterium, by containing carrying the restructuring pTRV2 virus vector of plant fertility regulatory gene and the Agrobacterium of pTRV1 carrier, to be mixed with OD600 be 0.6～1.0 bacterium liquid;

(3) after the part coring of excision plant, root is immersed in the bacterium liquid of step (2);

(4) take out the plant after infecting, through cultivating, finally show as sterile proterties.

Preferably, the method that above-mentioned use VIGS carrier system is cultivated dicotyledonous male sterile plants, comprises the following steps:

(1) clone COI1 gene, builds the restructuring pTRV2-COI1 virus vector that carries COI1 gene;

(2) pTRV2-COI1 and pTRV1 carrier are imported to Agrobacterium GV3101, it is 0.6～1.0 bacterium liquid that the Agrobacterium GV3101 that contains respectively pTRV2-COI1 and pTRV1 carrier is mixed with to OD600 in the ratio of 1:1;

(3) after the part coring of excision plant, root is immersed in the bacterium liquid of step (2);

(4) take out the plant after infecting, through plantation, vernalization, after blooming, finally show as sterile proterties.

Beneficial effect

1. the cultivation that VIGS technology is applied to male sterile plants of the invention, the sterile strain of plant of having set up a highly effective creates technical system.Utilizing this technical system to cultivate in the practice of sterile material, sterile rate has exceeded 96%, can reach 98%.

2. the male sterile line of the invention has the feature of unstable heredity, can be not hereditary in offspring, and therefore, in producing, field can not occur to spread because of uncontrollable biotechnology the gene contamination or the Biosafety that cause.

3. the present invention can quickly breeding male sterile plants, can select arbitrarily optimum kind as sterile parent, do not exist as nucleus, desired extensive guarantor's relation in cytoplasmic male sterility system, therefore parent selects freedom, assembly cross combination arbitrarily, is conducive to select in excellent excellent, filters out stronger advantage combination.Meanwhile, the present invention is simple to operate, and the time is short.Can be transplanted to field indoor seedling root immersion after infecting, after there is no the field operation of artificial emasculation and chemical emasculation, field workload is few, greatly cost-saving, improves breeding efficiency.Improve the breeding objective of the heterotic utilising efficiency of plant and realization " excellent+high-quality of mixing ".

Accompanying drawing explanation

The pcr amplification figure (514bp) of Fig. 1 embodiment 2 rape C OI1 gene cDNA fragments: Marker:DNA molecular weight standard; 2,3: the pcr amplification product of tobacco COI1 gene fragment;

The bacterium colony PCR of Fig. 2 embodiment 2pTRV2-COI1 vector construction identifies figure: Marker:DNA molecular weight standard; 1-6: the pcr amplification product that pTRV2-COI1 carrier in different bacterium colonies is identified;

Fig. 3 embodiment 2pTRV2-COI1 plasmid enzyme restriction is identified figure: Marker:DNA molecular weight standard; The NcoI of 1,2:pTRV2-COI1 plasmid and EcoICRI double digestion are identified; The EcoICRI of 3,4:pTRV2-COI1 plasmid and SmaI double digestion are identified;

Fig. 4 embodiment 2 turns the bacterium colony PCR detection figure of pTRV2-COI1 carrier Agrobacterium: Marker:DNA molecular weight standard; 1-3: the pcr amplification that turns the different bacterium colonies of pTRV2-COI1 carrier Agrobacterium detects;

The expression level of COI1 gene in Fig. 5 embodiment 2COI1 gene silencing rape: contrast the rape infecting containing empty carrier Agrobacterium for using; L1-5 is that 5 use contain the rapeseed plants that COI1 genophore Agrobacterium is infected;

Fig. 6 embodiment 2VIGS infects the fruit pod phenotype of rape: left side is for contrast rape fruit pod, normally solid; Right side is the fruit pod that VIGS infects rape, without fruit;

Fig. 7 embodiment 2VIGS infects the sterile phenotype of brassica napus inflorescence, and its fruit pod is all normally not solid;

The sterile phenotype that Fig. 8 embodiment 3 causes with the reticent tobacco COI1 of VIGS gene;

Fig. 9 VIGS system carrier pTRV1 and pTRV2 structural representation.

Embodiment

Below by embodiment, the present invention is specifically described; be necessary to be pointed out that at this following examples are only used to further illustrate the present invention; can not be interpreted as limiting the scope of the invention, these those skilled in the art can make some nonessential improvement and adjustment to the present invention according to the invention described above content.

Embodiment 1

1. build the carrier of the reticent rape C OI1 of VIGS gene

The acquisition of 1.1 rape C OI1 gene cDNAs

Cultivate Brassica Napus Seedling, in the time that growing to 10cm left and right, it extracts total RNA of stem stalk and cotyledon by TRIZOL method, obtain cDNA corresponding to the total RNA of rape by reverse transcription, take a morsel cDNA as template, forward primer is: AAATCTAGAGTGTCTCCGAACCATAGGC, reverse primer is: TCTCTCACCTCTCCAAGCTG, pcr amplification obtains one section of rape C OI1 gene cDNA fragment (SEQ ID NO.1 (1181) is to (1694)) that size is 514bp.

The acquisition of the 1.2 pTRV2 carriers containing rape COI1 gene

By the cDNA fragment of XbaI enzyme cutting COI1 gene, with XbaI and EcoICRI double digestion plasmid pTRV2, the enzyme system of cutting is respectively: 1.5 μ L Buffer (Buffer3), and 1 μ L XbaI, 5 μ L COII gene cDNA fragments, add water and are supplemented to 15 μ L; 1.5 μ L Buffer (Buffer MultiCore), 0.5 μ L Xba1,0.5 μ L EcoICRI0.1 μ L BSA, 3 μ L pTRV2 plasmids, add water and be supplemented to 15 μ L. after 2 hours endonuclease reactions, agarose gel electrophoresis, cuts glue and reclaims to obtain COI1 fragment and carrier segments.Cutting glue with following scheme reclaims:

Ultraviolet lamp incision glue, puts into 1.5ml centrifuge tube, adds sol solutions by 300 μ L/1OOmg blob of viscoses, and centrifuge tube is placed in to ready 50 ℃ of about 10min of preheating water bath, melts completely to blob of viscose.The solution having melted is proceeded to glue and reclaim in adsorption column, the centrifugal 1min of 8krpm, abandons liquid.In adsorption column, add 500 μ L washingss, leave standstill 1min, the centrifugal 1min of 12000rpm, abandons liquid, repeating step 4.Sky gets rid of (the centrifugal 1min of adsorption column 12000rpm), and adsorption column is proceeded to a clean centrifuge tube, adds 26-30 μ L ddH ₂o is in central membrane place, and room temperature leaves standstill 4min, and the centrifugal 1min of 12000rpm, is recycled product.

Recovery COI1 fragment and pTRV2 carrier segments are spent the night in 4 ℃ of connections, must be containing the pTRV2 carrier of rape COI1 gene, called after pTRV2-COI1, linked system is: 1 μ L T4DNA ligase enzyme Buffer (10x) 1 μ L T4DNA ligase enzyme, 1 μ L PEG4000,6 μ L COI1 fragments, 1 μ L carrier segments.

2pTRV2-COI1 carrier connects product heat shock and transforms bacillus coli DH 5 alpha.

2.1 transform

Get competence intestinal bacteria (DH5 α) at-80 ℃ of refrigerators, the about 10min of ice-water bath thaws, and connection product is added and mixed.After ice bath 30min, heat shock 60s in 42 ℃ of water-baths, ice bath 1-2min at once, adds 900-1000 μ L LB solution, 37 ℃ of 180rpm shaking tables about 1h that recovers.5krpm5min is centrifugal, inhales and abandons 900 μ L supernatant liquors, remaining resuspended, is coated with LB flat board (containing the Kan of 30mg/L), is inverted incubated overnight (about 14h) for 37 ℃.

2.2 picking positive colonies

Carrying out bacterium colony PCR evaluation through picking list bacterium colony on the flat board of incubated overnight, carry out primary dcreening operation.Through electrophoresis run glue confirm after, by the bacterium liquid that contains correct plasmid (adding 50% glycerine in 1:1 ratio) be stored in-20 ℃ for subsequent use.Extract plasmid by following scheme:

The bacterium liquid shaking is sub-packed in to an even number centrifugal 4min of 10ml sterile test tube 7krpm, abandon supernatant liquor, in test tube, add 200 μ L sol I (4 ℃) to mix, add again 200 μ L sol II (room temperature), turn upside down and mix (approximately 10 times), solution becomes sticky thick (as clear nasal mucus).In test tube, add 250 μ L sol III (room temperature) again, turn upside down and mix (approximately 10 times), occur cotton-shaped or block white precipitate, it is limpid that solution becomes.By centrifugal above-mentioned test tube 11krpm6min, get supernatant liquor and proceed in purification column, centrifugal 8krpm30s, abandons liquid.In purification column, add 650 μ L washingss, centrifugal 8krpm30s, abandons liquid.Purification column sky gets rid of (13k rpm2min is centrifugal), and purification column is proceeded to clean 1.5ml centrifuge tube, adds 30 μ L ddH to purification column ₂o (being added on tunica fibrosa), room temperature leaves standstill 4min, and centrifugal 13krpm2min obtains plasmid.

Finally, extraction plasmid being carried out respectively to the evaluation of NcoI-EcoICRI double digestion and EcoICRI-SmaI double digestion identifies.It is as follows respectively that enzyme is cut identification system: 1.5 μ L Buffer, and 0.5 μ L NcoI, 0.5 μ L EcoICRI, 0.1 μ L BSA, 3 μ L pTRV2-COII plasmids, add water and are supplemented to 15 μ L; 1.5 μ L Buffer, 0.5 μ L Sma1,0.5 μ L EcoICRI, 0.1 μ L BSA, 3 μ L pTRV2 plasmids, interpolation water is supplemented to 15 μ L. enzymes and cuts after 1 hour, and agarose gel electrophoresis evaluation enzyme is cut result.Enzyme is cut the positive plasmid that result is consistent with expection.Finally, transfer to biotech firm's sequence verification.

3. Agrobacterium GV3101 competence preparation

(1) streak culture 28 ℃ of Agrobacterium GV3101 2 days

(2) choose 1 single colony inoculation in 5mlYEB (having added 5 μ L Rif), 28 ℃ of incubated overnight

(3) get bacterium liquid GV31013ml and be inoculated in 200mlYEB liquid nutrient medium, 28 shake bacterium to OD ≈ 0.6 (about 4h)

(4) pack the bacterium liquid shaking into centrifugal bottle centrifugal 5krpm11min, abandoning liquid, to add (ice process 2h) distilled water resuspended, and the centrifugal 11min of 5krpm, abandons liquid

(5) in centrifugal bottle, add the resuspended standing 10min of ice-cold 0.1mM CaCl2, the centrifugal 11min of 5000rpm, abandons supernatant liquor

(6) in centrifugal bottle, add the ice-cold 85mL CaCl2 solution that contains 15% glycerine of 2mL

(7) with the packing of 1.5ml centrifuge tube (100 μ L/ pipes, carry out mark on centrifuge tube), put into rapidly liquid nitrogen and process several minutes

(8) competent cell of handling well is taken out from liquid nitrogen and be placed in-80 ℃ of preservations

4. transform Agrobacterium

(1) get competence Agrobacterium GV31013 part of-80 ℃ of preservations in thawing on ice

(2) to adding gently respectively plasmid in the bacterium liquid of every a Agrobacterium: 5 μ L pTRV2-COI1,1 μ L pTRV1,1 μ L pTRV2 (empty carrier), carefully mixes

(3) by the above-mentioned bacterium liquid ice-water bath 5min mixing

(4) ice-water bath bacterium liquid after treatment liquid nitrogen is processed 5min (liquid nitrogen liquid level exceeds bacterium liquid level)

(5) 37 ℃ of water-bath heat shock 5min

(6) in above-mentioned bacterium liquid after treatment, add the YEB of 900 μ L room temperatures placements in 28 ℃ of 200rpm shaking table recovery 1h

(7) the centrifugal 5min of bacterium liquid 6krpm after recovery, abandons supernatant 900 μ L

(8) the resuspended YEB flat board (1 μ L Kan/1ml YEB, 1 μ L Rif/1ml YEB) that is applied to of remaining bacterium liquid

Be inverted for (9) 28 ℃ and cultivate about 2d, to the bacterium colony that grows diameter 1-2mm

Picking list bacterium colony carries out bacterium colony PCR evaluation and carries out primary dcreening operation, run through electrophoresis the Agrobacterium that obtains the pTRV2-COI1 carrier that contains the reticent rape C OI1 of VIGS gene after glue is confirmed, called after: GV-COI1, and by the Agrobacterium called after that contains pTRV2 empty carrier: GV-C, the Agrobacterium called after by containing pTRV1 carrier: GV-1.

Embodiment 2

1. experiment Brassica Napus Seedling sowing, vernalization, grows seedlings

In sowing rape two No. 11, adopt the plastic optical fibre thing that fixes, MS liquid nutrient medium is nutriment, and stem stalk grows to choline dichloride that 5cm left and right sprays 1/500 dilution to suppress the overgrowth of Brassica Napus Seedling. and after first quarter moon, start vernalization, vernalization condition is 9h on daytime, 9 ℃; Night 15h, 40 ℃.12 ℃ of recovery 2d 9h on daytime after vernalization 14d, night 15h.The 25 ℃ of hot-house cultures of rear immigration of having recovered.

2. infect Brassica Napus Seedling with the Agrobacterium that contains VIGS carrier, cultivate the rape strain of reticent COI1 gene

2.1 infect and prepare with Agrobacterium

Respectively by GV-C, GV-1 and GV-COI1 in 6ml YEB substratum, add 6 μ L Rif, 6 μ L Kan, and transfer three single bacterium inoculations, in 28 ℃, 220rpm. shakes bacterium overnight incubation; Then, in 50ml YEB substratum, add 50 μ L Rif, 50 μ L Kan, 5 μ L Syringylethanones and 500 μ L incubated overnight bacterium liquid, then 28 ℃, 220rpm. is cultured to obviously thicken (about 12h).

2.2 high densitys infect

Shake the centrifugal 5min of each Agrobacterium bacterium liquid 5krpm of cultivation by two, abandon supernatant, respectively add the fresh YEB liquid nutrient medium of 20ml resuspended, then by A liquid: 10ml GV-C and 10ml GV-1; B liquid: 10ml GV-COI1 and 10ml GV-1, mix respectively.Respectively by Brassica Napus Seedling 100 strains, cut off part root system with scissors, be immersed in A liquid completely; By Brassica Napus Seedling 100 strains, cut off part root system with scissors, immerse B liquid completely, be placed in camera bellows spend the night (about 12h).

2.3 lower concentrations infect

The Brassica Napus Seedling that high density was infected takes out from high-concentration bacterial liquid, adds respectively a small amount of fresh MS to cultivate 10h. at camera bellows separately

3. soaked the cultivation of the rapeseed plants of root processing

The Brassica Napus Seedling that soaks root processing is planted by individual plant, with vermiculite and vegetable mould by volume 1:1 do cultivating soil, be respectively every group of rape called after GV-C and GV-COI1 by treatment solution A liquid B liquid, move into greenhouse cultivation.With tap water pouring, water 4-5 40ml Glan (Hoagland) nutritive medium suddenly therebetween.

Results and analysis:

1. the acquisition of rape C OI1 gene cDNA fragment

Obtain cDNA corresponding to the total RNA of rape by reverse transcription, the cDNA that takes a morsel obtains by pcr amplification the rape C OI1 gene cDNA fragment that a size is about 514bp as template, as Fig. 1.

After 2.pTRV2-COI1 carrier conversion bacillus coli DH 5 alpha, the PCR of plasmid identifies

Enzyme is cut to rape C OI1 gene fragment and cut being connected after product conversion bacillus coli DH 5 alpha of pTRV2 carrier with enzyme, on the flat board of incubated overnight, picking list bacterium colony carries out bacterium colony PCR evaluation.Electrophoresis result, as Fig. 2, conforms to expection, shows to obtain positive colony.

3. the enzyme of the positive colony that couple PCR identifies is cut evaluation

Previous step is accredited as to positive clone, extracts pTRV2-COI1 vector plasmid wherein, proceed enzyme and cut evaluation, electrophoresis result is as Fig. 3, and enzyme is cut result and conformed to expection, is further indicated as positive colony.

4. pair positive colony obtaining order-checking

Sequencing result conforms to the gene order of expection, shows to obtain rape pTRV2-COI1 carrier.

The bacterium liquid PCR that 5.pTRV2-COI1 carrier vector transforms after Agrobacterium identifies

PTRV2-COI1 carrier transforms after Agrobacterium GV3101, carries out bacterium liquid PCR and identifies, electrophoresis result, as Fig. 4, has object band, shows to obtain the Agrobacterium positive colony of pTRV2-COI1 carrier.

6.COI1 the COI1 gene expression dose in gene silencing rape obviously declines

Rape soaks and infects and normal growth after 6 weeks through root at Seedling Stage, detect to contrast and with the expression level of COI1 gene in the reticent COI1 vector for transgenic rape of VIGS plant, show that the expression level of COI1 gene in reticent COI1 vector for transgenic rape plant is lower than 20% of contrast, remarkable inhibition has been received in its expression.This explanation VIGS method success is reticent COI1 gene in rape.

7. the rape of goal gene silence shows sterile symptom

After rape soaks through root at Seedling Stage and infects, normal growth is bloomed, to productive phase, adjoining tree can be normally solid, and the plant of goal gene COI1 silence fruit pod is failed normal development, show as sterile, as Fig. 6 (the single fruit pod of the reticent plant of adjoining tree and goal gene), Fig. 7.It is A liquid that the root of adjoining tree infects liquid, consists of GV-C+GV-1, i.e. the mixed solution of the Agrobacterium of empty carrier pTRV2 and assistant carrier pTRV1 Agrobacterium.It is B liquid that the plant root of goal gene COI1 silence infects liquid, and it consists of GV-COI1+GV-1, contains the Agrobacterium of recombinant vectors pTRV2-COI1 and the mixed solution of assistant carrier pTRV1 Agrobacterium of goal gene COI1.Experimental result shows that the gene silencing system of virus induction successfully makes rape sterile by expection, cultivates rapeseed male sterility strain, and the male parent can be used as in rapeseed breeding is used.VIGS infects the statistical conditions of infertility rape plant in rape in table 1.

Table 1VIGS infects the statistics of infertility rape plant in rape

VIGS processes	GV-C+GV-1	GV-COI1
			Infect rape strain number	150	150
Infertility rape strain number	0	149

Embodiment 3

The corresponding sequence of tobacco COI1 gene ((444) .. (952) of SEQ ID NO.2) is built into VIGS carrier, and infects the male sterile plants that has also obtained tobacco after tobacco.As shown in Figure 8, the tobacco of reticent COI1 gene is because the heteroplasia of stamen has caused sterile.

Claims (8)

1.VIGS carrier system is in the application of cultivating in dicotyledonous male sterile plants.

2. VIGS carrier system as claimed in claim 1 is in the application of cultivating in dicotyledonous male sterile plants, and described dicotyledons is cress.

3. VIGS carrier system as claimed in claim 1 or 2, in the application of cultivating in dicotyledonous male sterile plants, is characterized in that: described VIGS carrier system derives from Tobacco rattle virus ,comprise helper viral vector pTRV1 and expressing viral vector pTRV2.

4. VIGS carrier system as claimed in claim 3, in the application of cultivating in dicotyledonous male sterile plants, is characterized in that: described expressing viral vector pTRV2 contains plant fertility regulatory gene.

5. VIGS carrier system as claimed in claim 4 is in the application of cultivating in dicotyledonous male sterile plants, and described plant fertility regulatory gene is one or more in COI1 gene, MSP1 gene, TDR gene or UDT1 gene.

6. the VIGS carrier system as described in as arbitrary in claim 3-5, in the application of cultivating in dicotyledonous male sterile plants, is characterized in that: described pTRV1 carrier comprises following structure: be followed successively by the RNA polymerase (RdRp) that left margin (LB), 2 × 35S promoter, 2 × 35S promoter drive the Tobacco rattle virus RNA expressing to rely on, rich halfcystine 16K albumen, motion albumen (MP), independently shear ribozyme (Rz), transcription terminator (NOS _t), right margin (RB); Described pTRV2 carrier comprises following structure: be followed successively by left margin (LB), 2 × 35S promoter, 2 × 35S promoter and drive the Tobacco rattle virus coat protein (CP) expressed, can insert the multiple clone site (MCS) of goal gene, independently shear ribozyme (Rz), transcription terminator (NOS _t), right margin (RB).

7. VIGS carrier system as claimed in claim 6, in the application of cultivating in dicotyledonous male sterile plants, is characterized in that the structure of described VIGS carrier system as shown in Figure 9.

8. right to use requires the method that the arbitrary described VIGS carrier system of 3-7 is cultivated dicotyledonous male sterile plants, comprises the following steps:

(1) clone plant fertility regulatory gene, builds the restructuring pTRV2 virus vector that carries plant fertility regulatory gene;

(2) will carry the restructuring pTRV2 virus vector of plant fertility regulatory gene import Agrobacterium, be 0.6～1.0 bacterium liquid by be mixed with OD600 containing the Agrobacterium of restructuring pTRV2 virus vector of carrying plant fertility regulatory gene;

(3) after the part coring of excision plant, root is immersed in the bacterium liquid of step (2);

(4) take out the plant after infecting, through cultivating, finally show as sterile proterties.

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