CN103820490B - A kind of method that uses the gene silencing system of virus induction to cultivate male sterile plants - Google Patents

Abstract

The invention provides VIGS carrier system in the application of cultivating in dicotyledonous male sterile plants. The sterile strain of plant that the present invention has set up a highly effective based on VIGS technology creates technical system, sterile rate has exceeded 96%, can reach 98%, and there is the feature of unstable heredity, can be not hereditary in offspring, can not occur to spread because of uncontrollable biotechnology the gene contamination or the bio-safety that cause. Meanwhile, the present invention is simple to operate, and the time is short, and field workload is few, greatly cost-saving, improves breeding efficiency.

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 ShuangziLeaf plant.

Background technology

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

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 environment temperature. Under initial bloom stage cryogenic conditions, be prone toTrace-pollen, the generation of micro mist will force maternal self-fertility, and F1 generation seed restoring degree is reduced, and on producing, application exists oneDetermine risk. For example, for rape, cytoplasmic sterility system micro mist problem is that breeding circle both at home and abroad is not yet thoroughly captured at presentTechnical barrier is also the large hidden danger of rape three line for hybrid seed production ubiquitous. Nucleus sterile line sterility is thoroughly stable, is not encircledBorder condition impact, restorer is many, easily obtains strong advantage combination. The weak point of this system is to exist in its sterile line 50% canEducate strain, in parent propagation and cenospecies produce, want artificial removal's sterile line in 50% fertile plant, cause production cost increase,Once and removal not in time, there will be a certain proportion of sterile strain in cenospecies. Through improvement, people be bred as recessive andDominant genic male sterile three is to have solved the difficulty that needs to pull out 50% fertile plant in the breeding of core sterile parent and cross breeding seed processTopic, but there is the problem that maintainer is less, parent's seed selection is more difficult in this system. Chemical emasculation refers at male plant Organ DifferentiationBefore or in growth course, spray interior absorption medicament, through a series of physiological and biochemical procedures, to stop the formation of pollen or to suppress flowerThe normal development of powder, and the male sterility causing. Utilizing male sterilant to cultivate kind is a kind of novel rapeseed breeding method,Application is at home and abroad comparatively extensive, but this systematic cross kind is owing to being subject to external condition impact, field complex operation, Er QieyouIn problems such as residues of pesticides, also there is certain limitation in large-area applications on producing.

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

The gene silencing (virusinducedgenesilencing, VIGS) of virus induction is also that one can be used for adjustingControl the biotechnology of plant endogenous gene expression dose. The gene silencing that VIGS causes is a kind of PTGS (post-Transcriptionalgenesilencing, PTGS) phenomenon is ubiquitous inherent immunity mechanism in plant.VIGS technology is by insert genes of interest fragment in viral vectors, and infects host plant, utilizes natural existence in plantInherent immunity mechanism, by endogenous genes of interest mRNA degraded, plant is occurred under genes of interest afunction or expressionThe phenotype of falling. Compared with plant transgenic technology, VIGS, without cultivating transfer-gen plant, and has easy and simple to handle, acquisition tableThe advantages such as type is quick, have been widely used in and plant disease-resistant, environment stress, cell signalling and the phase such as grow at presentThe research of correlation gene function. 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 limitation, can to utilize the existing high-quality kind of educatingMatter resource, quickly breeding sterility is the stable and good male sterile line of proterties thoroughly, and has that field is simple to operate, the production of hybrid seedsThe pollination control system that 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 vectorPTRV1 (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 left margin (LB), 2 × 35S promoter, 2 × 35S promoterThe RNA polymerase (RdRp), rich cysteine 16K albumen, the motion albumen that drive the Tobacco rattle virus RNA expressing to rely on(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 drive the Tobacco rattle virus coat protein (CP) expressed,MCS (MCS), independently shear ribozyme (Rz), transcription terminator (NOS_t), right margin (RB). As shown in Figure 9. UtilizingWhen the reticent plant gene of VIGS method, utilize MCS (MSC) between pTRV2 support C P and Rz by target fragment structureIn the pTRV2 carrier of building in. PTRV1 and pTRV2 carrier all have the border, left and right of T-DNA, for infecting plant process generalObject fragment in carrier imports plant cell, to start copying of Tobacco rattle virus. PTRV1 is containing the RNAl of TRV virusCDNA clone, by virus replication is assembled necessary; PTRV2 is containing TRV viral RNA 2cDNA clone and MCS (MCS).

Above-mentioned VIGS carrier system, with plant fertility controlling gene, is mainly recessive male sterility gene, is preferablyCOI1 gene (Coronatine-insensitivel), MSP1 gene (MultipleSporocyte), TDR gene (tapetumDegenerationretardation) and UDT1 gene (undevelopedTapetuml) etc., can shadow after gene silencingRing that microsporocyte growth, tapetal development, pollen bag and exposore are grown and the gene that causes male-sterile character.

Above-mentioned dicotyledon is preferably crucifer.

The sequence of rape C OI1 gene is SEQIDNO.1, and wherein nucleotides (1181) to the sequence fragment of (1694) canFor the whole rape C OI1 of silence gene. The sequence of tobacco COI1 gene is SEQIDNO.2, and wherein nucleotides (444) extremely(952) can be for the whole tobacco COI1 of silence gene. SEQIDNO.3 is the Tobacco rattle virus RNA1 in pTRV1 carrierSequence fragment, SEQIDNO.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 controlling gene, builds the restructuring pTRV2 viral vectors that carries plant fertility controlling gene;

(2) the restructuring pTRV2 viral vectors, the pTRV1 carrier that carry plant fertility controlling gene are imported to Agrobacterium, will containCarry the restructuring pTRV2 viral vectors of plant fertility controlling gene and the Agrobacterium of pTRV1 carrier be mixed with OD600 and 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 viral vectors that carries COI1 gene;

(2) pTRV2-COI1 and pTRV1 carrier are imported to Agrobacterium GV3101, by contain respectively pTRV2-COI1 andIt is 0.6～1.0 bacterium liquid that the Agrobacterium GV3101 of pTRV1 carrier is mixed with 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, has set up a highly effectiveThe sterile strain of plant create technical system. Utilizing this technical system to cultivate in the practice of sterile material, sterile rate has exceeded96%, can reach 98%.

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

3. the present invention can quickly breeding male sterile plants, can select arbitrarily optimum kind as sterile parent, noExist as nucleus, desired extensive guarantor's relation in cytoplasmic male sterility system, therefore parent selects freedom, assembly arbitrarilyCross combination, 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. In chamberIn seedling root is soaked after infecting and can be transplanted to field, after do not have the field of artificial emasculation and chemical emasculation to graspDo, field workload is few, greatly cost-saving, improves breeding efficiency. Improve the heterotic utilization ratio of plant and realizationThe breeding objective of " excellent+high-quality of mixing ".

Brief description of the drawings

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

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

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

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 of COI1 gene in Fig. 5 embodiment 2COI1 gene silencing rape: contrast as using containing empty carrier agriculture barThe rape that bacterium is infected; 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 isVIGS infects the fruit pod of 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.

Detailed description of the invention

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

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 it grows to 10cm left and right, extract total RNA of stem stalk and cotyledon by TRIZOL method, by insteadTranscribe and obtain cDNA corresponding to the total RNA of rape, the cDNA that takes a morsel is as template, and forward primer is:AAATCTAGAGTGTCTCCGAACCATAGGC, reverse primer is: TCTCTCACCTCTCCAAGCTG, pcr amplification obtains oneSize is one section of rape C OI1 gene cDNA fragment (SEQIDNO.1 (1181) is to (1694)) of 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, enzyme is cut systemBe respectively: 1.5 μ LBuffer (Buffer3), 1 μ LXbaI, 5 μ LCOII gene cDNA fragments, add water and are supplemented to 15 μ L;1.5 μ LBuffer (BufferMultiCore), 0.5 μ LXba1,0.5 μ LEcoICRI0.1 μ LBSA, 3 μ LpTRV2 matterGrain, add water be supplemented to 15 μ L. after 2 hours endonuclease reactions, agarose gel electrophoresis, cut glue reclaim COI1 fragment and carryBody fragment. Cutting glue with following scheme reclaims:

Uviol 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 inAbout 10min in ready 50 DEG C of preheating water baths, melts completely to blob of viscose. The solution having melted is proceeded to glue and reclaim adsorption columnIn, the centrifugal 1min of 8krpm, abandons liquid. In adsorption column, add 500 μ L cleaning solutions, 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 LddH₂O 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 DEG C of connections, must carry containing the pTRV2 of rape COI1 geneBody, called after pTRV2-COI1, linked system is: 1 μ LT4DNA ligase Buffer (10x) 1 μ LT4DNA ligase, 1 μ LPEG4000,6 μ LCOI1 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 Escherichia coli (DH5 α) at-80 DEG C of refrigerators, the about 10min of ice-water bath thaws, and connection product is added mixedEven. After ice bath 30min, heat shock 60s in 42 DEG C of water-baths, ice bath 1-2min at once, adds 900-1000 μ LLB solution, 37 DEG CThe 180rpm shaking table about 1h that recovers. 5krpm5min is centrifugal, inhales and abandons 900 μ L supernatants, remaining resuspended, is coated with LB flat board (containing 30mg/L'sKan), be inverted incubated overnight (about 14h) for 37 DEG C.

2.2 picking positive colonies

Carrying out bacterium colony PCR qualification through picking list bacterium colony on the flat board of incubated overnight, carry out primary dcreening operation. Run glue through electrophoresis trueAfter recognizing, by the bacterium liquid that contains correct plasmid (adding 50% glycerine in 1:1 ratio) be stored in-20 DEG C for subsequent use. Extract by following schemePlasmid:

The bacterium liquid shaking is sub-packed in to an even number centrifugal 4min of 10ml sterile test tube 7krpm, abandons supernatant, in test tube, addEnter 200 μ LsolI (4 DEG C) and mix, then add 200 μ LsolII (room temperature), turn upside down and mix (approximately 10 times), solution becomes stickyThick (as clear nasal mucus). In test tube, add 250 μ LsolIII (room temperature) again, turn upside down and mix (approximately 10 times), occur cotton-shapedOr block white precipitate, it is limpid that solution becomes. By centrifugal above-mentioned test tube 11krpm6min, get supernatant and proceed in purification column, centrifugal8krpm30s, abandons liquid. In purification column, add 650 μ L cleaning solutions, centrifugal 8krpm30s, abandons liquid. Purification column sky gets rid of (13kRpm2min is centrifugal), purification column is proceeded to clean 1.5ml centrifuge tube, add 30 μ LddH to purification column₂O (being added on tunica fibrosa),Room temperature leaves standstill 4min, and centrifugal 13krpm2min obtains plasmid.

Finally, extraction plasmid is carried out respectively to the qualification of NcoI-EcoICRI double digestion and EcoICRI-SmaI double digestion mirrorFixed. It is as follows respectively that enzyme is cut identification system: 1.5 μ LBuffer, 0.5 μ LNcoI, 0.5 μ LEcoICRI, 0.1 μ LBSA, 3 μ LPTRV2-COII plasmid, adds water and is supplemented to 15 μ L; 1.5 μ LBuffer, 0.5 μ LSma1,0.5 μ LEcoICRI, 0.1 μ LBSA, 3 μ LpTRV2 plasmids, interpolation water is supplemented to 15 μ L. enzymes and cuts after 1 hour, and agarose gel electrophoresis qualification enzyme is cut result. EnzymeCut the positive plasmid that result is consistent with expection. Finally, transfer to biotech firm's sequence verification.

3. Agrobacterium GV3101 competence preparation

(1) Agrobacterium GV3101 line cultivate 28 DEG C 2 days

(2) choose 1 single colony inoculation in 5mlYEB (having added 5 μ LRif), 28 DEG C of incubated overnight

(3) get bacterium liquid GV31013ml and be inoculated in 200mlYEB fluid 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,The centrifugal 11min of 5krpm, abandons liquid

(5) in centrifugal bottle, add the resuspended standing 10min of ice-cold 0.1mMCaCl2, the centrifugal 11min of 5000rpm, abandons supernatant

(6) in centrifugal bottle, add the ice-cold 85mLCaCl2 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 numberMinute

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

4. transform Agrobacterium

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

(2) to adding gently respectively plasmid in the bacterium liquid of every a Agrobacterium: 5 μ LpTRV2-COI1,1 μ LpTRV1,1 μLpTRV2 (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 DEG C 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 DEG C 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 LKan/1mlYEB, 1 μ LRif/1mlYEB) that is applied to of remaining bacterium liquid

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

Picking list bacterium colony carries out bacterium colony PCR qualification and carries out primary dcreening operation, runs after glue is confirmed and obtains and contain the reticent rape of VIGS through electrophoresisThe Agrobacterium of the pTRV2-COI1 carrier of COI1 gene, called after: GV-COI1, and by the Agrobacterium life that contains pTRV2 empty carrierBy name: 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 fluid nutrient medium is nutriment, stem stalk grows to 5cmLeft and right sprays the cycocel of 1/500 dilution to suppress the overgrowth of Brassica Napus Seedling. and after first quarter moon, start vernalization, vernalization condition is whiteIt 9h, 9 DEG C; Night 15h, 40 DEG C. 12 DEG C of recovery 2d 9h on daytime after vernalization 14d, night 15h. The 25 DEG C of greenhouses of rear immigration of having recoveredCultivate.

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 6mlYEB culture medium, add 6 μ LRif, 6 μ LKan, and transferThree single bacterium inoculations, in 28 DEG C, 220rpm. shakes bacterium overnight incubation; Then, in 50mlYEB culture medium, add 50 μ LRif,50 μ LKan, 5 μ L acetosyringones and 500 μ L incubated overnight bacterium liquid, then 28 DEG C, 220rpm. is cultured to obviously and thickens (approximately12h)。

2.2 high concentrations infect

Shake the centrifugal 5min of each Agrobacterium bacterium liquid 5krpm of cultivation by two, abandon supernatant, respectively add the fresh YEB Liquid Culture of 20mlBasic weight is outstanding, then by A liquid: 10mlGV-C and 10mlGV-1; B liquid: 10mlGV-COI1 and 10mlGV-1, mix respectively. PointNot 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 with scissorsPart root system, immerses B liquid completely, is placed in camera bellows spend the night (about 12h).

2.3 low concentrations infect

The Brassica Napus Seedling that high concentration was infected takes out from high-concentration bacterial liquid, adds respectively a small amount of fresh MS separately secretlyCase is cultivated 10h.

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 fertile soil by volume 1:1 cultivateSoil, is respectively every group of rape called after GV-C and GV-COI1 by treatment fluid A liquid B liquid, moves into greenhouse cultivation. With running water pouring,Water 4-5 40ml Glan (Hoagland) nutrient solution 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 one as template by pcr amplificationBar size is about the rape C OI1 gene cDNA fragment of 514bp, as Fig. 1.

The PCR qualification of plasmid after 2.pTRV2-COI1 carrier conversion bacillus coli DH 5 alpha

Enzyme is cut to rape C OI1 genetic 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 qualification. Electrophoresis result, as Fig. 2, conforms to expection, shows to obtain sunSex clone.

3. the enzyme of the positive colony of pair PCR qualification is cut qualification

Previous step is accredited as to positive clone, extracts pTRV2-COI1 vector plasmid wherein, proceed enzyme and cut mirrorFixed, 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.

5.pTRV2-COI1 carrier vector transforms the bacterium liquid PCR qualification after Agrobacterium

PTRV2-COI1 carrier transforms after Agrobacterium GV3101, carries out bacterium liquid PCR qualification, and electrophoresis result, as Fig. 4, has objectBand, 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, to contrast and with the reticent COI1 base of VIGSBecause the expression of COI1 gene in rapeseed plants detects, show the expression water of COI1 gene in reticent COI1 vector for transgenic rape plantFlat lower than 20% of contrast, remarkable inhibition has been received in its expression. This explanation VIGS method success is reticent COI1 base in rapeCause.

7. the rape of genes of interest silence shows sterile symptom

After rape soaks through root at Seedling Stage and infects, normal growth is bloomed, and to productive phase, adjoining tree can be justOften solid, and the plant of genes of interest COI1 silence fruit pod is failed normal development, show as sterile, as Fig. 6 (adjoining tree and orderThe single fruit pod of gene silencing plant), Fig. 7. It is A liquid that the root of adjoining tree infects liquid, consists of GV-C+GV-1, emptyThe mixed liquor of the Agrobacterium of carrier pTRV2 and assistant carrier pTRV1 Agrobacterium. The plant root of genes of interest COI1 silence infectsLiquid is B liquid, and it consists of GV-COI1+GV-1, contain genes of interest COI1 recombinant vector pTRV2-COI1 Agrobacterium withThe mixed liquor of assistant carrier pTRV1 Agrobacterium. Experimental result shows that the gene silencing system of virus induction successfully makes oil by expectionDish is sterile, cultivates rapeseed male sterility strain, and the male parent can be used as in rapeseed breeding is used. VIGS infects sterile oil in rapeThe statistical conditions of dish plant are 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 SEQIDNO.2) is built into VIGS carrier, andInfect the male sterile plants that has also obtained tobacco after tobacco. As shown in Figure 8, the tobacco of reticent COI1 gene sending out due to stamenEducate extremely caused sterile.

Claims (2)

1. VIGS carrier system is cultivated a method for dicotyledonous male sterile plants, it is characterized in that: described VIGS carrier isSystem derives from Tobacco rattle virus, and described dicotyledon is rape, said method comprising the steps of:

(1) clone COI1 genetic fragment, builds the restructuring pTRV2-COI1 viral vectors that carries COI1 gene,

Described COI1 genetic fragment is SEQIDNO.1<1181>～<1694>;

Described pTRV2 carrier comprises following structure: be followed successively by left margin (LB), 2 × 35S promoter, the driving of 2 × 35S promoterThe Tobacco rattle virus coat protein (CP) expressed, can insert the MCS (MCS) of genes of interest, independently shear ribozyme(Rz), transcription terminator (NOS_t), right margin (RB);

(2) pTRV2-COI1 and pTRV1 carrier are imported to Agrobacterium GV3101, will contain pTRV2-COI1 respectively and pTRV1 carriesIt is 0.6～1.0 bacterium liquid that the Agrobacterium GV3101 of body is mixed with OD600 in the ratio of 1:1; Described pTRV1 carrier comprises followingStructure: the Tobacco rattle virus RNA that is followed successively by left margin (LB), 2 × 35S promoter, 2 × 35S promoter driving expression relies onRNA polymerase (RdRp), motion albumen (MP), rich cysteine 16K albumen, independently shear ribozyme (Rz), transcription terminator（NOS_t), right margin (RB);

(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.

2. the method that a kind of VIGS carrier system as claimed in claim 1 is cultivated dicotyledonous male sterile plants, its feature existsIn: the structure of described VIGS carrier system is as shown in Figure 9.