CN101473226B - Phage-display plant defense peptides directed against phakopsora pachyrhizi and uromyces appendiculatus - Google Patents

Abstract

A method for the identification of peptides having an affinity for the surface of fungi is provided as is a method for the identification of peptides capable of affecting the development of a fungus. Also provided are compositions comprising peptides identified using the method of the present invention. In addition, isolated polynucleotides, vectors, expression cassettes and transformed cells capable of expressing peptides identified by the method of the present invention are provided.

Description

Phage display plant defense peptides to yam bean layer rest fungus and wart top uromyce

Related application

The application is the part continuation application of the Patent Application No. 09/829,549 submitted April 10 calendar year 2001, and the right of priority of the U.S. Provisional Patent Application that requires to submit on April 10th, 2000 number 60/195,785.The rights and interests that No. the 60/704th, 933, the U.S. Provisional Patent Application that the application also requires to submit on August 2nd, 2005.All above-mentioned applications are introduced this paper with the mode of reference.

Sequence table

The application is with the sequence table of accurately reproducing the computer-readable form of sequence described herein.

Technical background

The disclosure relates to uses display technique of bacteriophage to differentiate the peptide that combines with the disease fungus class, the peptide that more specifically, belong to phytophthora (Phytophthora), layer rest fungus (Phakapsora) belongs to and the disease fungus class of monospore rest fungus (Uromyces) genus combines.Use degenerate oligonucleotide to make up peptide phage display storehouse at random.Bacteriophage at its surface expression peptide contacts with the fungi of different life stages, and separate, bacteriophage that amplification combines and differentiate peptide.In case identify, can screen peptide and be used to differentiate and be characterized in the binding site on the fungi based on antifungal activity.

Phytophthora is important economically disease-causing organism in the U.S., and it causes the massive losses of variety of crops important on a lot of farming.Soyabean phytophthora (Phytophthora sojae) is the second important pathogen (Doupnik, Plant Dis.77:1170-1171,1993) of U.S. soybean.Phytophthora blight of pepper (Phytophthora capsici) has widely, and host range also limits the production of the vegetable crop of high value, Solanum the most significantly.The control pathogen is difficulty especially, need handle whole zone with living killing compound usually.Though effectively, Environmental costs and the growing concern of financial cost to this disposal route needs other alternative control method.

Phytophthora class (Phytophthora) is to be adapted to the obligate parasite of long-term surviving in soil under the non-existent situation of host plant.Egg spore or chlamydospore low-density ground are present in the soil and pathogen can be survived.Be prone in the presence of the infected plant, pathogen is grown through a series of generation infection and growth step disease cycle, meticulous adjusting fast.Through release, encystation, rudiment and the infection of zoospore, the pathogen that is begun by egg spore or chlamydospore is grown and is seemed at first to have seemed directly and work as.But the process of life stage is the meticulous adjusting that receives ambient signal, and especially those come from the signal of host plant.

Zoospore is to diffuse to the most important life stage in root infection position.Main predisposing infection area is positioned at the apical meristem of root, and cell extends herein.Signal (the Carlile that the effluent that discharges from the cell that extends moves to this position chemotaxis as the guiding zoospore; InPhytophthora:Its Biology, Taxonomy, Ecology and Pathology (phytophthora: its biology, taxonomy, ecology and pathology); Erwin et al.; Eds., APS Press, 1983; Deacon and Donaldson, Mycol Res.97:1153-1171,1993).The chemotactic response of zoospore changes with the composition of root effluent and is species specific.For example, the zoospore of phytophthora blight of pepper, Phytophthora cactorum bacterium (P.cactorum) and other kind attracted to (Hickman, Phytopathology, 60:1128-1135,1970 in sugar and the amino acid whose array; Khew andZentmyer, Phytopathology, 63:1511-1517,1973), but the zoospore of soyabean phytophthora attracted to (Norris et al., Plant Physiol, 117:1171-1178,1998) in the specific isoflavone compounds.Though chemical attraction cutter reason really is unknown,, Deacon and Donaldson (Mycol.Res.; 97:1153-1171,1993) and Carlile (inPhytophthora:Its Biology, Taxonomy; Ecology and Pathology, Erwin et al., eds.; APS Press, 1983) summarized test, shown participation at the chemoreceptor on zoospore surface.

Zoospore the time is enclosed in the capsule near the root surface at the response environment signal.The packing of the zoospore of palm mould bacterium (P.palmivora) and other phytophthora class forms, and for example, possibly receive influence (Griffith et al., Arch.Microbiol, 149:565-571,1988 of local Ca ion concentration; Warburton and Deacon, Fungal Genetics Biol, 25:54-62,1998).Packing forms and also can cause by the chemical inhibitor of high concentration or by the root cell wall composition.For example, the zoospore of soyabean phytophthora is enclosed in (Morris and Ward, Physiol Mol Plant Pathol, 40:17-22,1992) in the capsule in the presence of the isoflavones compound of high concentration.Contrast therewith; Melon and fruit pythium spp (Pythium aphanidermatum) zoospore is enclosed in (Longman and Callow in the capsule when the fucosido with Chinese celery root cell surface contacts with the galactosyl residue; Physiol.Mol Plant Pathol, 30:139-150,1987; Estrada-Garcia et al., J.Exp.Bot.41:693-699,1990).Deacon and Donaldson (Mycol Res., 97:1153-1171,1993) point out that the packing in the presence of the high concentration attractant forms infecting potential is harmful to, and therefore in time passing and be not selected.Yet they advise that the decoy that can fully concentrate the root surface is to make zoospore tend to form packing behind contact root surface residue.

When contacting with root, zoospore forms packing with specific direction, so, formed germ tube towards the root direction.If zoospore with form packing before root contacts, germ tube can be gone up in any direction and form, and must redirect in order to locate root and to infect this plant.The acceptor of the cell surface on germ tube is considered to that the root orientation process is relevant therewith.For example, people such as Morris (Plant Physiol., 117:1171-1178,1998) have explained that the growth of the soyabean phytophthora young is to the low concentration of the isoflavone compounds that is derived from soyabean phytophthora, nontoxic orientation response.It is directed towards the mycelia of host's root direction that Zentmyer (Science, 133:1595-1596,1961) has reported camphor tree phytophthora (P.cinnamomi), but the characteristic of attractant compound is not had explanation.

After the infection, mycelia passes plant tissue at iuntercellular and/or Intracellular growth, and it depends on kind (Stossel et a1., Can.J.Bot., 58:2594-2601,1980 of pathogen; Coffeyand Wilson, in Phytophthora:Its Biology, Taxonomy, Ecology andPathology, Erwin et al., eds., APS Press, 1983; Enkerli et al., Can.J.Bot., 75:1493-1508,1997; Hardham and Mitchell, Fungal Gen.Biol., 24:252-284,1998; Murdoch and Hardham, Protoplasma, 201:180-193,1998).Through some phytophthora class, comprise phytophthora infestans (Coffey and Wilson, inPhytophthora:Its Biology, Taxonomy; Ecology and Pathology, Erwin et al., eds., APS Press; 1983), phytophthora blight of pepper (Jones et al., Phytopathology, 64:1084-1090,1974) and soyabean phytophthora (Stossel et al.; Can.J.Bot, 58:2594-2601,1980), form haustorium.Mycelia all forms with cell membrane with the host cell wall with haustorium and contacts closely.Though lack direct evidence, by inference, the acceptor of cell surface is very important for the perception plant signal.The circumstantial evidence of cell surface receptor comes to be observed such as divide vesicle (Coffey and Wilson, in Phytophthora:ItsBiology, the Taxonomy that occurs in the haustorium distal tip; Ecology and Pathology, Erwin et al., eds.; APS Press, 1983).Heath (Can.J.Bot., 73 (Suppl.): S131-S139,1995) has discussed the possible incident of the terminal growth of hypha,hyphae, comprises the communication through ion channel and vesicle function and surrounding environment.

As stated, fingerprint evidence is to the cell surface receptor behavior and the protrusion of growing step mould to the triggering epidemic disease.Therefore, cell surface receptor can provide pathogen growth and the infective method thus destroyed.Because the only limited time of zoospore locatees, contacts and penetrate with the effectively mobile infection site of the root end of growth, the delay or the destruction of therefore growing can have very big influence.This time restriction derives from the change of root tissue infectibility, because when the mature tissue in elongated region, it becomes and obviously is difficult for infected influence (English andMitchell, Phytopathology, 78:1478-1483,1988).

" merging bacteriophage " is thread bacteriophage carrier, and wherein, external peptide and albumen are cloned in the phage ghost gene and are represented as the part bacteriophage coat protein.The shell gene that bacteriophage is merged in generation commonly used is pVIII gene and pIII gene.The pVIII of about 3900 copies forms the major part of tubulose virus body protein shell.Each pVIII coat protein becomes low angle with the virion major axis, and its C-end is embedded near the inside of DNA and its N-end is exposed to external environment condition.5 copies of pIII coat protein are positioned at the terminal of each virion and participate in being attached on the colibacillary pIII bacteriophage and re-assemblying virus after infecting and duplicating.The peptide of showing as part pVIII is restricted in its matrix of on the virion shell, showing.On the contrary, because the terminal position of pIII albumen, the peptide of showing as part pIII has more pliability property.Specific bacteriophage can be configured to the peptide that is shown as 6 to 15 amino acid lengths.With at random or degenerate oligonucleotide insert coat protein gene and enable to produce the bacteriophage in display random peptide storehouse.The typical storehouse of showing comprises many as 10 ⁸10 to 100 copies of individual random series peptide.Therefore, phage display is helpful to the rare peptide that screening has required combination characteristic.

The random peptide library of phage display has been used to separate the part of the cell surface receptor on the mammalian cell.For example, from the phage display storehouse peptide is separated, this phage display storehouse with stride film and integrate plain glycoprotein and combine and relate to cell-extracellular matrix and cell-cell interaction (O ' Neil et al, Proteins, 14:509-515,1992; Smith et al., J Biol Chem., 269:32788-32795,1994; Healy et al., Biochemistry, 34:3948-3955,1995).The peptide of phage display is blocked the extracellular molecules of cell and definition and the adhesion of other cell (Koivunen et al., J Biol Chem., 268:20205-20210,1993 specifically; Koivunen et al., J Cell Biol, 124:373-380,1994; Healy et al., Biochemistry, 34:3948-3955,1995; Pasqualini et al., Nature Biotech., 15:542-547,1997).The random peptide library of phage display also has been used to select to distinguish the peptide (Pasqualini and Ruoslahti, Nature, 380:364-366,1996) of brain tissue and nephridial tissue.In vivo, the affinity selectivity of the peptide at random of phage display also has been used to select the peptide (Pasqualini et al., Nature Biotech., 15:542-547,1997) with the vascular endothelial cell selective binding of special tumor tissues.When these peptides and anticarcinogen merged and be injected into tumor-bearing mice, these peptides were successfully with the drug targeting tumor vessel and stop the tumour of progressive stage to grow (Arap etal., Science, 279:377-380,1998).

The phage display method only is applied to phytopathogen under condition of limited.The phage display method almost has been exclusively used in antibody (Susi et al., Phytopathology, 88:230-233,1998 of differential plant viral diagnosis; Ziegler et al., Phytopathology, 88:1302-1305,1998; Griep et al., J Plant Pathol., 105:147-156 1999; Tothet al., Phytopathology, 89:1015-1021,1999).Phage display is used to be chosen in single situation (the Gough et al. that the epitope that exposes with the surface on the young and the spore of disease epidemic disease epidemic disease mould (Phytophthora infestans) has the antibody of affinity; J.Immunol.Methods; 228:97-108,1999).Antibody fragment phage display, single chain variable fragment (Fv) to separating is estimated, because it possibly influence spore or young behavior.Antagonist is tested for sporangial antifungal activity, but does not find that it can detected antifungal activity.

Yam bean layer rest fungus (Phakopsora pachyrhizi) is to produce soybean (big shield beans, Glycinesmax) fungi of rust.This pathogen has propagated into all other soybean Production Regional in the world from the Asia.During autumn in 2004, yam bean layer rest fungus arrives the U.S..At present, in any soybean varieties, all there is not known durable resistance.Wart top uromyce (Uromycesappendiculatus) is to go up the fungi that produces rust beans (Kidney bean, Pheaseolus vulgaris).Breeding man is just making great efforts to discern the controlled gene that is made as rust resistance in the soybean.Yam bean layer rest fungus occurred owing to having reported in Brazil, produced the fungi of soybean rust, the U.S. soybean producer has reckoned with its arrival.The yam bean layer rest fungus that arrives the U.S. in the last autumn this expectation that terminated, and the farmer must possibly annual situation about occurring react to this new disease at present.Farmer's worry is based on when not having the successful implementation control measure, in the world the report of the loss in other zone 10% to 80%.

Because also do not find lasting, the natural resistance of rust after surpassing 18,000 U.S. soybean kinds having tested, the soybean producer worrying, and, pathogen, yam bean layer rest fungus possibly infect any cultigen of output potentially.In the expection that the rust pathogen is arrived, carried out a large amount of research with definite effective fungicide, and obtained to be used for the urgent governmental approval of soybean.Traditional screening and breeding method have been determined does not have main resistant gene to above-mentioned pathogen, and the situation of wart top uromyce (U.appendiculatus) and yam bean layer rest fungus especially.

Antifungal agent possibly be defence front for many years, before the resistance of new resistant gene or other form is determined.

Traditionally, antifungal agent is also of no use produces most soybean.Therefore, has only finite information about the cost of this disease control practice and possible economic vitality thereof.In the Missouri State and other state, these problems have caused for the uncertain estimation of area that can soybean be replaced with alternative crops.

In order to protect the soybean farmer and to guarantee that product satisfies the needs of country, must develop alternative antifungal agent as early as possible.The disclosure has proposed the novel rust resistance based on biotechnology.This technology comprises the develop and field of defense peptides in the genetically modified plants, for example soybean.This technology has influenced key areas beans (Kidney bean) rust pathogen, wart top uromyce equally.

As the fungi of inducing rust, wart top uromyce and yam bean Phakopsora rest fungus (Uredinales) order in basidiomycetes (Basidiomycetes) guiding principle.Wart top uromyce produces 5 spore stages on single host plant.Yam bean layer rest fungus mainly duplicated through the uredospore on the single host plant (uredospores).Uredospore is responsible for propagating fast fungi.Except soybean, yam bean layer rest fungus can infect many beans.

The uredospore of wart top uromyce penetrates through the gas opening of leaf.The difference of yam bean layer rest fungus is that the uredospore that germinates directly penetrates through the leaf cuticular cellulose.Usually, the uredospore germination that drops on leaf surfaces is to produce the infection pad (appresorium) attached to the surface.In these two types, appresorium all produces the hyphal peg that penetrates plant.After penetrating, each fungi development becomes the linear structure (mycelia) in the iuntercellular growth through leaf texture.Mycelia gets into host cell under the situation of not killing host cell.There, they form spherical structure (haustorium), from the leaf cell that lives, draw nutrition.Soon, each fungi forms the uredium that produces extra spore after the infection.

The phage display storehouse of combination provides a large amount of arrays of peptide at random, and selects the part (O ' Neil et al., 1992) to protein of interest matter by it.Phage display peptide library is filobactivirus clone's a potpourri, its each on virosomal surface, show single external peptide sequence (Cwirla, 1990; Scott and Smith, 1990).The peptide of showing and its coding DNA physical connection in this phage genome.Therefore, peptide can easily and apace be transferred to other carrier system or display systems by discriminated union.Typical storehouse comprises 10 ⁹Plant peptide variant at random.

Random peptide library can be used for separating for the important part of the cell surface molecule of mammalian cell.For example, eluriate, and stride the film glucoprotein, integrate element, have the peptide of affinity from the storehouse, to be isolated (O.Neil et al., 1992 through carry out biology to the molecule of purifying; Smith et al., 1994; Healy et al., 1995).Some peptide comes to light and blocks cell and the extracellular molecules of definition and the adhesion of other cell.The combination of peptide and the validity of inhibition are special (Koivunen et al., 1993 to the peptide sequence motif; Koivunen et al., 1994; Healy et al., 1995; Pasqualini et al., 1995), and the validity of selecting is special (Pasqualini and Ruoslahti, 1996) to the certain organs tissue.

Therefore, the quick and effective method that needs the peptide of screening and phytopathogen specific bond.In case identify, peptide can further be estimated the ability of its prevention pathogenic infection plant, and; Suitable peptide can directly be used for plant, be used to handle soil; Perhaps, randomly, can with in the sequence introduced plant of encoded peptide so that plant has immunity or resistance to pathogen.In the case, can develop economy and the environmentally safe effective method of control phytopathogen.

Summary of the invention

The present invention has overcome the problems referred to above and through the art technology that resistance development is provided to the pathogen in the plant.On the one hand, make soybean resistance arranged to the soybean rust that does not have durable resistance at present.In another example, identical common technology can provide the Kidney bean that common rust is had resistance.

In one example, differentiate that the surface of phytopathogen is had the method for the peptide of affinity.In the method, made up through the degenerate oligonucleotide that encoded peptide is provided and to have comprised the storehouse of peptide at random.This oligonucleotides is inserted in the suitable carriers, and said carrier also can transfection host cell at the peptide of its surface expression coding.The carrier of host cell infectious form that increases with the carrier transfection is to produce the peptide storehouse on carrier.Then the carrier in expression of peptides storehouse is contacted with the target pathogen and it is combined with pathogen.Remove the carrier that unconjugated carrier and wash-out combine with pathogen.Then the carrier of wash-out is increased in proper host cell and separate the oligonucleotides that inserts.Then through any suitable method to this oligonucleotide sequencing, and, derive the amino acid sequence of this peptide from this oligonucleotide sequence.

Relating in one aspect to of means of the present disclosure as the peptide of novel soybean and beans resistance factor and the method for selection thereof.Under the situation that need not to know specific pathogenic target in the pathogen, peptide can be differentiated and selected easily.Because in fact these peptides are not must be present in crude soya bean or the beans, do not expose wherein before the pathogen.Therefore, the validity that is used for the peptide of soybean and beans can extend to various pathogen populations, and its effectiveness is likely durable.

The classic method that the phage display peptide is selected is based on the elutriation to the storehouse of special interested purifying molecule, like Barbas et al., and 2001 described instances.The difference of we's science of law and prior art is to screen the purifying target molecule that need not to know in advance particular target or high concentration to the storehouse of full cell.

In aspect described one, when showing as the part of scafffold proteins, the micromolecule that is called peptide can provide rust resistance in plant transformed.In one example, can be according to means disclosed herein, select these peptides through binding affinity to the infectiousness structure of the young (spore that promptly germinates) of wart top uromyce and yam bean layer rest fungus.This binding affinity can suppress the further growth of spore and cause a disease.Therefore, peptide shows as and suppresses causing a disease of these fungies.In concrete instance, cytokinin oxidase can be modified as the scafffold proteins of showing selected peptide in the plant.

Through after a while, the pathogen population can adapt to selected existence of giving the peptide of resistance.Yet,, can select new defense peptides to change the challenge of pathogen population with reply fast if the selection of peptide and screening are easily.It is quick and simple that the concrete advantage of disclosure means comprises that peptide selection method and phenotype are estimated; The selection of peptide need not be known the pathogenic target in the pathogen; The effectively recovery of the high percent of peptide; Differentiate fast the ability of required new defense peptides, the ability that the framing scaffold peptide is used for the ability of susceptible plants tissue and modifies required framing scaffold peptidic constructs fast.

Antifungal composition is provided on the other hand; Said composition comprises at least a peptide, and it is selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ ID NO:24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ ID NO:4).

Be recombinant nucleotide on the other hand; It comprises the sequence of encoded peptide, and said peptide is selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ ID NO:24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ ID NO:4).

Be recombinant vector on the other hand; It comprises the nucleotide sequence of encoded peptide, and said peptide is selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ ID NO:24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ ID NO:4).

Also having on the one hand is to use the carrier cell transformed; Said carrier comprises the nucleotide sequence of encoded peptide, and said peptide is selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ IDNO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ IDNO:24), EFRKNYPSAAPLIPR (SEQ ID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ IDNO:4).

Expression cassette is provided on the other hand, and it comprises as the component that is operably connected, promoter; The nucleotide sequence of encoded peptide, said peptide are selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ ID NO:24), EFRKNYPSAAPLIPR (SEQID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ ID NO:4); And transcription stop signals sequence.

Other aspect provides the recombinant that comprises expression cassette plant, and this expression is closed and comprised promoter; The nucleotide sequence of encoded peptide, said peptide are selected from ADPPRTVST (SEQ ID NO:7), ADRPSMSPT (SEQ ID NO:8), ADITDPMGA (SEQ ID NO:20), AVGTHTPDS (SEQ ID NO:21), AVSPNVHDG (SEQ ID NO:22), LTRCLVSTEMAARRP (SEQ ID NO:24), EFRKNYPSAAPLIPR (SEQID NO:31), LFXCYPPCTYSYCLS (SEQ ID NO:33) and AAPDLQDAM (SEQ ID NO:4); And transcription stop signals sequence.

The characterizing method that the pathogenic surface is had the peptide of affinity is provided on the other hand, and this method comprises: random peptide library is provided, and process through the degenerate oligonucleotide that encoded peptide is provided in this peptide storehouse; Oligonucleotides is inserted in the suitable carriers, and said carrier makes host cell at said peptide of its surface expression and transfection; And with carrier transfection proper host cell the carrier of the infectious form that increases on carrier, to produce the peptide storehouse.

Then the carrier in expression of peptides storehouse is contacted with interested phytopathogen and it is combined with this pathogen.In conjunction with after, remove unconjugated carrier and with the carrier that combines from this pathogen wash-out.The carrier of wash-out is increased in proper host cell and is separated in the said oligonucleotides that inserts in the carrier of wash-out.Then, the peptide of being encoded by this oligonucleotides is produced out, contacts with interested phytopathogen, and is observed communicable influence.In one embodiment, to the oligonucleotide sequencing of this separation, derive the amino acid sequence of said peptide from said nucleotide sequence, and said peptide prepares through chemosynthesis.In another embodiment, prepare peptide through the oligonucleotides that separates being inserted the expression vector that is used to transform the suitable host cell.Then transformed host cells is remained under the condition of the expression that is suitable for peptide.

In order to reclaim the peptide that combines with the groupy phase of uncertain surface protein, also to full cell screening combinatorial libraries.Can be to the peptide of interested phenotype evaluation through this screening recovery.For example, we have selected to have with the active zoospore of mould phytopathogen (epidemic disease is mould) peptide of affinity.Phenotypic screen has produced through inducing immature packing to destroy the subclass (Bishop-Hurley et al., 2002) of the peptide of spore normal development.We have also shown, when being shown as the phage display form or when as free molecule and when synthetic, the peptide that affinity is selected destroys zoospore and grows (Laskey et al., 2001; Bishop-Hurley et al., 2002).

Brief Description Of Drawings

These and further feature, aspect and advantage will become through following explanation, appended claim and accompanying drawing and be more readily understood.Wherein:

Fig. 1 shows the composition of the f8 peptide sequence that is divided into 6 families.The left side is a dendrogram, and the right is the title and the sequence of peptide.

Fig. 2 shows the encystation effect that the response of phytophthora blight of pepper zoospore contacts with the said f8 phage display peptide of given variable concentrations.Percent value is represented the mean value of two tests.The encystation number percent that does not comprise the contrast zoospore population of bacteriophage changes between 0 to 10%.

Fig. 3 shows the encystation effect that the response of phytophthora blight of pepper zoospore contacts with the said f8 and the f88-4 phage display peptide of given variable concentrations.

Fig. 4 shows combining of said phage display peptide and phytophthora blight of pepper zoospore.The mean value of 3 tests of numeric representation.

Fig. 5 shows the specificity that said phage display peptide combines with phytophthora blight of pepper.The mean value of 3 tests of numeric representation.

Fig. 6 is the collection of illustrative plates of plasmid pJE-7.AOX-P is an alcohol oxidase promoter, Mat-α. be the secretion sequence of mat-α, CKX1 is cytokinin oxidase 1 sequence, and Pc87 is the peptide of example, and AOX-TT is the alcohol oxidase terminator sequence.Below the figure for coding Pc87, demonstration (+) chain (5 ' AG CTA GCA GAT AGA CCA TCA ATG TCA CCA ACATAG T 3 '; SEQ ID NO:46) and the double-stranded sequence of (-) chain (5 ' CT AGA CTA TGT TGG TGA CATTGA TGG TCT ATC TGC T 3 ', SEQ ID NO:47).

Fig. 7 shows the amino acid sequence (SEQ ID NO:48) that is contained in the example embolus among the pJE-7; Wherein, Underlined nucleotide is mat-α secretion sequence (fracture by the Kex2 enzyme appears at last underlined spermine acid), then is cytokinin oxidase 1 sequence.Two amino acid (KL) that add double underline are added with the assisting building fused protein.The amino acid sequence of the peptide Pc87 of example shows with boldface type.

Fig. 8 is the method flow diagram that shows a kind of method of differential plant defense peptides, and said peptide can be used for transforming plant and gives resistance to the rust pathogen.

Fig. 9 shows near the encystation effect (left side) of the zoospore that the root apex zone of conversion is and near the encystation binding mode (right side) of the normal zoospore the wild type tip of a root, and the cytokinin oxidase (CKX) of the peptide that affinity is selected is showed in the root secretion of this conversion.

Figure 10 shows the inhibiting effect (left side) of phage display peptide clone 19 (SEQ ID NO:50, the tables 3) of affinity selection to the growth of the wart top uromyce young, and normal uredospore germinates and growth (right side) in the presence of non-selected phage library.

Definition

" secretion sequence " be meant guide new synthetic secretory protein or memebrane protein to and through the cell membrane of endoplasmic reticulum or through the bacterium inner membrance from tenuigenin to pericentral siphon or from mitochondrial matrix to inner space or from the sequence of chloroplast stroma to thylakoid.This sequence merges the gene of expressing to the heterologous host and has guaranteed the secretion of recombinant protein matter from host cell.

" young " is meant the packing (germination back 5-8 hour) of the new germination with abiogenous germ tube

" TBS " is meant Tris BS (50mM Tris-HCl (Tri(Hydroxymethyl) Amino Methane Hydrochloride), pH 7.5,150mM NaCl).

" recombination of polynucleotide " is meant polynucleotide, and it does not contain one or whole two nucleotide sequences that polynucleotide flank in the organism genome of natural generation is connected, and said polynucleotide are derived from said organism genome.This term comprises; For example; Polynucleotide and the fragment thereof of incorporating plasmid or virus, prokaryotes or the Eukaryotic genomic DNA of carrier or expression cassette, self-replicating into, or conduct is independent of the polynucleotide and the fragment thereof of the individual molecule existence of other polynucleotide.It also comprises the recombination of polynucleotide as the part of hybridization polynucleotide, for example, and the recombination of polynucleotide of coded polypeptide sequence.

" IPTG " is isopropylthiogalactoside.

" TU " is meant transduced unit.

" NAP damping fluid " is 80mM NaCl, 50mM NH ₄H ₂PO ₄, and use NH ₄It is 7.0 that OH regulates pH.

" NZY-Tc " comprises 1%NZ amine A (typtone type nutrient solution; Humko-SheffieldChemical, Norwich, N.Y.), to regulate pH be 7.0 bacterial growth nutrient solution for 0.5% yeast extract, 0.5%NaCl, use NaOH.

" PCR " is meant the PCR.

" polynucleotide " here used and " oligonucleotides " interchangeable use and refer to the nucleotide of random length, ribonucleotide or deoxyribonucleotide, (2 or a plurality of monomer) form of polymerization.Though nucleotide combines through phosphodiester bond usually, said term also comprises the polynucleotide that contains the neutral amide backbone key of being made up of the amino-ethyl glycine unit.Said term only refers to the primary structure of molecule.Therefore, this term comprises the DNA and the RNA of double-stranded and strand.

It also comprises the modification of known type, and for example, mark methylates; " cap " replaces the nucleotide of one or more natural generations with analog, modifies between nucleotide, for example; Have the modification (for example, methyl phosphorodithioate, phosphotriester, phosphoramidate, carbamate etc.) of uncharged key, comprise the modification of suspended portion, (comprise like protein; For example, nuclease, toxin, antibody, signal peptide, poly-L-Lysine etc.), have the modification (for example acridine, psoralen etc.) of intercalator; Contain the modification (for example, metal, radioactive metal, boron, oxidisability metal etc.) of sequestrant, contain the modification of alkylating agent; Modification (for example, different nucleic acid of α etc.) with modifier keys, and the form of polynucleotide unmodified.Polynucleotide not only comprise sense strand but also comprise antisense strand.

" sequence " is meant the linear precedence that monomer occurs in polymkeric substance, for example, and order or nucleotide the order in polynucleotide of amino acid in polypeptide.

" peptide ", " protein " are with " polypeptide " interchangeable use and be meant by two or more amino acid that are connected through the peptide bond mode and form compound.

" recombinant protein matter " be meant, no matter comprise the natural still one-level amino acid sequence of variation, the protein that the gene that carries through recombinant DNA molecules in the express cell obtains, this cell are different from gene and/or protein by the cell of natural discovery.In other words, the host of said gene and its expression is an allos.Should be understood that any change of gene, comprise the polynucleotide that add coding affinity purifying fragment, make this gene become non-natural based on the purpose of this definition, therefore, this gene can not be in any cell " natively " find.

" NIg peptide " is meant the peptide that is not immunoglobulin (Ig), generally acknowledged immunoglobulin (Ig) zone or comprises the zone of immunoglobulin (Ig).For example, the strand variable region of immunoglobulin (Ig) is excluded outside this definition.

" pure basically " or " basic purifying " is meant that material does not contain the protein of other pollution, nucleic acid and other is derived from the biomaterial in organism source.Purity can be measured through standard method; And common be at least about 40% pure, more common be at least about 50% pure, be generally at least about 60% pure, be more typically at least about 70% pure, often be at least about 75% pure, more often be at least about 80% pure, typically be at least about 85% pure; More typically be at least about 90% pure, be preferably at least about 95% pure, more preferably about 98% pure, and pure at least 99% in preferred embodiment also.Analysis can be weight or molar percentage, through evaluations such as example gel decoration method, AAS or end-labellings.

Detailed Description Of The Invention

Below provide detailed description to put into practice the inventive method to help those skilled in the art.Even so, this detailed description should not be construed as excessive restriction the present invention, because under the situation of the spirit of the discovery that does not depart from property of the present invention and category, those of ordinary skills can make in the embodiment of this discussion and revise and change.

All publications of quoting in this application, patent, patented claim, database and other be with reference to all introducing in this mode with reference, as each independent publication, patent, patented claim, database and other with reference to specifically and individually being expressed as through with reference to introducing.

The increasing concern yam bean of soybean producer layer rest fungus, the cause of soybean rust, the Production Regional and the propagation that will get into the North America in the near future.This concern results from the world some zone and surpasses the estimation of 50% crop loss.Increase along with the rust fungi of Brazil's discovery more than north latitude 5 degree recently for this sick concern.Probably in the near future, the spore of yam bean layer rest fungus will be brought into the U.S. (Rizvi, 2004) through north wind in vogue.Because there is not known to the effective disease resistance gene of rust, present disease control plan to rely on the application of the protection germifuge of repetition.Below the disclosure other selection is provided, uses new peptide technology to differentiate and send the defense peptides that combines with the infectiousness structure of yam bean layer rest fungus and relevant rust pathogen and develop to stop rust.Defense peptides can be designed in plant, express and be delivered to pathogenic infection and the position of trooping.Plant, for example soybean and broad bean can be designed to express these peptides to produce the plant system that rust is had unique resistance.

Therefore, the scheme of exploitation is differentiated the peptide that combines with the communicable yam bean layer rest fungus spore and the young, estimates the validity that the peptide that combines suppresses spore-germination and conk, the resistance potentiality of candidate's defense peptides in introducing and the test plants.

The rust pathogen produces spore (uredospore), infects leaf, stem and the seed skin of soybean.These sporocyst winds are moved and are dropped on the plant, and they germinate on this plant and penetrate the germ tube (being defined as juvenile stage) of plant tissue with generation.After penetrating, fungi further grows (Bonde etc., 1976 through producing the filamentous growth that in being organized in iuntercellular and cell, spreads; Koch and Hoppe, 1988).Through after a while, tissue is trooped and is caused the generation of other infectiousness spore, and these spores discharge in wind and move to other plant.

Though imagination some resistant genes to soybean rust, major part is in the open air under the condition or failed in the greenhouse inoculation test.In case pathogen arrives new geographic area, the raiser faces the situation that in fact in commercially available soybean varieties or improved line, does not have available rust resistance completely.For foreknowable future, the raiser need each season of growth period property use germifuge.

The use routine techniques is found new resistant gene and this gene is bonded to a lot of years of expection needs in the commercially available soybean line in soybean or its close relative plant.Following open this process of advantageously having accelerated.Its possibility is from present peptide is technological, Plant Transformation is technological and development of molecular biology obtains, and these technical combinations are used for the disclosed development of this paper.The peptide technology of combination can filter out and the infectiousness structure knot of phytopathogen is incorporated in before the plant infection or destroys the peptide of its growth between infection period a large amount of, diversified peptide set.The instrument of molecular biology and Plant Transformation can be structured in the delivery system of these defense peptides of infection site expression of plant.

Defense peptides may be defined as the infectiousness structure with the plant and animal pathogen, and for example spore combines and destroys the peptide of this pathogen normal development.In conjunction with and to destroy or stop the peptide of pathogen function be to grow very important pathogen surface protein and obtain this effect regulating through combining.The surface protein that is elected to be this purposes can be, for example, cell membrane forms and the required enzyme of plant growth, or possibly, controls the protein to the picked-up of the requisite mineral of growing.Method disclosed herein, exemplarily explanation can effectively be screened to comprising 1,000,000,000 or the pathogen of the multiple peptide set of more combinations of peptide at random.Said technology is applied to defeat yam bean layer rest fungus and related diseases substance especially.

Mode through following instance will show how to differentiate and infectiousness yam bean layer rest fungus spore and the young and the relevant or similar peptide that combines of the spore of pathogen.The set and the affirmation that produce peptide combine with the infectiousness spore or the germ tube (young) of pathogen.In order to differentiate the peptide of combination, with spore or the young and the set of peptide at random, be commonly referred to the storehouse, mix mutually, this at random peptide show that on bacteriophage (virus) particle for example, like Wilson etc., 1998 is said.Through the method, exemplary illustration, the spore and the young can be exposed to 1,000,000,000 at random in the peptide.A variety of widely suitable peptide storehouses are well known in the art.In some storehouse, peptide only comprises 8 amino acid, and in other storehouse, each peptide comprises 15 amino acid.

After the exposure that mixes and suitably cultivate, washing can be removed the bonding unstable peptide of all and spore and the young.Can handle the peptide that the said spore and the young adhere to release, increase its quantity, and repeat screening process.After several screening steps repeatedly, wherein, each step has been confirmed the binding affinity of binding peptide in the last step, reclaims the bonding very firm a variety of peptides of confirming as with protein and spore and germ tube surface of other component.In an example, 3 or 4 step sizing steps are enough to provide this result's.

As long as the binding affinity of peptide such as above-mentioned method are confirmed, just can estimate the validity that binding peptide suppresses spore-germination and conk.In case a lot of peptides are differentiated out, just can select the influential peptide of pathogen function.What cherish a special interest is the peptide that stops spore-germination and young growth.Any of these two kinds of destructive behaviors all will cause the pathogenic infection and the disease that stop or reduce.That high throughput assay can be used for detecting is mould in epidemic disease, stop in yam bean layer rest fungus or the similar pathogen or a large amount of peptide of the ability of slow down spore-germination or plant growth.Experience shows that the peptide of the test of significant proportion possibly grow influential to pathogen.

In case identify defense peptides, under not by the situation of the enzymatic degradation of plant, it be delivered to endophytic infection place.Obtain this a kind of method of sending and be to be attached to by natural plant said peptide on that produce and the protein that in the tissue of being trooped, produce by pathogen.In an example, cytokinin oxidase successfully is used for this purpose.This protein is produced by natural plant, and the adjusting of its involved in plant growth hormone (basic element of cell division).Defense peptides is attachable on this protein, and is used to change the pathogen function.In an example, this realizes in epidemic disease is mould.

Defense peptides can with close by natural secretion of cell in cytokinin oxidase and other protein blend in space between cells, wherein they can contact with the rust pathogen of trooping.Perhaps, this fusion can comprise the protein that belongs to the vegetable cell wall fraction.These protein also are placed in iuntercellular and the cell and the synergistic position of pathogen.Commercially available protein group database is that the candidate's peptide carrier protein that can be used as the CKX substitute provides diversified gene order.Can use the structure of these carrier proteins of conventional structure protein group Algorithm Analysis of the optimal representation that is used for defense peptides or displaying.

Protein-peptidic constructs of candidate can at first be expressed in yeast, Pichia pastoris and secretion.This host promotes to concentrate or protein purification or polypeptide, and tests their inhibiting effect to pathogen spore-germination or young growth.

As long as the best pattern of having confirmed best defense peptides and on carrier protein, having showed just can confirm plant, in soybean or broad bean, form genetic constructs.The Plant Transformation of foreign gene and the method for expression are well known in the art.

Pernicious especially and pathogen danger like yam bean layer rest fungus, are limited by government regulation.

Through utilizing initial peptide selection scheme to adopt the substitute of rust pathogen or the complicacy that analog can be avoided this danger and regulations.Hollier and King, 1985 have reported the fungal pathogens that causes the southern corn rust, pile handle rest fungus (Puccinia polysora) more.This pathogen not restriction obtains easily, and can under the condition of code test chamber, operate.

In one embodiment, the nucleotide sequence of the peptide through 6 to 15 amino acid lengths of will encoding is inserted into and makes up the peptide storehouse in the suitable carriers, although can be used for the sequence of longer encoded peptide.By the peptide of nucleotide coding in fact can be completely random or can limit it and form to satisfy the needs on the structure or on the function.For example, rather than restriction, the halfcystine bridge can insert in the peptide.In one embodiment, the immunoglobulin domain that nucleotide sequence is not encoded immunoglobulin (Ig) (antibody) or generally acknowledged is like the variable region.Can use arbitrary carrier of expressing the oligonucleotides that inserts.Preferred use cause cell or virus surface or in the cell of cell or virus the carrier in compartment or organelle surface expression peptide storehouse.In the case, the peptide of expression can be used for and potential target molecule or cell interaction, and said molecule or cell contact with the surface that comprises said peptide.As conspicuous to those of ordinary skills; If the surface expression of peptide compartment or organelle in cell; Said potential target also must be present in the cell, and perhaps compartment in organelle or the cell must be exposed to external environment condition through the dissolving such as cell.

The method that produces oligonucleotides and be inserted into carrier is known to those of ordinary skills, and only in this brief overview.The most common ground, tris phosphite method synthetic oligonucleotide (Tetrahedron Lett.22:1859-1862,1981 on solid support of using Beaucage and Caruthers; Simultaneously referring to U.S. Patent number 4,973,679 and 4,458,066).A large amount of solid supports can be used, and comprises controlled hole beaded glass, polystyrene copolymer, silica gel and cellulose paper.The preparation of oligonucleotides start from 3 of first nucleotide '-bonding of oh group and solid support.The solid support that comprises nucleotide can get from commercial source.Oligonucleotides from 3 ' position to 5 ' position is synthetic, and, chain through soluble 5 '-on protection 3 ' phosphate structural unit, activation or the phosphoramidite fixing oligonucleotides 5 '-nucleophillic attack of hydroxyl prolongs.And then the intermedium dinucleotide phosphite ester that forms must be oxidized to more stable phosphate before chain elongation.Repeat this process up to the nucleotide that has added requirement.There is commercially available automation equipment to come synthetic oligonucleotide.In addition, a lot of trade companies provide the customization oligonucleotides synthetic service.

The carrier system of any peptide in can the expression of peptides storehouse all can be used in the practice here, and a lot of carrier system be known in the art (referring to, Wilson and Findlay for example, Can.J Microbiol, 44:313-329,1998).When peptide was showed as part pVIII, suitable bacteriophage system comprised 8 types, 88 types and 8+8 type.When using pVIII, suitable bacteriophage system comprises 3 types, 33 types and 3+3 type.When peptide was inserted among the pVI, suitable bacteriophage system comprised 6 types, 66 types and 6+6 type.In addition, can use phage t7 and bacteriophage T8 carrier system.In a preferred embodiment, the peptide in the library is expressed as the coat protein fusion with the trichobacteria bacteriophage, so that said peptide is expressed also and can be interacted with target molecule or cell surface receptor in virosomal surface.In a preferred embodiment, used the f8-1 library, wherein, the coat protein of 8-mer peptide and pVIII merges at random.In another preferred embodiment, use the f88-4 library, wherein, the coat protein of 15-mer peptide and pVIII merges at random.Phage display in the f88-4 library have no amino acid to have the peptide of preference.Bacteriophage in the f8-1 library is no preference, except at one of primary alanine and deputy four residues.All other positions are all taken by arbitrary amino acid.

The method of producing the f8-1 phage display peptide library formerly describe (referring to, Petrenko etc., Prot.Engineering, 9:797-801,1996 and the list of references wherein quoted).Heterologous peptides is showed in said library on each copy of 3900 copies of main coat protein pVIII.The expression of peptide need not bringing out by IPTG.The degenerate oligonucleotide that is used for the 8-aminoacid insertion is: GCA GNN (NNN) ₇, wherein N is arbitrary nucleotide.Therefore, first amino acid is that the alanine (A) and second amino acid are valine (V), alanine (A), aspartic acid (D), glutamic acid (E) or glycocoll (G).Remaining amino acid completely random is arranged in the peptide.

The method of likewise, producing the f88-4 phage display peptide library is also formerly described (Zhong etc., J.Biol.Chem.269:24183-24188,1994; Smith and Scott, Methods inEnzymology, 217:228-257,1993; Smith, Gene, 128:1-2,1993 and the list of references wherein quoted).15-amino acid heterologous peptides is showed in this library on 150 to 300 copies of main coat protein pVIII.The remainder of 3900 copies of pVIII subunit is derived from wild type pVIII.Therefore, this phage genome has two kinds of pVIII genes of two kinds of dissimilar pVIII molecules of coding.A kind of pVIII is a recombinant of showing allos 15-mer peptide, and another kind is the wild type pVIII that is present in usually on the bacteriophage.The expression of reorganization pVIII is driven by IPTG induction type tac promoter/operon.Because the existence of two kinds of pVIII genes, said f88 virion is made up of the wild type and the form of inlaying of reorganization pVIII subunit.

The oligonucleotide sequence that is used for 15-mer aminoacid insertion thing is (NNK) ₁₅, wherein N is that A, T, C or G and K represent G or T.Therefore, the zone around this 15-mer aminoacid insertion thing is: LVPMLSFA (X) ₁₅PAEGDDPAKA (SEQ ID NO:1), wherein X is by any amino acid of codon NNK coding.

Phage particle can be used for screening the peptide at random of on virion, expressing based on the binding ability of itself and compound of interest and cell.In a preferred embodiment, phage display peptide library is used to screen the peptide that combines with phytopathogen.In another preferred embodiment, the binding ability of peptide and disease fungus class is screened.Also have another preferred embodiment, phage display peptide and phytophthora are belonged to the ability that the member combines screen.When research during more than the pathogen of single life stage, preferably study each life stage because along with the change of developing stage, quantity, type and with the affinity of binding site on significant difference possibly appear.

For example, when studying the member of phytophthora genus, with about 10 ⁵To 10 ⁶Organism and about 10 ⁸To 10 ⁹The phage display peptide mix mutually, and hatch one period that enough makes its combination.Conspicuous for those of ordinary skills, depend on factor such as the kind of pathogen, bacteriophage and peptide, can use other concentration of organism and displayed polypeptide according to the disclosure.Under some situation, the preincubate displayed polypeptide needs with identical organic other life stage, and purpose is to differentiate the peptide that only combines with specific life stage.After hatching, organism is through repeatedly wash the peptide that combines to remove unconjugated and insecurely.To the situation of the mould zoospore of epidemic disease, use the LiCl solution of about 50mM to wash.After the washing, the phage display peptide of elution of bound, the bacteriophage of amplification wash-out preferably under low pH, and in suitable hosts.In one embodiment, the host is hungry K91 Escherichia coli.The method of amplification bacteriophage is being known in the art and is being found in Escherichia coli, for example, and Smith and Scott, Methods in Enzymology, 217:228-257,1993; Ausubel et al.eds., Short Protocols in Molecular Biology, 2nd ed., Wiley&Sons, 1995; And Sambrook et al., Molecular Cloning, Cold SpringHarbor Laboratory Press, 1989.In one embodiment, at least once repeat screening process so that the phage display peptide enrichment of high-affinity.In another embodiment, repeat screening process 3 times.

In case identify the bacteriophage that the peptide of high-affinity is showed; This bacteriophage of increasing, preferably in Escherichia coli, and standard method is used in the separation of this phage DNA; The method of for example in following document, seeing: Smith and Scott; Methods in Enzymology, 217:228-257,1993; Ausubel et al.eds., Short Protocols in Molecular Biology, 2nd ed., Wiley & Sons, 1995; And Sambrook et al., Molecular Cloning, 2nd ed., Cold SpringHarbor Laboratory Press, 1989.In case isolate phage DNA, use with the identical restriction enzyme that is used to insert oligonucleotides with the oligonucleotides that inserts from dna cleavage, the fragment of restriction enzyme is then separated from the residue of DNA.Then, can use any standard method to oligonucleotide sequencing.Order-checking can be carried out through any suitable method, for example, and dideoxy sequencing method (Sanger et al.; Proc.Natl.Acad.Sci.USA, 74:5463-5467,1977), Maxam-Gilbert method (Maxam and Gilbert; Proc.Natl.Acad.Sci.USA; 74:560-564,1977) or its any change, comprise the use automatic sequencing.In one embodiment, and use ABI Prism 377 type automatic sequencers (Applied Biosystems, Foster City, Calif.).In case known the sequence of oligonucleotides, used genetic code can easily derive the amino acid sequence of the peptide of coding.

The phage display peptide of high-affinity can further screen with regard to its growth, growth and/or communicable ability that changes pathogen.In this embodiment, hatch one period that it is combined of phage display peptide and target pathogen.In conjunction with after, can be observed the change of pathogen on the ability of its growth or infection host.In one embodiment, the zoospore that belongs among the member of about 200 phytophthoras combines with the distilled water pnagus medius displayed polypeptide with the serial dilution of constant volume twice in the double dish.The concentration range of bacteriophage can change, but usually 1 to 10 * 10 ⁹Between virion/μ l.In each screening step, all comprise the negative control that does not comprise bacteriophage.At room temperature, be generally after 20 minutes the incubation time, be determined at the quantity of the zoospore that encysts under each bacteriophage concentration.Use the method can reasonably select to define the peptide of character and its kind or inducing of the distinctive receptor-mediated functional response of life stage are estimated the encystation of this functional response such as zoospore.The peptide of the growth of disturbing pathogen of coming to light can be used for preventing or limits the infection of this pathogen to the host.

This method also can be used for being characterized in the peptide of bind receptor on the pathogenic surface.In this embodiment, the peptide that is labeled shows that bacteriophage (test bacteriophage) is with different organic and cultivate at the cell of different developmental phases.Then, the RA of the bacteriophage of mark can combine to measure with the Scatchard analysis through competition.In the competition binding analysis, the test bacteriophage of constant density is combined with the target pathogen, add the unlabelled stimulation bacteriophage under the finite concentration scope then.This stimulation bacteriophage can be identical or different with the test bacteriophage.Wash the target pathogen then removing the bacteriophage that combines specifically non-or insecurely, and measure the amount of the test bacteriophage of combination through the amount that measurement is present in the mark on the target cell.The degree of competition can be passed through the combination (IC of the test bacteriophage of needs inhibition 50% ₅₀) the concentration of stimulation bacteriophage test.The result of competition test can be used for measuring the variation of cell surface receptor quantity, type and affinity in time.

The reorganization oligonucleotides is in the scope of disclosed means, and it comes to light through method described herein, and coding has the peptide of antifungal activity.These reorganization oligonucleotides can be used for producing the recombination of polynucleotide that is used as clone or expression vector usually, although other purposes is possible.Cloning vector is the dna molecular as the self-replacation of transfer DNA fragment to host cell.Three kinds of prevailing types of cloning vector are bacterial plasmid, bacteriophage and other virus.Expression vector is so that be inserted in the cloning vector that protein will transcribed and be translated as to the coded sequence of privileged site through design.

Cloning vector and expression vector all comprise the nucleotide sequence that carrier is duplicated in one or more suitable host cells.In cloning vector, this sequence normally makes carrier can be independent of host cell chromosome and duplicates, and comprises origin of replication or autonomously replicating sequence.The origin of replication of a lot of bacteriums and virus is well-known to those skilled in the art and includes, but are not limited to the viral starting point of pBR322 plasmid starting point, 2 μ plasmid starting points and SV40, polyomavirus, adenovirus, VSV and BPV.

Oligonucleotide sequence of the present disclosure can be used to produce anti-fungus peptide through the recombinant expression carrier that use contains oligonucleotide sequence.The expression vector that is fit to comprises chromosomal, achromosomal and synthetic dna sequence dna; For example SV 40 derivants, bacterial plasmid, phage DNA, baculoviral, yeast plasmid, the carrier and the viral DNA of deriving by the combination of plasmid and phage DNA, for example cowpox, adenovirus, bird pox virus and Pseudorabies virus.In addition, can use any in the host reproducible and viable other carrier.

Can interested nucleotide sequence be inserted in the carrier through several different methods.In modal method, use usually the method that details in the document known to those skilled in the art and below for example: Sambrook et al., Molecular Cloning; A Laboratory Manual; 2nded., Cold Spring Harbor Press, (1989) and Ausubel et al.; Short Protocols inMolecular Biology; 2nd ed., John Wiley & Sons 50 (1992) can insert suitable restriction endonuclease sites with sequence.

In expression vector, interested sequence may be operably coupled to by host cell identification to instruct the mRNA synthetic expression control sequenc or the promoter that are fit to.Promoter is the non-translated sequence that is usually located at from 100 to 1000 base-pairs (bp) of the upstream from start codon of structural gene, and promoter is in its control the transcribing and translating of adjusting nucleic acid down.That promoter is divided into induction type usually or composing type.Inducible promoter is such promoter, and it is as to the reaction of some variation in the environment, and the for example existence of nutriment or do not exist or variation of temperature impels in its control and causes the increase of DNA transcriptional level down.In contrast, constitutive promoter is kept constant relatively transcriptional level.

When nucleotide sequence was in sense with another nucleotide sequence and concerns, it was operably connected.For example, presequence or secretion guide's DNA is operably connected with the DNA of polypeptide, if this polypeptid DNA is expressed as the preceding albumen of participating in the polypeptide secretion; Promoter is operably connected with coded sequence, if this promoter influences transcribing of sequence; Perhaps ribosome binding site is operably connected with coded sequence, if this ribosomes is placed in the site of convenient translation.Usually, the sequence that is operably connected is an adjacency, and for secretion guide's situation be adjacency and in reading mutually.In conjunction with being that splicing through at the restriction enzyme position realizes.If there is not suitable restriction site, then can use well known to a person skilled in the art synthetic oligonucleotide joint or connector (adapter or linker).Sambrook et al.，Molecular Cloning，A LaboratoryManual，2nd ed.，Cold Spring Harbor Press，(1989)and Ausubel et al.，ShortProtocols in Molecular Biology，2nd ed.，John Wiley&Sons(1992)。

The common promoter that is used for expression vector includes, but not limited to LTR or SV40 promoter, Escherichia coli lac or trp promoter and phage PL promoter.Effectively the inducible plant promoter comprises heat shock promoter (Ou-Lee et al. (1986) Proc.Natl Acad.Set USA 83:6815; Ainley et al. (1990) Plant Mol.Biol.14:949), be derived from nitrate inducible promoter (Back et al. (1991) Plant Mol Biol.17:9), hormone induction type promoter (Yamaguchi-Shinozaki et al. (1990) the Plant Mol.Biol.15:905 of spinach nitrite reductase gene; Kares et al. (1990) Plant Mol.Biol.15:905) and photoinduction type promoter (Kuhlemeier et al. (1989) the PlantCell 1:471 relevant with the LHCP gene family with the little subunit of RuBP carboxylase; Feinbaum et al. (1991) Mol.Gen.Genet.226:449; Weisshaar etal. (1991) EMBOJ.10:1777; Lam and Chua (1990) Science 248:471; Castresana et al. (1988) EMBOJ.7:1929; Schulze-Lefert et al. (1989) EMBOJ.8:651).Can use that known controlling gene in protokaryon or eukaryotic is expressed, well known to a person skilled in the art other promoter.Expression vector also can comprise the ribosome binding site and the transcription terminator of translation initiation.This carrier also can comprise and be used for the sequence that amplification gene is expressed.

Expression vector and cloning vector can, and normally, comprise and select gene or selection marker thing.Typically, this gene code is for the existence and the necessary protein of growth of suppressed by vector transformed host cells.The instance of the mark that is fit to comprises resistance and the Escherichia coli of eukaryotic dihyrofolate reductase (DHFR) or the neomycin resistance to tetracycline or ampicillin.Selection marker thing in the plant comprises the resistance to bleomycin, gentamicin, glyphosate, hygromycin, kanamycins, methotrexate, phleomycin, phosphinothricin (phosphinothricin), actinospectacin, streptomysin, sulfonamide and sulfonylureas.Maliga et al., Methods in Plant Molecular Biology (molecular biology of plants method), Cold Spring Harbor Press, 1995, p.39.

In addition, expression vector also can comprise be operably connected with the nucleotide sequence of protein, be mark sequence as other protein of coding of mark.Its result is hydridization or the fused protein that comprises two connections and different protein.Said marker protein can provide for the recombinant protein matter that is produced by expression vector, for example, and mark immunity or biology enzyme.Suitable mark includes, but not limited to alkaline phosphatase (AP), myc, hemagglutinin (HA), β-glucuronidase (GUS), luciferase and green fluorescent protein (GFP).

Polynucleotide sequence of the present disclosure also can be the part of expression cassette; This expression cassette comprises at least; Operationally 5 ' to the direction of 3 ' position, connect, such as the polynucleotide of the adjusting sequence of promoter, the disclosed peptide of code book and in host cell acting transcription stop signals sequence.Said promoter can be any type described herein, for example, and tissue-specific promoter, seed specific promoters, plasmid specificity promoter etc.Said expression cassette can further comprise the target that is operably connected, the secretion peptide-coding region that transports the transmission of the protein that maybe can guide generation.Said expression cassette also can further comprise the nucleotide sequence of coding screening mark and/or purification part.

More specifically, the disclosure comprises recombinant precursor, and it contains the polynucleotide sequence of the separation of the disclosed anti-fungus peptide of code book.But said construct package carrier, for example plasmid vector or viral vectors, said sequence is inserted into wherein forward or backwards.This recombinant precursor can further comprise the adjusting sequence, comprises, for example, the promoter that is operably connected with sequence.Many suitable carriers and promoter are known to those skilled in the art and are commercially available.

The further embodiment of the disclosure relates to the transformed host cell that comprises construct, and this construct contains oligonucleotide sequence of the present disclosure.Said host cell can be more senior eukaryotic, for example mammalian cell or vegetable cell, or the eukaryotic of even lower level, and yeast cells for example, perhaps the host can be prokaryotic, for example bacterial cell.Said construct is introduced host cell can pass through accomplished in many ways; Comprise: the transfection, 1 of calcium phosphate transfection, the mediation of DEAE-glucosan; 5-dimethyl-1,5-phenodiazine 11 methylene gather Methobromide, bioplast fusion, liposome, directly nucleus, scraping loading and electroporation are arrived in microinjection.In plant, can use multiple diverse ways will transform/expression vector is incorporated into plant protoplast, cell, callus, leaf dish etc., to produce genetically modified plants.These methods comprise; The protoplast transformation of for example, the conversion of agrobacterium-mediation, particle gun transmission, microinjection, electroporation, polyglycol-mediation, (Potrykus (1991) the Annu.Rev.Plant Physiol Plant Mol.Biol.42:205) such as conversions of liposome-mediation.

The peptide that expression through polynucleotide of the present disclosure produces can obtain through following steps: through any aforesaid method transformed host cell, and the host cell of under the condition of being fit to, growing; Induce the expression of polynucleotide and separate protein of interest matter.If protein is retained in the host cell, then can obtain this protein through the dissolving of host cell, and if protein is secreted protein, then can from nutrient culture media, separate.But the certain methods purification of protein is arranged and be known those of ordinary skills.These methods comprise deposition, for example ammonium sulfate precipitation or precipitation with alcohol, acid extractants; Negative ion or cation-exchange chromatography, cellulose phosphate chromatography, hydrophobic interaction chromatography; Affinity chromatography, hydroxyapatite, lectin chromatography; Electrophoresis, isoelectric focusing and immune precipitation under the high performance liquid chromatography (HPLC), natural or sex change condition.

. perhaps, can be by the peptide of polynucleotide encoding of the present disclosure through the chemical synthesis generation of the synthetic or classical solution peptide of use solid-phase peptide synthetic (it is synthetic to be also referred to as the liquid phase peptide).In the liquid phase that oligomer is supported was synthetic, the product of growth was attached on the big soluble polymer group.Therefore, based on relatively very big dimensionally huge different of product and unreacted reactant that invest on the polymkeric substance, the product of each synthesis step can separate from unreacted reactant.Reaction can be taken place in the solution of homogeneous phase, and eliminated and the synthetic relevant loaded down with trivial details purification step of traditional liquid phase.The synthetic spontaneous liquid phase that also can adapt to peptide of the liquid phase of oligomer support is synthesized.

Synthetic for solid-phase peptide, this process causes that suitable amino acid whose assembled in sequence is the peptide of required sequence, and the end of the peptide of growth is connected with insoluble carrier.Usually, the c-terminus of peptide is connected with polymer phase, and when the agent of dissociating is handled, can discharge from said polymkeric substance.In common methods, amino acid combines with resin particle, and, produce peptide through the amino acid that progressively adds protection with the step-by-step movement that produces amino acid chain.Usually use the modification of describing by Merrifield this technology (referring to, for example, Merrifield, J.Am.Chem.Soc.96:2989-93,1964).In automatic solid phase method; (for example be loaded into organic coupling agent through amino acid with c-terminus; PAM, 4-oxo aminomethyl phenyl acetamide methyl) go up and synthetic peptide, said organic connection covalently is connected on the insoluble polystyrene resin with divinyl benzene crosslinked.Can protect terminal ammonia through stopping with tertbutyloxycarbonyl.Hydroxyl and carboxylic group can be protected through utilizing the O-benzyl group to stop usually.In the automatic peptide compositor, accomplish and synthesize, a lot of commercially available compositors are arranged.After synthetic, can slough product from resin.Usually use hydrofluorite or TFMS to slough blocking group according to existing method (for example, Bergot and McCurdy, Applied BiosystemsBulletin, 1987).After separation and the purifying, obtain about yield of 60% to 70% usually.The purifying of accomplishing the product peptide be through; For example; For example make the peptide crystallization the methyl butyl ether from organic solvent; Be dissolved in distilled water then, and use dialysis (if the molecular weight of peptide is greater than about 500 dalton) or high back voltage liquid chromatography (for example, using 0.1% trifluoroacetic acid and acetonitrile C18 post) as solvent if the molecular weight of peptide less than 500 dalton.The peptide of purifying can store for future use by freeze-drying and with dry state.Can use common high pressure liquid chromatography (HPLC) and electrojet mass spectrum (ES-MS) analytical approach to accomplish the analysis of gained attitude.

Usually, comprising the genetically modified plants of the cell that contains disclosure polynucleotide can be through any preceding method production; The vegetable cell that screening transforms in selective medium; Make by the plant of plant transformed cytothesis with the generation differentiation; And the selection plant transformed, it expresses the protein by polynucleotide encoding of the present disclosure with desired level.The concrete grammar that transforms multiple dicotyledon and obtain genetically modified plants has well write down (Gasser and Fraley, Science244:1293,1989 in document; Fisk and Dandekar, Scientia Horticulturae 55:5,1993; And the document of quoting).

Conversion and plant regeneration are succeedd in multiple monocotyledon.Instantiation is following: asparagus (Asparagus officinalis; Bytebier et al. (1987) Proc.Natl.Acad.Sci.USA 84:5345), barley (Hordeum vulgarae; Wan and Lemaux (1994) PlantPhysiol.104:37), corn (Zea mays; Rhodes et al. (1988) Science 240:204; Gordon-Kamm et al. (1990) Plant Cell 2:603; Fromm et al. (1990) Bio/Technology 8:833; Koziel et al. (1993) Bio/Technology 11:194), oat (Avena sativa; Somers et al. (1992) Bio/Technology 10:1589), orchard grass (Dactylisglomerata; Horn et al. (1988) Plant Cell Rep.7:469), rice (Oryza sativa, including indica and japonica varieties; Toriyama et al. (1988) Bio/Technology 6:10; Zhang et al. (1988) Plant Cell Rep.7:379; Luo and Wu (1988) Plant Mol.Biol Rep.6:165; Zhang and Wu (1988) Theor.Appl.Genet.16:835; Christou et al. (1991) Bio/Technology 9:957), rye (Secalecereale; Dela Pena et al. (1987) Nature 325:274), Chinese sorghum (Sorghum bicolor; Cassas et al. (1993) Proc.Natl Acad.ScI USA 90:11212), sugarcane (Saccharumspp.; Bower and Birch (1992) Plant is J.2:409), Festuca Arundinacea (Festuca arundinacea; Wang et al. (1992) Bio/Technology 10:691), turfgrass (Agrostis palustris; Zhong et al. (1993) Plant Cell Rep.13:1) and wheat (Triticum aestivum; Vasilet al. (1992) Bio/Technology 10:667; Weeks et al. (1993) Plant Physiol.102:1077; Becker et al. (1994) Plant J.5:299).

In a preferred embodiment, adopt the recombination of polynucleotide of the disclosed anti-fungus peptide of code book to transform plant, wherein these polynucleotide form the peptide by plant secretion.In another preferred embodiment, the root of genetically modified plants secretes antimycotic peptide.The plant of secretion anti-fungus peptide can make up through said method, has used the expression cassette of the secretion sequence introducing that will guide the peptide secretion.Perhaps, can adopt nucleotide sequence to transform plant, this is nucleotide sequence coded by anti-fungus peptide of the present disclosure and usually by the fused protein of the protein of plant secretion structure.For example, can produce fused protein at anti-fungus peptide and cytokinin oxidase.But cytokinin oxidase is the defensive enzyme that shows as the foreign cell mitogen of degraded disturb plant Growth Control.Through the anti-fungus peptide and the cytokinin oxidase gene zone of control secretion are merged, the antibiotic peptide will be transformed plant secretion and gone out, thereby the protection of avoiding the pathogen fungi is provided.

Before fused protein was used to transform plant, the method that can use phage display of the present disclosure was screened the activity of the fused protein that comprises anti-fungus peptide.Usually, fusion can be the structure that comprises following material: anti-fungus peptide; The secretion control section of protein, for example cytokinin oxidase; And pVIII or pIII bacteriophage coat protein.The phage display fused protein that then, can use method screening of the present disclosure so to make up combines and causes limiting the fused protein of pathogenic variation to select those and target pathogen fungi.

Embodiment

The purpose of following examples provides the explanation of application of the present disclosure.Following examples are not in order to define or otherwise limit scope of the present invention fully.

Embodiment 1: the generation of fungal species and zoospore

The fungal bacterial strain that uses is phytophthora blight of pepper (P.capsici) (ATCC 15399), soyabean phytophthora (P.sojae) (bacterial strain 7-6-1; Classification 25) (A.F.Schmitthenner, Ohio State University) and phytophthora parasitica (Phytophthora parasitica).All cultures all remain on butter bean agar plate (soyabean phytophthora) or corn meal agar under 15 ℃ as mycelium, and dull and stereotyped (Difco is USA) on (phytophthora blight of pepper and phytophthora parasitica).Through (5mm * (Campbell Soup Co. makes the mycelium copy on agar plate USA) 5mm) to be transferred to 10%V8

vegetable juice that comprises clarification with hyphal peg.There are 3 bolts to grow 3 to 6 days down on each plate at 25 ℃.Through repairing said flat board and under 25 ℃, illumination, cultivating, in phytophthora blight of pepper, can cause sporangial generation.After 1 to 2 day, through the said dull and stereotyped release that can cause zoospore in 20 to 30 minutes of aseptic water washing.The generation of phytophthora parasitica zoospore is identical with the generation of phytophthora blight of pepper, before under 25 ℃, illumination, cultivating, washs said dull and stereotyped 2 minutes with sterilized water.The release that from the soyabean phytophthora sporangium, causes zoospore be through with sterilized water with 30 minutes interval washing plate 4 times.Zoospore discharged in 2 to 4 hours.After it discharges, filter zoospore to remove spore softgel shell and mycelial fragment through 4 layers of cheese cloth.The sample eddy current of suspending liquid was counted packing with hemacytometer at microscopically to cause encystation in 30 seconds then.

Embodiment 2: as the K91kan Bacillus coli cells of the hunger of conversion unit and the preparation of Titerinz bacteriophage

Before library screening, according to disclosed method (Smith & Scott, Methods inEnzymology, 217:228-257,1993; Yu and Smith, Methods in Enzymology, 267:3-27,1996), bacteriophage is carried out concentration determination as the tetracycline conversion unit (TU) in K91 BluKan (kalamycin resistance) Escherichia coli of hunger.Conversion unit is to measure the communicable effective ways of bacteriophage, and is typically expressed as the TU/ml of bacteriophage.In brief, K91BluKan cell (Smith and Scott, Methods in Enzymology, 217:228-257,1993) in the super broth (superbroth) of 20ml, under 37 ℃, commove (about 170rpm) is grown to index stage in mid-term (OD ₆₀₀About 0.45).Then this cell is softly being shaken other 5 minutes of cultivation down so that the F pIII regeneration of any pruning.In the Sorvall SS34 rotor under 4 ℃, with this cell in aseptic 50ml round bottom spiral cover (Oak Ridge) pipe with 2,200rpm centrifuging 10 minutes.Topple over and supernatant, then this cell is suspended among the 80mM NaCl of 20ml again, place the 125ml culture flask and under 70rpm, 37 ℃, softly shook 45 minutes.As above this cell of centrifuging also is suspended in the cold NAP buffer solution of 1ml more then.Hungry cell is preserved under 4 ℃ and was kept infectiousness 3 to 5 days.

Bacteriophage is carried out concentration determination as the conversion unit (TU) of hungry cell in Escherichia coli K91BluKan (as above preparation).Use the TBS/ gel bacteriophage to be carried out assay determination as thinning agent.Each bacteriophage dilution of 10 microlitres is deposited on the inwall of the aseptic disposable pipe of 15ml that becomes 10 ° of angles settings with surface level with the drop form.The Escherichia coli K91BluKan cell of 10 microlitres hunger is added each bacteriophage drop and at room temperature it cultivated 10 minutes to allow the time of the concentrated cell of phage-infect.After 10 minutes, the 1ml super broth that will comprise the 0.2mg/ml tetracycline be added to said cell and shake down, 37 ℃ cultivated 20 to 40 minutes.For the amplification of f88-4/15mer bacteriophage, this super broth also comprises 1mM IPTG to induce the expression of recombinant pVIII.Then the cell that infects is launched (each flat board of 200ml) on the Luria-Bertani that comprises the 40mg/ml tetracycline (LB) flat board.Then should flat board about 24 hours 37 ℃ of cultivations.

Embodiment 3: combine the selection of the zoospore of bacteriophage

With in the f8-1 library 10 ¹¹The aliquot of conversion unit (TU) (Petrinko et al., ProteinEngineering, 9:797-801,1996) at room temperature is added among the 50mM LiCl of 4ml 10 ⁶In the new phytophthora blight of pepper zoospore that discharges, under room temperature, the soft stirring, cultivated 30 minutes then.Except the situation (Soj clone) of the zoospore that uses soyabean phytophthora, to the identical method of f88-4 library use.Comprise the zoospore 10 times that combines bacteriophage with the 50mL LiCl of 150 μ l washing, then with 45 seconds of 1000xg centrifuging to remove unconjugated bacteriophage.After 10 washings, with the bacteriophage of the elution buffer of 200 μ l (0.1N HCl, enough making pH is 2.2 glycocoll, the 1mg/ml bovine serum albumin) elution of bound.Escherichia coli K91BluKan cell through infecting aforesaid hunger increases the bacteriophage of wash-out.Then through bacteriophage that be described below and this amplification of the method purifying polyglycol deposition, and like Smith and the described method of Scott it is suspended in (Methods in Enzymology, 217:228-257,1993) in the TBS damping fluid again.Purifying bacteriophage aliquot aliquot is used further to be used for 3 affinity purifying and 2 amplification step altogether as stated in the new zoospore that discharges subsequently.Like Smith and Scott said (Methods inEnzymology, 217:228-257,1993), take turns the selective enrichment that percentage yield monitoring after the selection combines the bacteriophage of zoospore through calculating each.This calculating is to be used for total bacteriophage (being expressed as conversion unit) of zoospore and to measure from the total production of the bacteriophage (as conversion unit) of zoospore regeneration and carry out through calculating, and representes this result with number percent.After each screening of taking turns from zoospore the yield of the bacteriophage of wash-out be 10 ^-4% to 10 ^-5Between the %, show that said method is successful on screening and bacteriophage that zoospore combines.Zoospore is intact, spherical behind the washing step, encystation almost do not occur during being illustrated in screening process.

Embodiment 4: the bacteriophage purifying

By the Escherichia coli K91BluKan cell of phage-infect overnight growth in 20ml super broth (tetracycline that contains 40mg/ml), under 37 ℃, 170rpm.With this nutrient solution in the SS34 rotor with 5,000xg centrifuging 10 minutes is to precipitate this Bacillus coli cells (containing bacteriophage).Remove supernatant and be placed in the new round bottom spiral cover pipe, the ratio with the every ml supernatant of 150l adds PEG/NaCl (16.7% polyglycol/3.3M NaCl) with the deposition bacteriophage then.Under 4 ℃ with the bacteriophage deposition of spending the night, then through in the 50ml of SS34 rotor round bottom spiral cover pipe with 10, the 000rpm centrifuging precipitated in 20 minutes.The deposition bacteriophage is suspended in the Tris buffer saline (TBS) of 1ml again.It is deposited once more and under 4 ℃, spend the night through the PEG/NaCl that adds 150ml.Be suspended among the TBS again with the bacteriophage deposition and with this deposition through the centrifuging on desk centrifuge.

Embodiment 5:DNA separates, checks order and analyzes

The DNA that will be used for checking order according to the method (Methods in Enzymology, 217:228-257,1993) of Smith and Scott separates from independent phage clone.(Applied Biosystems, Foster City Calif.) check order from 3 ' end to single stranded DNA according to manufacturers protocol to use ABI Prism377 type automatic sequencer.The primer that is used for f8 clone is 5 '-GGAGCCTTTAATTGTATCGG-3 ' (SEQ ID NO:2).The primer that is used for f88 clone is 5 '-AGT AGC AGA AGC CTG AAG A-3 ' (SEQ ID NO:3).

Use ExPASy molecular biology server (network address: Http:// www.expasv.ch/) " translation " program dna sequence dna is translated.Use canonical algorithm (promptly) FASTA (Lipman andPerson, Science, 227:1435-1441; 1985) and BLAST (Altschul et al., J.Molecular Biol., 215:403-410; 1990) order to sequence be stored in sequence library (GenBank, EMBL, dbEST; SwissProt, PIR) nucleic acid and protein sequence compare.

Use ClustalW (Thompson et al., Nuc.Acid Res., 22:4673-4680; 1994) and PAM250 weight table peptide sequence is compared, and use TreeView (Page, ComputerApplic.Biosci.; 12:357-358,1996) to observing dendrogram.The peptide sequence (table 1) of 19 expections of f8-mer dna sequence encoding that obtain.Most peptides comprise the amino acid residue that is contemplated to strong alpha-helix former (being Glu, Ala and Leu) and alpha-helix saboteur (being Gly and Pro).Although lack shared motif, ClustalW multisequencing comparison program is used for the form bunch collection of similar peptide with dendrogram.The dendrogram that is made up by the peptide that matees shows that the f8-mer peptide sequence can be grouped into 6 principal home groups, shown in Fig. 1 and table 1.Sequence by the f88-4/15mer library is selected is as shown in table 2.

Embodiment 6: encystation detects

Separate the phage clone of selecting (Methods inEnzymology, 217:228-257,1993) according to the method for Smith and Scott, and be suspended in the distilled water again rather than TBS except bacteriophage, use polyglycol that it is carried out purifying twice as stated.Through at A ₂₆₉Measure absorbance and calculate the concentration (Smith and Scott, Methods in Enzymology, 217:228-257,1993) of this virion.The water droplet and bacteriophage cultivations that will comprise about 20 μ l of about 400 new zoospores that discharge, this bacteriophage are 1 * 10 through the twice serial dilution so that it comprises the bacteriophage concentration that contains peptide ¹⁰, 5 * 10 ⁹, 2.5 * 10 ⁹Or 1.25 * 10 ⁹Virion/μ l water droplet.Negative control does not obtain bacteriophage and is used for monitoring the amount at the spontaneous encystation of zoospore population.Cultivate under the room temperature after 20 minutes, use the microscope of 100x magnification that the quantity of the zoospore of encystation is counted.Virion concentration according to Smith and Scott (Methods in Enzymology, 217:228-257,1993) calculating bacteriophage.

The effectiveness of f8 inducing peptide prematurity parcel changes (Fig. 2) along with sequence family and bacteriophage concentration.Concentration is 1 * 10 ¹⁰During virion/μ l (64 μ M), the family of a lot of peptides is effective to inducing packing.Yet concentration is 2.5 * 10 ⁹During virion, between the peptide of peptide that produces high-level encystation effect (Pc42, Pc78, Pc87 and Pc64) and the low-level encystation effect of generation (Pc15B, Pc56 and Pc45), has 3 to 5 times difference.Concentration is 1.25 * 10 ⁹When (8 μ M), the family of all peptides is all to inducing the encystation effect to have MIN effectiveness.Wild type phage also produces the encystation effect to the phytophthora blight of pepper zoospore; Yet in all tests, the part of zoospore that is produced encystation by the bacteriophage of selecting is greater than by 2 to 7 times of the parts of the encystation of wild type phage.Phytophthora blight of pepper bacteriophage that select, that contain peptide is distinctive with the ability that zoospore encysts to phytophthora blight of pepper in the egg.When soyabean phytophthora and phytophthora parasitica zoospore and 1 * 10 ¹⁰During the containing the peptide bacteriophage and cultivate of virion/μ l, almost do not observe encystation, wherein said concentration produces almost 100% encystation to the phytophthora blight of pepper zoospore.Obtain similar result for the f88-415mer peptide.Ability and f-8 that representational 15mer peptide produces the prematurity encystation clone the more as shown in Figure 3 of Pc87.

Embodiment 7: binding specificity

The ability of combination phytophthora blight of pepper zoospore that relatively has the phage display peptide of high and low encystation inducibility.Select phage clone Pc87 and Pc45 as the representativeness clone (Fig. 2) who induces high and low-level encystation effect respectively at random.Introduce phage vector as control treatment.Phage clone is through the coli-infection amplification and as stated by purifying.For each association reaction, 5 * 10 ¹⁰The bacteriophage of TU and 200,000 phytophthora blight of pepper zoospore are cultivated together.Select to carry out association reaction and washing like the bacteriophage among the embodiment 3.Bacteriophage from zoospore population wash-out is carried out concentration determination Escherichia coli K91BluKan cell also represent as whole conversion units.Use similar process to confirm whether the bacteriophage of selecting combines with the phytophthora blight of pepper packing.

Phage vector combines the phytophthora blight of pepper zoospore with clone Pc45 and Pc87 differently.After 30 minutes hatch, surpass 10 ⁷The bacteriophage Pc87 of TU by wash-out, and only has an appointment 50 from the zoospore, and the bacteriophage Pc45 of 000TU or phage vector are under the same conditions by wash-out (Fig. 4).In addition, distinctive in conjunction with to stage of zoospore the time: as to be less than 10 ⁴Pc87TU is combined (Fig. 5) by wash-out-observe approximately identical background in the control vector bacteriophage from the packing.

Embodiment 8: the structure of secretion fusion

Through synthetic oligonucleotides being connected to the restriction endonuclease sites of carrier pJE-6, HindIII and XbaI, the DNA that makes up carboxyl terminal merges the interested peptide of encoding.Plasmid vector pJE-6 is that (Pichiapastoris expression construct pROM-46 Calif.) makes up, like aforementioned (Cregg et al., Bio/Technology, 11:905-910,1993 for Invitrogen, Carlsbad by coming from plasmid pPICZ-alpha; Rosenfeld, Methods in Enzymology, 306:154-169,1999).Plasmid pROM-46 is with restriction endonuclease (HindIII) digestion, and is flat with Klenow enzyme and dNTP ' s benefit, connects with the T4 dna ligase again.These steps have been eliminated the HindIII restriction site that is present in the pPICZ-alpha plasmid sequence, and plasmid is designated as pJE-4.To replace terminator codon to adopt restriction site HindIII at the series jump of coded sequence 3 ' end through PCR.This plasmid is appointed as pJE-6.The synthetic oligonucleotide of the peptide of encoded representation (Pc87, ADRPSMSPT, SEQ IDNO:8) is connected with plasmid pJE-6 with HindIII and XbaI digestion.This plasmid is appointed as pJE-7 (Fig. 6).The order-checking of pJE-7 plasmid is inserted with definite, and the result is as shown in Figure 7.

Table 1

Table 2

As stated, the protein framing scaffold can be designed to when peptide is transformed into plant, show said peptide.For purposes of illustration, cytokinin oxidase (CKX) can be used as peptide transmission framing scaffold.Be derived from the CKX family of corn member (Morris, 1997) can, for example, as the transmission molecule.CKX is that endogenous ground produces, and has the peptide signal sequence from emiocytosis, and is present at proteinase under the situation in iuntercellular zone by abundant glycosylation so that stability (Morris et al., 1999) to be provided.

Based on the three-dimensional structure of known CKX, like Malito et al., 2004 reports can be engineered to the terminal displayed polypeptide at the C-that exposes with CKX.For example, can express framing scaffold construct and from yeast, it is secreted.In initial test, the inhibition ability of selected peptide is significant, and obtains 80% to 90% zoospore encystation.In the control group of water, phytophthora zoospore encystation is 25% or lower (Fang et al., 2004).For example, this construct capable of using produces the tomato root of hair, and in this case, finds that scafffold proteins is secreted to rhizosphere, and wherein, scafffold proteins was induced zoospore encystation at said rhizosphere at it before the root surface aggregation, had therefore stoped infection.As shown in Figure 9.

Except above work but through constructed expansion, from the combinatorial libraries of the uredospore strong bonded of wart top uromyce select the peptide of 15-mer.This fungi is as the substitute of yam bean layer rest fungus or the analog through selecting, wherein, near yam bean layer rest fungus part by strict control to keep this pathogen.This screening identifies a lot of peptides that behind initial spore-germination, suppress growth, and is for example, shown in figure 10.Said peptide is effective in the concentration of micromole's level.The growth of defense peptides in CKX can make it can transfer to the surface that penetrates the hypha,hyphae between leaf cells.

Embodiment 9: peptide selection method is learned

Fig. 8 is the method flow diagram that shows the scheme 800 of peptide selection.At first, initial phage display peptide library can mix the peptide of having an effect with phytoprotein and other factor to remove in step 802 with plant extracts.The product of so removing peptide is designated as word bank 1.

Word bank 1 mixes 804 with the uredospore that germinates or the young through the known biological technology of eluriating.In conjunction with the phage display peptide from the young, reclaim 806.The concentration of the phage display peptide that each reclaims increases through the amplification in the Escherichia coli 808.The reclaiming 810 of phage display peptide amplification back also mixes 812 to guarantee to remove the peptide of having an effect with phytoprotein and other factor once more with plant extracts.The product of so removing peptide is designated as word bank 2.

Step 814 determines whether that above step carried out enough number of times guarantee to select the stringency of candidate's plant defense peptides.From word bank 2 beginning, word bank 3,4 and 5 is through repeating step 806 to step 812 n time generation repeatedly continuously.Using under the biological situation about eluriating, make the word bank of up-to-date generation repeatedly at every turn, for example, word bank 2 mixes with the uredospore or the young that germinate.In conjunction with the phage display storehouse from the young, reclaim.The concentration of each phage display peptide that reclaims is through increasing in Escherichia coli.

According to processing procedure design, in case said process has repeated n time, it suppresses the ability that young (uredospore that promptly germinates) grow the phage clone test that can select at random from word bank n (or from arbitrary other n sub-banks) 816.Showing successfully inhibiting candidate's peptide can be used as the fusion construct that plant defense peptides is combined with plant surface GFP (for example CKX) and provides 818; In intermediate host (for example Pichia),, and finally be used to transform 822 soybean or arable land broad bean to give rust resistance by amplification 820.

The specific embodiments of preceding method can be described A to E by 5 parts.

Part A relates to through removing the peptide of having an effect with plant component and prepares word bank 1.Pinto leaf and the 7ml damping fluid (pH 7.0 for 20mM Tris-HCl, 5mM EDTA) of 3.0g are mixed, then this potpourri is ground so that homogenate to be provided.In the 2ml microtubule, with this homogenate of 0.9ml and 0.1ml 10 ¹⁴Bacteriophage (f88-4/15-mer) merges so that total 10 to be provided ¹³Virion.Under about 23 ℃ room temperature, the gained potpourri was hatched 30 minutes at rail mounted oscillator (about 500rpm).Use conventional hydro-extractor under at a high speed with centrifugal 5 minutes of the potpourri of hatching.With supernatant recovery, preservation and then centrifugal.This supernatant is used for the bacteriophage amplification.

Part B relates to the biology of the first round and eluriates.Cultivated 5 hours under 20C, in 0.01% polysorbas20 and the 25ppm B-ionone from about 1.5 hundred ten thousand spores of selected pathogen.Add 10 ¹²The TU bacteriophage.This potpourri is assigned in 2 microtubules that cumulative volume is 3ml.The microtubule that will comprise spore/phage mixture was hatched 30 minutes at (about 500rpm) under about 23 ℃ room temperature, on the rail mounted oscillator.The potpourri that centrifuging is hatched; Remove supernatant; The water that adds 0.5ml; And residue merged in the microtubule.With the material centrifuging that merges; Remove supernatant; And with this sediment of 1ml water washing.Step with centrifugal separation and washing repetition 10 times with this supernatant removal.The elution buffer adding of 150 μ l is comprised in the said sediment 11 of bacteriophage and spore.Said elution buffer was mixed and at room temperature leaves standstill this potpourri 5 minutes.Then with this potpourri centrifuging and preserve supernatant.75 μ l elution buffers are added and mix to carry out foregoing other centrifuging.Merge all supernatants.Use 210 μ l elution buffers to carry out repetition.The 1M Tris-HCl (pH 9.0) that adds 44 μ l with in the supernatant of wash-out.The supernatant of neutralization is increased to produce word bank 1.

Portion C relates to preparation word bank 2.Use the word bank 1 of biological amplification of eluriating of the first round among the part B to produce word bank 2.Its completion is through initial f88-4/15-mer storehouse in use word bank 1 rather than the part A, but imitates the scheme of part A in others.

Part D relates to through use and comprises that 2,3 and 4 more biological rounds of eluriating of taking turns produce other word bank.Part B is repeated 3 times:

1. take turns biological the elutriation to the 2nd, use from 4.2 * 10 of the 2nd word bank ¹¹The TU bacteriophage is to produce word bank 3;

2. take turns biological the elutriation to the 3rd, use from the 2nd secondary pollutant and eluriate 5.6 * 10 of amplification ¹¹The TU bacteriophage is to produce word bank 4; And

3. take turns biological the elutriation to the 4th, use from the 3rd secondary pollutant and eluriate 5.1 * 10 of amplification ¹¹The TU bacteriophage is to produce word bank 5.

To take turns biological bacterium colony collection, order-checking of eluriating and test its inhibition from the 4th young growth.

Part E relates to the inhibition of test to the uredospore growth.With 3 * 10 ¹²The spore (about 250) that germinates of 30 μ l in the 20C night incubation.Estimate fungi plumule pipe growth (being young growth) and with water and f88 as contrast, promptly have no the independent bacteriophage of displayed polypeptide, compare.

Embodiment 10: suppress the peptide that wart top uromyce is grown through stoping the young (spore of germination) to grow

Can repeat the methodology of embodiment 9 to pathogen wart top uromyce.Table 3 and table 4 demonstration can be used for suppressing the peptide from uredospore (young) growth of these pathogen germinations.

Table 3

Demonstration is to the very strong inhibiting peptide of the growth of wart top uromyce:

The phage display peptide clone Sequence Serial ID number:

Pp 15 ADPCHMPPRMPPLPI 49

Pp 19 NHVSTLKTRHRLIPF 50

Pp 18 SSNAPPLSYPPLLVP 51

Pp 31 TMARPIPTFLPPPSL 52

Pp 6 TVAPTTHRHYVWSMD 53

Pp 16 VFTPMNLSPPFMQPP 54

Table 4

Demonstration is to the medium inhibiting peptide of the growth of wart top uromyce:

The phage display peptide clone Sequence Serial ID number:

Pp 55 AAGPNIPPPHRASTW 55

Pp 28 AHLYSGASLYRVYRS 56

Pp 56 GPPSILLAIGTLSLT 57

Pp 50 LSSPYACALFVVKGA 58

Pp 39 RGWSVSHHSLLMPVP 59

Pp 21 RSTASPQALNPLVAS 60

Pp 53 SLFFEVSRMLVRLLS 61

Pp 2 SRWWRCVTMTQPCTT 62

Pp 37 VVALRWGWSPLRPPG 63

Embodiment 11: use cytokinin oxidase as the protein framing scaffold to transmit the position that selected peptide to wart top uromyce infects beans (Kidney bean) tissue

With CKX such as above-mentioned modification to merge the defense peptides in table 3 and the table 4.The conversion of Kidney bean of carrying out agrobacterium-mediation is to provide the plant of expressing every kind of peptide.Plant transformed is exposed in the uromyce of wart top.The ratio that infects and the order of severity and check plant are relatively to confirm the effectiveness of defense peptides to pathogen.

Embodiment 12: use cytokinin oxidase as the protein framing scaffold to transmit the position that selected peptide to yam bean layer rest fungus infects soybean (big shield beans) tissue

With CKX such as above-mentioned modification to merge the defense peptides in table 3 and the table 4.The conversion of soybean of carrying out agrobacterium-mediation is to provide the plant of expressing every kind of peptide.Plant transformed is exposed in the yam bean layer rest fungus.The ratio that infects and the order of severity and check plant are relatively to confirm the effectiveness of defense peptides to pathogen.

Conclusion

According to detailed description of the present invention and the foregoing description, can understand, realized several aspect of the present invention.

Should be understood that through explanation and embodiment and described the present invention in detail, purpose is to make others skilled in the art be familiar with the present invention, its principle and its practical application.The description of the specific embodiments that preparation that the present invention is concrete and method are not limited to propose is treated these descriptions and embodiment and would rather say so according to appended claims and equivalent thereof.When some above embodiment with describe comprise some about the present invention can active mode conclusion the time, the present inventor also is not intended to receive those conclusions and effect constraint, and only as possible explanation it is proposed.

Should further understand, the specific embodiments that the present invention proposes does not also mean that and is exhaustively or limits of the present inventionly that and according to previous embodiment and detailed description, it is much replaced, modifies and changes those of ordinary skills will be conspicuous.Therefore, the intent of the present invention is to comprise that all that drops on the spirit of following claim and replacement, modification and the variation in the category.

List of references

Following document is incorporated this paper by reference into, and is as this paper that the disclosed degree of its full content is the same:

Barbas, C.F., III.Burton, D.R., Scott, J.K., Silverman, GJ. (2001) .PhageDisplay (phage display) .A Laboratory Manual.Cold Spring harborLaboratory Press.

Bishop-Hurley; S.L., Schmidt, FJ.; Erwin; A.L., and Smith, A.L. (2005) .Peptides Selected for Binding to a Virulent Strain of Haemophilusinfluenzae by Phage Display Are Bactericidal (for the peptide of the virulent strain of the combination haemophilus influenzae selected through phage display is bactericidal) .Antimicrob.Agents Chemother.492972-2978.

Bishop-Hurley, S., Schmidt, FJ.; Smith, G.P., Morris, R.O.; Elder, J., Roop; P., and English, J.T.2002.Phage display peptides that disrupt life-stageprogression in Phytophthora (destroying the phage display peptide that phytophthora belongs to the life stage progress) .App.Env.Micro.68:3315-3320.

Bonde; M.R., Melching, J.S.; And Bromfield; K.R.1976.Histology of thesuscept-pathogen relationship between Glycine max and Phakopsorapachyrhizi, the cause of soybean rust (histology of the suscept between big shield beans and the yam bean layer rest fungus-pathogen relation, the root of soybean rust) .Phytopathology66:1290-1294.

Cwirla; S.E., Peters, E.A.; Barrett; R.W., and Dower, WJ. (1990) the .Peptideson phage:a vast library of peptides for identifying ligands (peptide on the bacteriophage: a large amount of peptides storehouse of differentiating part) .Proc.Natl.Acad.Sci.87:6378-6382.

Fang; Z, Mounter, S.Simon; M.; Schmidt, FJ., and English; J.T. (2004) .Protein scaffold design for delivery of peptides that induce encystment ofPhytophthora capsici zoospores (the protein framing scaffold design of phytophthora blight of pepper zoospore encystation is induced in transmission) .Abstract presented at the NSF PhytophthoraMolecular Genetics Workshop.New Orleans, LA.

Healy, J.M., Murayama; O., Maeda, T.; Yoshino, K., Sekiguchi; K., andKikuchi, M. (1995) .Peptide ligands for integrin V3 selected from random phage display libraries (being selected from the peptide part that random phage is showed the plain V3 of integration in storehouse) .Biochemistry 34:3948-3955.

Hollier; C.A., and King.S.B.1985.Effects oftemperature and relativehumidity on germinability and infectivity of Puccinia polysorauredospores (temperature and relative humidity are to uredinial germinating power of many heaps handle rest fungus and communicable influence) .Plant Dis.69:937-939.

Koch; E.; And Hoppe, H.H.1988.Development of infection structures by thedirect-penetrating soybean rust fungus (Phakopsora pachyrhizi Syd.) Onartificial membranes (the directly exploitation of the infection structure of infiltration soybean show bacterium (yam bean layer rest fungus) on artificial rust) .J.Phytopathol.122:232-244.

Koivunen; E.; Gay; D.A., Ruoslahti, E. (1993) .Selection of peptides bindingto the 51 integrin from phage display library (selecting to integrate the plain peptide that combines) .J.Biol.Chem.268:20205-20210 with 51 from the phage display storehouse

Koivunen; E.; Wang; B., and Ruoslahti, E. (1994) .Isolation of a highlyspecific ligand for the 51 integrin from phage display library (separate 51 from the phage display storehouse and integrate plain high specific part) .J.Cell Biol.124:373-380.

Laskey, J.T., Bishop-Hurley; S., Mounter, S.A.; English; J.T., and Schmidt, F.J. (200) .Phage-display peptides that disrupt developmental progressionof Phytophthora species (destroy phytophthora and belong to the phage display peptide that growth is carried out) .Phytopathology 91:S53.

Morris; R.O.1997.Hormonal regulation of seed development (hormone regulation of seed development), ppl 17-149in:Larkins, B.A.; And Vasil; I.K., eds.Cellular andMolecular Biology of Plant Seed Development.Kluwer Academic Publ., Boston.

Morris R.O.; Bilyeu K.D.; Laskey J.G., Cheikh N.N. (1999) Isolation of agene encoding a glycosylated cytokinin oxidase from maize (from corn, separating glycosylation cytokinin oxidase encoding gene) .Biochemical&BiophysicalResearch Communications 255:328-333.

O ' Neil, K.T., Hoess; R.H., Jackson.S.A., Ramachandran; N.S., Mousa, S.A.; And DeGrado, W.F. (1992) .Identification of novel peptide antagonists for GPIIb/IIIa from a conformationally constrained phage display library (differentiating new GPIIb/IIIa peptide antagonists) .Proteins 14:509-515. from the phage display storehouse that conformation retrains

Pasqualini; R.; Koivunen; E., Ruoslahti, E. (1995) .A peptide isolated fromphage display libraries is a structural mimic of an RGE-binding site onintegrins (peptide that separates from the phage display storehouse is a structural simulation thing of integrating the plain RGE of combination position) .J.Cell Biol.130:1189-1196.

Pasqualini, R., and Ruoslahti, E.1996.Organ targeting in vivo using phagedisplay peptide libraries (using the intracorporeal organ target of phage display peptide library) .Nature.380:364-366.

Rizvi，A.2004.Soybean Rust Colloquium，Annual Meeting of the SouthernSoybean Disease Workers，St.Louis，MO.

Scott, J.K., and Smith, G.P. (1990) .Searching for peptide ligands with anepitope library (utilizing epi-position storehouse screening peptide part) .Science 249:386-390.

Smith, J.W., Hu; D., Satterthwait, A.; Pihz-Sweeney; S., and Barbas III, CF. (1994) .Building synthetic antibodies as adhesive ligands for integrins (foundation) .J.Biol.Chem.269:32788-32795. as the synthetic antibody of integrating plain adhesion ligand

Wilson; D.R.; And Finlay; B.B.1998.Phage display:applications, innovations, and issues in phage and host biology (phage display: the application in bacteriophage and host living beings, new method and problem) .Can.J.Microbiol.44:313-329

Sequence table

<110>University of Missouri

ENGLISH，JAMES T

SCHMIDT，FRANCIS J

STACEY，GARY

ZHIWEI，FANG

< 120>to the phage display plant defense peptides of yam bean layer rest fungus and wart top uromyce

<130>Matter 438962

<150>10/829,549

<151>2001-04-10

<150>60/195,785

<151>2000-04-10

<160>63

<170>PatentIn version 3.3

<210>1

<211>33

<212>PRT

< 213>type 88 trichobacteria bacteriophages

<220>

< 221>variant

<222>(9)..(23)

< 223>x is that wherein N is A, T, C or G by arbitrary amino acid of codon NNK coding;

And K is appointed as G or T

<400>1

Leu Val Pro Met Leu Ser Phe Ala Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa

1 5 10 15

Xaa Xaa Xaa Xaa Xaa Xaa Xaa Pro Ala Glu Gly Asp Asp Pro Ala Lys

20 25 30

Ala

<210>2

<211>20

<212>DNA

< 213>artificial

<220>

< 223>primer

<220>

< 221>composite character

<222>(1)..(20)

< 223>primer

<400>2

ggagccttta attgtatcgg

20

<210>3

<211>19

<212>DNA

< 213>artificial

<220>

< 223>primer

<400>3

agtagcagaa gcctgaaga

19

<210>4

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>4

Ala Ala Pro Asp Leu Gln Asp Ala Met

1 5

<210>5

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>5

Ala Asp Arg Leu Asn Ser Asp Ala Gly

1 5

<210>6

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>6

Ala Asp Arg Pro Ser Thr Thr Ser Leu

1 5

<210>7

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>7

Ala Asp Pro Pro Arg Thr Val Ser Thr

1 5

<210>8

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>8

Ala Asp Arg Pro Ser Met Ser Pro Thr

1 5

<210>9

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>9

Ala Asp Arg Thr Ser Asn Ala Ser Thr

1 5

<210>10

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>10

Ala Asp Lys Ser Tyr Ile Pro Ser Ser

1 5

<210>11

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>11

Ala Val Arg Asn Pro Ser His His Ser

1 5

<210>12

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>12

Ala Asp Pro Thr Pro Arg Gly His Ser

1 5

<210>13

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>13

Ala Asp Pro Thr Arg Gln Pro His Ser

1 5

<210>14

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>14

Ala Glu His Gln Asn Ser Ala Gly Pro

1 5

<210>15

<211>10

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>15

Ala Asp Ala Arg Ser Ala Gly Ala Ile Ser

1 5 10

<210>16

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>16

Ala Asp Ser Lys Asn Ala Gly Pro Met

1 5

<210>17

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>17

Ala Glu Thr Lys Phe Ser Gly Ser Ala

1 5

<210>18

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>18

Ala Asp Pro Lys Gly Ser Gly Val Thr

1 5

<210>19

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>19

Ala Gly Leu Thr Ser Pro Asn Asp Met

1 5

<210>20

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>20

Ala Asp Ile Thr Asp Pro Met Gly Ala

1 5

<210>21

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>21

Ala Val Gly Thr His Thr Pro Asp Ser

1 5

<210>22

<211>9

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>22

Ala Val Ser Pro Asn Val His Asp Gly

1 5

<210>23

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>23

Val Ala Ala Phe Ser Leu Val Trp Ala Thr His Leu Met Leu Ser

1 5 10 15

<210>24

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>24

Leu Thr Arg Cys Leu Val Ser Thr Glu Met Ala Ala Arg Arg Pro

1 5 10 15

<210>25

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>25

Ser Ala Pro Tyr Leu Pro Tyr Phe Asp Leu Leu His Phe Pro Ile

1 5 10 15

<210>26

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(14)..(14)

< 223>x=unknown amino acid

<400>26

Pro Ser Ser Tyr Glu Ala Ser Arg Arg Pro Glu His Trp Xaa Phe

1 5 10 15

<210>27

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>27

Ser Ala Thr Asp Thr Thr Leu Pro Met Met Thr Ala Ile Arg Ser

1 5 10 15

<210>28

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

<221>VARIANT

<222>(9)..(9)

< 223>x=unknown amino acid

<400>28

Thr Arg Leu Ser Pro Met Glu Ser Xaa Ala Met Leu Leu Ala Pro

1 5 10 15

<210>29

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>29

Leu Leu Pro Val Ser Pro Pro Phe Ala Pro Asn Ala Ser Ser Thr

1 5 10 15

<210>30

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>30

Met Ser Asn Phe Pro Thr Ser His Ala Pro Cys Pro Val Glu Ile

1 5 10 15

<210>31

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>3l

Glu Phe Arg Lys Asn Tyr Pro Ser Ala Ala Pro Leu Ile Pro Arg

1 5 10 15

<210>32

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(2)..(2)

< 223>x=unknown amino acid

<400>32

Pro Xaa Val His G1y Ser Ile Pro Leu Thr Pro Pro Leu Gly Phe

1 5 10 15

<210>33

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(3)..(3)

< 223>x=unknown amino acid

<400>33

Leu Phe Xaa Cys Tyr Pro Pro Cys Thr Tyr Ser Tyr Cys Leu Ser

1 5 10 15

<210>34

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(14)..(14)

< 223>x=unknown amino acid

<400>34

Met Ser Asn Phe Pro Thr Ser His Ala Pro Cys Pro Val Xaa Ile

1 5 10 15

<210>35

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>35

Pro Glu Trp Lys Ser Ser Trp Ser Pro Cys Thr Pro Arg Cys Pro

1 5 10 15

<210>36

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(11)..(11)

< 223>x is M or I

<400>36

Ala Met Ser Arg Trp Leu Arg Pro Arg Glu Xaa Asn Ala Pro Pro

1 5 10 15

<210>37

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(6)..(6)

< 223>x=unknown peptide

<220>

< 221>variant

<222>(10)..(10)

< 223>x=unknown peptide

<400>37

Thr His Thr Thr Phe Xaa Val Thr Val Xaa Leu His Glu Pro Pro

1 5 10 15

<210>38

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(6)..(6)

< 223>x=unknown peptide

<220>

< 221>variant

<222>(10)..(10)

< 223>x=unknown peptide

<400>38

Thr His Thr Thr Phe Xaa Val Thr Val Xaa Leu His Glu Pro Pro

1 5 10 15

<210>39

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>39

Pro Thr Leu Gly Arg Phe Asn Arg Pro Ser Cys Ser Ile Ile Val

1 5 10 15

<210>40

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>40

Ala Pro Gln Cys His Pro His Leu Pro Phe Asp Met Ile His Val

1 5 10 15

<210>41

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<220>

< 221>variant

<222>(12)..(12)

< 223>x=unknown peptide

<400>41

Asn His Asn Ser Leu Pro Ala Gln Tyr Leu Val Xaa Ile Leu Arg

1 5 10 15

<210>42

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>42

Asp Gln Pro Cys Thr Pro Ser Pro Asp Val Ser Phe Tyr Arg Ser

1 5 10 15

<210>43

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>43

Val Ala Ala Pro Ser His Trp Leu Lys Pro Ser Leu Asp Cys Phe

1 5 10 15

<210>44

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>44

Asn Pro Leu Tyr Lys Asn Pro Pro Pro Arg Val Ala Met Cys Leu

1 5 10 15

<210>45

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>45

Leu Ile Phe Arg Tyr Ala Pro Pro Pro Leu Phe Leu Arg Pro Pro

1 5 10 15

<210>46

<211>36

<212>DNA

< 213>artificial

<220>

< 223>the DNA+ chain of encoded peptide Pc87

<400>46

agctagcaga tagaccatca atgtcaccaa catagt

36

<210>47

<211>36

<212>DNA

< 213>artificial

<220>

< 223>the DNA-chain of encoded peptide Pc87

<400>47

ctagactatg ttggtgacat tgatggtcta tctgct

36

<210>48

<211>611

<212>PRT

< 213>artificial

<220>

< 223>peptide Pc87 at random

<220>

< 221>signal

<222>(1)..(1)

< 223>Mat-α secretion sequence

<220>

< 221>domain

<222>(1)..(1)

< 223>cytokinin oxidase 1

<220>

< 221>domain

<222>(1)..(1)

< 223>connector

<220>

< 221>domain

<222>(1)..(1)

< 223>peptide Pc87 at random

<400>48

Met Arg Phe Pro Ser Ile Phe Thr Ala Val Leu Phe Ala Ala Ser Ser

1 5 10 15

Ala Leu Ala Ala Pro Val Asn Thr Thr Thr Glu Asp Glu Thr Ala Gln

20 25 30

Ile Pro Ala Asp Ala Val Ile Gly Tyr Ser Asp Leu Glu Gly Asp Phe

35 40 45

Asp Val Ala Val Leu Pro Phe Ser Asn Ser Thr Asn Asn Gly Leu Leu

50 55 60

Phe Ile Asn Thr Thr Ile Ala Ser Ile Ala Ala Lys Glu Glu Gly Val

65 70 75 80

Ser Leu Glu Lys Arg Leu Ala Ala Gly Thr Pro Ala Leu Gly Asp Asp

85 90 95

Arg Gly Arg Pro Trp Pro Ala Ser Leu Ala Ala Leu Ala Leu Asp Gly

100 105 110

Lys Leu Arg Thr Asp Ser Asn Ala Thr Ala Ala Ala Ser Thr Asp Phe

115 120 125

Gly Asn Ile Thr Ser Ala Leu Pro Ala Ala Val Leu Tyr Pro Ser Thr

130 135 140

Gly Asp Leu Val Ala Leu Leu Ser Ala Ala Asn Ser Thr Pro Gly Trp

145 150 155 160

Pro Tyr Thr Ile Ala Phe Arg Gly Arg Gly His Ser Leu Met Gly G1n

165 170 175

Ala Phe Ala Pro Gly Gly Val Val Val Asn Met Ala Ser Leu Gly Asp

180 185 190

Ala Ala Ala Pro Pro Arg Ile Asn Val Ser Ala Asp Gly Arg Tyr Val

195 200 205

Asp Ala Gly Gly Glu Gln Val Trp Ile Asp Val Leu Arg Ala Ser Leu

210 215 220

Ala Arg Gly Val Ala Pro Arg Ser Trp Asn Asp Tyr Leu Tyr Leu Thr

225 230 235 240

Val Gly Gly Thr Leu Ser Asn Ala Gly Ile Ser Gly Gln Ala Phe Arg

245 250 255

His Gly Pro Gln Ile Ser Asn Val Leu Glu Met Asp Val I1e Thr Gly

260 265 270

His Gly Glu Met Val Thr Cys Ser Lys Gln Leu Asn Ala Asp Leu Phe

275 280 285

Asp Ala Val Leu Gly Gly Leu Gly Gln Phe Gly Val Ile Thr Arg Ala

290 295 300

Arg Ile Ala Val Glu Pro Ala Pro Ala Arg Ala Arg Trp Val Arg Phe

305 310 315 320

Val Tyr Thr Asp Phe Ala Ala Phe Ser Ala Asp Gln Glu Arg Leu Thr

325 330 335

Ala Pro Arg Pro Gly Gly Gly Gly Ala Ser Phe Gly Pro Met Ser Tyr

340 345 350

Val Glu Gly Ser Val Phe Val Asn Gln Ser Leu Ala Thr Asp Leu Ala

355 360 365

Asn Thr Gly Phe Phe Thr Asp Ala Asp Val Ala Arg Ile Val Ala Leu

370 375 380

Ala Gly Glu Arg Asn Ala Thr Thr Val Tyr Ser Ile Glu Ala Thr Leu

385 390 395 400

Asn Tyr Asp Asn Ala Thr Ala Ala Ala Ala Ala Val Asp Gln Glu Leu

405 410 415

Ala Ser Val Leu Gly Thr Leu Ser Tyr Val Glu Gly Phe Ala Phe Gln

420 425 430

Arg Asp Val Ala Tyr Ala Ala Phe Leu Asp Arg Val His Gly Glu Glu

435 440 445

Val Ala Leu Asn Lys Leu Gly Leu Trp Arg Val Pro His Pro Trp Leu

450 455 460

Asn Met Phe Val Pro Arg Ser Arg Ile Ala Asp Phe Asp Arg Gly Val

465 470 475 480

Phe Lys Gly Ile Leu Gln Gly Thr Asp Ile Val Gly Pro Leu Ile Val

485 490 495

Tyr Pro Leu Asn Lys Ser Met Trp Asp Asp Gly Met Ser Ala Ala Thr

500 505 510

Pro Ser Glu Asp Val Phe Tyr Ala Val Ser Leu Leu Phe Ser Ser Val

515 520 525

Ala Pro Asn Asp Leu Ala Arg Leu Gln Glu Gln Asn Arg Arg Ile Leu

530 535 540

Arg Phe Cys Asp Leu Ala Gly Ile Gln Tyr Lys Thr Tyr Leu Ala Arg

545 550 555 560

His Thr Asp Arg Ser Asp Trp Val Arg His Phe Gly Ala Ala Lys Trp

565 570 575

Asn Arg Phe Val Glu Met Lys Asn Lys Tyr Asp Pro Lys Arg Leu Leu

580 585 590

Ser Pro Gly Gln Asp Ile Phe Asn Lys Leu Ala Asp Arg Pro Ser Met

595 600 605

Ser Pro Thr

610

<210>49

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>49

Ala Asp Pro Cys His Met Pro Pro Arg Met Pro Pro Leu Pro Ile

1 5 10 15

<210>50

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>50

Asn His Val Ser Thr Leu Lys Thr Arg His Arg Leu Ile Pro Phe

1 5 10 15

<210>51

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>51

Ser Ser Asn Ala Pro Pro Leu Ser Tyr Pro Pro Leu Leu Val Pro

1 5 10 15

<210>52

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>52

Thr Met Ala Arg Pro Ile Pro Thr Phe Leu Pro Pro Pro Ser Leu

1 5 10 15

<210>53

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>53

Thr Val Ala Pro Thr Thr His Arg His Tyr Val Trp Ser Met Asp

1 5 10 15

<210>54

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>54

Val Phe Thr Pro Met Asn Leu Ser Pro Pro Phe Met Gln Pro Pro

1 5 10 15

<210>55

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>55

Ala Ala Gly Pro Asn Ile Pro Pro Pro His Arg Ala Ser Thr Trp

1 5 10 15

<210>56

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>56

Ala His Leu Tyr Ser Gly Ala Ser Leu Tyr Arg Val Tyr Arg Ser

1 5 10 15

<210>57

<2ll>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>57

Gly Pro Pro Ser Ile Leu Leu Ala Ile Gly Thr Leu Ser Leu Thr

1 5 10 15

<210>58

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>58

Leu Ser Ser Pro Tyr Ala Cys Ala Leu Phe Val Val Lys Gly Ala

1 5 10 15

<210>59

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>59

Arg Gly Trp Ser Val Ser His His Ser Leu Leu Met Pro Val Pro

1 5 10 15

<210>60

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>60

Arg Ser Thr Ala Ser Pro Gln Ala Leu Asn Pro Leu Val Ala Ser

1 5 10 15

<210>61

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>61

Ser Leu Phe Phe Glu Val Ser Arg Met Leu Val Arg Leu Leu Ser

1 5 10 15

<210>62

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>62

Ser Arg Trp Trp Arg Cys Val Thr Met Thr Gln Pro Cys Thr Thr

1 5 10 15

<210>63

<211>15

<212>PRT

< 213>artificial

<220>

< 223>peptide embolus at random

<400>63

Val Val Ala Leu Arg Trp Gly Trp Ser Pro Leu Arg Pro Pro Gly

1 5 10 15

Claims (10)

1. polynucleotide; It comprises first coded sequence of coded plant defense peptides; Said peptide is selected from SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and combination thereof.

2. polynucleotide as claimed in claim 1, further the plant polypeptide of coding and the fusion of said plant defense peptides is to offer said plant defense peptides to target pathogen, and wherein said plant polypeptide is CKX family protein member.

3. polynucleotide as claimed in claim 2, the peptide of wherein said first coded sequence coding SEQ ID NO:50.

4. the recombinant vector that comprises the said polynucleotide of claim 1.

5. comprise the cell that claim 1 reaches polynucleotide, said polynucleotide have been introduced into said cell through transfection.

6. give the method for plant anti-fungal infection ability, comprising:

(a) with in the polynucleotide introduced plant; These polynucleotide comprise first coded sequence of coded plant defense peptides, and said plant defense peptides is selected from SEQ ID NO:49, SEQ ID NO:50, SEQ IDNO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, SEQ ID NO:62, SEQ ID NO:63 and combination thereof;

(b) make said plant defense peptides in said plant, express so that the function of said defense peptides performance anti-fungal infection.

7. method as claimed in claim 6, wherein said plant is a soybean.

8. method as claimed in claim 6, wherein said polynucleotide are introduced into said plant through conversion.

9. like the said method of claim 6; Wherein said polynucleotide also comprise the coded sequence of encoded peptide transmission framing scaffold; Said peptide transmission framing scaffold is a maize cell mitogen oxidase, and the expression of wherein said peptide transmission framing scaffold promotes the transmission and/or the displaying of said plant defense peptides.

10. method as claimed in claim 6, the peptide of wherein said first coded sequence coding SEQ IDNO:50.

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