CN104928301A - Pathogenesis key target gene of Verticillium dahlia thiamine translocator and interference vector and application thereof - Google Patents
Pathogenesis key target gene of Verticillium dahlia thiamine translocator and interference vector and application thereof Download PDFInfo
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Abstract
The invention discloses a pathogenesis key target gene of a Verticillium dahlia thiamine translocator and an interference vector and application thereof and belongs to the field of the cloning and application of the pathogenesis key target gene of Verticillium dahlia. A five-segment target gene sequence of the thiamine translocator gene of Verticillium dahlia is introduced into Nicotiana benthamiana to allow transient expression of dsRNA by means of virus-induced gene silencing, and a three-segment pathogenesis key target gene capable of evidently improving plant disease resistance is screened. The RNAi technology is further utilized to construct the stably inherited interference vector of the three-segment target gene sequence, used to transform Nicotiana benthamiana; a transgenic tobacco plant obtained has evidently enhanced resistance on Verticillium dahlia, high resistance is shown, and the transgenic tobacco plant even reaches the immune level. The pathogenesis key target gene segment of the thiamine translocator gene of Verticillium dahlia and the RNA interference vector are suitable for the application to improve the ability of the pants to resist Verticillium dahlia and to cultivate novel transgenic plants resisting Verticillium dahlia.
Description
Technical field
The present invention relates to the crucial target gene of the verticillium dahliae VitB1 translocator course of disease, also relate to the interference carrier containing the crucial target gene of the described VitB1 translocator course of disease, the invention still further relates to the crucial target gene of the described verticillium dahliae VitB1 translocator course of disease or interference carrier raising plant to the application in verticillium dahliae disease resistance, belong to clone and the Application Areas of verticillium dahliae course of disease key gene.
Background technology
Verticillium dahliae (Verticillium dahliae) is that a kind of soil passes tracheomycosis evil, infect scope widely, the 200 diversified economy crops comprising xylophyta, herbaceous plant and vine can be infected, cause great financial loss to China and even the world.In view of the hazardness that it is serious, probe into the focus that verticillium dahliae pathogenesis and the interaction mechanism between host and pathogenic bacteria are research always.How improving the disease resistance of host plant body to verticillium dahliae is problem anxious to be resolved.
The verticillium dahliae Microsclerotia remained in soil is subject to the induction of plant secretion liquid and sprouts, the Microsclerotia sprouted without any need for adsorption structure directly surely grow the surface in roots of plants and penetrate in the cortex of epidermic cell and remove (Reusche M, Thole K, Janz D, Truskina J, Rindfleisch S, Dr ü bert C, Polle A, Lipka V, Teichmann T:Verticillium infection triggersVASCULAR-RELATED NAC DOMAIN7-dependent de novo xylemformation and enhances drought tolerance in Arabidopsis.Plant Cell 2012, 24:3823 – 3837, Zhao P, Zhao Y-L, Jin Y, Zhang T, Guo H-S:Colonizationprocess of Arabidopsis thaliana roots by a green fluorescent protein-taggedisolate of Verticillium dahliae.Protein Cell 2014,5:94 – 98.).The mycelia penetrating into plant epidermis cell grows and extends to the vascular tissue of root with iuntercellular through cell walls in the cell of root, change the endogenous cycle border of plant soma, form a kind of symbiotic environment (Pegg, G.F., and Brady, B.L. (2002) .Verticillium Wilts. (Wellingford, UK:CABIPublishing); Klosterman SJ, Atallah ZK, Vallad GE, Subbarao KV:Diversity, pathogenicity, and management of Verticillium species.Ann Rev Phytopathol2009,47:39 – 62.).In symbiotic environment, plant materials hinders mycelial invasion, and mycelium utilizes the nutritive substance in symbiotic environment to maintain generation and release (the Fradin EF of mycelial invasion and spore, Thomma BP:Physiology and molecular aspects of Verticilliumwilt diseases caused by V.dahliae and V.albo-atrum.Mol Plant Pathol 2006,7:71 – 86.).Be discharged into fascicular spore upwards to be transported by the transpiration of plant materials, the spore germination being transported to vascular bundle wall forms germ tube, permeates to plant soma, surely grows, infects and breed.
So far, verticillium dahliae pathogenesis still rests on the desk study stage.Verticillium dahliae is grown surely in host plant body, how to adapt to the intracellular environment in pin main body and how to absorb nutritive ingredient in host cell also do not report so far.Therefore, explore beautiful greatly wheel and invade reproduction mechanisms in a mechanism of causing a disease process and seem more important, search out and participate in protein factor crucial in absorption process, " cdna reverse group " provides a technical strategies for the disease-resistant novel material kind of cultivation.
VitB1 is also VITMAIN B1, is a kind of vitamin B group of solubility, plays an important role in the matter and energy metabolic process of cell as much important coenzyme, is the necessary nutritive substance of organism.VitB1 the nourishing and growing of fungi, invade, requisite nutritive ingredient in reproductive growth process.By major way (the Mojzita D. that the VitB1 translocator on plasma membrane will be fungi microbe acquisition VitB1 in the VitB1 transporte to cells outside environment, Hohmann S.Pdc2coordinates expression of the THI regulon in the yeast Saccharomycescerevisiae.Molecular genetics and genomics, 2006,276 (2): 147-161.).VitB1 translocator (Thiamine transporter) is a plasmalemma protein, belong to the folic acid translocator family of reduced form, comprise film and embed Binding Capacity albumen, two ATP associated proteins and a translocator subunit (Lagarde, W.H., Underwood, L.E., Moats ?Staats, B.M., & Calikoglu, A.S. (2004) .Novel mutation in the SLC19A2gene in an African ?American female with thiamine ?responsive megaloblastic anemiasyndrome.American Journal of Medical Genetics Part A, 125 (3), 299-305.).Mainly the VitB1 of extraneous lower concentration is transported in organism, carries out metabolism for body.
The at present relevant research of the VitB1 translocator of verticillium dahliae yet there are no any report, and from VitB1 transporter gene, screening obtains the crucial target gene of the course of disease and will have great importance to the disease resistance of verticillium dahliae for improving plant.
Summary of the invention
Technical problem to be solved by this invention is to provide the crucial target gene of the VitB1 translocator course of disease of verticillium dahliae (Verticillium dahliae) and the rna interference vector containing the crucial target gene of the described VitB1 translocator course of disease;
Another technical problem to be solved by this invention is applied to by the rna interference vector of crucial for described VitB1 translocator course of disease target gene to improve plant to the disease resistance of verticillium dahliae.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
First the present invention discloses the crucial target gene of the VitB1 translocator course of disease of verticillium dahliae (Verticillium dahliae), and its nucleotides sequence is classified as shown in SEQ ID No.1, SEQ ID No.2, SEQ IDNo.3, SEQ ID No.4 or SEQ ID No.5; Preferably, the crucial target gene of the described VitB1 translocator course of disease, its nucleotides sequence is classified as shown in SEQ ID No.2, SEQ ID No.4 or SEQ IDNo.5.
The invention also discloses the rna interference vector containing the crucial target gene of the described VitB1 translocator course of disease and the host cell containing described rna interference vector.
The crucial target gene of the VitB1 translocator course of disease of the present invention can be applied to and improve plant to the disease resistance of verticillium dahliae, comprises the following steps: (1) builds the rna interference vector containing the crucial target gene of the described VitB1 translocator course of disease; (2) constructed rna interference vector is transformed in plant or vegetable cell; (3) screening obtains the transgenic plant of improving verticillium dahliae disease resistance.
Rna interference vector of the present invention can be applied to and improve plant to the disease resistance of verticillium dahliae, comprises the following steps: described rna interference vector is transformed in plant or vegetable cell by (1); (2) screening obtains the transgenic plant of improving verticillium dahliae disease resistance.
The present invention further discloses a kind of method of cultivating the transgenic plant new variety of anti-verticillium dahliae, comprise the following steps: (1) builds the rna interference vector containing the crucial target gene of the described VitB1 translocator course of disease; (2) constructed rna interference vector is transformed in plant or vegetable cell; (3) screening obtains the transgenic plant new variety improved verticillium dahliae disease resistance.
The scheme of described conversion and the type of visual for the scheme of described Nucleotide introduced plant plant for transforming or vegetable cell is changed.The appropriate method of described Nucleotide introduced plant cell is comprised: microinjection, electroporation, Agrobacterium-medialed transformation, direct gene transfer etc.
Plant of the present invention is the host plant of verticillium dahliae, be preferably farm crop, comprising: in tobacco, cotton, tomato, potato, muskmelon, watermelon, cucumber or peanut any one or multiple.
5 sections of target-gene sequence Thit1 (shown in SEQ ID No.1) of verticillium dahliae VitB1 transporter gene, Thit2 (shown in SEQID No.2), Thit3 (shown in SEQ ID No.3), Thit4 (shown in SEQ ID No.4), Thit5 (shown in SEQ ID No.5) import in this life cigarette by virus induced gene silencing technology (VIGS) by the present invention, make its transient expression dsRNA, and carry out verticillium dahliae inoculation, observe the susceptible symptom of this life cigarette plant, and disease index analysis is carried out to it.Found that, the disease resistance importing this life cigarette plant of 5 sections of target fragments all significantly improves, connecing bacterium after the 12nd day, import interference base because of the susceptible symptom of this life cigarette plant not obvious, only blade occurs here withering below, there is not susceptible symptom in blade above, and plant entirety does not occur here withering, withered symptom.Simultaneously, also there are differences between the susceptible symptom of this life cigarette plant of importing 5 sections of target genes, the susceptible symptom of this life cigarette plant of quiding gene fragment Thit2, Thit4, Thit5 is lower than this life cigarette plant of quiding gene fragment Thit1, Thit3, and the disease resistance of plant to verticillium dahliae is stronger.Disease index statistics finds, this uncured tobacco of injection target gene fragment, disease index is all remarkable in zero load.
The present invention is on the basis filtering out three fragment gene fragment Thit2 of disease-resistant phenotype obvious verticillium dahliae VitB1 transporter gene, Thit4, Thit5, utilize Gateway technology, build the genetic stability interference carrier of VitB1 translocator three sections of target-gene sequences, transform this life cigarette, obtain genetically modified tobacco plant.The disease resistance experimental result turning this life cigarette plant of target gene shows, the disease resistance of Transformation of tobacco plant to verticillium dahliae of the three sections of different target genes (VdThit-1 (shown in SEQ ID No.5), VdThit-2 (shown in SEQ ID No.4), VdThit-3 (shown in SEQ ID No.2)) obtained significantly improves compared with wild-type, show as high resistance (disease index is below 15%) respectively, disease-resistant (disease index is at 15-30%), disease-resistant.
After turning this life cigarette plant inoculation verticillium dahliae of target gene, substantially there is not susceptible symptom in the transgenic tobacco plant obtaining VdThit-1 interference fragment (shown in SEQ ID No.5), basically identical with the adjoining tree not connecing bacterium, plant shows resistance, even reaches immunity.The transgenic tobacco plant simultaneously obtaining VdThit-2 (SEQ ID No.4 shown in) or VdThit-3 (shown in SEQ ID No.2) interference fragment only over-ground part base portion occurs here two to three blades wither, do not occur susceptible symptom with upper part, plant shows disease-resistant even high resistance.
Turn fungal biomass detected result in this life cigarette plant of target gene to show, in wild-type tobacco plants, a large amount of verticillium dahliaes detected, biomass is more than 5 times that turn target gene tobacco plant; And in the tobacco plant turning target gene, detect that the biomass of verticillium dahliae is little, and especially at the transgenic tobacco plant obtaining VdThit-1 interference fragment, trace verticillium dahliae biomass only detected, basically identical with the adjoining tree of not infecting.
To sum up, (the genetic stability interference carrier of VdThit-1 (shown in SEQ ID No.5), VdThit-2 (shown in SEQ ID No.4), VdThit-3 (shown in SEQ IDNo.2) transforms this life cigarette to the VitB1 translocator that the present invention obtains three sections of target-gene sequences, and pole significantly improves the disease resistance of tobacco to verticillium dahliae.Proceeded in other crops, be expected to the new test materials obtaining high resistance verticillium dahliae, can be applied in the practice production of the breeding for disease resistance such as cotton, tomato, potato, muskmelon, watermelon, cucumber, peanut.
Technical solution of the present invention compared with prior art, has following beneficial effect:
The present invention utilizes VIGS technology screening to 3 sections of target fragments of verticillium dahliae VitB1 transporter gene, RNAi technology is utilized to obtain the interference carrier of above-mentioned three VitB1 transporter gene fragments further, transform this life cigarette, obtain the tobacco plant of high resistance verticillium dahliae, especially the disease resistance obtaining the tobacco plant of VdThit-1 target fragment is more remarkable, even reaches immune grade.3 sections of target fragments of verticillium dahliae VitB1 transporter gene of the present invention and rna interference vector can be applied to and improve plant to the disease resistance of verticillium dahliae, cultivate the transgenic plant new variety of anti-verticillium dahliae.
the term definition arrived involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.
Term " polynucleotide " or " Nucleotide " mean the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside, ribonucleoside or ribonucleotide and polymkeric substance thereof.Except nonspecific restriction, otherwise the nucleic acid of the known analogue containing natural nucleotide contained in described term, and described analogue has the binding characteristic that is similar to reference nucleic acid and carries out metabolism in the mode of the Nucleotide being similar to natural generation.Unless other specific restriction, otherwise described term also means oligonucleotide analogs, and it comprises PNA (peptide nucleic acid(PNA)), DNA analogue used in antisense technology (thiophosphatephosphorothioate, phosphamide acid esters etc.).Unless otherwise, otherwise the specific nucleic acid sequence sequence that also impliedly contains its conservative varient (including, but is not limited to degenerate codon replace) of modifying and complementary sequence and clearly specify.Particularly, the 3rd sequence replaced through mixing base and/or deoxyinosine residue by producing one of them or more than one selected (or all) codon replaces to realize degenerate codon (people such as Batzer, Nucleic Acid Res.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); The people such as Rossolini, Mol Cell.Probes 8:91-98 (1994)).
Term " recombinant host cell " or " host cell " mean the cell comprising Nucleotide of the present invention, and no matter use which kind of method to carry out inserting to produce recombinant host cell.Host cell can be prokaryotic cell prokaryocyte or eukaryotic cell.
Term " RNA disturbs (RNA interference, RNAi) " means the phenomenon of being induced the silenced gene expression with former sequence by external source or endogenic double-stranded RNA in cell.
Accompanying drawing explanation
Fig. 1 is the carrier figure of TRV1 and TRV2;
Fig. 2 is the course of disease curve of this life of VIGS-Thit instantaneous disturbance cigarette plant;
Fig. 3 is Gateway interference carrier information; Wherein, A:pDONR207 carrier information; B:pK7GWIWG2 (I), 0 carrier information;
Fig. 4 is the Molecular Identification of this uncured tobacco genetic transformation seedling; Wherein, A: be primer with VdThit-1-F/R, turns the PCR Molecular Detection of the Transformation of tobacco plant of VdThit-1 target gene; 1-6 (transgenic tobacco plant) electrophoresis path increases VdThit-1 target fragment, and Wt (WT lines) electrophoresis path does not increase, and M is DNA marker; B: be primer with VdThit-2-F/R, turns the PCR Molecular Detection of the Transformation of tobacco plant of VdThit-2 target gene; 1-6 (transgenic tobacco plant) electrophoresis path increases VdThit-2 target fragment, and Wt (WT lines) electrophoresis path does not increase, and M is DNA marker; C: be primer with VdThit-3-F/R, turns the PCR Molecular Detection of the Transformation of tobacco plant of VdThit-3 target gene; 1-6 (transgenic tobacco plant) electrophoresis path increases VdThit-3 target fragment, and Wt (WT lines) electrophoresis path does not increase, and M is DNA marker;
Fig. 5 is the Disease-resistance Analysis and the fungal biomass detection analysis that turn target gene tobacco plant; Wherein, A: the disease index statistical study turning target gene tobacco plant; B: the fungal biomass turning target gene tobacco plant detects.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, any restriction is not formed to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments or replacement all fall into protection scope of the present invention.
1, material
Bacterial strain and plasmid: verticillium dahliae Vd991 (Plant Protection institute, Chinese Academy of Agricultral Sciences; letter Gui Liang researcher is so kind as to give); agrobacterium tumefaciens agrobacterium tumefaciens GV3101; carrier TRV1, TRV2 (Tsing-Hua University professor Liu Yule is so kind as to give); carrier pDONR207, pK7GWIWG2 (I), 0 for preserving in the present inventor laboratory.
The Agrobacterium injection of the structure of embodiment 1VIGS-Thit instantaneous disturbance carrier, this life cigarette and the inoculation of verticillium dahliae
1, experimental technique
With verticillium dahliae total serum IgE for template, design 5 couples of VitB1 transporter gene (VDAG_01137, Broad Institute) target gene primer (primer sequence is in table 1), the frag info of amplification VitB1 transporter gene, length is about 250bp.5 sections of fragments that amplification obtains are connected to the multiple clone site region (Fig. 1) of carrier TRV2, obtain recombinant vectors TRV2-Thit1, TRV2-Thit2, TRV2-Thit3, TRV2-Thit4, TRV2-Thit5, electroporated Agrobacterium GV3101, mix with the Agrobacterium GV3101 bacterial strain 1:1 of TRV1 (carrying TRV virus another part gene information), utilize syringe to be injected in by the Agrobacterium mixed in the tender leaf of this life cigarette seedling of 3 to 5 true leaves.Simultaneously using TRV2-PDS carrier and TRV2 empty carrier as positive and negative control, transformation Agrobacterium GV3101, with this life of Agrobacterium GV3101 bacterial strain 1:1 hybrid injection cigarette of TRV1.After 20 days (positive plant occur albefaction after), adopt and dip in root method inoculation verticillium dahliae, observe the susceptible symptom of this life cigarette plant, and disease index analysis is carried out to it.
Table 1 primer sequence
2, experimental result
5 sections of verticillium dahliae VitB1 transporter gene fragments (Thit1 (shown in SEQID No.1), Thit2 (shown in SEQ ID No.2), Thit3 (shown in SEQ ID No.3), Thit4 (shown in SEQ ID No.4), Thit5 (shown in SEQ ID No.5)) of the present invention clone import in this life cigarette and give expression to dsRNA (young leaves of photobleaching appears in this life cigarette top of inoculation TRV2-PDS carrier), inoculate this life cigarette injected with verticillium dahliae spore suspension.Found that: import interference base because of this life cigarette plant susceptible symptom significantly lower than zero load inject this life cigarette plant.Connecing bacterium after the 12nd day, import interference base because of the susceptible symptom of this life cigarette plant not obvious, only, blade occurs here withering, above blade there is not susceptible symptom, plant entirety does not occur here withering, withered symptom; And inject with zero load and inoculate equivalent spore suspension this life cigarette plant occur obvious susceptible symptom, plant general performance goes out here to wither, withered symptom.
Also there are differences between the susceptible symptom of this life cigarette plant of the 5 sections of target genes simultaneously imported, the susceptible symptom of this life cigarette plant of quiding gene fragment Thit2 (nucleotides sequence be classified as SED No.2 shown in), Thit4 (nucleotides sequence is classified as shown in SED No.4), Thit5 (nucleotides sequence is classified as shown in SED No.5) is lower than this life cigarette plant of quiding gene fragment Thit1 (shown in SEQ ID No.1), Thit3 (SEQ ID No.3 is shown), and the disease resistance of plant to verticillium dahliae is stronger.
After this life cigarette plant inoculates the 8th day, 10 days, 12 days, carry out disease index statistics, found that: to it compared with zero load, this uncured tobacco of injection target gene fragment, disease index all has significantly lower than zero load; Along with the increase of number of days, disease index rises gradually, but also all remarkable in unloaded (Fig. 2).
To sum up result shows, the disease resistance of this life cigarette plant to verticillium dahliae of 5 sections of VitB1 translocator interference significantly strengthens, and plant shows as disease resistance; Disease resistance simultaneously between 5 sections of target fragments also there are differences, and the disease resistance of this life cigarette plant of quiding gene fragment Thit2, Thit4, Thit5 is extremely remarkable, and plant shows as high disease resistance.Therefore, this test, by VIGS technology screening, obtains the target fragments that can improve the verticillium dahliae VitB1 transporter gene of Tobacco resistance.
The genetic stability interference carrier of embodiment 2 VitB1 translocator target-gene sequence builds and transforms this life cigarette
1, experimental technique
The clone of 1.1 target fragments and the structure of interference carrier
The present invention is on embodiment 1 basis, in order to obtain the transfer-gen plant containing target gene dsRNA of genetic stability, the present invention utilizes Gateway technology, the three fragment gene fragment VIGS-Thit5 (nucleotides sequence is classified as shown in SED No.5) of the obvious VitB1 transporter gene of disease-resistant phenotype selecting embodiment 1 to screen, VIGS-Thit4 (nucleotides sequence is classified as shown in SED No.4), VIGS-Thit2 (nucleotides sequence is classified as shown in SED No.2) is target gene, and length is about 250bp.Design the primer VdThit-1-F/R that BP site is contained at three sections of two ends, VdThit-2-F/R, VdThit-3-F/R (primer sequence is in table 1), attB1 Post section sequence (aaaaaagcaggct) is added at 5 ' end, attB2 site sequence (aagaaagctgggt) is added, for the structure (primer information is in table 1) of Gateway interference carrier at 3 ' end.With the total serum IgE of verticillium dahliae for template, the target fragment of amplification three sections of VitB1 translocators respectively, be primer (primer information is in table 1) with complete attB site sequence attB-F/R again, with object fragment above for template, increase with the target fragment in complete attB site, build RNAi carrier for Gateway method.Build Gateway interference carrier and need following two reactions: (1) BP reacts: the entry vector of employing is pDONR
tM207 (Fig. 3), have Kan resistance and ccdB lethal gene, have attP recombination site; (2) LR reaction: the interference carrier of employing is pK7GWIWG2 (I), and 0 (Fig. 3), has Spe resistance and ccdB lethal gene, have attR recombination site.BP and LR operation is carried out with reference to BP reaction and LR reaction kit.
By BP reaction and LR reaction, VitB1 three sections of target fragments that clone obtains are connected to Gateway interference carrier pK7GWIWG2 (I), on 0, form the plant conversion carrier containing fungal target gene.The interference carrier transformation Agrobacterium LBA4404 that electric shocking method will obtain containing target fragment, for the genetic transformation of the plant such as this life cigarette.
1.2 the genetic transformation of this uncured tobacco and Molecular Identification
To plant the aseptic tobacco on MS minimum medium, removing blade edge and main vein, be cut into the segment of 0.4 × 0.6cm size.By the explant that cuts at above-mentioned OD
600be soak 5min in the Agrobacterium bacterium liquid of 0.1 ~ 0.2, the bacterium liquid of plant material surface is blotted with aseptic filter paper, vanelets is placed in be covered with one deck filter paper tobacco Bud polarization substratum (MS+NAA 0.2mg/L+6-BA 2mg/L) on carry out Dual culture, 25 DEG C of light culture 3 days.
Tobacco explants through Dual culture is transferred to and cultivates containing in antibiotic resistant buds screening culture medium (MS+NAA 0.2mg/L+6-BA 2mg/L+Kan 100mg/L+Carb 500mg/L), periodicity of illumination is that 16h illumination/8h is dark, can sprout after 2 ~ 3 weeks.When resistant buds grows to 1 ~ 2cm height, cut budlet and proceed to root induction in root media (MS+Kan 100mg/L+Carb 500mg/L), after 1 ~ 2 week, just have Adventitious root initiation.
After obtaining the transformed plant of tobacco, Molecular Identification is carried out to it.The total genome extracting transformation seedlings is template, with VdThit-1-J-F/R or VdThit-2-J-F/R or VdThit-3-J-F/R for primer (primer sequence is in table 1), carry out PCR Molecular Detection, the plant of test positive seedling carries out succeeding transfer culture, to obtain the positive tobacco plant isozygotied, it is next step disease resistance Preparatory work of experiment.
The disease resistance test of 1.3 transgenic tobacco plants and the detection of fungal biomass and Molecular Detection
Respectively (each target gene respectively selects three different strains) tobacco seed of wild-type tobacco and acquired three sections of different target genes is cultivated under sterile environment, when seedling grows to 7-9 sheet true leaf, with off-the-shelf 10
6the wild-type verticillium dahliae bacterial strain spore suspension of spore/ml inoculates the transgene tobacco seedling of acquired nine groups of different strains of different target gene separately, often group arranges 3 groups (10 seedling) and repeats, and within 12 days, observes the susceptible situation of plant later and adds up the disease index of plant.
Then the change of the biomass of verticillium dahliae in host is analyzed, this test is respectively by susceptible wild-type with turn the material that ground 1cm stem section that target gene obtains this life cigarette plant detects as qPCR fungal biomass, extract DNA, using the actin of this life cigarette (primer is in table 1) gene as reference gene, the ribosomal RNA gene (Z29511) of fungi is as the detection gene of fungal biomass, primer Vd-F/Vd-R (see table 1) is ITS1 and the ITS2 region based on ribosomal RNA gene, detect the biomass of fungi in susceptible raw cigarette, from molecular biology angle determination transgenic tobacco plant to the disease resistance grade of verticillium dahliae.
2, experimental result
The acquisition of 2.1 turns of target gene tobacco plants
Transformed by the genetic stability of this agriculture bacillus mediated life cigarette, the interference carrier containing VitB1 transporter gene is converted into this uncured tobacco, finally obtain transgene tobacco through kantlex screening.Carry out PCR detection to the transgenic tobacco plant obtained, every section of target gene all obtains multiple different positive transformants strain (Fig. 4 A-C), and random choose 3 transgenic lines carry out next step Disease-resistance Analysis respectively.
The Disease-resistance Analysis of 2.2 turns of target gene tobacco plants and the Molecular Detection of fungal biomass
The disease resistance experiment turning this life cigarette plant of target gene finds: the Transformation of tobacco plant of three sections of different target genes of acquisition (VdThit-1 (nucleotides sequence is classified as shown in SED No.5), VdThit-2 (nucleotides sequence is classified as shown in SEDNo.4), VdThit-3 (nucleotides sequence is classified as shown in SED No.2)) to the disease resistance of verticillium dahliae compared with wild-type, disease resistance significantly improves, show as high resistance (disease index is below 15%) respectively, disease-resistant (disease index is at 15-30%), disease-resistant (Fig. 5).The present invention, using this life of wild-type cigarette and the host of this life cigarette plant as verticillium dahliae turning target gene, inoculates the spore (5 × 10 of same concentrations
6spore/ml) suspension observed discovery after the 8th day, the 10th day, the 12nd day: substantially there is not susceptible symptom in the transgenic tobacco plant obtaining VdThit-1 interference fragment (nucleotides sequence is classified as shown in SED No.5), basically identical with the adjoining tree not connecing bacterium, plant shows resistance, even reaches immunity (Fig. 5 A).Obtain VdThit-2 (nucleotides sequence is classified as shown in SED No.4) simultaneously, or the transgenic tobacco plant of VdThit-3 (nucleotides sequence be classified as SED No.2 shown in) interference fragment only over-ground part base portion occur here two to three blades wither, do not occur susceptible symptom with upper part, plant shows disease-resistant even high resistance (Fig. 5 A).And wild-type tobacco plants occur obvious susceptible symptom, in the 12nd day plant occur here withering, dead symptom.
Turn fungal biomass in this life cigarette plant of target gene and the raw cigarette plant of susceptible of wild type strain to detect and find: in the tobacco plant of wild-type, a large amount of verticillium dahliaes detected, biomass is more than 5 times (Fig. 5 B) turning target gene tobacco plant.And in the tobacco plant turning target gene, detect that the biomass of verticillium dahliae is little, especially at the transgenic tobacco plant obtaining VdThit-1 interference fragment (nucleotides sequence is classified as shown in SED No.5), trace verticillium dahliae biomass only detected, substantially the same with the adjoining tree of not infecting (Fig. 5 B).
Above result shows, the transgenic tobacco plant obtaining VdThit-1, VdThit-2, VdThit-3 interference fragment shows significant disease resistance to verticillium dahliae, plant reaches more than disease-resistant level, and the transgenic tobacco plant especially obtaining VdThit-1 interference fragment reaches the high resistance even level of immunity to verticillium dahliae disease resistance.
To sum up, verticillium dahliae VitB1 transporter gene 5 sections of target-gene sequences to be imported in this life cigarette by virus induced gene silencing technology (VIGS) and make its transient expression dsRNA by the present invention, and carry out verticillium dahliae inoculation, find that the disease resistance of this life cigarette plant of importing 5 sections of target fragments all significantly improves, and wherein have the disease resistance pole of 3 sections of target sequence to significantly improve.The present invention utilizes RNAi technology on above-mentioned experiment basis, build the genetic stability interference carrier of VitB1 translocator three sections of target-gene sequences, transform this life cigarette, obtain genetically modified tobacco plant, it significantly strengthens the disease resistance of verticillium dahliae, especially the disease resistance transforming the tobacco plant of the VdThit-1 gene fragment of acquisition is the strongest, shows high resistance, even reach immune grade to the disease resistance of verticillium dahliae.Therefore, the present invention obtains the genetic stability interference carrier of VitB1 translocator three sections of target-gene sequences, transform this life cigarette and improve tobacco to the disease resistance of verticillium dahliae, proceeded in other crops, be expected to the new test materials obtaining high resistance verticillium dahliae, be applied in the practice production of the breeding for disease resistance such as cotton, tomato, potato, muskmelon, watermelon, cucumber, peanut.
Claims (10)
1. the crucial target gene of the VitB1 translocator course of disease of verticillium dahliae (Verticillium dahliae), is characterized in that: its nucleotides sequence is classified as shown in SEQ ID No.1, SEQ ID No.2, SEQ IDNo.3, SEQ ID No.4 or SEQ ID No.5.
2., according to the crucial target gene of the VitB1 translocator course of disease described in claim 1, it is characterized in that: its nucleotides sequence is classified as shown in SEQ ID No.2, SEQ ID No.4 or SEQ ID No.5.
3. the rna interference vector containing the crucial target gene of the VitB1 translocator course of disease described in claim 1 or 2.
4. the host cell containing rna interference vector described in claim 3.
5. the crucial target gene of the VitB1 translocator course of disease described in claim 1 or 2 is improving plant to the application in the disease resistance of verticillium dahliae.
6. according to application according to claim 5, it is characterized in that, comprise the following steps: (1) builds the rna interference vector containing the crucial target gene of the VitB1 translocator course of disease described in claim 1 or 2; (2) constructed rna interference vector is transformed in plant or vegetable cell; (3) screening obtains the transgenic plant of improving verticillium dahliae disease resistance.
7. rna interference vector described in claim 3 is improving plant to the application in the disease resistance of verticillium dahliae.
8. according to application according to claim 7, it is characterized in that, comprise the following steps: rna interference vector described in claim 3 is transformed in plant or vegetable cell by (1); (2) screening obtains the transgenic plant of improving verticillium dahliae disease resistance.
9. cultivate a method for the transgenic plant new variety of anti-verticillium dahliae, it is characterized in that, comprise the following steps: (1) builds the rna interference vector containing the crucial target gene of the VitB1 translocator course of disease described in claim 1 or 2; (2) constructed rna interference vector is transformed in plant or vegetable cell; (3) screening obtains the transgenic plant new variety improved verticillium dahliae disease resistance.
10. according to the application described in claim 5 or 7, it is characterized in that, described plant comprises farm crop; Preferably, described plant comprises: in tobacco, cotton, tomato, potato, muskmelon, watermelon, cucumber or peanut any one or multiple.
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