CN1254381A - Expression of fungicide-binding polypeptides in plants for producing fungicide tolerance - Google Patents

Abstract

The invention relates to a method for producing fungicide-tolerant plants by expressing a fungicide-binding antibody therein.

Description

In plant, express mycocide in conjunction with polypeptide to produce the mycocide tolerance

The present invention relates to prepare the method for anti-mycocide plant in conjunction with polypeptide by expressing external source mycocide in plant or in the plant organ.The invention still further relates to corresponding nucleic that is coded in polypeptide, antibody or antibody moiety that the mycocide binding characteristic is arranged in the transgenic plant and the purposes that transforms plant itself.

Known genetic engineering method allows that foreign gene is special to be transferred in the Plant Genome.This method is called conversion, and the plant that obtains is transgenic plant.At present, transgenic plant are used for many fields of biotechnology.As anti-insect plant (Vaek etc., vegetable cell 5 (1987), 159-169), antivirus plant (Powell etc., science 232 (1986), 738-743) and anti-ozone plant (Van Camp etc., biotechnology 12 (1994), 165-168).The example of the improvement proterties that obtains by genetic engineering has: the fruit (Oeller etc. storage period of improvement, science 254 (1991), 437-439), the starch yield (Stark etc. that improve in the potato stem tuber, science 242 (1992), 419), starch ingredients changes (Visser etc., Mol.Gen.Genet.225 (1991), 289-296) change (Voelker etc. with lipidic component, science 257 (1992), 72-74) and the high molecular synthetic (Poirer etc. of external source, science 256 (1992), 520-523).

The important goal of the work of carrying out in the plant molecular genetics field is to produce herbicide tolerant.Herbicide tolerant is characterised in that the consistency of plant or plant organ and used weedicide improve (with regard to type or level).This can realize by number of ways.The enol pyruvoyl shikimic acid of the target enzyme of known method combined utilization metabolic gene such as pat gene and careless ammonium phosphine resistance (WO8705629) or antiweed such as resistance glyphosate-3-phosphate synthase (WO9204449), and the resistance plant that in cell and tissue culture, uses weedicide to select the tolerant plants cell and obtain, as in about acetyl-CoA carboxylase inhibitor, describe (US5162602, US5290696).

Antibody is the albumen as the immunity system component.The common trait of all antibody be they three-dimensional globosity, light chain and heavy chain formation and with high specificity with molecule or molecular moiety structure bonded basic capacity (Alberts etc.,: Molekularbiologie, derZelle[cellular elements biology], the 2nd edition, 1990, VCH Verlag, ISBN 3-527-27983-0,1198-1237).According to these features, antibody is used for multiple-task.Application can be divided in the animal and human's body that produces antibody uses antibody, and promptly so-called in-situ applications and ex-situ use, promptly behind the cell that produces antibody and organism separation antibody, use antibody (Whitelam und Cockburn, TIPS the 1st volume, 8 (1996), 268-272).

Is the work (nature 256 (1975) 495-97) that Kohler and Milstein carry out with somatic hybridization clone (hybridoma) as the basis at very special antigenic antibody source.This method allows unified structure and is produced by the so-called monoclonal antibody that cell fusion method produces.The splenocyte of immune mouse and murine myeloma cell merge.This can make the hybridoma infinite multiplication.Simultaneously, emiocytosis is at the used antigenic specific antibody of immune mouse.Splenocyte provides the ability that produces antibody and the myeloma cell makes cell indeterminate growth and secretory antibody constantly.As the clone, because each hybridoma is from single B cell, all antibody molecules of generation have identical structure, comprise antigen binding site.Because can infinitely obtain the antibody of single, known specificity and common structure, this method has promoted the application of antibody greatly.Monoclonal antibody is widely used in immunodiagnostics and therapeutics.

In recent years, so-called phage display method has been used to prepare antibody, has avoided immunity system and immune animal repeatedly.The affinity of antibody and specificity obtain in-vitro measurements (Winter etc., the immunity comment 12 (1994) academic year, 433-455; Hoogenboom, TIBTech, the 15th volume (1997), 62-70).Contain the encoding antibody variable region, i.e. the gene fragment of antigen binding site sequence and bacteriophage coat protein gene fusion.Then, with the phage-infect bacterium that contains such fusion gene.The phage particle that obtains has the shell that contains antibody sample fusion rotein, antibodies district directed outside.Such phage display library can be used for separating the antibody fragment that contains hope and with certain antigen-specific bonded phage.Each phage that is separated in this way produces the mono-clonal antigen consistent with monoclonal antibody in conjunction with polypeptide.The antigen binding site gene of each phage uniqueness can be separated to and be used to make up the gene of complete antibody from phage DNA.

In the crop protection field, antibody is particularly useful as the ex-sita analysis tool of antigenic quality and quantity detection.This is included in (Sharp etc. in the tap water, (1991) ACS Symp Ser., 446 (Pestic.Residues Food Saf.) 87-95), the detection of plant constituent, weedicide or mycocide in (WO9423018) or plant or the plant organ in the pedotheque, and with antibody auxiliary as the binding molecule purifying.

Hiatt etc., nature, 342 (1989), 76-78 has described first and prepared immunoglobulin (Ig) in plant.Scope comprise single-chain antibody to the polymer secretory antibody (J.Ma and Mich Hein, 1996, Annuals New York Academy of Sciences, 72-81).

More recent trial utilizes antibody in-situ conservation plant to avoid the pathogenic agent invasion and attack, especially virus disease, be (Tavlacloraki etc., nature 366 (1993), the 469-472 that realizes at the special antibody of virus capsid protein or its part by in vegetable cell, expressing; Voss etc., molecular breeding 1 (1995), 39-50).

Similar methods also is used for protective plant and avoids nematode infections (Rosso etc., biological chemistry and biophysical research communication, 220 (1996) 255-263).Also have examples of applications in the pharmacology, wherein the expressed in situ of antibody in plant is used for oral immunisation (Ma etc., science 268 (1995), 716-719; Mason and Arntzen, Tibtech the 13rd volume (1996), 388-392).Through port, larynx or digestive tube provide that plant forms to health or from plant or plant organ, the antibody that is suitable for consuming, can produce effective immunoprotection.And, in plant, expressed single-chain antibody at lower molecular weight plant hormone dormin because the combination of dormin in the plant, observed the plant hormone available reduce (Artsaenko etc., plant magazine 8 (5) (1995), 754-750).

On the agronomy in the important farm crop chemical control of fungi need the mycocide of the plant-less toxicity effect of highly selective.The phytotoxic effect of mycocide can be according to the inhibition of plant-growth, the photosynthesis of reduction and the output that therefore reduces.Yet, in some cases, be difficult to develop selectivity plant destructive mycocide enough, that in all important farm crop, can use and not cause providing in any farm crop output.Import that mycocide resistance or tolerance crop plants help to address this problem and can handle so far also and can not or only when production loss can be accepted, develop the new purposes of anti-mycotic agent in farm crop under the possible situation.

Developing anti-mycocide crop plants by tissue culture or seeds mutagenesis and natural selection is restricted.On the one hand, phytotoxic effect must be can be detected on the tissue culture level, and on the other hand, has only these plants to operate by tissue culture technique, successfully regenerates from cell cultures and puts in order the strain plant.And, mutagenesis and select after, that plant may show is undesirable, must pass through, in addition in some cases repeated backcross could remove do not wish feature.And, by hybridizing the plant that the resistance that imports will be confined to same species.

For above-mentioned reasons, separation resistance encoding gene and the genetic engineering method that it imports crop plants is more superior than traditional plant breeding method in the target mode.

Up to the present, by molecular biology method development herbicide tolerant or antiweed crop plants need understand weedicide in plant mechanism of action and find to produce gene to Herbicid resistant.Many weedicides of present commercial use work by the enzyme of blocking-up indispensable amino acid, lipid or pigment biosynthesizing step.Change the gene of these enzymes and the gene of these changes is imported crop plants and produce herbicide tolerant by making weedicide no longer be subjected to the bonded mode.Selectable example is in nature, and for example table of discovery reveals the similar enzyme that weedicide is had natural resistance in microorganism.From such microorganism, be cloned into the gene that this gives resistance, it is cloned into suitable carriers again, through successfully transforming, in the crop plants of herbicide sensitive, express (WO96/38567) then.

One of purpose of the present invention is that development is novel, the general genetic engineering method of preparation mycocide tolerance transgenic plant.

The part that we are surprised to find by express allogenic polypeptide, antibody or antibody that the mycocide binding characteristic is arranged in plant has reached this purpose.

The present invention at first relates to preparation mycocide binding antibody and clone's genes involved or gene fragment.

The first step be preparation suitable with mycocide bonded antibody.Particularly by using the antigen immune vertebrates, in most cases mouse, rat, dog, horse, donkey or goat are realized for this.In the case, this antigen be by functional group with such as the high molecular carrier of bovine serum albumin (BSA), chicken ovalbumin, keyhole limpet hemocyanin (KLA) or other carrier is relevant or link coupled has the compound of Fungicidally active.Behind the repetitive administration antigen, monitor immune response, be separated to suitable antiserum(antisera) thus with ordinary method.At first, this method produces the polyclonal serum that contains different specific antibodies.For directed in-situ applications, essential gene order of separating single, the special monoclonal antibody of coding.Number of ways can be used for this purpose.The cell that the first method utilization produces antibody merges with tumour cell and produces the Hybridoma Cell Culture that continues to produce antibody, at last, and by selecting the clone that the clone who obtains causes producing the homogeneous of specific monoclonal antibody.

The part of antibody or antibody promptly, the cDNA of so-called single-chain antibody (SCFV) is separated to from such monoclonal cell system.These cDNA sequence clones comprise and carry out functional expression in the plant to expression cassette and protokaryon and eukaryote.

Optionally, may select antibody, and these antibody combine the product that then its catalysis is transformed into no fungicidal properties with the mycocide molecule by phage display library.The method for preparing catalytic antibody is at Janda etc., science 275 (1997) 945-948, and chemistry is selected katalysis in the associating antibody library; Catalytic antibody, 1991, Ciba Foundation Symposium159 obtains among the Wiley-Interscience Publication describing.In theory, clone this catalytic antibody gene and in plant, express and to cause producing anti-mycocide plant.

The present invention is specifically related to encoding sequence coding mycocide and prepares the mycocide tolerant plants in conjunction with the expression cassette of polypeptide or its functional analogue and with these expression cassettes.This nucleotide sequence can be dna sequence dna or cDNA sequence.Be suitable for inserting according to the encoding sequence of expression cassette of the present invention for example those from hybridoma, coding has mycocide binding characteristic polypeptide to make the host produce dna sequence dna to plant enzyme inhibitor resistance thus.

And expression cassette according to the present invention contains the regulation and control nucleotide sequence that the control encoding sequence is expressed in host cell.In preferred embodiments, expression cassette according to the present invention comprises the upstream, promptly at 5 ' of encoding sequence-end; If promotor and downstream i.e. 3 ' end polyadenylation signal and suitable, other with the polypeptide of mycocide binding characteristic is arranged and/or passes on the controlling element that the intermediate encoding sequence of peptide can be operatively connected.Be interpreted as referring to promotor, encoding sequence, terminator and suitable if can be operatively connected, the sequence arrangement of other controlling element makes when encoding sequence obtains expressing that controlling element can be by the performance function of being thought.The preferred sequence that can be operatively connected is but is not limited to, guarantee the target sequence and the translational enhancer of apoplast, plasma membrane, vacuole, plastid, plastosome, endoplasmic reticulum (ER), nuclear, liposome or other compartment Subcellular Localization, for example from 5 ' leader sequence (Gallie etc. of tobacco mosaic virus (TMV), nucleic acids research 15 (1987), 8693-8711).

In theory, the suitable promotor according to expression cassette of the present invention is any promotor that can guarantee exogenous gene expression.The preferred promotor of using is specifically from the promotor of plant or from the promotor of plant virus.Particularly preferably be CaMV35S promotor (Franck etc., cell 21 (1980) 285-294) from cauliflower mosaic virus.This promotor contains a plurality of recognition sequences of transcribing effector, and their acting in conjunction causes the permanent constitutive expression of quiding gene (Benfey etc., EMBO's magazine, 8 (1989) 2195-2202).

Also can comprise chemically inducible promoter according to expression cassette of the present invention, the expression of allogenic polypeptide in plant is controlled at concrete time point by means of it.Such promotor is PRP1 promotor (Ward etc. for example, molecular biology of plants 22 (1993), 361-366 can be by the promotor (WO95/1919443) of Induced by Salicylic Acid, obtained describing and can using in other in reference by dormin inductive promotor (EP335528) or by acetate or pimelinketone inductive promotor (WO9321334).

Other particularly preferred promotor is that those guarantee expression promoter in tissue that phytotoxicity Fungicidally active therein plays a role or the plant organ.Guarantee that the specific expressed promotor of leaf deserves particular mention.Must mention potato kytoplasm FBPase promotor or potato ST-LST promotor (Stockhans etc., EMBO's magazine 8 (1989) 2445-245).

Under seed-specific expression promoter helps, the stably express that can make single-chain antibody up to the whole solvable seeds of transgene tobacco seed proteic 0.67% (Fiedler and conrad biology/technology 10 (1995), 1090-1094).Because the seed in that sow or the germination process also can be expressed and also is that the object of the invention is wished, according to the present invention, the such germination and the promotor of seed specific also are preferred controlling elements.Therefore, expression cassette according to the present invention for example contains, the promotor of seed specific (preferred USP or LEB4 promotor), and the LEB4 signal peptide is detained signal with gene and the ER that expresses.With reference to single-chain antibody (ScFV gene), in Fig. 1, illustrate the structure of expression cassette with the diagrammatic form.

According to for example T.Maniatis E.F.Fritsch and J.Sambrook, molecular cloning: laboratory manual, cold spring harbor laboratory, cold spring port (1989), T.J.Silhavy, M.L, Berman and L.W.Enquist, the gene fusion experiment, cold spring harbor laboratory, the cold spring port, NY (1984) and Ausubel, F.M. etc., contemporary molecular biology method, routine reorganization and clone technology that GreenePublishing Assoc and Wiley-Interscience (1987) describes, expression cassette according to the present invention is with suitable promotor and suitable polypeptid DNA and preferably, inserts the special transit peptides DNA of coding chloroplast(id) between promotor and the polypeptid DNA and polyadenylation signal and merges and produce.

When particularly preferred sequence allows to navigate to apoplast, plastid, folliculus, plasma membrane, plastosome, endoplasmic reticulum (FR) or does not have suitable operating sequence, stay in the compartment of formation, i.e. (Kermode, Crit.Rev.Plant Sci.15 in the cytosol, 4 (1996), 285-423).Location in ER and the cell walls has proved for quantitative albumen accumulation in transgenic plant especially favourable (Schouten) etc., molecular biology of plants 30 (1996), 781-792; Artsaenko etc., plant magazine 8 (1995) 745-750).

The present invention also relates to encoding sequence coding mycocide in conjunction with fusion rotein, meromixis albumen is the expression cassette of the transit peptides of control polypeptide transportation.Particularly preferably be the special transit peptides of chloroplast(id), it is to scale off in conjunction with the polypeptide portion enzyme from mycocide mycocide is transported to the chloroplast(id) of plant in conjunction with polypeptide after.Particularly preferred transit peptides or its functional similarity thing (for example, the transit peptides of rubisco small subunit or ferredoxin NADP oxydo-reductase) from plastid transketolase (TK).

The polypeptid DNA of preparation expression cassette needs according to the present invention or polypeptide cDNA preferably come by means of polymerase chain reaction (PCR) amplification.Known DNA cloning method with PCR, Innis etc. for example, PCR method, methods and applications guide, Academic Press (1990).Detect the dna fragmentation of PCR generation with the polysaccharase mistake in the construct of avoiding to express with sequential analysis easily.

The coding mycocide that inserts can synthesize preparation or natural acquisition or contain synthetic and n DNA mixture of ingredients in conjunction with the nucleotide sequence of polypeptide.Generally speaking, preparation contains the synthesizing ribonucleotide sequence of the codon that plant likes.Codon by expressed proteins in the high albumen of the frequency of occurrences and the most of interest plant species is determined the codon that plant is liked.When preparation during expression cassette, can operate a plurality of dna fragmentations to obtain in suitable meaningful reading and to be equipped with the nucleotide sequence of correct reading frame.For dna fragmentation is connected to each other, connector or joint are added on the fragment.

Should provide to transcribe meaning according to promotor of the present invention and terminator zone, and have the joint that contains one or more restriction sites or polylinker to insert this sequence.In principle, joint has 1-10, and 1-8 is individual usually, preferably 2-6 restriction site.In the regulation and control zone, the size of joint is less than 100bp usually, is less than 60bp usually, but or even 5bp.Promotor according to the present invention is the born or homologous of host plant, or is external source or different with it.Expression cassette according to the present invention comprises the sequence and the transcription termination region according to promotor of the present invention, any hope of transcribing meaning with 5 '-3 '.Multiple terminator can exchange mutually.

And, can adopt the operation that suitable restriction site is provided or removes unnecessary DNA or restriction site.If insert, deletion, substitute as conversion and transversion if possible, can adopt vitro mutagenesis, " primer [sic] repairings ", restriction or connection.With regard to suitable operation such as restriction, " chewing-back " or mend flat to obtain to provide segmental complementary terminal with regard to " flush end " to connect.

According to the present invention, for successfully the particularly important is add the special ER make average expression level improve three times to four times be detained signal SEKDEL (Schuoten, A. etc., molecular biology of plants 30 (1996), 781-792).Other delay signal of the naturally occurring ER of being positioned in plant and animal albumen also can be used for making up this expression cassette.

Preferred polyadenylation signal is the plant polyadenylation signal, preferably consistent with T-DNA edge sequence polyadenylation signal from Agrobacterium tumefaciems, especially the gene 3 of Ti-plasmids pTiACH5 T-DNA edge sequence (octopine synthase) (Gielen etc., EMBO's magazine 3 (1984) 835, et seq.) or its functional analogue.

For example can comprise according to expression cassette of the present invention, constitutive promoter (preferably CaMV35S promotor), LeB4 signal peptide, the gene that will express and ER are detained signal.With reference to single-chain antibody (scFv gene), in Fig. 2, represented the structure of expression cassette with schematic form.Aminoacid sequence KDEL (Methionin, aspartic acid, L-glutamic acid, leucine) preferably is detained signal as ER.

There is the amalgamation and expression box of mycocide binding characteristic polypeptide preferably to be cloned into carrier coding, for example is suitable for transforming the pBin19 of Agrobacterium tumefaciems.Transform plant, especially farm crop with known method with the edaphic bacillus that has transformed such carrier, for example tobacco by as callus leaf or leaf are partially immersed in the edaphic bacillus solution, is cultivated in suitable substratum subsequently.It is known transforming plant with edaphic bacillus, and particularly F.F.White is used for the carrier that higher plant gene shifts, transgenic plant, the 1st volume, engineering and application, S.D.Kung and R.Wu edit, Academic Press, 1993, the 15-38 page or leaf and S.B.Gelvin, the molecular genetics that shifts from edaphic bacillus to plant T-DNA edge sequence, transgenic plant, the 49-78 page or leaf.Can be in known manner from callus leaf or blade-section cell transformed regeneration of transgenic plant, these transgenic plant contain and are incorporated into the gene that mycocide binding characteristic polypeptide is arranged according to the expression of expression cassette of the present invention.

For with the DNA conversion host plant of coding mycocide in conjunction with polypeptide, expression cassette according to the present invention is incorporated in the recombinant vectors with insertion, this carrier DNA contains extra function conditioning signal, for example is used to the sequence of duplicating or integrating.Suitable carriers obtains in (1993) the 6/7th chapter 71-119 pages or leaves describing especially at " molecular biology of plants method and biotechnology " (CRC Press).

Utilize above-mentioned reorganization and clone technology, expression cassette according to the present invention can be cloned into and allow in the appropriate carriers that they for example increase in intestinal bacteria.Suitable cloning vector is pBR332, pUC series, M13mp series and pACYC184 particularly.Especially suitable is the binary vector that can duplicate in intestinal bacteria and edaphic bacillus, for example pBin19 (Bevan etc., (1980), nucleic acids research 12,8711).

The present invention relates in addition using according to expression cassette of the present invention and transforms plant, vegetable cell, plant tissue or plant organ.The preferred purpose of using is the adjusting to the mycocide resistance with plant toxicity activity.

According to the selection of promotor, expression can occur in the leaf specifically, in the seed or in other plant organ.Such transgenic plant, its reproductive material and vegetable cell, plant tissue or plant organ are the other themes of the present invention.

Change foreign gene over to Plant Genome and be called conversion.In this process, above-mentioned conversion and be used for instantaneous or stable conversion from the method for plant tissue or vegetable cell aftergrowth.Suitable method has the protoplast transformation taken in by the polyoxyethylene glycol mediated dna, with the biolistic[sic of particle gun] method, electroporation, dried embryo incubation, microinjection and agrobacterium-mediated transgenosis in containing dna solution.The method of mentioning obtains describing, as, B.Jenes etc., gene transfer technique exists: transgenic plant, the 1st volume engineering and application, editor: S.D.Kung and R.Wu.Academic Press (1993) 128-143 and at Potrykus, Annu, Rev.Plant.Physiol.Plant.Molec.Biol.42 (1991) 205-225) in.The construction that will express preferably is cloned into the carrier that is suitable for transforming Agrobacterium tumefaciems, for example pBin19 (Bevan etc., nucleic acids research, 12 (1984) 8711).

The edaphic bacillus that has transformed according to expression cassette of the present invention can transform plant with known method, especially farm crop such as cereal, corn, soybean, paddy rice, cotton, beet, Canola, Sunflower Receptacle, flax, potato, tobacco, tomato, rape, clover, lettuce and various shrub, tree, nut and Vitis kind, coffee for example, the fruit tree is as apple, pears or cherry, nutwood such as English walnut or Semen Caryae Cathayensis and the grape vine that is even more important, for example, in suitable substratum, cultivate subsequently by callus leaf or leaf are partially immersed in the edaphic bacillus solution.

According to the present invention, although suitable sequence function still likely on the function of coding mycocide in conjunction with polypeptide is the nucleotide sequence difference.Therefore, functional analogue comprises the varient and be suitable for the artificial sequence oligodeoxynucleotide that vegetable codon use of artificial sequence oligodeoxynucleotide as obtaining with chemosynthesis of naturally occurring sequence described herein.

Particularly, functional analogue is interpreted as the nature or the artificial mutation of the coding mycocide binding characteristic polypeptide of initial separation, and this sudden change still shows the function of hope.Sudden change comprises substituting, add, delete, exchange or inserting of one or more nucleotide residues.Therefore, the present invention also comprises by modifying the nucleotide sequence that this nucleotide sequence obtains.The purpose of this modification can be, for example, further limit in the encoding sequence or other, for example, insert a plurality of cleavage sites of Restriction Enzyme.

Other functional analogue is to compare miopragia or those stronger varients with beginning gene or gene fragment.

And, as long as the mycocide resistance that can induce aforesaid hope to avoid phytotoxic effect to crop plants, artificial DNA sequence all is fit to.Such artificial DNA sequence can have mycocide to obtain evaluation in conjunction with albumen active and that make up by the moulding or external selection of molecule by for example anti-translation.Suitable especially is to use the dna encoding sequence that anti-translation peptide sequence obtains according to the special codon of host plant.Transformed other known of plant by computer-aided analysis, the expert who is familiar with the plant genetics method is easy to determine that specific codon uses.

The suitable similar nucleotide sequence according to the present invention that must mention in addition is the sequence of encoding fusion protein, and wherein fusion rotein partly is that the mycocide of non-plant origin is in conjunction with similar portions on polypeptide or its function.For example, the second section of fusion rotein can be the other polypeptide that enzymic activity is arranged, or borrows its auxiliary antigenic peptide sequence (for example myc-tag or his-tag) that can detect the scFvs expression.Yet it is the modulin sequence preferably, for example, instructs the polypeptide that the mycocide binding characteristic is arranged to arrive the signal or the transit peptides of the action site of wishing.

Yet, the present invention also relates to expression product prepared in accordance with the present invention and transit peptides and the fusion rotein of mycocide binding characteristic polypeptide arranged.

With regard to the object of the invention, resistance/tolerance refers to the resist ability of mycocide with plant toxicity activity of the artificial plant that obtains.It comprises at least and during the plant generation inhibitor is partly reached insensitivity especially completely.

The phytotoxicity action site of mycocide is leaf texture normally, so external source mycocide can provide enough protections in conjunction with the specific expression of polypeptide leaf.Yet a people is readily appreciated that the phytotoxicity effect of mycocide need not be limited in leaf texture, also can tissue-specific mode realize in other all organs of plant.

In addition, external source mycocide has superiority in conjunction with the constitutive expression of polypeptide.In addition, but abduction delivering is also desirable.

For example, the external efficient of in the substratum that contains series concentration gradient mycocide, breeding or determine the tool mycocide binding characteristic express polypeptide of transgene expression by the root meristematic tissue by the seed germination experiment.In addition, can be in type of detection and all altered mycocide tolerance of being tried plant of level in the greenhouse experiment.

The present invention relates to the reproductive material of the transgenic plant, transgenic cell, tissue, organ and these plants that have transformed according to expression cassette of the present invention in addition.Particularly preferably be transgenic crop such as cereal, corn, soybean, paddy rice, cotton, beet, Canola, Sunflower Receptacle, flax, potato, tobacco, tomato, rape, clover, lettuce and various shrub tree, nut and Vitis kind, coffee for example, fruit is set as apple, pears or cherry, nutwood such as English walnut or Semen Caryae Cathayensis and the grape vine that is even more important.

The mycocide with phytotoxicity effect of available inhibition plant enzyme is handled transgenic plant, vegetable cell, plant tissue or plant organ, and not successful thus plant transformed, vegetable cell, plant tissue or plant organ are dead or destroyed.The example of suitable activeconstituents is strobilurins, specifically is the metabolite and the functional deriv of methyl methoxy imino--α-(O-tolyloxy)-O-tolyl acetic ester (BAS 490F) and these compounds.The DNA that coding has mycocide binding characteristic polypeptide and insertion expression cassette according to the present invention also can be used as selective marker.

The present invention has advantage, particularly with regard to farm crop, in case induced the selection resistance of crop plants to mycocide with phytotoxicity effect, such mycocide just can even be used for these farm crop with the control harmful fungoid with higher utility ratio, otherwise will cause plant destruction.Compound in the following colony can be mentioned as the example that has the mycocide of plant toxicity activity like this, rather than restriction:

Sulphur; Dithiocarbamate and derivative thereof such as iron (III) dimethyldithiocarbamate, the zinc dimethyldithiocarbamate, zinc vinyl bisdithiocarbamic ester, magnesium vinyl bisdithiocarbamic ester, MnZn vinyl diamines bisdithiocarbamic ester, tetramethyl-thiuram disulphide [sic], zinc (N, N '-vinyl bisdithiocarbamic ester) ammonia mixture, zinc (N, N '-propylene bisdithiocarbamic ester) ammonia mixture, zinc (N, N '-propylene bisdithiocarbamic ester), N, two (thiocarbamoyl) disulphide of N '-polypropylene; Nitro-derivative such as dinitrobenzene (1-methylheptyl) phenylisocrotonic acid ester, the 2-second month in a season-butyl-4,6-dinitrophenyl-3,3-dimethacrylate, the 2-second month in a season-butyl-4,6-dinitrophenyl isopropyl carbonate, di-isopropyl 5-nitro isophthalic ester; Heterocycle material such as 2-heptadecyl-2-tetrahydroglyoxaline acetic ester, 2,4-two chloro-6-(O-the chloroanilino)-second month in a season-triazine, O, O-diethyl phthalimido phosphono sulfo-thing, two (dimethylamino) phosphoryls of 5-amino-1-[]-3-phenyl-1,2, the 4-triazole, 2,3-dicyano-1, the 4-anthraquinone dithio, 2-sulfo--1,3-two sulphur hydrocarbon [4,5-b] quinoline, methyl isophthalic acid-(butyl carbamyl)-2-benzoglyoxaline carbaminate, the amino benzoglyoxaline of 2-methoxycarbonyl, 2-(2-furyl) benzoglyoxaline, 2-(4-triazolyl) benzoglyoxaline, N-(1,1,2,2-tetrachloro ethylenebis dithiocarbamate) tetrahydric phthalimide, N-trichloromethylthio tetrahydric phthalimide, N-trichloromethylthio phthalic imidine, N-dichloro fluoro methyl sulfo--N ', N '-dimethyl-N-phenyl sulfo group diamide, 5-oxyethyl group-3-trichloromethyl-1,2, the 3-thio biphosphole, 2-sulfo-cyanato-methyl-thio-benzothiazole, 1,4-two chloro-2, the 5-dimethoxy-benzene, 4-(2-chloro-phenyl-hydrazono-)-3-methyl-5-isoxazolidinone, pyrimidine-2-sulfo-[sic] 1-oxide compound, 8-alkyl quinoline or its mantoquita, 2,3-dihydro-5-carboxylic acylaniline-6-methyl isophthalic acid, the 4-oxathiin, 2,3-dihydro-5-carboxylic acylaniline-6-methyl isophthalic acid, 4-oxathiin-4, the 4-dioxide, 2-methyl-5,6-dihydro-4H-pyrans-3-carboxylic acylaniline, 2-methyl furan-3-carboxylic acylaniline, 2,5-dimethyl furan-3-carboxylic acylaniline, 2,4,5-dimethyl furan-3-carboxylic acylaniline, N-cyclohexyl-2,5-dimethyl furan-3-carboxylic acid amides, N-cyclohexyl-N-methoxyl group-2,5-dimethyl furan-3-carboxylic acid amides, 2-toluyl aniline, 2-phenyl-iodide formylaniline, N-formyloxy-N-morpholine-2,2,2-three chloroethyl acetals, piperazine-1, two basic couples-1-(2 of 4-, 2,2-three chloroethyls) methane amide, 1-(3,4) dichlorobenzene amido-1-formyloxy amino-2,2, the 2-ethylene dichloride; Amine is as 2,6-dimethyl-N-three decyl morpholines or its esters, 2,6-dimethyl-N-ring decyl morpholine or its esters, N-[3-(right-the tert-butyl phenyl)-2-methyl-propyl]-cis-2,6-dimethyl-morpholine, N-[3-be right-the tert-butyl phenyl)-the 2-methyl-propyl] piperidines, (8-(1, the 1-dimethyl ethyl)-and N-ethyl-N-propyl group-1,4-Er Evil spiral [4.5] decane-2 vulkacit H; Azole such as 1-[2-(2,4 dichloro benzene base)-4-ethyl-1,3-dioxolane-2-base ethyl]-1H-1,2, the 4-triazole, 1-[2-(2,4 dichloro benzene base)-4-just-propyl group-1,3-dioxolane-2-base ethyl]-1H-1,2, the 4-triazole, N-(just-propyl group)-N-(2,4,6-Trichlorophenoxy ethyl)-N '-imidazolyl urea, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-azido--1-yl)-2-butanone, 1-(4-chlorophenoxy)-3,3-dimethyl-1-(1H-1,2,4-azido--1-yl)-the 2-butanols, (2RS, 3RS)-1-[3-(2-chloro-phenyl-)-2-(4-fluorophenyl)-oxyethane-2-ylmethyl]-1H-1,2, the 4-triazole, 1-[2-(2, the 4-dichlorophenyl)-amyl group]-1H-1,2, the 4-triazole, 2,4 '-two fluoro-α-(1H-1,2,4-triazolyl-1-methyl) diphenyl-methyl alcohol, 1-((two (4-fluorophenyl) methyl-silicane base) methyl)-1H-1,2, the 4-triazole, 1-[2RS, 4RS; 2RS, 4SR]-4-bromo-2-(2,4 dichloro benzene base) tetrahydrofuran base]-1H-1,2, the 4-triazole, 2-(4-chloro-phenyl-)-3-cyclopropyl-1-(1H-1,2,4-azido--1-yl)-butane-2-alcohol, (+)-4-chloro-4-[4-methyl-2-(1H-1,2,4-azido--1-ylmethyl)-1,3-dioxolane-2-yl] phenyl-4-chloro-phenyl-ether, (E)-(R, S)-1-(2, the 4-dichlorophenyl)-4,4-dimethyl-2-(1H-1,2,4-azido--1-yl) penta-1-alkene-3-alcohol, 4-(4-chloro-phenyl-)-2-phenyl-2-(1H-1,2,4-triazolyl methyl) butyronitrile, 3-(2, the 4-dichlorophenyl)-6-fluoro-2-(1H-1,2,4-azido--1-yl) quinazoline-4 (3H)-ketone, (R, S)-2-(2, the 4-dichlorophenyl)-1-H-1,2,4-azido--1-yl) hexane-2-alcohol, (1RS, 5RS; 1RS, 5SR)-5-(4-chlorophenylmethyl)-2,2-dimethyl-1-(1H-1,2,4-azido--1-ylmethyl) cyclopentanol, (R, S)-1-(4-chloro-phenyl-)-4,4-dimethyl-3-(1H-1,2,4-azido--1-ylmethyl) pentane-3-alcohol, (+)-2-(2, the 4-dichlorophenyl)-3-(1H-1,2, the 4-triazolyl) propyl group-1,1,2,2-tetrafluoro ether, (E)-and 1-[1-[4-chloro-2-trifluoromethyl) phenyl] imino-)-2-propoxy-ethyl]-the 1H-imidazoles, the own nitrile of 2-(4-chloro-phenyl-)-2-(1H-1,2,4-azido--1-ylmethyl), α-(2-chloro-phenyl-)-α-(4-chloropropyl)-5-rubigan, 5-butyl-2-dimethylamino-4-hydroxyl-6-methylpyrimidine, two (right-chloro-phenyl-)-3-piconols, 1, two (3-ethoxy carbonyl-2-thioureido) benzene of 2-, 1, two (3-methoxycarbonyl-2-thioureido) benzene of 2-; Strobilurines such as methyl-E-methoxyimino-[α-(O-tolyloxy)-O-tolyl] acetic ester, methyl-[E]-2-{2-[6-(2-cyano-benzene oxygen) pyrimidine-4-base oxygen base] phenyl }-3-methoxy acrylate, methyl-E-methoxyimino-[α-(2-Phenoxyphenyl)] ethanamide, methyl-E-methoxyimino-[α-(2, the 5-dimethyl phenoxy)-O-tolyl] ethanamide; Anilino-pyrimidine such as N-(4,6-dimethyl pyrimidine-2-yl) aniline, N-(4-methyl-6-(1-proyl) pyrimidine-2-base) aniline, N-[4-methyl-6-cyclopropyl pyrimidine-2-base] aniline; Phenylpyrrole class such as 4-(2,2-two fluoro-1,3-benzo dioxole-4-yl)-pyrroles-3-nitrile; Cinnamide such as 3-(4-chloro-phenyl-)-3-(3, the 4-Dimethoxyphenyl) acryloyl morpholine; And various mycocides such as Cyprex acetic ester; 3-[3-(3; 5-dimethyl-2-oxygen cyclohexyl)-and 2-hydrocarbon ethyl] glutarimide; the N-methyl-or N-ethyl-(4-trifluoromethyl);-2-[3 '; 4 '-Dimethoxyphenyl] benzamide [sic]; phenyl-hexafluoride; methyl-N-(2; the 6-3,5-dimethylphenyl)-N-(2-furoyl)-DL-L-Ala salt; DL-N-(2; the 6-3,5-dimethylphenyl)-N-(2 '-methoxy ethanoyl) alanine methyl ester; N-(2; the 6-3,5-dimethylphenyl)-N-chloracetyl-D; the amino butyrolactone of L-2-; DL-N-(2; the 6-3,5-dimethylphenyl)-N-(phenylacetyl) alanine methyl ester; 5-methyl-5-vinyl-3-(3; the 5-dichlorophenyl)-2; 4-dioxy-1; the 3-oxazolidine; 3-[3; 5-dichlorophenyl-(5-methyl-5-methoxyl methyl)]-1; 3-oxazolidine-2; 4-diketone [sic]; 3-(3; the 5-dichlorophenyl)-1-sec.-propyl carbamyl glycolylurea; N-(3; the 5-dichlorophenyl)-1; 2-dimethylcyclopropane-1; the 2-dicarboximide; 2-cyanogen-[N-(B aminocarbonyl)-2-methoxyimino] ethanamide; N-(3-chloro-2,6-dinitrobenzene-4-trifluoromethyl)-5-trifluoromethyl-3-chloro-2-aminopyridine.

The identical derivative of these mycocide functions has same function scope at plant pathogenic fungi as these materials of specifically mentioning, and littler, equate or more obvious plant toxicity activity.

Illustrate rather than restriction the present invention by following example: general cloning process:

The purifying of for example restricted cutting of the step of Zhi Hanging within the scope of the present invention, agarose gel electrophoresis, dna fragmentation, nucleic acid is transferred to being connected of nitrocellulose and nylon membrane, dna fragmentation, Bacillus coli cells conversion, microbial culture, phage operation and recombinant DNA sequence analysis according to press of (1989) cold spring harbor laboratory such as Sambrook, the carrying out that ISBN 0-87969-309-6 describes.

Hereinafter used bacterial isolates (intestinal bacteria XL-I Blue) is from Stratagene.Edaphic bacillus bacterial strain (the Agrobacterium tumefaciems that are used for Plant Transformation.The C58C1 that contains plasmid pGV2260 or pGV3850Kan) by descriptions (nucleic acids research 13 (1985) 4777) such as Deblaere.Optionally, available edaphic bacillus strain LBA4404 (clontech) or other suitable strain system.Carrier pUC19 (Yanish-Perron, gene 33 (1985) 103-119), pBluscript.SK-(Stratagene), pGEM-T (Promega), pZero (Invitrogen), pBin19 (Bevan etc., nucleic acids research 12 (1984) 8711-8720) and pBinAR (Hofgen and Willmitzer, plant science 66 (1990) 221-230) be used to clone purpose.The sequential analysis of recombinant DNA

(Sanger etc., institute of NAS newspaper 74 (1977) 5463-5467), are measured the sequence of recombinant DNA molecules with the laser fluorescence dna sequencing device of pharmacia company with the Sanger method.The preparation of expression of plants box

Form with EcoRI-KpnI fragment (the Nucleotide 6909-7437 that is equivalent to cauliflower mosaic virus) (Franck etc., cell 21 (1980) 285) is inserted plasmid pBin19 (Bevan etc., nucleic acids research 12,8711 (1984)) with 35S CaMV promotor.The polyadenylation signal of the gene 3 of Ti-plasmids pTiACH5 (Gielen etc., EMBO's magazine 3 (1984) 835) T-DNA edge sequence; Nucleotide 11749-11939 is separated to the PvuII-HindIII fragment, add the SphI joint after; Be cloned into the PvuII site between the SphI-HindIII cleavage site of carrier.Produced plasmid pBinAR (Hofgen and Willmitzer, plant science 66 (1990) 221-230).Application Example embodiment 1

Because the mycocide non-immunogenicity, they must with solid support material coupling, for example KLH.The base if molecule responds, then directly coupling; If no, when synthesis of antifungal agents, introduce functional group or in building-up process the selective reaction precursor these molecules are coupled on the carrier molecule in simple reactions steps.At " the immunochemistry handbook " of Miroslavic Ferencik, 1993, Chapman ﹠amp; Hall, antigen one chapter 20-49 page or leaf is found the example of linked reaction.

The carrier molecule (antigen) of injecting this modification repeatedly is used for immunity, for example, and the Balb/c mouse.In case detect q.s with ELISA (enzyme-linked immunosorbent assay) with antigen bonded antibody, just take out the splenocyte of these animals and merge to cultivate hybrid with the myeloma cell.Use in addition " mycocide modify BSA " as the antigen among the ELISA to distinguish at haptenic immune response with at the immune response of KLH.

With preparing monoclonal antibody with the currently known methods similar methods, for example at " practical immunology " Leslie Hudson and Frank Hay, Blackwell Scientific Publications, 1989 or at " monoclonal antibody: theory and practice " James Goding, 1983, AcademicPress, Inc, or at " monoclonal antibody practical guide ", J.Liddell and A.Cryer, 1991 John Wiley ﹠amp; Sons; Or Achim Moller and Franz Emling " Monoklonale Antikorper gegen TNF und deven Verwendung " [at monoclonal antibody and the application thereof of TNF] described method.European patent specification EP-A260610.Embodiment 2

The starting point of this research is the monoclonal antibody of discerning mycocide BAS 490F specifically and high-affinity being arranged.Selected hybridoma cell line is characterised in that excretory has high-affinity at the monoclonal antibody of mycocide antigen BAS 490F and can obtain the distinguished sequence (Berek, C. etc., nature 316, (1985) 412-418) of this immunoglobulin (Ig).Should at the monoclonal antibody of BAS 490F the starting point that single chain antibody fragments (the anti-BAS 490F of scFv-) makes up.

At first, separating mRNA and be transcribed into eDNA from hybridoma.This cDNA is as the template of variable immunoglobulin gene VH of amplification and VK, and wherein the Auele Specific Primer of heavy chain is VHlBACK and VH FOR-2, and the light chain primer is VK2BACK and MJK5 FONX (Clackson etc., nature 352, (1991) 624-628.Isolating variable immunoglobulin (Ig) is the starting point that single chain antibody fragments (the anti-BAS 490F of scFv-) makes up.In fusion PCR subsequently, three composition VH, VK and linker fragment are joined together in the PCR reaction, thereby the anti-BAS 490F of ScFv-obtains amplification (Fig. 3).

In the bacterium system, express the back the anti-BAS 490F of the scFv-gene that makes up is carried out Function Identification (antigen-binding activity).In order to reach this purpose, use Hoogenboom, nucleic acids research such as H.R. 19 (1991), the method for 4133-4137 is synthesized the anti-BAS 490F of scFv-with solvable antibody fragment in intestinal bacteria.Measure activity and the specificity (Fig. 4) that detects the antibody fragment that makes up with ELISA.

In order to allow antibody fragment seed-specific expression in tobacco, with the downstream of the anti-BAS490F gene clone of scFv-in the LeB4 promotor.Isolating LeB4 promotor shows the seed-specific expression (Baumlein, H. etc., Mol.Gen.Genet.225, (1991) 121-128) of various exogenous genes strictness in tobacco from Vicia fqba.The anti-BAS 490F of scFv-polypeptide is transported to endoplasmic reticulum and causes the segmental stable accumulation of lot of antibodies.In order to reach this purpose, scFv anti-BAS 490F gene and signal peptide sequence merge guaranteeing that it enters endoplasmic reticulum, are detained signal SEKDEL with ER and merge to guarantee that polypeptide is retained among the ER (Wandelt etc., 1992) (Fig. 5).

The expression cassette that makes up is cloned into binary vector pGSGLUC1 (Saito etc., 1990) and is transferred to the edaphic bacillus strain by electroporation is among the EHA101.The tobacco that reorganization edaphic bacillus clone is used for subsequently transforms.Each construct 70-140 strain tobacco that regenerates.After the self-pollination, from the seed of regenerated rotaring gene tobacco plant results different developmental phases.From these seeds, obtain to be in the soluble proteins in the water buffer system after the extracting.Analyze transgenic plant and show that it is 1.9% that scFv-anti-BAS 490F gene and ER are detained the maximum accumulation of the anti-BAS 490F of scFv-albumen that obtains in the fusion permission mature seed of signal SEKDEL.

The anti-BAS 490F of the scFv-gene size that makes up is about 735bp.Variable domains merges each other with the VH-L-VL order.

Identify special selectivity in the ripe tobacco seed extract with direct ELISA.The value that obtains clearly illustrates that the albumen extract contains activated antibody fragment on the function.Embodiment 3

Under USP promotor control, single chain antibody fragments seed-specific expression and gathering in transgene tobacco seed cell endoplasmic reticulum.

The starting point of this research is at the single chain antibody fragments of mycocide BAS 490F (the anti-BAS 490F of scFv-).Carry out the Function Identification (antigen-binding activity) of the anti-BAS 490F of the scFv-gene of this structure after expressing after in the bacterium system, expressing and in the tobacco leaf.Measure activity and the specificity that detects the antibody fragment that makes up with ELISA.

In order to allow antibody fragment seed-specific expression in tobacco, with the downstream of the anti-BAS490F gene clone of scFv-in the USP promotor.Isolating USP promotor shows the seed-specific expression (Fiedler, U. etc., molecular biology of plants 22, (1993) 669-679) of various exogenous genes strictness in tobacco from Vicia faba.The anti-BAS 490F of scFv-polypeptide is transported to endoplasmic reticulum and causes the segmental stable accumulation of lot of antibodies.In order to reach this purpose, scFv-anti-BAS 490F gene and signal peptide sequence merge guaranteeing that it enters endoplasmic reticulum, are detained signal SEKDEL with ER and merge to guarantee that polypeptide is retained among the ER (Wandelt etc., 1992) (Fig. 1).

The expression cassette that makes up is cloned into binary vector pGSGLUC1 (Saito etc., 1990) and is transferred to the edaphic bacillus strain by electroporation is among the EHA101.The tobacco that reorganization edaphic bacillus clone is used for subsequently transforms.After the self-pollination, from the seed of regenerated rotaring gene tobacco plant results different developmental phases.From these seeds, obtain to be in the soluble proteins in the water buffer system after the extracting.Analyzing single chain antibody fragments that fusion that transgenic plant show that scFv-anti-BAS 490F gene and ER are detained the dna sequence dna of signal SEKDEL causes with BAS 490F binding affinity being arranged under the control of USP promotor has just synthesized as far back as the 10th day of seed development.Embodiment 4

In order to obtain the wide expression of antibody fragment in plant, especially in leaf, with the anti-BAS 490F of scFv-gene clone in CaMV35S promotor downstream.Expression (Benfey and Chaua, science 250 (1990), the 956-966 of this strong constitutive promoter mediate foreign gene in all plant tissues.The anti-BAS 490F of scFv-albumen allows stable being accumulated in the leaf of lot of antibodies fragment that will obtain to the transhipment of endoplasmic reticulum.At first, with the anti-BAS 490F of scFv-gene with guarantee to enter the signal peptide sequence of endoplasm meat and guarantee product be retained in ER among the ER be detained signal KDEL and merge (Wandelt etc., plant magazine 2 (1992), 181-192).The expression cassette that makes up is cloned into binary vector pGSGLUC1, and (Saito etc., vegetable cell breeding 8 (1990) 718-721) and by electroporation is transferred to edaphic bacillus bacterial strain EHA101.The tobacco that reorganization edaphic bacillus clone is used for subsequently transforms.About 100 strain tobacco plants of regenerating.Take off the leaf that is in different developmental phases from the regenerated transgenic tobacco plant.From these seeds, obtain to be in the soluble proteins in the water buffer system after the extracting.Analysis subsequently (Western engram analysis and ELISA measure) shows that the antigen that biologic activity is arranged that obtains is more than 2% in conjunction with the maximum accumulation of the anti-BAS 490F of scFv-polypeptide from leaf.In the green leaf of growth fully, identify the high expression level value, but in old and feeble leaf material, also detected antibody fragment.Embodiment 5

By means of the synthetic oligonucleotide, coding obtains pcr amplification at the cDNA fragment of the single-chain antibody of BAS 490F.

The pcr amplification of single-chain antibody cDNA carries out on the DNA thermal cycler of Perkin Elmer company.Reaction mixture contains the relevant oligonucleotide of 8ng/ μ l single-stranded template cDNA, 0.5 μ M, 200 μ M Nucleotide (pharmacia), 50mM KCl, 10mM Tris-Hcl (pH8.3 25 ℃ time the, 1.5mM Mgcl ₂) and 0.02 μ/nl Taq polysaccharase (Perkin Elmer).Reaction conditions is provided with as follows:

Annealing temperature: 45 ℃

Denaturation temperature: 94 ℃

Elongating temperature: 72 ℃

Cycle number: 40

The result obtains the fragment of about 735 base pairs, and it is connected among the carrier pBluescript.Connect mixture and be used for transformed into escherichia coli XL-I Blue, plasmid obtains amplification.About the application and the optimization of polymerase chain reaction, referring to: Innis etc., 1990, PCR method, methods and applications guide, Academic Press.Embodiment 6 expresses the preparation of transgene tobacco that coding has the single-chain antibody cDNA of mycocide binding characteristic

Plasmid pGSGLUC1 is transformed among the Agrobacterium tumefaciems C58C1:pGV2260.Be used in 1: 50 diluent transformation of tobacco of overnight culture plant (tobacco cultivation kind Samsun NN) of the conversion male edaphic bacillus clone who cultivates in the Murashige-Skoog substratum (2MS substratum) that contains 2% sucrose.In culture dish, the blade of germ-free plant (each about 1cm ²) in 1: 50 edaphic bacillus diluent incubation 5-10 minute.Cultivated 2 days containing on the 2MS substratum of 0.8%Bacto-Agar lucifuge then.Under 16 hours illumination/8 hour dark, continue to cultivate after 2 days, on the MS substratum that contains 500mg/l cefotaxime (cefotaxim-Sodium), 500mg/l kantlex, 1mg/l benzyladenine (BAP), 0.2mg/l naphthylacetic acid and 1.6g/l glucose, continue to cultivate with the rhythm and pace of moving things weekly.The branch of growth is transferred on the MS substratum that contains 2% sucrose, 250mg/l cefotaxime and 0.8%Bacto-Agar.Embodiment 7 is at the stable accumulation of single chain antibody fragments in endoplasmic reticulum of mycocide BAS 490F

The starting point of this research is the single-chain antibody at mycocide BAS 490F (the anti-BAS 490F of scFv-) of expressing in tobacco plant.Measure quantity and the activity of identifying the anti-BAS 490F of synthetic scFv-polypeptide with Western engram analysis and ELISA.

The form of wanting expressed exogenous gene and LeB4 signal peptide (N-end) and ER delay signal KDEL (C-end) to merge with translation is expressed under the control of CaMV35S promotor the anti-BAS 490F of scFv-gene might be expressed in endoplasmic reticulum.The transhipment of the anti-BAS 490F of scFv-polypeptide in endoplasmic reticulum allows the stable accumulation of a large amount of active antibody fragments.Behind the results leaf material, that it is frozen in-20 ℃ (1), freeze-drying (2) or under the greenhouse dry (3).From blade to be checked by extracting obtain to be in the water damping fluid soluble proteins and with the anti-BAS 490F of affinity chromatography purification scFv-polypeptide.The anti-BAS 490F of the scFv-of equivalent amount of purified polypeptide (that freeze, freeze dried and exsiccant) is used to identify the activity (Fig. 6) of antibody fragment.Fig. 6 A represents from the antigen-binding activity of the anti-BAS 490F of the scFv-polypeptide of fresh (1), freeze-drying (2) and dry (3) blade purifying.Fig. 6 B represents the proteic representative amount of the anti-BAS 490F of scFv-(about 100ng) that ELISA measures that is used for by Western engram analysis mensuration.The size of molecular weight of albumen standard has been represented in the left side.The discovery antigen-binding activity is basic identical.Embodiment 8

In order to verify the mycocide tolerance that produces the rotaring gene tobacco plant that mycocide binding characteristic polypeptide is arranged, handle these tobacco plants with the BAS 490F of various amounts.In the greenhouse, all experiments all prove compared with the control, express more height endurability and the more unconspicuous phytotoxic effect of the plant performance of the anti-BAS 490F of scFv-gene to mycocide BAS 490F.

Claims (28)

1. one kind by expressing external source mycocide prepares the mycocide tolerant plants in conjunction with polypeptide method in plant.

2. method according to claim 1, wherein external source mycocide is single chain antibody fragments in conjunction with polypeptide.

3. method according to claim 1, wherein external source mycocide is complete antibody or its fragment in conjunction with polypeptide.

4. method according to claim 1, wherein mycocide is methyl methoxy imino--α-(O-tolyloxy)-O-tolyl acetic ester (BAS 490F).

5. according to any described method among the claim 1-3, wherein plant is list or dicotyledons.

6. method according to claim 5, wherein plant is a tobacco.

7. according to any described method, wherein allogenic polypeptide constitutive expression in plant among the claim 1-6.

8. according to any described method among the claim 1-6, wherein induce the expression of allogenic polypeptide in plant.

9. according to any described method among the claim 1-6, wherein allogenic polypeptide is expressed in leaf.

10. according to any described method among the claim 1-6, wherein allogenic polypeptide is expressed in plant seed.

11. an expression of plants box is made up of in conjunction with gene, ER delay signal and the terminator of polypeptide promotor, signal peptide, encoding exogenous mycocide.

12. expression cassette according to claim 11, wherein used constitutive promoter are the CaMV35S promotors.

13. expression cassette according to claim 11, wherein the gene that will express is the gene of single chain antibody fragments.

14. expression cassette according to claim 11, wherein with the gene that combines polypeptide with mycocide that other functional protein such as enzyme, toxin, chromophore and conjugated protein translation fusion form are used or gene fragment as the gene that will express.

15. expression cassette according to claim 11 wherein wants the polypeptide expressed gene to obtain from hybridoma or by other recombination method such as antibody phage methods of exhibiting.

16. the purposes of expression cassette according to claim 11, be used for dicotyledonous or monocotyledonous conversion make plant seed or leaf specifically constitutive expression external source mycocide in conjunction with polypeptide.

17. purposes according to claim 16, wherein expression cassette is changed over to bacterial isolates and be used to transform the recombinant clone of gained dicotyledonous or monocotyledons make plant seed or leaf specifically constitutive expression external source mycocide in conjunction with polypeptide.

18. the purposes of expression cassette according to claim 11 is as selective marker.

19. the purposes according to claim 17 or the 18 conversion plants that obtain is used to prepare mycocide in conjunction with polypeptide.

20. method that imports vegetable cell, callus, complete plant and plant protoplast conversion plant by the mycocide of will encoding in conjunction with the gene order of polypeptide.

21. method according to claim 20 wherein transforms by edaphic bacillus, Agrobacterium tumefaciems carry out particularly.

22. method according to claim 20 wherein transforms by electroporation and is undertaken.

23. method according to claim 20 wherein transforms by microprojectile bombardment methods and is undertaken.

24. mycocide is in conjunction with the production method of polypeptide, by express the gene of the such polypeptide of coding, isolated polypeptide then in plant or vegetable cell.

25. contain the plant of expression cassette according to claim 11, expression cassette is wherein given the mycocide tolerance.

26. plant according to claim 25 has tolerance to methyl methoxy imino--α-(O-tolyloxy)-O-tolyl acetic ester (BAS 490F).

27. the method for a controlling plant pathogenic fungi in transgenosis mycocide resistance crop plants comprises at forming the crop plants of mycocide in conjunction with polypeptide or antibody and uses mycocide.

28. mycocide that methyl methoxy imino--α-(O-tolyloxy)-O-tolyl acetic ester (BAS 490F) has high binding affinity in conjunction with polypeptide or antibody, is according to claim 24ly obtained preparation.

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