CN1232502A - Antibodies against avirulence/pathogenicity proteins of plant pathogens - Google Patents
Antibodies against avirulence/pathogenicity proteins of plant pathogens Download PDFInfo
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- CN1232502A CN1232502A CN97194125.4A CN97194125A CN1232502A CN 1232502 A CN1232502 A CN 1232502A CN 97194125 A CN97194125 A CN 97194125A CN 1232502 A CN1232502 A CN 1232502A
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- C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
- C07K16/12—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria
- C07K16/1203—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from bacteria from Gram-negative bacteria
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
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Abstract
The subject invention pertains to materials and methods that provide plants with resistance to plant pathogens and pests. Antibodies and aptamers that immunoreact or bind and inhibit the action of protein expression products from avirulence and/or phatogenicity genes, including, but not limited to, the Xanthomonas avr/pth family of such genes, are described. The antibodies of the subject invention function by blocking the action of the primary protein products of avr/pth genes by intercepting and denaturing them prior to their translocation to the plant nucleus. The method of the subject invention concerns transforming a plant with polynucleotide molecules that encode the antibodies. Expression of the antibodies in the plant confers resistance from pathogens and pests. The subject invention also pertains to polynucleotide molecules encoding the subject antibodies, as well as plants and plant tissue transformed with the polynucleotide molecules encoding the subject antibodies.
Description
The present invention finishes under government supports, is subsidized by USDA, and license number is 86-CRCR-1-2234,58-43YR-5-3 and 58-7B30-3-465.Government has certain right in the present invention.
Background of invention
Nontoxicity (avr) gene is determined in many different microorganism phytopathogens, and predicted will the discovery in virus and insect.The avr gene is at first at nineteen forty-two (Flor, 1942) describe and be proved to be or hint by Flor and be present at least 27 kinds of different plant hosts/parasite systems, comprise rest fungus, ustilago, bunt, mildew, shot hole, nematode, insect, bacterium, virus, and even the caused disease of other plant (Day, 1974).
The avr gene often is a pathogenic gene.Opposite with toxicity, the idea that insect and pathogenic agent should be carried avirulent encoding gene is a mystery always, and has proposed to explain several different effective hypothesis (Gabriel and Rolfe, 1990) of this mystery.Recent work has shown that the whole family of avr gene also is pathogenic (pth) gene (Swarup etc., 1991,1992; Yang etc., 1994; Gabriel etc., 1993).This Xanthomonas campestris avr/pth gene family comprises the avr gene of the maximum number of having cloned at present and having checked order.
All member's codings of Xanthomonas campestris avr/pth gene family have the albumen (Yang and Gabriel, 1995) of nucleus signal for locating (NLSs).These genes have been proved the pathogenic of several different Xanthomonas campestris have been absolutely necessary.The function of the NLSs of genes encoding is to instruct protein to enter plant nucleolus.The summary of the supposition peptide sequence of having delivered of other known avr/pth gene discloses the NLSs that these genes are also encoded and supposed.Wherein foremost is the yellow branch of fungal pathogens born of the same parents' avr4 (Joosten etc., 1996), explains the hrpN (Wei etc., 1992) of starch Erwinia by oneself, and from hrpZ family (pathotype syringae, the glycinea and the tomato of pseudomonas syringae; Preston etc., 1995).The prototype Avr/Pth albumen that carries NLSs is PthA and Avrb6, and it has represented Xanthomonas campestris avr/pth gene family.Recently, use pthA and avrb6 to prove, citrus channel (pthA) and fusarium wilt of cotton symptom need the NLSs (Yang and Gabriel, 1995 and the Gabriel laboratory, do not deliver) of these genes encodings respectively.In addition, avr Bs3 (the Bonas etc. that delivering, 1989), avrXal (Hopkins etc., 1992) dna sequence dna and in this laboratory of Florida university to avrB4, pthB, pthC, in all dna sequence dnas of not delivering that obtain in the work of pthP and pthN, can observe NLSs much at one.In fact, in all members of the Xanthomonas campestris avr/pth gene family of being surveyed, the NLS coding region is guarded.The Asia citrus channel disease of having delivered of not delivering with this laboratory that studies show that needs pthA or homologue (Swarup etc., 1992), False citrus channel disease needs pthB or homologue, Mexico Lime cancrosis needs pthC or homologue, bacteroidal willow channel disease needs pthC or homologue, and common beans blight needs pthP or homologue (Yang etc., 1996).Known avrXa7 also is a member of Xanthomonas campestris avr/pth gene family, and it helps pathogenic (the bacterium blight of rice of rice Xanthomonas; Leach etc., 1996).By our expansion to pthA and avrb6 working result, seem at all known avr/pth genes to be pathogenic required or help pathogenic (citrus channel, the fusarium wilt of cotton, the bacterium blight of rice, the willow channel, form of ownership with the common bacteria blight) in the example, all needs from pathogenic agent, to export, import vegetable cell and be transported to plant nucleolus.In addition, attack the many of its host, if not maximum microorganisms, fungi, virus and even insect pest pathogenic as if give the credit to the steps necessary that albumen is imported into plant nucleolus.
(WO 91/15585,1991) such as De Wit has described the method for protective plant opposing pathogenic agent, and this method is incorporated in the Plant Genome that comprises the corresponding resistant gene by the polymerized nucleoside acid sequence of the proteic non-toxic gene of special initiation of will encoding.Non-toxic gene is so that genetic expression occurs over just the mode of the position of pathogenic infection plant regulates and control.The purpose of De Wit method is to come the activated plant defensive raction by non-toxic gene being provided and making it transcribe and translate into active protein product.In De Wit method, there are not explanation or suggestion non-toxic gene or its product to be checked or to disturb.On the contrary, the objective of the invention is to help under the pathogenic situation of condition to suppress or to check the normal function of non-toxic gene product at the non-toxic gene product.
The transfer of gene in plant of the antibody of the anti-effectively specific target virus of coding described.Tavladoraki etc. (1993) have at first confirmed the effective use of resistant to viral disease antibody in plant.But, do not have explanation or suggestion in the art in plant, expressing to phytopathogen nontoxicity or the immunoreactive antibody of pathogenicity proteins.
Show as antibody, nucleic acid and albumen often have identical high-affinity and a molecular specificity level with other molecule bonded ability.A kind of brand-new genetic technique develops in conjunction with the ability of the nucleotide sequence of feature (similar to antibody) around separate extremely rare tool sepcific ligands from big stochastic sequence storehouse.Method therefor is to repeat to select and the amplification scheme, is called SELEX sometimes and (refers to the phylogeny by the part of index concentration; With reference to Tuerk﹠amp; Gold[1990]; Gold[1995]).The tool sepcific ligands that screens in conjunction with the molecule of feature be called as " aptamers " (from Latin aptus, assembling; With reference to Szostak, J.W.1992).Though term aptamer is used for describing nucleic acid molecule at first, it is applicable to that too albumen is (with reference to Tuerk﹠amp; Gold, 1990; Colas etc., 1996).
Described can the arrestin function coding RNA or the gene (Conrad etc. [1996] of albumen aptemers (can combine) with the target protein high-affinity; Colas etc. [1996]).For target protein bonded specificity, the function of aptemers is similar to antibody function.Aptemers does not express in plant and does not have explanation or suggestion to express in plant at the aptamers in conjunction with phytopathogen nontoxicity or pathogenicity proteins in the art.
The invention summary
The theme invention relates to the antibody of expressing in the plant, and wherein antibody is to the immune response of the avr/pth of phytopathogen and insect elementary (albumen) gene product.The Avr/Pth albumen of these genes encodings typically comprises NLSs, and it plays an important role instructing albumen to enter in the plant nucleolus.Avr/Pth albumen is output actively from the pathogenic agent of invading, and is input to plant cytoplasm, and is transported to plant nucleolus.By with the anti-Avr/Pth antibodies of having expressed, the easy inactivation of Avr/Pth albumen, thereby the steps necessary that disease produces in the blocking-up plant.
The theme invention also relates to the nucleic acid molecule of encoding antibody or aptemers, and antibody and aptemers are attached to Avr/Pth albumen or contain on the albumen of NLSs.
The theme invention also relates to the method that protective plant is not subjected to the pathogenic organisms infringement.Avr/pth gene or coding NLSs expression of gene provide in plant comprising any bacterium, fungi, virus, the resistance of insect or pathogenic required other biological pathogenic agent.
The theme invention also relates to antibody multimer nucleic acid molecule plant transformed and the plant tissue with coding anti-Avr/Pth of the present invention and aptemers.Another aspect of the present invention is the plant and the plant tissue of expressing anti-Avr/Pth or anti-Avr/Pth aptemers antibody.The accompanying drawing summary
Fig. 1 represents the Western trace with the thick molten thing of Xanthomonas campestris cell of the anti-PthA antiserum(antisera) of rabbit polyclonal mark.Swimming lane 1, oranges and tangerines Xanthomonas campestris strain 3213 (Gabriel etc., 1989); Swimming lane 2,3213, sign-sealing derivative (Yang etc., 1995) of Xcl.2 (pthA ∷ npt-sac); Swimming lane 3, xanthomonas campestris 3048 (Gabriel etc., 1989); Swimming lane 4, transconjugant 3048/pZit 45 (PthA
+) (Swarup etc., 1992).The derivative that PthA or its block represented in asterisk.Roughly top band is dual in the swimming lane 1.The sequence summary
SEQ ID No.1 invents operable PCR primer according to theme.
SEQ ID No.2 invents operable PCR primer according to theme.
SEQ ID No.3 invents operable PCR primer according to theme.
SEQ ID No.4 invents operable PCR primer according to theme.
SEQ ID No.5 invents operable PCR primer according to theme.
SEQ ID No.6 invents operable PCR primer according to theme.
SEQ ID No.7 invents operable PCR primer according to theme.
SEQ ID No.8 invents operable PCR primer according to theme.Detailed Description Of The Invention
The theme invention relates to and the immunoreactive antibody of the protein product of avr/pth gene.Typically, the avr/pth gene product has nucleus signal for locating (NLSs), and its function is to instruct gene product to enter plant nucleolus.The antibodies of theme invention is to by on the Avr/Pth albumen of aptamers and avr/pth genes encoding and make its inactivation, so prevention albumen is transported to plant nucleolus.The antibody and the aptamers of theme invention also can be used for purifying Avr/Pth albumen or be used to identify the proteic immunohistology research of Avr/pth.The avr/pth gene product that is comprised in the invention scope comprises all viruses, microorganism, insect, the avr/pth gene of fungi and plant origin.
By using Avr/Pth albumen, or its fragment, or the suitable host animal of NLS peptide immunization, anti-Avr/Pth or anti-NLS antibody can be produced.In preferred embodiments, inoculate animal with the total length Avr/Pth protein immunization of purifying.On the other hand, can be with the amino of corresponding full-length proteins, carboxyl or middle partly immunization animal.Can be from suitable pathogenic agent such as Xanthomonas campestris purifying or clone's albumen or peptide, perhaps with peptide synthesizer or same way as synthetic proteins or peptide.Also can produce antibody (Yang, Y. and Gabriel, D.W., 1995) at containing the peptide of NLS aminoacid pattern as: K-R/K-X-R/K.
From the antibody secreting cell of immunization animal, produce hybridoma according to standard method.Produce hybridoma and monoclonal antibody method in this area well-known (Kohler and Milstein, 1995).The hybridoma of the antibody of screening generation and Avr/Pth albumen and/or NLS reactive polypeptide.In order to determine antibodies to proteic which zone, the avr/pth gene that the available constraints enzyme is handled the clone produces different gene fragments.These may corresponding genes 5 ', 3 ', or the fragment of region intermediate can be by subclone, expression with dried antibody screening program.Preferably, screening has the highest antibody in conjunction with affinity to albumen.
Use the polymerized nucleoside acid sequence of standard molecular biological technique clones coding anti-Avr/Pth antibody of the present invention from selected hybridoma.Typically, separating mRNA and reverse transcription become cDNA from the hybridoma of being selected.In preferred embodiments, by using polymerase chain reaction (PCR) and, from the polymerized nucleoside acid sequence of the cDNA sequence amplification encoding antibody of selected hybridoma to the special PCR primer of immunoglobulin variable light chain and heavy chain district.Then, clone's antibody gene sequence is integrated in the suitable expression vector and is used for transforming plant or plant tissue.Method with heterologous gene conversion plant is well-known in the art.In preferred embodiments, with several different antibodies gene-transformed plants or plant tissue, plant can produce antibody and can be incorporated into and be selected from Avr/Pth albumen amino like this, on the site of carboxyl and region intermediate.Can select induction type ground or composing type ground to express anti-Avr/Pth antibody according to used expression system in the theme invention plant transformed.The antibody of expressing in the plant also is called " plantibody " herein.The effect of the main antibody that plant transformed can produce by plant prevents to express the causal organism invasion of avr/pth gene, and wherein " plantibody " is attached on the protein product of avr/pth gene, thus the function of arrestin product.
The antibody of theme invention can use any animal that has suitable fusion partners clone, prepares from hybridoma.In the embodiment of enumerating, produce hybridoma with mouse lymphocyte.Other animal is such as rat, monkey, sheep, rabbit, and goat and pig also can be used for producing hybridoma.
Term used herein " antibody " is meant immunoglobulin (Ig) or its fragment that is attached on antigen or the haptenic epitope.In a preferred embodiment of the invention, antibody be divalence and comprise immunoglobulin variable light chain (V at least
L) and variable heavy chain (V
H).The theme invention also comprises the unit price form of antibody.
Term used herein " aptamer " is meant Nucleotide or the albumen that has with protein target molecule high affinity and specific binding capacity.
Each member's of measured so far Xanthomonas campestris avr/pth gene family predicted amino acid sequence identity is greater than 95%; Therefore, can easily prepare according to theme invention can be in conjunction with majority, if not all, and the proteic antibody of avr/pth gene family.Any Xanthomonas campestris Avr/Pth albumen can be suppressed by antibody with other pathogenicity proteins that enters vegetable cell and be within the scope of the present invention.The Avr/Pth albumen that the present invention includes can include, but not limited to AvrB4, Avrb6, Avrb7, AvrBs3, AvrXa7, AvrXa10, AvrB101, AvrB102, PthA, PthB, PthC, PthN, PthPC and PthP.
Another aspect of the present invention is the polynucleotide molecule that contains the nucleotide sequence of code book invention antibody or aptamers.Polynucleotide molecule can be made up of RNA or DNA.Preferably, polynucleotide molecule is made up of DNA.Theme invention polynucleotide molecule can be incorporated in the suitable polymerized nucleoside acid vectors, and as clone or expression vector, all these are well-known to those skilled in the art.Those skilled in the art can select carrier, make in the plant theme polymerized nucleoside acid sequence transform and expression reaches at utmost.
The theme invention also relates to the method for opposing to the morbific biological protection plant of plant.Method mentioned herein comprises makes plant have the ability that produces antibody or aptamers, this antibody or aptamers are attached to and suppress to invade biogenic proteic function, and this albumen is to biology causes a disease or the generation disease is absolutely necessary by invading in plant.In preferred embodiments, with the polynucleotide molecule conversion plant of encoding antibody or aptamer, this antibody or aptamer can be incorporated on the Avr/Pth albumen of causal organism avr/pth genes encoding.In one embodiment, with a plurality of different polynucleotide molecules conversion plants of coding different antibodies and/or aptamers, this antibody and/or aptamers have the different binding specificity to target protein.
What theme was invented is with the antibody of coding theme invention and/or polynucleotide molecule plant transformed or the plant tissue of aptamers on the other hand.Any conversion plant or plant tissue make allogeneic dna sequence DNA be incorporated in the Plant Genome and express method that this DNA produces theme antibody or aptamer all within the scope of the present invention in vegetable cell.The plant of theme invention comprises unifacial leaf and dicotyledonous kind.General those skilled in the art can easily select and utilize specific vegetation type and optimal clone of kind and method for transformation.Plant tissue in the scope of the invention can be exemplified as, but is not limited to protoplasma, vegetable cell, plant seed and seedling tissue.
Below be that explanation is used to implement the material of theme invention and the embodiment of method.These embodiment should not think to limit the detailed record of not preparing as material of the present invention and all possible modification of method.The preparation of embodiment 1 antibody
Be cloned into the pET-19b expression vector (Novagen, Inc., Madison, pthA encoded protein product WI) is purified as immunogen, produces the antibody be attached on the Avr/Pth albumen.The pET-19b carrier produces the gene fusion product of the translation of the terminal leader sequence of N-with 10 histidine residues, and these histidine residues are as affinity purification " mark ".Mixed the divalent cation (Ni that does not move by use
++) the support resin, the affinity chromatography purifying PthA albumen of " Histidine in conjunction with " resin (Novagen).Albumen elutriant from affinity column is further purified by protein electrophoresis on the preparation sds page, downcuts about 130kDa band and electroelution albumen from gel.Electroelution albumen from preparation SDS/PAGE gel is used for preparing the antibody special to PthA albumen.
Inoculate rabbit to produce the polyclonal antibody of anti-PthA with the PthA protein immunization of purifying.For the Western trace, the Xanthomonas campestris cell is grown in the PYGM nutrient solution, and centrifugal cell harvesting also boiled 2 minutes in sample buffer (50mM Tris-HCl, pH6.8,10% glycerine, 2%SDS, 0.1% tetrabromophenol sulfonphthalein).Lysate is 11, centrifugal 5 minutes of 600xg, and electrophoresis on the 8%SDS-polyacrylamide gel, and isolating albumen is transferred on the nitrocellulose filter.Film is surveyed with anti-PthA antibody and is developed the color with alkaline phosphorus enzyme enzyme link coupled goat-anti-rabbit antibody (Sigma Chemical Co.).Fig. 1 represents the Western trace with the Xanthomonas campestris cell lysate of the polyclonal antibody labeling of the anti-PthA of rabbit.
In another embodiment, use the PthA albumen of the histidine mark of purifying as described above, can produce 5 ' end of anti-pthA gene product, the mouse monoclonal antibody of 3 ' end and repeating unit.The mouse hybridoma reacts with the proteic different piece of PthA earlier and screens.Clone and the expression among pET-196 of pthA5 ' end from original promotor to Stu I site; The peptide of 240 amino acid whose suppositions of its coding.Encode respectively 660 amino acid and 260 amino acid whose supposition peptides the pthA gene from the Stu I to Hinc II site the middle part part and from 3 ' end of Hinc II, the translation product of the fusion that also can be cloned into respectively in the suitable pET carrier and express.Thick leach protein goods can be expressed and therefrom obtain to these three kinds of histidine mark genetic fusants in intestinal bacteria or other appropriate host.Then protein product is used for the hybridoma that ELISA detects the antibody that screens secretion and fusion protein immunization reaction.Optionally, protein product is purifying on " Histidine combination " resin, is used for antibody screening then.Shown PthA proteicly or amino by ELISA screening, or carboxyl or region intermediate have the antibody of kickback to separate dna sequence dna with clones coding antibody.The embodiment preparations with dna sequence dna aptamer 2 encoding antibodies
According to ELISA and Western trace The selection result, produce with PthA proteic or amino, or carboxyl, or the hybridoma of the monoclonal antibody of region intermediate reaction can be used as the source of antibody gene, this antibody gene is used for transforming plant or plant tissue.The method of using is deferred to Danielsson﹠amp; The method of Borrebaeck (1992) is incorporated herein by reference.
The hybridoma of being selected ties up in the substratum growth and (Biotecx Labs Houston) extracts whole RNA with Ultraspec RNA separation system.Behind the quantitative RNA of spectrophotometer, with the cDNA copy of reversed transcriptive enzyme and the synthetic mRNAs of widow (dT) primer.Then, use is increased corresponding to the special cDNA of immunoglobulin (Ig) (Ig) mRNA light chain and heavy chain in conjunction with the polymerase chain reaction (PCR) of following PCR primer, and these PCR primers have designed the special antibody gene of mouse that is used for increasing, and (following primer sequence is from 5 ' to 3 ' expression; The restriction site underscoring):
Amplification variable heavy chain (V
H) primer of conserved regions:
5 ' primer (SEQ ID NO.1)
(C/G)AG?GT(C/G)(A/C)A(A/G)CTG?CAG(C/G)AG?TC(A/T)GG?3′
PstⅠ
3 ' primer (SEQ ID NO.2)
TGA?GGA?GAC?GGT?GAC?CGT?GGT?CCC?TTG?GCC?CC?3′
BstEⅡ
The primer of variable light chain (VL) conserved regions that increases
5 ' primer (SEQ ID NO.3)
GAC?ATC?GAG?CTC?ACC?CAG?TCT?CCA?3′
SacⅠ
3 ' primer (SEQ ID NO.4)
CCG?TTT?CAG?CTC?GAG?CTT?GGT?CCC?3′
XhoⅠ
Selectable 3 ' primer (SEQ ID No.5 and SEQ ID NO.6)
If (can not use when obtaining the PCR product) with first 3 ' primer
5′CCC?GAA?TTC?TTA?GAT?CTC?CAG?CTT?GGT?CCC?3′(SEQ?ID?NO.5)
5′CCC?AAG?CTT?GAC?ATT?GTG?ACC?CAG?TCT?CCA?3′(SEQ?ID?NO.6)
With standard pcr amplification antibody cDNA.Preferably the antibody gene of expressing is formed bivalent molecule.(Holliger etc., 1993) are cloned and expressed to the bivalent antibody fragment in bacterium.Making up the bivalent antibody fragment makes the C-terminal N-terminal direct and light chain in variable heavy chain district merge; Therefore, these two ends are impossible from connection.At V
HAnd V
LBehind the segmental first time pcr amplification, determine the correct dna sequence dna of fragment end by the PCR order-checking.Carry out the pcr amplification second time in order to correct sequence information for basic primer.Primer also can mix special restriction restriction enzyme site to be simplified to pET-226 (Novagen, Madison, Wisconsin) transcribe/clone in the expression vector, and Kozak is provided conserved sequence, ATG translation initiation site and a terminator codon in the reading frame: the 2nd takes turns V
H5 ' primer (SEQ ID NO.7), 5 ' TCG GAT CCG GAA ACC ATG (C/G) AG GT (C/G) is A (A/G) CTG CAG (C/G) AGTC (A/T) GG 3 ' (A/C)
BamH I Kozak Pst I the 2nd is taken turns V
L3 ' primer (SEQ ID NO.8), 5 ' TCC CCG GGT ACC TCA CCG TTT CAG CTC GAG CTT GGT CC3 '
The initial used identical V of pcr amplification of Sma I Kpn I * * * Xho I * * * translation stop codon
H3 ' and V
L5 ' primer also can be used for the 2nd and takes turns pcr amplification.
Line style V
H-carrier-V
LConstruct is mended through Klenow and gentle flat terminal the connection is formed final closed loop molecule.The BamH I can be cloned into again in the suitable expression vector and according to known technology to Xho I restriction fragment and be used to transform plant then.For example, Bam H I can be cloned among the coli expression carrier pET-226 to Xho I restriction fragment, form reading frame endomixis body, this syzygy coding peIB leader sequence (being used for exporting) and a protein carboxyl groups end (being used for protein purification) polyhistidine tag from intestinal bacteria.After in pET-226, producing gene fusion, available Nde I/Xho I will comprise that the whole construct of pel B leader sequence is cloned among the pQY29.2 again, in providing a leader sequence, this sequence allows little anti-Avr/Pth antibody peptide to output to plant cell wall.For the bacterial gene for any clone provides plant promoter, this laboratory has made up carrier pQY29.2 (Yuan and Gabriel do not deliver).In this respect, any suitable carriers that contains plant promoter and transcription terminator and polyadenylic acid signal equally reasonably well satisfies.Carrier pQY29.2 is at an ATG translation initiation codon and have the polylinker of an open reading frame that inserts for the clone from the downstream of the 35s promotor of cauliflower mosaic virus.3 ' transcription terminator of a rouge alkali synthetase and polyadenylic acid signal are positioned at the opposite side of polylinker.Carrier be designed with the 2nd plasmid pGN 100 (Bogusz etc. that carry a function gus reporter gene, 1990) merge, and final and the 3rd plasmid pGA472 (An etc., 1985) merges and is provided for the border, the left and right sides of the neomycin resistance gene and the T-DNA of Plant Transformation.Last construct is transferred among the Agrobacterium tumefaciens strain AGL1 (Lazo etc., 1991) from intestinal bacteria by the bacterium joint with standard method.Because BamH I and Kpn I site are single on pQY29.2, the VH of amplification and VL polynucleotide can be to insert with respect to the promotor of pQY29.2 plasmid and the correct direction of 3 ' transcription terminator of rouge alkali synthetase.Having in the pQY29.2 carrier of mobile pel B leader sequence polynucleotide constructs also can prepare and be used for transforming plant.
Picture Turk﹠amp; Gold (1990); Szosbak (1992); Gold (1995); (1996) such as Colas etc. (1996) and Conrad are described, and the method for producing and select aptamers has been well-known in this area; Each method is hereby incorporated by.The DNA of the anti-Avr/Pth antibody of embodiment 3 usefulness coding transforms plant
Use standard material and method, with polynucleotide molecule conversion plant and the plant tissue of immunoreactive anti-Avr/Pth antibody of coding or aptamers.For example, Agrobacterium tumefaciens have been widely used for transforming plant (Smith etc., 1995 is described) with allogeneic dna sequence DNA.Allogeneic dna sequence DNA is integrated in the Ti-plasmids of Agrobacterium tumefaciens and plasmid is led back in the bacterium again.Typically by wound location inoculation, want plant transformed then with infectation of bacteria at plant or plant tissue.The Ti-plasmids that will contain allogeneic dna sequence DNA is then transferred in the plant nucleolus, and the DNA that has shifted in nucleus is integrated in the host cell genome.
Method (the Lorz etc. that plant and plant tissue also can absorb with the protoplasma as allogeneic dna sequence DNA, 1985) and by the fine-grained particles that has wrapped the DNA that will be imported plant with high speed bombard (Klein etc., 1998) transform plant and plant tissue, the partickle bombardment method is particularly suitable for and is widely used in monocotyledons and transforms the particularly conversion of grass.
Be to be understood that embodiment described herein and embodiment only are for illustrative purposes, various preferred plans, modification or will be proposed to those skilled in the relevant art and be included in the spirit and scope of this application and in the additional claim scope according to its variation.
Quote in full following reference herein as a reference.Reference
An, the European molecular biology magazine of G. etc. (1985) " the new cloning vector that is used for the higher plant conversion ", 4:277-284.
Bogusz, D. etc. (1990) " it is specific expressed that the non-leguminous plant hemoglobin gene keeps organ in the heterologous transgene plant " vegetable cell 2:633-641.
Bonas, the common molecular genetics 218:127-136 of U. etc. (1989) " non-toxic gene of the pathogenic mutation of the yellow born of the same parents bacterium of bird rape capsicum spot disease, the heredity of avrBs3 and constitutional features ".
Colas, P. waits the natural 380:548-550 of (1996) " heredity of the peptide aptamers of the kinases 2 of identification and inhibition cyclin dependent is selected ".
Conrad, R.C. waits (1996) " external selection of protein-bonded nucleic acid aptamers " Enzymology method 267:336-367.
Danielsson, L., C.A.K.Borrebaeck (1992) " from the Ig variable region DNA of unicellular amplification rearrangement " antibody engineering: practical guide, 89-102 page or leaf.
Day, the genetics of P.R. (1974) host-parasite mutual relationship, W.H.Freeman and Co., San Francisco.
Flor, H.H. (1992) " pathogenic heredity in flax grid rust " plant pathology 32:653-669.
Gabriel, the toxic molecule mechanism of D.W. etc. (1993) " virulent gene that the host of Xanthomonas campestris is special " bacterium, 141-158 page or leaf.
Gabriel, D.W., B.G.Rolfe (1990) " working model of specific recognition in the plant-microorganism mutual relationship " plant pathology annual report 28:365-391.
Glod, L. (1995) oligonucleotide of diagnosis and therapeutant " be used to study, " journal of biological chemistry 270:13581-13584.
Holliger, P. etc. (1993) " disome: little divalence and bispecific antibody fragment " institute of NAS reports 90:6444-6448.
Hopkins, the molecular biology 5:451-459 of C.M. etc. (1992) " evaluation of the non-toxic gene family of the pathogenic mutation of rice Xanthomonas paddy rice " plant-microorganism mutual relationship.
Joosten, the natural 367:384-386 of M.H.A.J. etc. (1994) " host is owing to single base pair in the non-toxic gene changes the resistance of forfeiture to the tomato fungal pathogens ".
Klein, T.M. etc. (1988) " influence the factor that gene enters maize cell by high speed particle " Bio/Technology 6:559-563.
Kohler, G., the natural 256:495-497 of C.Milstein (1975) " cultured continuously of the fused cell of the predetermined specific antibody of secretion ".
Lazo, G.R. etc. (1991) " the Arabidopsis gene library of the DNA transformed competence colibacillus in the edaphic bacillus " physiotechnology 9:963-967.
Leach, defensive raction induces D.Mills and H.Kunoh, eds. academic press, New York (in the printing) in J.E. etc. (1996) " the molecule aspect of pathogenic and host-resistance: the needs of the signal conduction " paddy rice.
Preston, the molecular biology 8:717-732 of G. etc. (1995) " pseudomonas syringae cloves mutation of causing a disease; the HrpZ albumen of glycinea and tomato is by the operon coding that contains Yersinia ysc homologue and at tomato, and in soybean, do not cause anaphylaxis " plant-microorganism mutual relationship.
Smith, R.H., E.E.Hood (1995) " monocotyledonous Agrobacterium tumefaciens transform " farm crop science 35 (2): 301-309.
Swarup, S. etc. (1991) " oranges and tangerines Xanthomonas campestris pathogenic gene seat makes the pathogenic mutation bacterial strain of several xanthomonas campestrises can cause the damage of cancer sample on citrus " plant pathology 81:802-809.
Swarup, the molecular biology 5:204-213 of S. etc. (1992) " a kind of oranges and tangerines Xanthomonas campestris pathogenic gene, pthA, the autonomous nontoxicity of multiple-effect ground coding on non-host " plant-microorganism mutual relationship.
Szostak, J.W. (1992) " external genetics " biological chemistry science trend 17:89-93.
Tavladoraki, the natural 366:469-472 of P. etc. (1993) " express a kind of have the transgenic plant of the single-chain Fv antibody of function to protect virus attack specifically ".
Tuerk, C.and Gold, L. (1990) " phylogeny of the part by index concentration: the RNA part of phage T4DNA polysaccharase " science 249:505-510.
Ward, E.S (1992) " is host expresses and antibody purification fragment with intestinal bacteria " antibody engineering: practical guide, 121-137 page or leaf.
Wei, Z.M. etc. (1992) " separate the anaphylactoid primosome that the starch Erwinia produces, Harpin by phytopathogen " science 257:85-88.
The molecular biology 7:345-355 of Yang.Y. etc. (1994) " the host specificity feature of the pathogenic mutation of the oranges and tangerines Xanthomonas campestris of leaf and xanthomonas campestris high mallow and release increase are respectively to be determined by the repeating unit of 102 base pairs of pthA and avr6 " plant-microorganism mutual relationship.
Yang, Y., D.W.Gabriel (1995) " reorganization provides the mechanism that the new host specificity develops in the gene of single phytopathogen gene " bacteriology magazine 177:4963-4968.
Yang, Y., the molecular biology 8:627-631 of D.W.Gabriel (1995) " Xanthomonas campestris nontoxicity/pathogenic gene family coding has the plant nucleolus target signal of function " plant-microorganism mutual relationship.
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PCT patent No. .91/15585 is published on October 17th, 1991.
Sequence table (1) essential information
(ⅰ) application information people
(A) applicant's title: University of Florida
(B) street name: 223 Grinter Hall
(C) city: Gainesville
(D) state/province's name: Florida
(E) country: US
(F) postcode (ZIP): 32611
(G) phone: (352) 392-8929
(H) fax: (352) 392-6600
(ⅱ) denomination of invention: the material and the method that suppress plant insect and pathogenic agent
(ⅲ) sequence number: 8
(ⅳ) contact address
(A) contact person: Saliwanchik﹠amp; Saliwanchik
(B) street name: 2421 N.W.41st street, Suite A-1
(C) city: Gainesville
(D) state name: Florida
(E) country: USA
(F) postcode (ZIP): 32606
(ⅴ) computer-reader form
(A) media type: floppy disk
(B) computer: IBM PC compatible
(C) operating system: PC-DOS/MS-DOS
(D) software: PatentIn Release#1.0, version #1.25
(ⅵ) present application materials:
(A) application number: US
(B) applying date: on March 22nd, 1996
(C) classification
(ⅷ) lawyer/proxy information
(A) title: Lloyd, Jeff
(B) registration number: 35,589
(C) reference/number of recording: UF-159C1
(ⅸ) telecom information
(A) phone: (352) 375-8100
(B) fax: (352) 372-5800 (2) is about the information of SEQ ID NO:1
(ⅰ) sequence signature
(A) length: 22 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:1NAGGTNNANC TGCAGNAGTC NGG 23 (2) information about SEQ ID NO:2
(ⅰ) sequence signature
(A) length: 32 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:2TGAGGAGACG GTGACCGTGG TCCCTTGGCC CC 32 (2) information about SEQ ID NO:3
(ⅰ) sequence signature
(A) length: 24 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:3GACATCGAGC TCACCCAGTC TCCA 24 (2) information about SEQ ID NO:4
(ⅰ) sequence signature
(A) length: 24 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:4CCGTTTCAGC TCGAGCTTGG TCCC 24 (2) information about SEQ ID NO:5
(ⅰ) sequence signature
(A) length: 30 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:5
CCCGAATTCT TAGATCTCCA GCTTGGTCCC 30 (2) information about SEQ ID NO:6
(ⅰ) sequence signature
(A) length: 30 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:6CCCAAGCTTG ACATTGTGAC CCAGTCTCCA 30 (2) information about SEQ ID NO:7
(ⅰ) sequence signature
(A) length: 41 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:7TCGGATCCGG AAACCATGNA GGTNNANCTG CAGNAGTCNG G 41 (2) information about SEQ ID NO:8
(ⅰ) sequence signature
(A) length: 38 bases
(B) type: nucleic acid
(C) chain: strand
(D) topological framework: line style
(ⅱ) molecule type: DNA (synthesizing)
(ⅹ ⅰ) sequence description: SEQ ID NO:8
TCCCCGGGTA?CCTCACCGTT?TCAGCTCGAG?CTTGGTCC 38
Claims (15)
1. antibody, aptamer or its Fab, wherein said antibody or aptamer are attached on the nontoxicity or pathogenicity proteins of phytopathogen.
2. the antibody of claim 1, wherein said albumen comprises a NLS at least.
3. antibody according to claim 1 or aptamer, wherein said albumen is by the genes encoding that is selected from the colony that comprises avr and pth gene.
4. the antibody of claim 3 or aptamer, wherein said gene is selected from the colony that comprises Xanthomonas campestris avr and pth gene.
5. antibody according to claim 1 or aptamer, wherein said albumen are selected from and comprise Avr4, hrpN, hrpZ, AvrB4, Avrb6, Avrb7, AvrBs3, AvrXa7, AvrXa10, AvrB101, AvrB102 and PthA, PthB, PthC, PthN, the colony of PthPC and PthP.
6. antibody according to claim 1 or aptamer, wherein said antibody comprises V
LAnd V
HImmunoglobulin domains.
7. antibody according to claim 1 or aptamer, wherein said antibody is monovalent or divalence.
8. antibody according to claim 1 or aptamer, wherein said antibody is expressed in vegetable cell.
9. the polynucleotide molecule that contains the nucleotide sequence of coding antibody according to claim 1 or aptamer.
10. a protective plant is avoided the method for phytopathogen or insect infringement, and this method comprises to be advanced in this Plant Genome the polynucleotide molecule of claim 9 is chimeric.
11. polynucleotide molecule plant transformed with claim 9.
12. plant according to claim 11, wherein said plant is a dicotyledons.
13. plant according to claim 11, wherein said plant is a monocotyledons.
14. polynucleotide molecule plant transformed tissue with claim 9.
15. plant tissue according to claim 14, wherein said plant tissue are selected from and comprise protoplastis, vegetable cell, plant seed and seedling tissue.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US62253896A | 1996-03-25 | 1996-03-25 | |
| US08/622,538 | 1996-03-25 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN1232502A true CN1232502A (en) | 1999-10-20 |
Family
ID=24494560
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN97194125.4A Pending CN1232502A (en) | 1996-03-25 | 1997-03-25 | Antibodies against avirulence/pathogenicity proteins of plant pathogens |
Country Status (6)
| Country | Link |
|---|---|
| EP (1) | EP0889960A1 (en) |
| CN (1) | CN1232502A (en) |
| AU (1) | AU2548597A (en) |
| BR (1) | BR9708258A (en) |
| IL (1) | IL126250A0 (en) |
| WO (1) | WO1997035980A1 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1114616C (en) * | 1999-08-10 | 2003-07-16 | 南京农业大学 | Exciter for inducing allergic reaction of plant and preparation method and use thereof |
| CN103210087A (en) * | 2010-09-06 | 2013-07-17 | 淡马锡生命科学研究院有限公司 | Molecular interaction between Xa10 and AvrXa10 |
| CN106973944A (en) * | 2017-05-09 | 2017-07-25 | 福建农林大学 | Applications of the TALE albumin As vrXa7 on c itrus canker disease is prevented and treated |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1056866A1 (en) * | 1998-02-25 | 2000-12-06 | Wisconsin Alumni Research Foundation | CULTIVAR SPECIFICITY GENE FROM THE RICE PATHOGEN $i(MAGNAPORTHE GRISEA), AND METHODS OF USE |
| FR2835665B1 (en) | 2002-02-04 | 2004-04-02 | Canon Kk | CODING AND DECODING OF DIGITAL SIGNALS |
| WO2004013321A1 (en) * | 2002-07-26 | 2004-02-12 | Degussa Ag | Esterase esta (xc4a) of xanthomonas vesicatoria |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP0648266B1 (en) * | 1992-07-01 | 2006-02-08 | Cornell Research Foundation, Inc. | Elicitor of the hypersensitive response in plants |
| US5981730A (en) * | 1994-04-13 | 1999-11-09 | The General Hospital Corporation | RPS gene family, primers, probes, and detection methods |
-
1997
- 1997-03-25 AU AU25485/97A patent/AU2548597A/en not_active Abandoned
- 1997-03-25 WO PCT/US1997/004924 patent/WO1997035980A1/en not_active Application Discontinuation
- 1997-03-25 IL IL12625097A patent/IL126250A0/en unknown
- 1997-03-25 BR BR9708258A patent/BR9708258A/en not_active Application Discontinuation
- 1997-03-25 EP EP97917024A patent/EP0889960A1/en not_active Withdrawn
- 1997-03-25 CN CN97194125.4A patent/CN1232502A/en active Pending
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1114616C (en) * | 1999-08-10 | 2003-07-16 | 南京农业大学 | Exciter for inducing allergic reaction of plant and preparation method and use thereof |
| CN103210087A (en) * | 2010-09-06 | 2013-07-17 | 淡马锡生命科学研究院有限公司 | Molecular interaction between Xa10 and AvrXa10 |
| CN103210087B (en) * | 2010-09-06 | 2015-04-29 | 淡马锡生命科学研究院有限公司 | Molecular interaction between Xa10 and AvrXa10 |
| CN106973944A (en) * | 2017-05-09 | 2017-07-25 | 福建农林大学 | Applications of the TALE albumin As vrXa7 on c itrus canker disease is prevented and treated |
Also Published As
| Publication number | Publication date |
|---|---|
| BR9708258A (en) | 1999-08-03 |
| EP0889960A1 (en) | 1999-01-13 |
| AU2548597A (en) | 1997-10-17 |
| WO1997035980A1 (en) | 1997-10-02 |
| IL126250A0 (en) | 1999-05-09 |
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