CN1370233A

CN1370233A - ERS-genes, method of screening for chemical compounds capable of inducing ERS in plants

Info

Publication number: CN1370233A
Application number: CN00809155.2A
Authority: CN
Inventors: K－H·克格尔
Original assignee: BASF SE
Current assignee: BASF SE
Priority date: 1999-05-21
Filing date: 2000-05-19
Publication date: 2002-09-18
Also published as: CA2370332A1; AU5674800A; IL146234A0; WO2000071748A2; WO2000071748A3; EP1179070A2; JP2003505013A; BR0010829A

Abstract

This patent application discloses new genes the expression of which is inducible by SAR inducing compounds, and in a particular embodiment by both SAR inducing compounds and ISR inducing compounds. These genes enable the establishment of novel methods for testing chemical compounds and natural products for their capability to induce an enhanced resistance status (ERS), and particularly SAR or ISR, in plants and for screening a multiplicity of chemical compounds, for compounds or compositions having the capability to induce ERS in plants. This disclosure also enables the use of recombinant DNA molecules comprising these DNA molecules in transgenic plants to confer enhanced tolerance to disease and pest organisms.

Description

ERS gene, screening can be in plant the method for the compound of inducing ERS

Background of invention

The present invention relates to a kind of detection compound and in plant, induce method at the ability of pathogenic bacteria enhanced resistance state (ERS), also relate to aforesaid method screening can be in plant purposes on the compound of inducing ERS.On the other hand, the invention still further relates to the dna molecular of coded polypeptide or its part, it is expressed in can be induced by the ERS inducing compounds and can use under their natural genotypic environments according to methods analyst of the present invention, the invention further relates to transgenic plant, the plant part or the seed that comprise said dna molecular are by the polypeptide/albumen of this dna molecule encode or its part and specific recognition with in conjunction with the antibody of this polypeptide/albumen or its part.

The enhanced resistance state (ERS) of plant is the result of biology and/or chemical induction resistance phenomenon, and this resistance phenomenon is particularly including systemic acquired resistance (SAR) and inductive system resistance (ISR) by the bacillus radicicola mediation.

At last decade, cogent data have been accumulated, be that salicylic acid (SA) (for example is absolutely necessary to the foundation of dicotyledons to the systemic acquired resistance of wide spectrum pathogenic bacteria, Yang etc., gene and growth 11, thereby cause the enhanced resistance state (ERS) of those plants 1621-1639 (1997)).

Opposite with SAR, cause other plant defense reaction that is summarized as term ISR of ERS, for example (plant pathology academic year comments 36 to the resistance of bacillus radicicola mediation, 453-483 (1998) and comprise proteinase inhibitor 1 and the wound response of the jasmonic of 2 genetic expressions mediation, be can not be by Whitfield's ointment or other SAR inducing compounds inductive, sometimes even be subjected to the inhibition (for example Doares etc., plant physiology 108,1741-1746 (1995)) of Whitfield's ointment or derivatives thereof.

United States Patent (USP) 5,614,395 disclose a kind of auxiliary method of screening the agricultural chemicals of inducing SAR down the genetic improvement plant that contains mosaic gene, and described mosaic gene produces reaction to the compound of inducing SAR, and it is disclosed as a kind of wheat cdna such as WCI-1.This method just is limited on the floristics to this mosaic gene sensitivity, especially is limited on the wheat.

International Patent Application WO 98/00023 has proposed a kind of by detecting the expression of plant defense gene such as PDF 1, the method of the active compound of screening induction of resistance, some genes disclosed herein are to be induced by phytopathogen such as Alternaria brassicicola or Botrytiscinera, but can not be induced the agricultural chemicals of SAR such as Whitfield's ointment or dichloro-isonicotinic acid to induce.

Up to now, the report and the reactivity of the corresponding gene pairs SAR of sequence of the present invention inducing compounds as yet.Thereby, also there is not motivation to remove to utilize the naturally occurring expression level of gene of the present invention or the ERS inducing compounds that the product screening comprises the SAR inducing compounds.

From EP 0 734 530, the embodiment of EP 0 816 309 and EP 0 818 431 learns, the generation of synthetic combinatorial libraries.Yet, just may screen this library up to now so that the compound that selection has medicinal character.Therefore, very desirable is a kind of high-throughout screening method of exploitation, to produce important compound as farming research, especially to screen the method for ERS inducing compounds.

Summary of the invention

The present invention is according to the most wonderful discovery: a kind of new gene separates existence with traditional pathogenic relevant (PR) gene, its expression can be induced by the SAR inducing compounds, in a specific embodiment, can induce by SAR inducing compounds and ISR inducing compounds.

This discovery can be set up some novel methods, in plant, induce the ability of enhanced resistance state (ERS), especially SAR or ISR with detection compound and natural product, and screen multiple compound, to obtain the compound or the composition that in plant, have the inducing ERS ability.This discovery makes it possible to use in transgenic plant the recombinant DNA molecules that comprises these dna moleculars to give plant to disease and pest organism enhanced tolerance.

The present invention relates to have following (A) to the dna molecular of (H) sequence; These sequence coded polypeptides or its part; its expression induced by ERS-induced compounds: A: (346bp) ACATGAAGAG CAGCGACGGC AAGGTCTACG ACTCCTTCAC CATCCACAGG 50GATTACCGCG ACGTGCTCAG CTGCGTGCAC GACAGCTGCT TCCCCACCAC 100GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200AGCTGCACAA TTTATATGTG CTAGTAAAAA GATCATGCAA GAGGTGGGTG 250 TATGCTCGTT ATATATGCTT TGTAACTCCT TCATGTCATA TTWTTATGGG 300 TTAATAAAAA CATCCTTTAT CAAAAAAAAA AAAAAaAAAA GCTTGT 350B: (292bp) ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50 GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100 GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGAA TGAAATCGTA 150 TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200 TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250 CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GCC: (387bp) ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50 GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100 ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150 AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200 CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250 MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300 GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350 TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGTE: (512bp) ACAGAAGCTT GGACAGTTCC ACTCGGAAGC TTCGGTTCGC GCTACCGTTC 50 CCGATGCTCG CCTACCCATT CTACTTGTGG TCAAGGAGTC CAGGGAAGTC 100 AGGCTCGCAT TTCCACCCGA GCAGCGATTT GTTCCAGCCG AACGAGAAGA 150 ACGACATACT GACGTCGACG ACATGCTGGC TTGCCATGGC TGGCCTGCTC 200 GCTGGGCTCA CTGCCGTGAT GGGCCCCCTT CAGATACTCA AGCTCTACGC 250 CGTCCCCTAC TGGATTTTTG TTATGTGGCT GGACTTTGTC ACCTACCTGC 300 ACCACCACGG CCACAACGAC AAGCTTCCCT GGTATCGCGG AAAGGCATGG 350 AGCTATCTGC GCGGGGGCCT GACAACGCTC GACAGGGACT ACGGGTGGCT 400 CAACAACATC CACCACGACA TCGGGACTCA CGTGATCCGC CATCTTTTCC 450 CGCAAATCCC GCATTACCAT CTAGTGGAGG CGACCGAGGC GGCGAACAGS 500 TGCTAGGGAA GTF: (484bp) ACAAGCTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGTGG TAAACAACAA 150 CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200 TTATTATACA AAAAATTGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250 TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300 TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350 TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400 AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGACAAA 450 ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500 TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550 ACTTTCTTCG CAAGATTGCC AACATAAACC TTGTG: (305bp) GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100 CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGACA 150 TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200 CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCC ACATTGGCTA 250 GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300 TAAGTH (1035bp) CAGGATCATA GCTACAGGCG ACAATGCCCG GTCATCGACA ATCGCTGGCA 50 GCGGCTTCTC TGAAGCTACC ATCTCTTCTG CTTCTGTGGA TCTTTAGCTG 100 GAACTGGGGG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATGACGGAGC 150 TTCAGAAACA TGTCTCCTTT TTCGACCGGA ACAAGGATGG CTTCATCACT 200 CCTACAGAAA CCATCCAAGG GTTTGTTGCA ATCGGTTGCG AGTATGCATT 250 TGCTACTGCT GCCTCTGCCG CCATTCACGG TGCCCTTGCT CCTCAAACAA 300 CCCCGGCTGG TACACCACTG CCTCACTTGA CAATATACGT AGAGAATATC 350 CACAAAGCTA TGCATGGAAG TGATCCAGGT GTATATGATG CCAAAGGAAG 400 GTTTCTTCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450 GACCAGATGC GTTGACTCTT GCGGAGATGC ATGTGATGCT CTTTGCAAAA 500 CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550 ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600 TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650 CACCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700 AGTTTGGATA TGGAAAGGTT TGTCCCGAAG AAGGTTTTCC TGCTATCTCC 750 AAATTCAACT AGAGTTTATT TTCTTTTCCT CCAAGTTGTA ATTGGTTTTA 800 TAAGACCTTC ATAGCCGATC AATACAACGA AGCAAGTTGG ATATATTTCC 850 CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATGAGC 900 GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950 GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKGAK GAWAAWATTT 1000 ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA The present invention also relates to a method for detecting compounds in plants inducing enhanced resistance state (ERS) the ability of the method; Said method comprises: (a) plant or plant part are contacted with compound; This plant or plant part comprise the genetic fragment with sequence (A) to (H) that expression can be induced by the SAR inducing compounds; This genetic fragment coded polypeptide / protein or its part: and (b) expression of the said genetic fragment of analysis, wherein the expression of said genetic fragment shows that said compound has the ability of inducing ERS....

In a specific embodiment, be can not be by the phytopathogen inductive by the expression of SAR inducing compounds inductive said gene fragment (A) to (H).In another preferred embodiment, the segmental expression of said gene also can be induced by the ISR inducing compounds in addition.

In addition, according to method of the present invention, at different floristics, such as grain such as wheat or barley, and screening ERS inducing compounds in the rice; The existence of the ERS inducing compounds in the working sample; The concentration of the ERS inducing compounds in the working sample; The relative capacity that compares two inducing compounds inducing ERS in plant; The ERS that changes compound with detection compound and chemical composition induces the ability of effect.

Another aspect of the present invention relates to the test kit that is used for the inventive method.

Another aspect of the present invention relates to a kind of being used for and is provided at the compound method that plant has the inducing ERS ability, comprises; (a) produce a kind of synthetic combinatorial libraries of different compounds and, screen the compound in above-mentioned library (b) according to screening method of the present invention.Another aspect of the present invention is a new compound, and it is obtainable under the help of this method, and new compound is as the purposes of agricultural chemicals.

The present invention relates to coded polypeptide/proteic dna molecular, its expression can be according to methods analyst of the present invention; About the fragment of above-mentioned dna molecular, this fragment can be used as probe or primer; Also relate to the microorganism transformed, transgenic plant, plant part or the seed that comprise above-mentioned dna molecular; Relate to the polypeptide of above-mentioned dna molecule encode and albumen and specific recognition and in conjunction with aforementioned polypeptides and proteic antibody.

These and other purposes of the present invention and feature will become very clear from the detailed description of hereinafter statement.

Detailed description of the preferred embodiments

The ability of now having found the ERS of compound inducing plant can detect by (a) with (b) effectively:

(a) plant or plant part are contacted with compound, this plant or plant part include by coded polypeptide/albumen of the present invention or and the part (A) to (H) gene fragment, its expression can be induced by the SAR inducing compounds, especially can be induced by SAR inducing compounds and ISR inducing compounds; With

(b) expression of the above-mentioned gene fragment of analysis.

By using aforesaid method, can screen a large amount of compounds and composition, have the compound and/or the composition of the ability of inducing plant ERS with evaluation.In a specific embodiment, for example, with combinatorial library such as EP 0 734 530, exemplify among EP 0 816 309 and the EP 0 818431 explanation combinatorial library form or with the form of the library of receiving natural compounds and/or composition or set or to contain the form in the library that natural compounds and synthetic compound and composition form, a large amount of above-claimed cpd and compositions can be provided.

Perhaps, aforesaid method also can be used in the working sample can be in plant the existence of the compound of inducing ERS, wherein superincumbent step (a) lining, sample with equivalent replaces compound contact plant or plant part, so, the segmental expression of said gene show in this sample, exist one or more can be in plant the compound of inducing ERS.

In a specific preferred embodiment, can be by SAR inducing compounds inductive and the preferably expression in above-mentioned plant by SAR inducing compounds and ISR inducing compounds inductive said gene fragment, can not be by naturally occurring pathogenic bacterium inducing.As if if plan to implement method of the present invention in the environment that phytopathogen exists, this feature is valuable especially.This will be in the greenhouse or the feelings row of field when using plant to implement method of the present invention.In these cases, above-mentioned feature will guarantee the existence of the segmental any expression of observed said gene owing to compound with inducing ERS ability in plant or composition.If in cell and tissue culture system, use this method according to the present invention, can utilize above-mentioned concrete feature, but and it is inessential.

In the application's context, term " naturally occurring phytopathogen " comprises pathogenic microorganism, especially plant pathogenic fungi, bacterium and the virus of all kinds.

Plant and plant part, the especially leaf, root and the seed that comprise all kinds in the term " plant " that above and hereinafter uses and " part of plant ".Above-mentioned plant or plant part preferably are selected from down group: plant protoplast, vegetable cell, plant tissue, callus tissue, the plantlet of growth, plant leaf, jejune whole strain plant, sophisticated whole strain Plants and Seeds.More particularly, above-mentioned plant or plant part, especially vegetable cell or plant leaf are or from monocotyledons, such as cereal, especially a kind of plant of wheat or Triticum, be the plant of Triticum more preferably, and paddy rice also can derive from dicotyledons.

The gene fragment of step " a " can be to have any gene fragment of sequence (A) to (H) or its part, and its expression can be induced by the SAR inducing compounds, is preferably induced by SAR inducing compounds and ISR inducing compounds.This definition comprises the gene fragment that any naturally occurring gene fragment that shows these characteristics and non-natural exist, its sequence and above-mentioned naturally occurring gene fragment and varient thereof and contain the above-mentioned gene fragment of one or more parts and the sequence of the gene fragment of varient has a large amount of homologys, wherein all these gene fragments and varient keep the characteristics show above, be that its expression is induced by the SAR inducing compounds, preferably induce by SAR inducing compounds and ISR inducing compounds.

As already mentioned above, in a specific embodiment of the present invention, the expression of said gene fragment or varient can not be by naturally occurring pathogenic bacterium inducing, the expression of these gene fragments or varient can be induced by the SAR inducing compounds, and preferably by SAR inducing compounds and ISR inducing compounds inductive.

In another embodiment, said gene fragment (A) to (H) is a kind of gene fragment, its nucleotide sequence show with nucleotide sequence (A) to (H), the perhaps very big homology of its varient, the above given nucleotide sequence that perhaps comprises one or more parts, the sequence or its variant that have substantial homology with it.Therefore, this term also comprise can with the sequence of the most of hybridization of top given nucleotide sequence, especially under the condition of low stringency.

Mean the replacement of naturally occurring gene fragment (A) one or more Nucleotide to the nucleotide sequence of (H) at the term " its varient " that above and hereinafter uses, variation, modify, insert, disappearance or interpolation, the expression of this gene fragment is induced by the SAR inducing compounds, preferably induce by SAR inducing compounds and ISR inducing compounds, for example, a kind of gene fragment that contains top given nucleotide sequence, if the expression of its varient also can be used the SAR inducing compounds and be induced, preferably induce by SAR inducing compounds and ISR inducing compounds.This term comprises the allelic variation body of abiogenous gene fragment (A) to (H) especially, and its expression is induced by the SAR inducing compounds, preferably by SAR inducing compounds and ISR inducing compounds inductive.This term also comprises synthetic varient, and it comprises or is made up of nucleotide sequence in essence, and these nucleotide sequences can be hybridized with naturally occurring parental gene fragment in large quantities.This hybridization is preferably carried out under the condition of low stringency, in another embodiment, carries out between low stringency and high tight degree condition, in unusual special embodiment, even carries out under the condition of the tight degree of height.As a kind of rule, the low stringency condition may be defined as 3 times SSC, and temperature is a room temperature to 65 ℃, and high tight degree condition is 0.1 times SSC, and temperature is 68 ℃.SSC is the sodium-chlor of 0.15M, the abbreviation of 0.015M trisodium citrate damping fluid.

At least comprise and the homology of parental gene sequence (A) at the phrase " a large amount of homologys " that above and hereinafter uses to the essential Nucleotide of (H), suppose that its expression also can be induced by the SAR inducing compounds, preferably induce by SAR inducing compounds and ISR inducing compounds.Typically, when 60% or more Nucleotide when being identical with naturally occurring parental gene fragment, promptly show as homology, being 65% more typically, being preferably 70%, preferably is 75%, even more preferably be 80% or 85%, particularly preferably be 90%, 95%, 98% or 99% or higher homology.

In one of the inventive method specific embodiment, the nucleotide sequence of coding indicator protein or polypeptide can be induced, preferably also be comprised by SAR inducing compounds and ISR inducing compounds inductive gene fragment (A) to (H) to its expression by the SAR inducing compounds, and this nucleotide sequence is fused to the coding region to the gene of arbitrary dna molecular of (H) with respect to (A).The result, this indicator protein or polypeptide expression are induced, are preferably induced by SAR inducing compounds and ISR inducing compounds by the SAR inducing compounds, and, when using the ERS inducing compounds, express simultaneously with the form of fusion rotein to the expression of the dna molecular of (H) with (A).In specific embodiment, above-mentioned nucleotide sequence coded glucuronidase, luciferase or the green fluorescent albumen (GFP) of coding indicator protein or polypeptide.In addition, indicator protein may be to give to the indefatigable enzyme of weedicide, as acetolactate synthestase.

Use SAR inducing compounds and the ISR inducing compounds measured according to method of the present invention can be summarized as general name " ERS inducing compounds ".This term comprises can provide plant to all types harmful organism all compounds with raising resistance feature, for example to harmful microorganism, resembles plant pathogenic fungi, and bacterium is with virus and to the insect such as insect, mite and nematode.As what set forth, it comprises and can the inducing plant system obtain resistance (SAR) and comprise all compounds that do not rely in the inducible system resistance (ISR) of salicylic defense mechanism.For example, by the dead reaction of activating cells (HR), the apposition of cell walls, the accumulation of PR genetic expression and/or plant protecting chemical, the ERS inducing compounds is the induction of resistance state in plant.

These application of compound cause processed plant to be protected, and avoid being subjected to harmful microorganism, plant pathogenic fungi for example, seriously the infecting of bacterium and virus and insect, mite and nematode.

The SAR inducing compounds comprises phenylformic acid, Whitfield's ointment, dichloro-isonicotinic acid, polyacrylic acid, aminobutyric acid, arachidonic acid and derivative thereof and the natural product that resembles harpin and other releaser.

Another group SAR inducing compounds is a phendioxin, 2, and the 3-thiadiazoles derivative for example resembles U.S. Pat 4,931, and 581 and US 5,229,384 is disclosed.

The reference compound of ISR inducing compounds is representative with jasmonic and its derivative such as methyl jasmonic.

Known ERS inducing compounds preferably is used as the reference compound that screens novel agrochemical.

By method of the present invention,, can easily measure indivedual members of the agrochemicals, particularly synthetic combinatorial libraries that comprise among other compound, especially the present invention by the analytical test compound.

The ERS inducing compounds, form that can be pure with solution or suspension, with pulvis or dust, or is used with other conventional dosage forms of using on the agricultural.These formulations can comprise solid or liquid vehicle, promptly with above-mentioned ERS inducing compounds bonded material, and promoting plant, tissue, the application on cell or tissue is cultivated, or improve storage, handle or transportation character.The example of suitable carrier comprises silicate, clay, resin, alcohol, ketone, aliphatics or aromatic series carbohydrate or the like.If make traditional wettable powder, aqueous suspension or emulsion concentrated solution, the formulation of ERS inducing compounds can comprise the tensio-active agent that one or more are traditional, ionic or non-ionic, as wetting agent, emulsifying agent or dispersion agent.

The ERS inducing compounds can be used with other formulation material individually or jointly, and for example, with adjuvant, weedicide, mycocide, sterilant, growth regulator or fertilizer use together.

As a kind of liquid dosage form, the ERS inducing compounds can be sprayed to the blade of plant, is sprayed on the seed or in the soil or in other growth or the cultivation medium before stem or branch or the sowing.

The ERS inducing compounds uses with certain amount, is enough to the inducing ERS effect after for some time.As a kind of rule, if be administered on the whole plants, compound is used with the concentration of every liter of soil volume 0.1 to 1000mg activeconstituents in the soak process of soil, perhaps sprays with the concentration of 0.1-100mg in every liter of spray solution.If in the cell or tissue culture systems, use, according to the volume of substratum, also can use lower concentration now.As a kind of index, 2 to 72 hours, especially enough detected reaction in 12 to 48 hours.

In principle; can come the reaction of monitoring plant with each traditional method that analyzing gene is expressed to compound effects; as Western trace or Northern engram analysis; for example; can can detect or monitor its gene by mensuration and be included in segmental indicator protein of said gene or polypeptide expression by a kind of SAR inducing compounds inductive gene its expression its enzymic activity.If this expression that must detect is tissue-specific, this analysis will be carried out in above-mentioned specific tissue.

If there is a kind of low expression level of composing type in the segmental part of said gene fragment or said gene, to under the non-existent situation of compound, detect its expression this plant or plant part, compare with the low expression level of above-mentioned composing type, the compound inductive is expressed the increase that will be measured as expression.The version of this detection method also is included in method of the present invention (b) step.

The expression analysis of said gene fragment (A) to (H) preferably carries out with the analysis of Northern trace type, especially use the cDNA of the gene fragment of digoxigenin labeled, for example, DNA with nucleotide sequence given in (A) to (H), or its fragment, most preferably be to use commercially available Northern trace test kit, the DIG High Prime labelling kit of for example German Boehinger Mannheim company.

In another preferred embodiment of the present invention, carry out the available reversed transcriptive enzyme-polymerase chain reaction of the expression of gene fragment (RT-PCR).

In another preferred embodiment of the present invention, for example, analysis by Western trace type, with antibody analyze the expression of above-mentioned gene fragment, to detect the segmental gene product of said gene, or a kind of specific indication polypeptide or albumen, this proteic gene order is represented the segmental part of said gene.

If the segmental detection of expression of said gene is based on a kind of detection of expression in the said gene fragment (A) to (H) and " additional " nucleotide sequence coding indicator protein or polypeptide that is contained in, this detection also can be passed through alternate manner, rather than by (for example using nucleic acid probe, to the Northern engram analysis) or antibody is (for example, to the Western engram analysis), depend on the type of the indicator protein of generation or polypeptide and decide.Indicator protein or polypeptide can be a kind of fluorescin or chemoluminescence albumen or polypeptide, as green fluorescent protein (GFP), thereby can detect the segmental expression of said gene by fluorescence or the chemoluminescence that detects above-mentioned this vegetable cell.Perhaps, described indicator protein or polypeptide can also show a kind of enzymic activity, and this enzymic activity is not in used plant of the natural the present invention of being present in or the plant part.Therefore, the activity that can analyze this enzyme detects indicator protein or polypeptide expression and the segmental expression of said gene.This analysis can be based on the disappearance of a kind of substrate of this enzyme in the determination and analysis substratum or pass through the formation of a kind of product of transformation assay of enzyme.In a specific embodiment, the above-mentioned product of this enzymatic conversion can be a kind of compound, because Enzymatic transformation, this compound can change color.An example with indicator protein of enzymic activity is a glycuronidase, and this indicator protein usually is not found in plant.In addition, indicator protein can show the activity of enzyme, and the patience to another chemokines such as weedicide or plant toxicity compound for example can be provided.In this case, when weedicide or plant toxicity compound exist, carry out expression analysis, expression is estimated according to patience to chemokines.

Analyze above-mentioned gene fragment (A) and can carry out-produce a kind of result in the following manner to the analytical procedure (b) of the expression of (H), whether this result only can measure expression and take place;-compare with other, can measure the expression (" semiquantitative determination ") that 2 or more sample show said gene fragment high level.-quantitative result about expression level (" quantitative assay ") is provided.

If implement analytical procedure (b) with the nucleic acid or the protein material that equate or prepare the plant of handling from step (a) of equal quantities or the plant part in fact carefully, thereby can semiquantitative determination or or even the segmental expression level of quantitative assay said gene, method of the present invention also can be used to: the compound concentrations of ERS that can inducing plant in the-working sample; Whether-mensuration compound or composition are induced reference compound to show stronger ERS than ERS and are induced effect; With-measure compound or composition whether or the ERS that on which kind of degree, can change compound induce effect.

The method of ERS compound concentration that can inducing plant in the working sample be characterised in that it comprises the following steps:

(i), comprise as top institute's steps outlined (a) and (b), wherein in step (a), plant or plant part contact with the sample of equal portions according to this method of the invention process;

(ii) by with the result of the corresponding test-results comparison step of measuring with the above-claimed cpd of concentration known (i), the concentration of ERS inducing compounds in the working sample.

Measure compound and whether induce for example Whitfield's ointment of reference compound than ERS, dichloro-isonicotinic acid, or jasmonic show stronger ERS induce effect this method be characterised in that it comprises the following steps:

(i), comprise as top institute's steps outlined (a) and (b), wherein in step (a), plant or plant part contact with the compound of specific concentrations according to this method of the invention process;

(ii) according to this method of the invention process, comprise as top institute's steps outlined (a) and (b), wherein in step (a), plant or plant part induce reference compound to contact with a certain amount of ERS, compare with the compound concentration in the step (i) volumetric molar concentration such as to cause;

(iii) by comparison step (i) and result (ii), the ERS inductive of mensuration compound is level relatively.

Because will finish any step (b) from what step (a) was handled for the plant of analyzing or the nucleic acid or the protein material of plant part material preparation with equivalent in fact, step (iii) will produce semiquantitative result, induce in the reference compound which can induce the segmental expression of said gene of higher degree so that realize to measure compound and ERS, therefore in plant, produce stronger ERS inducing action.Perhaps, also can assess these results, induce reference compound to compare, to realize the expression of qualitative assessment compound inductive relative extent with ERS.By the mode of photodensitometer or in liquid scintillation counter, analyze gel by analyzing the relevant portion that downcuts, this gel is used for separating the analysis of mixtures that contains radio-labeling detection compound (for example nucleotide probe or antibody), for example, by scanning analysis gel or film, the result that picture or its print implements this assessment.

Measure compound or composition whether or the ERS that on which kind of degree, can change agrochemicals induce effect method be characterised in that it comprises the following steps:

(i), comprise as top institute's steps outlined (a) and (b), wherein in step (a), plant or plant part contact with the compound or the composition of specific concentrations according to this method of the invention process;

(ii) as in the step (i) under the situation that compound or composition exist, according to this method of the invention process, comprise as top institute's steps outlined (a) and (b), wherein in step (a), plant or plant part contact with the farming thing of same specific concentrations.

(iii) by comparison step (i) and result (ii), the ERS that measures compound or composition change compound or composition induces the ability of effect.

Because in the method, also will finish any step (b) from what step (a) was handled for the plant of analyzing or the nucleic acid or the protein material of plant part material preparation with equivalent in fact, step (iii) will produce semiquantitative result, measure so that realize, it is the enhancing that compound or composition cause the said gene fragment expression, reduce or or even be suppressed, therefore will produce the ERS inducing action in the plant stronger or that weaken, perhaps even will cancel the inducing action of ERS.Perhaps, also can assess these results, compare separately with agrochemicals, with realize qualitative assessment agrochemicals and compound or with the expression of composition inductive relative extent.By the mode of photodensitometer or in liquid scintillation counter, analyze gel by analyzing the relevant portion that downcuts, this gel is used for separating the analysis of mixtures that contains radio-labeling detection compound (for example nucleotide probe or antibody), for example, by scanning analysis gel or film, the result that picture or its print implements this assessment.

Measure compound or composition whether or the ERS that on which kind of degree, can change agrochemicals induce the method for effect further to be used in one approach, this method is used to screen compound or the composition that the ERS that can change agrochemicals induces effect.

The invention still further relates to the use of the test kit that uses in the inventive method.The mentioned reagent box be characterised in that it comprises following composition:

(a) one or more plants or plant part are comprising gene fragment (A) to (H), and its expression can be induced by the SAR inducing compounds, preferably be induced by SAR inducing compounds and ISR inducing compounds; With

(b) method of detection said gene fragment expression.

In a specific embodiment, this test kit can comprise one or more plants or plant part, and the expression that is included in said gene fragment (A) to (H) wherein is can not be by the phytopathogen inductive.In another specific embodiment, this test kit can comprise one or more plants or plant part, and said gene fragment wherein also comprises the nucleotide sequence of coding indicator protein or polypeptide in addition.

If this test kit comprises one or more plant parts, as vegetable cell, available said plant part is fixed on the solid support.Suitable upholder can resemble agarose by glass, kinds of specific polysaccharides, or specific plastic substance polyacrylamide, polystyrene polyvinyl alcohol, silicon.The method of fixed cell and tissue and technology are that the technician grasps on the upholder of these types.In a specific technical scheme, screening method especially of the present invention, this test kit can comprise flat board, as microtiter plate, comprise a large amount of reacting holes, these reacting holes contain said plant part, especially vegetable cell, and it preferably is fixed on the bottom or wall in these holes.

The instrument that detects said gene fragment (A) to (H) expression comprises one or more nucleotide probes, for example the cDNA of certification mark or said gene cDNA fragment fragment.Detectable mark can be, fluorescence for example, chemoluminescence or radio-labeling, especially digoxin molecule.Perhaps, in order to use in the analysis of enzyme-linked immunosorbent assay or Western trace type, the instrument that detects the said gene fragment expression will comprise a kind of antibody, this antibodies specific ground identification and in conjunction with the gene product of said gene fragment.This antibody will carry a detectable mark, for example a kind of fluorescence or chemoluminescence dyestuff, the active part of a kind of radio-labeling or a kind of enzyme or structure, or have specific secondary antibody to detect this antibody to said one-level antibody by using, this secondary antibody also can distinguish over said one-level antibody and be provided individually in test kit.For (to) analyze the enzymic activity of indicator protein or polypeptide, in this case, said gene fragment also comprises the coding indicator protein separately or the nucleotide sequence of polypeptide, the expression of this gene fragment can be induced by the ERS inducing compounds, the instrument that detects the said gene fragment expression will comprise one or more reagent for the enzymic activity of analyzing indicator protein or polypeptide, the substrate that for example comprises a kind of indicator protein, optionally use and possible for detecting substrate through the reagent of enzymatic conversion as product with one or more essential cofactors.

In addition, above-mentioned instrument can comprise that one or more are used for the damping fluid of analytical reaction and/or the reagent that is used to measure, especially the reagent that supplies the display analysis result (for example, under the situation that detects attached to the enzymic activity on the antibody), they can provide independent of one another, can be independent of probe or antibody provides, or provide in the bonded mode or with the array mode of some mentioned component.

Perhaps, this test kit can comprise more integral part, especially as independent component, plant part and test compound or composition are carried out the damping fluid of incubation as one or more, the broken plant or the cyto-architectural factor of plant part, enable reaction detecting the segmental expression of said gene, or the working instructions of test kit.

In addition, the invention relates to provides the method with compound of inducing ERS ability in plant, and it comprises step:

(a) produce compound synthetic combinatorial libraries and

(b), screen the compound in above-mentioned library with the method that comprises the compound that above generalized step (a) and detection (b) can inducing ERSs in plant according to the present invention.The present invention also comprise have the inducing ERS ability, by the obtainable new compound of present method, and as the purposes of agrochemicals, in particular for the ERS in the inducing plant.About these formulation and application, with reference to relevant explanation early given in this specification sheets as the new agricultural compound of agrochemicals.

On the other hand, the present invention relates to the isolated DNA molecule of proteins encoded or polypeptide or its part, if said dna molecular is positioned under its natural genotypic environment, its expression is induced by the SAR inducing compounds, preferably induce, or relate to and its complementary nucleotide sequence by SAR inducing compounds and ISR inducing compounds.

In a specific embodiment of the present invention, isolated DNA molecule comprise Nucleotide series (A) to (H) or with its complementary nucleotide sequence.

According to method of the present invention, can measure the expression of these dna moleculars.

The invention still further relates to isolated DNA molecule, the nucleotide sequence of these dna moleculars and top given nucleotide sequence be homology basically, especially hybridize with the latter, or have the varient of the dna molecular of top given nucleotide sequence, perhaps relevant with above-mentioned dna molecular by sudden change.About the implication of term " homology basically " and " varient ", with reference to relevant explanation early given in this specification sheets.In the present context, specific technical scheme is to have the dna molecular allelic variant of nucleotide sequence (A) to (H) and have and (A) dna molecular of the nucleotide sequence of given nucleotide sequence hybridization in (H).Summarized as top, preferably, this hybridization is carried out under the condition of low stringency, in another and technical scheme, between low stringency and high tight degree condition, carry out, in unusual special technique scheme, even under the condition of the tight degree of height, carry out.As a kind of rule, the low stringency condition may be defined as 3 times SSC, and temperature is a room temperature to 65 ℃, and high tight degree condition is 0.1 times SSC, and temperature is 68 ℃.SSC is the sodium-chlor of 0.15M, the abbreviation of 0.015M trisodium citrate damping fluid.Another specific technical scheme is other dna molecular, the nucleotide sequence that these dna moleculars have not necessarily respectively with Nucleotide series (A) to (H) in a kind of sequence hybridization, but, because the degeneracy of genetic code, the given identical aminoacid sequence of aminoacid sequence in codified and (A) to (H) sequence.

In a specific technical scheme, the dna molecular of proteins encoded/polypeptide or its part, under intracellular natural genotypic environment, its expression is induced by the SAR inducing compounds, preferably induce by SAR inducing compounds and ISR inducing compounds, the genome that these dna moleculars are plant-derived, segmental that form or comprise nucleotide sequence (A) by using to the segmental dna hybrid probe of the dna molecular of (H) to the dna molecular of (H) by having a nucleotide sequence (A), can identify and isolate these dna moleculars.Remove the other plant of barley in screening, especially during the gene in other plant of Hordeum, also comprise monocotyledons, also comprise dicotyledons, the preferred fragment of said dna molecular comprises at least 15 Nucleotide, typically between 15 and 30 Nucleotide, more preferably be at least 25 Nucleotide, but if necessary, can comprise even the Nucleotide of higher quantity, as 50 or more Nucleotide.

Therefore, the present invention includes the fragment of dna molecular of the present invention, especially comprise isolated DNA molecule fragment with above-mentioned given nucleotide sequence, and as the purposes of hybridization probe, especially as the purposes of screening probe or primer, especially as the purposes of sequencing or PCR primer.The present invention also comprises the shorter fragment of mentioning than above-mentioned, for example comprises 8 to 15 Nucleotide, when especially they are as nucleotide probe or primer.During in particular as nucleotide probe, form with mark, for example radio-labeling or the mark that adheres to inactive report or indication molecule, digoxin molecule for example uses to comprise one or more dna moleculars segmental or that be made up of one or more fragments of the present invention of the present invention.Especially as the PCR primer, in fragment sequence of the present invention, use the dna molecular that comprises or form by one or more fragments of the present invention basically with a kind of form that comprises modified nucleotide sequences, this modified nucleotide sequences provides the product of suitable restriction site and any amplification behind PCR in above-mentioned primer, help the clone of above-mentioned amplified production.Although these nucleotide sequences are modified, under the PCR condition, the PCR primer will be hybridized with target dna molecule of the present invention specifically.

Dna fragmentation of the present invention for example,, may pass through to use suitable restriction enzyme, or prepare by chemosynthesis to the basis of (H) at nucleotide sequence (A).According to the present invention, preparation dna fragmentation and the various suitable technique that contain one or more these segmental dna molecular are technology that the technician knows.

Nucleotide fragments of the application of the invention or dna molecular are made the screening probe, in a different plant species, identified a corresponding gene, whether determine that especially whether whether it express by ERS inducing compounds inductive, by SAR inducing compounds inductive, may be very interesting by SAR inducing compounds and ISR inducing compounds inductive preferably.This may put into practice; for example; as what summarized among the embodiment; when an ERS inducing compounds exists; analyze said plant or plant part by culturing plants or plant part with by the mode of Northern-trace or the analysis of RNA dot blot, for example; by using with the special hybridization probe of nucleotide fragments (A) to (H), being used to identify said gene and when the ERS inducing compounds does not exist, comparing with the result of above-mentioned plant of cultivation or plant part.The plant of identifying by the method for describing can be used in the method for the present invention, this plant will comprise a kind of gene, this gene can be induced by the ERS inducing compounds, especially can be induced by the SAR inducing compounds, is preferably induced by SAR inducing compounds and ISR inducing compounds.

In another special technique scheme, the present invention relates to a kind of isolated DNA molecule, this dna molecular comprises dna molecular or the fragment that nucleotide sequence defined above and coding indicator protein or polypeptide merges.In an optimized technical scheme, if be connected on the one expressed sequence on expressing, the complete coded message of above-mentioned dna molecular will be expressed.Albumen or polypeptide that the indicator protein of another nucleotide segment coding of this of this dna molecular or polypeptide are a kind of easy detection, as passing through to use specific Nucleotide hybridization probe, by using special antibody or because this albumen or specific character such as fluorescence or the enzymic activity of polypeptide self.In a specific embodiment, can the encode plant AHAS enzyme of glycuronidase or a green fluorescent protein or a modified forms of said another nucleotide sequence part of this dna molecular.The generation of the separation of indication polypeptide or proteic suitable gene and the reorganization chimeric dna molecule of clone and above-mentioned definition is the known technology of technician.

The present invention also comprises DNA construct, it comprises the dna molecular and/or the fragment of one or more above definition, especially be used as the construct of nucleotide probe or PCR primer, and under expression control sequenc control, as being connected on operability ground under a said dna molecular and/or a segmental expression control sequenc control, comprise dna molecular and/or segmental construct.

Particularly as the use of nucleotide probe, to use the dna molecular that comprise or form by one or more dna moleculars of the present invention and fragment with a kind of form of mark, wherein said mark can be by additional inactive report or indication molecule, as fluorescence or phosphorescent molecules, the digoxin molecule, biotin molecule or their derivative are finished.

Particularly as the use of PCR primer, according to the present invention, in fragments sequence of the present invention, with the form that comprises additional nucleotide sequence or modified nucleotide sequences, use the DNA construct comprise or form by one or more dna moleculars of the present invention and fragment basically, this additional nucleotide sequence or modified nucleotide sequences provide the product of suitable restriction site and any amplification behind PCR in above-mentioned primer, help the clone of above-mentioned amplified production.Although these Nucleotide series are modified, under the PCR condition, the PCR primer will be hybridized with target dna molecule of the present invention specifically.In another special technique scheme, DNA construct is connected to said dna molecular with also comprising operability and/or a segmental segment table reaches control sequence.

The invention still further relates to and comprise one or more dna moleculars of the present invention and segmental carrier, plasmid, clay, virus, phage genome.

The invention still further relates to and contain dna molecular of the present invention, the dna molecular of the complementary sequence of fragment and construct and these dna moleculars, the rna transcription product of fragment and construct.

The whole bag of tricks and technology that dna molecular of the present invention, fragment and construct are separated, make up, synthesize and modify are that the technician knows, and all description are arranged, as (1989) such as Sambrook, and Ausebel etc.

In addition, the present invention relates to by dna molecular of the present invention, fragment, with carrier construction encoded polypeptides/albumen or its part, relate to some protein, this protein has or comprises a kind of aminoacid sequence, it and the corresponding aminoacid sequence of dna sequence dna (A) to (H) show at least 65%, typically, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, at least 95% homology particularly preferably, and relate to said proteinic polypeptide fragment, its condition are these segmental aminoacid sequences and corresponding " reference " fragment corresponding to the polypeptide of dna sequence dna (A) to (H) shows at least 65%, typically, at least 75%, preferably at least 80%, more preferably at least 85%, even more preferably at least 90%, at least 95% homology particularly preferably.

In the context of the present invention, term " polypeptide " comprises and typically comprises at least 18 amino acid, and at least 25 amino acid more specifically are particularly preferably more than 40 amino acid whose molecules.

In a specific technical scheme of the present invention, polypeptide and albumen are by the protein of dna molecule encode of the present invention or the fusion rotein of its part, and this fusion rotein has a kind of indicator protein or the polypeptide of having summarized as top.In another embodiment, the invention provides polypeptide/albumen or its part, be included in polypeptide/albumen or its part described in two sections of the fronts.

By cultivating host's biology of transforming with one or more said dna moleculars and, can obtaining these polypeptide/albumen or its part by method known in the art, optionally these polypeptide of purifying and albumen.Perhaps, by utilizing known peptide or albumen synthetic technology also can synthesize these polypeptide of preparation and albumen.

Polypeptide of the present invention and albumen can be used to prepare mono-clonal or polyclonal antibody, perhaps, if they show the activity of enzyme, can utilize the activity of this enzyme for multiple purpose.

On the other hand, the present invention also comprises mono-clonal or polyclonal antibody or identification specifically and in conjunction with a peptide species of the present invention or proteic antiserum(antisera).Polypeptide of the application of the invention or albumen can prepare corresponding antibody, according to the known method of technician host immunity are inoculated.

In addition, the present invention includes microorganism transformed, as bacterium, bacteriophage, virus, eukaryote such as fungi, yeast, protozoon, algae, and people, the animal and plant cell, they comprise a kind of dna sequence dna of reorganization, and this dna sequence dna comprises at least a dna molecular of the present invention, fragment or carrier construction.These microorganism transformed can be used as a kind of expression system, produce dna molecular of the present invention, the gene product of fragment or carrier construction.The representative microbial that is used for this purpose is a bacterium, and as intestinal bacteria, yeast is as yeast saccharomyces cerevisiae.The conversion microorganism of other type of the present invention as Agrobacterium, resembles agrobacterium tumefaciens, and it is by dna molecular of the present invention, and fragment or construct transform, and can be used to transform plant, thereby transgenic plant are provided.

Use dna molecular of the present invention, it all is the technology that the technician knows that fragment or construct transform method of microorganism, is included in the structure of expression vector under the control of composing type or inductive promotor, that contain dna sequence dna of the present invention.This promotor also can make information encoded organism, carries out tissue specific expression in the specific compartment as plant.

At last, the present invention relates to comprise the transgenic plant of recombinant DNA sequence, plant part or seed, this recombinant DNA sequence comprise one or more dna moleculars of the present invention.Term " plant " or " plant part " comprise all plant propagation materials, as plant protoplast, and vegetable cell, plant tissue, callus, the plantlet of growth, plant leaf, jejune whole strain plant, sophisticated whole strain plant.Transgenic plant, plant part or seed can comprise be incorporated in its genome or be positioned at extrachromosomal above-mentioned recombinant DNA sequence.The suitable gene or the clone of gene fragment, the structure of suitable conversion carrier and the genetic information that is included in the carrier are all known the technician to various technology and method that vegetable cell imports, and can produce transgenic plant of the present invention, plant part or seed.The technology that genetic information imports to vegetable cell comprises that direct DNA (for example shifts, by electroporation or by using high-molecular weight permeate agent and particle bombardment to import in the protoplastis, the DNA particle that wherein is wrapped is shot in the plant tissue), and use natural host/carrier system (for example Agrobacterium or plant virus).The extrachromosomal of recombinant DNA sequence kept, can use various specific virus such as tobacco mosaic virus (TMV) (TMV) or the potato virus X (PVX) of in its genome, inserting the dna sequence dna of reorganization.

The demonstration plant that is used to integrate recombinant DNA sequence of the present invention comprises dicotyledons and monocotyledons, and especially cereal class and crop are as potato, calona, rape, soybean, sugar beet and tobacco.

For the present invention being described, the elucidated hereinafter specific embodiment.These embodiment only are illustrative, are not to be interpreted as limited field of the present invention and fundamental principle by any way.Except illustrate herein and describe those, those skilled in the art will understand various change of the present invention from the description of the following examples and front.These changes also will fall in the scope of appending claims.

The embodiment method

The inhibition difference subtracts hybridization (SSH) and is used to identify a relevant cover difference formula expressing gene (Diatchenko etc., 1996).This technology is to deduct contrast effectively and drive common sequences in the cDNA colony of thing according to the abundance that is included in the average target cDNA in the contrast cDNA colony with by the mode of hybridization.In the PCR of a particular form, the target cDNA that the difference formula is expressed is optionally increased, but not the amplification of target cDNA is suppressed.

Deduction cDNA from non-inductive plant (driving thing), we have separated the cDNA fragment that poor formula is expressed from the barley plants (contrast) of chemical induction.Plant, pathogenic bacteria and inoculation

Plant, i.e. barley (Hordeum vulgare L.) cultivar Ingrid and Rorl-2 mutant A89, in 16 ℃, illumination (the 100 μ Es of 60% relative humidity and 16 hours ^-1m ^-2) in the growth case, grow.The contrast of chemical induction is seeded in the 10mm of the K1 strain isolated (Hinze etc., 1991) of the 3rd day the barley physiological strain (Erysiphe graminis DC:Fr.f.sp.hordei) after the chemical treatment with powdery mildew ^-2The conidium inoculation.Plant is activated

2, and the 6-dichloro-isonicotinic acid (DCINA, CGA 41396, gas Ba Jiaji (Coba GeigyAG), Basel, Switzerland), be applied to the Herba Hordei Vulgaris of 5 days seedling ages in the mode of soaking into soil with the formulation of 25% activeconstituents and wettable powder (WP) carrier (Metrauxet al.1991).The final concentration of compound is 5 mg/litre soil volumes.Suspension is prepared with aseptic tap water.The separation of total RNA

Be used for suppressing the poly (A) that difference subtracts hybridization and cDNA latter end rapid amplifying experiment (raceexperiment) in order to separate ⁺RNA separates total RNA and carries out according to the method (1991) of DUDLER etc. from 5 gram leaves.

For Northern analyzes, the handbook that provides is revised a little and (AGS) separated total RNA by use RNA washing agent (clean).Blade material grinds in liquid nitrogen, and the RNA de-sludging (clean) that adds 1.7 milliliters in the sample aliquot of 200 milligrams blade powder is extracted after the chloroform of damping fluid and 200 microlitres, and inclusion is through thorough mixing and shook 30 minutes.Sample is centrifugal in 20800xg (4 ℃, 30 minutes) again, and water is used isopyknic chloroform extracting again.After centrifugal (20800xg, 4 ℃, 30 minutes), RNA gets off with isopyknic isopropanol precipitating from top aqueous phase, and sample is in preserving 1 hour on ice, and then centrifugal in 20800xg (4 ℃, 30 minutes).Discard supernatant liquor, precipitation is washed once with 70% ethanol (20 ℃).After simple centrifugal, discard supernatant liquor up hill and dale, be deposited in drying under the room temperature, the RNA water dissolution of proper volume.Poly+RNA (poly (A) ⁺RNA) separation

Total RNA equivalent of different time points (be respectively chemical induction and control treatment after 12,24,48 hours) concentrated merge.The RNA that merges is as follows with dnase digestion: the RNA of every equal portions 750 micrograms is by Tutofusin tris-hydrochloric acid (pH7.5) of the 1M of adding 12.5 microlitres, the magnesium chloride of 50 microlitre 50mM, the ethylenediamine tetraacetic acid (EDTA) of the 25mM of 1 microlitre (pH7.5), 2.5 the RNA enzyme inhibitors of microlitre (40 unit/microlitres), the DNA enzyme I (10 units/microlitre) of 10 microlitres and water to 250 microlitre of proper volume.Reaction mixture in the air incubator 37 ℃ of following incubations 30 minutes.In order to extract RNA, add water and make volume compensation to 1250 microlitres, add isopyknic phenol (saturated), chloroform (3: 1), thorough mixing, under the 20800xg room temperature centrifugal 5 minutes with Tutofusin tris-hydrochloric acid (pH8.0).Water is transferred in the new test tube, added the sodium acetate (pH5.2) of the 3M of 0.1 times of volume and cold (20 ℃) 96% ethanol of 2.5 times of volumes then.Preserved 24 hours down sample mixed and in-20 ℃.In 20800xg, after 4 ℃ times centrifugal 1 hour, discard supernatant liquor, precipitate with cold (20 ℃) 70% washing with alcohol.Mixed solution in 20800xg, 4 ℃ centrifugal 15 minutes down, discards supernatant liquor again.Be deposited under the room temperature dry, and with the water dissolution of proper volume.

The RNA that separates poly-adenosine according to manufacturer's recommendation, with DYNABEADS mRNA purification kit (Dynal).The polyacrylamide sex change glue with 6% and the RNA of silver-colored chromoscopy poly-adenosine, method is as follows: be used for encapsulating or run glue, use the groove (BioRad) of Mini-PROTEAN II type, in 12 milliliters sol solution, add 5.The adenosine-5'-phosphosulfate(APS) (BioRad) of Temed of 6 microlitres (SERVA) and 72 microlitres 10% arrives glue then immediately, and makes its polymerization 2 hours.After 100 volts of prerunnings 45 minutes, total RNA of the RNA of the polyadenylation of 100 nanogram(ng)s and 250 nanogram(ng)s 5 times phosphonoacetic acid slow (PAA) in the liquid in 98 ℃ of sex change 5 minutes, then promptly in cooled on ice, and go up sample to glue.In 1 times tbe buffer liquid, under 60 volts, run glue.After the electrophoresis, glue incubation 3 times in fixed solution, 15 minutes, the water flushing was 4 times again, each 2 minutes, used moistening 1 time of the glycerine of 1.5% (volume/volume) again, 2 minutes.Mix with 7 milliliters staining fluid A with 13 milliliters staining fluid B.Glue is immersed in this mixed solution, displays up to band.By glue being transferred to termination reaction in the stop bath.Subtract the synthetic cDNA of hybridization (SSH) and produce subtracted library by suppressing difference

Use PCR-Select ^TMThe cDNA difference subtracts test kit (Clontech), suppresses difference and subtract hybridization between (" contrast ") of non-inductive of A89 (" ordering about thing ") and A89 chemical induction.Except that the modification of relevant hybridization step, all programs, as synthesizing of first and second cDNA chains, RsaI digestion is connected the rule of all recommending according to manufacturer and carries out with joint.To hybridization for the first time, a kind of disconnected " ordering about thing " and the mixture that has been connected " contrast " cDNA colony of one in two different joints are carried out sex change, and descend whole 8 hours of maintenance at 68 ℃.To the hybridization step second time, mix " ordering about thing-contrast " hybridization sample, add " ordering about thing " of new sex change, in 68 ℃ of following incubations 12 hours.Following PCR reaction with Adantage  cDNA polysaccharase mixture (Clontech) carries out subtracts the handbook operation that test kit provides according to difference, and all PCR and hybridization step carry out on Perkin-Elmer 2400 thermal cyclers.

Now, the enrichment of PCR mixture the is got up cDNA that about equally abundance difference formula expresses.Difference subtracts the clone of cDNA to the TA carrier

Though it is active that Adantage  cDNA polysaccharase mixture only shows a spot of check and correction, can directly carry out the adenosine tailing in the second time after the pcr amplification, contains " adenosine-pendant " to guarantee most of cDNA fragment.The Taq polysaccharase (Eurogentec) of the reaction mixture of 10 microlitres and dATP (final concentration of 0.4mM) and 1 unit was 70 ℃ of following incubations 15 minutes.Need not further increase, the difference of 1 microlitre and 3 microlitres subtracts cDNA and is connected to 50 nanogram(ng) Pt-Adv (Adantage ^TMPCR clones test kit, Clontech) on.Connect mixture and import to intestinal bacteria TOP10 F ' (Clontech) afterwards, the library coated plate is to the penbritin that contains 100 mcg/ml, on the agar plate of the 5-bromo-4-chloro-3-indoles-β-D-galactoside (X-Gal) of 50 mcg/ml and isopropylthio-D-galactoside (IPTG) of 280 μ M.Up to bacterium colony as seen bacterial growth can clearly pick out indigo plant/hickie.Described flat board is preserved down in 4 ℃.Bacterium colony PCR, oppositely northern trace and screening

In order to assess the cDNA of deduction effect and the real differential expression of screening, carry out bacterium colony PCR, PCR product trace to film and with article one chain cDNA of digoxigenin labeled hybridization, this cDNA derive from merging from inductive and non-inductive plant RNA as the polyadenylation of template extraction.

Altogether picking 480 whites and nattier blue single clone, at first be used for inoculating the agar plate of the penbritin that contains 100 mcg/ml, inoculate in the microtiter plate that contains 40 microlitre sterilized waters in 96-hole.Bacterium in the microtiter plate with Perkin-Elmer 9700 thermal cyclers by being heated to cracking in 98 ℃, 5 minutes.The PCR reaction mixture moves on in the microtiter plate in 96 holes with micropipet as follows: with providing enzyme, the dNTP of 200 μ M concentration, 1.5mM magnesium chloride, 0.5 the Taq polysaccharase (Eurogentec) of individual unit and the every kind of primer that is positioned at the inset two ends of 0.4 μ M (nested primers 1 and nested primers 2R) are with the lysate inset that 10 times PCR damping fluid (Eurogentec) amplification of usefulness standard is cloned in the reaction solution of 30 microlitres of 10 microlitres.By carry out circulation in PGR:94 ℃, 30 minutes with following condition; 35 circulations, each circulation 94 ℃, 15 seconds, 68 ℃, 30 seconds and 72 ℃, 60 seconds; And then circulation in 72 ℃, 5 minutes.

After the amplification, carry out prerunning, consider that minimum inset size is that 0.1 kilobase is right, selects among 360 clones some to do reverse northern analysis.Sample carries out parallel electrophoresis (glue A and glue B) on the equal portions of two 10 microlitres in 1.5% agarose/bromination second pyridine (EtBr), 1 times TBE.By the same applied sample amount of fluorescence monitoring that because of nucleic acid the accumulation of bromination second pyridine is produced under the ultraviolet lamp.Glue in 2 times SSC trace to nylon membrane (Boehringer) last 16 hour.Filter membrane A and B have the sodium hydroxide of 0.5M then in immersion, 1.5M the whatman test paper of sodium-chlor on sex change 2 times, each 5 minutes, the Tris/HCL (pH 7.4) of 0.5M is arranged in immersion then, 1.5M the whatman test paper of sodium-chlor in and 2 times, each 5 minutes.At last with 125mJ by crosslinked action (GS GeneLinker, BioRad) fixed dna.

For synthesizing of article one cDNA chain, the poly+RNA of 2 micrograms adds the oligomerization (dT of 2.22 micrograms ₁₅) primer is heated to 65 ℃ in the volume of 13 microlitres, 10 minutes, cooled on ice 5 minutes.Article one chain damping fluid (Boehringer) of 5 times, 7.5 the dNTPs stoste (dATP of 2mM of microlitre, dGTP, dCTP, 0.52mM dTTP, 0.28mM digoxin-dUTP (Boehringer)) and RNA enzyme inhibitors (Boehringer) water of 37.5 units add to 15 microlitres, join in the mixture of poly+RNA/primer.At room temperature incubation is after 2 minutes, by murine leukemia virus (MMuLV) reversed transcriptive enzyme (Boehringer) the beginning reverse transcription that adds 40 units.Mix this component fully, in 37 ℃ of following incubations 1 hour.By being heated to 95 ℃, 5 minutes termination reactions, then cooled on ice 5 minutes.In order to digest RNA, each adds the RNA enzyme A (RNaseA) of 1 unit and RNA enzyme H (RNaseH) in the synthetic liquid of article one chain, then in 37 ℃ of following incubations 1.5 hours.The control of article one cDNA chain of digoxigenin labeled

For the cDNA that expresses with reverse northern technology screening difference formula, digoxin is penetrated into the estimation of the intensity in inductive (" object of reference ") and non-inductive (" ordering about thing ") the strand cDNA colony and makes it equalization is very important.

Article one chain synthetic equal portions in the RNA of proper volume sample-loading buffer in 95 ℃ of sex change 5 minutes, again in cooled on ice and go up 1.5% agarose gel of sample to sex change, electrophoresis in the formaldehyde (37%) of the damping fluid and 5% (volume/volume) of 1 times 3-(N-morpholino) propanesulfonic acid MOPS.After the electrophoresis, the probe trace is continued 16 hours to the nylon membrane of the sodium phosphate buffer (pH6.5) of 25mM, use then 125mJ (the crosslinked machine of GS gene, BioRad) crosslinked.(Boehringer) detect the cDNA of digoxigenin labeled by chemiluminescence according to " the digoxin system user guide of filter hybridization ".Estimate the concentration of " object of reference " and " ordering about thing " probe of the high zinc mark in ground by comparison signal intensity.

The screening of the cDNA that the difference formula is expressed is carried out with following method.Filter membrane (A and B) in DiGEasy HyB (Boehringer) with (" the object of reference ") poly+RNA that derives from non-inductive (" ordering about thing ") and chemical induction respectively, through the strand cDNA of digoxigenin labeled 50 ℃ of hybridization 16 hours down.Filter membrane washs under room temperature 2 times with 2 times SSC/0.1%SDS, again with 0.1 times SSC/0.1%SDS in 50 ℃ of washings 2 times down.(Boehringer) detect according to " the digoxin system user guide of filter hybridization " by chemiluminescence.The separation of plasmid

Press (1989) method culturing bacterium such as Sambrook, plasmid separates with the plasmid Miniprep test kit (Genomed) of JETSTAR.Sequencing

Primer cycle sequencing test kit (Amersham) and LongRanger sol solution (FMC) with hot Sequenase mark carry out sequencing on Licor 4200 sequenators (MWG-RIOTECH).Reaction is provided with as follows: 2pmol primer (carrying out 5 ' mark) with IRD-800, and the template DNA of 1.0 micrograms, the dimethyl sulfoxide (DMSO) of 10% (volume/volume) (DMSO) adds water and adds to 21 microlitres.To the A of 1.5 microlitres, C adds the primer/template mixture of 4.5 microlitres in G or the T reagent.On Perkin-Elmer 2400 thermal cyclers, circulate: circulation in 95 ℃, 5 minutes with following parameters; 30 circulations, each circulation 95 ℃, 15 seconds, 61 ℃ (M13 oppositely-29) and 64 ℃ (M13 forward-21), 15 seconds, 70 ℃, 15 seconds.Reaction solution is by adding the 4 microlitre stop buffer termination reactions that test kit (Amersham) provides.Behind 30 minutes prerunning, to glue, electrophoresis runs glue to carry out according to the method that the MWG-rule of sequenator is recommended sample on the equal portions of 1-2 microlitre.Sequential analysis

E value 5e-42 is with the homology 98/102 (96%) of registration number AI622765 (corn (Zea mays) cDNA).

E value 3e-20 is with the homology 53/54 (98%) of registration number AU033069 (paddy rice (Oryza sativa) cDNA).blastn/nr

E value 2e-08 is with the homology 26/34 (76%) of registration number CAB36731 (the possible albumen of Arabidopis thaliana (Arabidopsis thaliana); similar to acid phosphatase lipase EC 3.1.3.2), positive rate 28/34 (81 %). clone B: (292 base pairs) ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGAA TGAAATCGTA 150TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GC homology: None clone C : (387 base pairs) ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGT homology : blastn / dbest (ESTs of unknown function)

E value 1e-118 is with the homology 340/382 (89%) of registration number C27470 (rice cDNA).

E value 1e-73 is with the homology 241/274 (87%) of registration number D39787 (rice cDNA).blastx/nr

E% 20value% 204e-44% 20is% 20with% 20registration% 20number% 20Y11384% 20% 20% 20 (% 20% 20to% 20the% 20Medicago% 20sativa% 20annexin% 20class% 20protein% 20of% 20seepage% 20water% 20pressure% 2C % 20dormin% 20and% 20olighydria% 20sensitivity% 20% 20)% 20% 20homology% 2088% 2F128% 20% 20% 20 (% 20% 2068% 25% 20% 20)% 20% 20.% E5% 85% 8B % E9% 9A% 86E% EF% BC% 9A% 20% 20% 20 (% 20% 20512% E4% B8% AA% E7% A2% B1% E5% 9F% BA% E5% AF% B9% 20% 20)% 20% 20ACAGAAGCTT% 20GGACAGTTCC% 20ACTCGGAAGC% 20TTCGGTTCGC% 20GCTACCGTTC% 2050CCGATGCTCG% 20CCTACCCATT% 20CTACTTGTGG% 20TCAAGGAGTC% 20CAGGGAAGTC% 20100AGGCTCGCAT% 20TTCCACCCGA% 20GCAGCGATTT% 20GTTCCAGCCG% 20AACGAGAAGA% 20150ACGACATACT% 20GACGTCGACG% 20ACATGCTGGC% 20TTGCCATGGC% 20TGGCCTGCTC% 20200GCTGGGCTCA% 20CTGCCGTGAT% 20GGGCCCCCTT % 20CAGATACTCA% 20AGCTCTACGC% 20250CGTCCCCTAC% 20TGGATTTTTG% 20TTATGTGGCT% 20GGACTTTGTC% 20ACCTACCTGC% 20300ACCACCACGG% 20CCACAACGAC% 20AAGCTTCCCT% 20GGTATCGCGG% 20AAAGGCATGG% 20350AGCTATCTGC% 20GCGGGGGCCT% 20GACAACGCTC% 20GACAGGGACT% 20ACGGGTGGCT% 20400CAACAACATC% 20CACCACGACA% 20TCGGGACTCA% 20CGTGATCCGC% 20CATCTTTTCC% 20450CGCAAATCCC% 20GCATTACCAT% 20CTAGTGGAGG % 20CGACCGAGGC% 20GGCGAACAGS% 20500TGCTAGGGAA% 20GT% E5% 90% 8C% E6% BA% 90% E6% 80% A7% EF% BC% 9Ablastn% 2Fnr

E value 0.0 is with the homology 467/496 (94%) of registration number D43688 (wheat (Triticum aestivum) is at the mRNA of the plastid omega-3-fatty acid desaturase (responsive to temperature type) of the leaf of light and dark place growth). clone F: (484 base pairs) ACAAGCTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGTGG TAAACAACAA 150CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200TTATTATACA AAAAAttGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGACAAA 450ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550ACTTTCTTCG CAAGATTGCC AACATAAACC TTGT homology: blastn / dbest (ESTs of unknown function)

E value 1e-37 is with the homology 207/248 (83%) of registration number AI600292 (corn cDNA).blastx/nr

E value 2e-14 is with the homology 40/73 (54%) of registration number CAB41335 (albumen that Arabidopis thaliana is possible has and Nicotiana plumbaginifolia rna binding protein 30 similaritys), positive rate 48/73 (64%). Clone G : (305 base pairs) GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGACA 150TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCC ACATTGGCTA 250GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300TAAGT homology: blastn / dbest (ESTs of unknown function )

E value 2e-13 is with the homology 103/125 (82%) of registration number C25074 (rice cDNA).blastx/nr

E value 1e-19 is with the homology 44/59 (74%) of registration number S61830 (corn is to the subtilisin / chymotrypsin inhibitor [serpin] of wound and fungal infection reaction), positive rate 50/59 (84%). Clone H: (1035 base pairs) caggATcATA GCTACAgGCg AcAATGCCCG GTCATCgACA ATCGcTgGCA 50 GCgGCTTCTC TGAAGcTACC ATCTCTTcTG CTTCTGTGGA TCTTTAGCTG 100 GAACTGGG gG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATgACGGAGC 150 TTCAGAAACA TgTCTCCTTT TTCGACCGGA ACAAGGATgG CTTCATcACT 200 CCTACAGAAA CCATCCAAGG GTtTGTtGCA ATCGGTTGCG AGTATgCATT 250 TGCTACTG cT GCCTCTGCCG CCATTCACGG TGCCcTTGCT CCTcAAAcaA 300 CCCCGGCTGG TaCaCCACTg CCTCACTtGA CAATATACGT AGAGAATAtC 350 CACAAaGCTA TGCATGGAAG tGaTtCaGGt gTATATGAtg CCAAaGGAaG 400 GTTTCTtCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450 GaCCAGATGC GTTGACTCTT GCGGAGATGCATGTGATGQFGCHJQFGCHJ \ VSDZXDXDZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZZDDDDZZZZZZZCTCTTTGCAAAA 500CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650CaCCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700AGTTtGGAtA tGGAAAGGtT tGtCCCGAAG AAGGTTTtCC tGCTATCtCC 750AAATTCAACT AGAGTTTATT tTCTTTtCCt CCAAGTtGTA ATtGGcTTTA 800TAAgACCTtC ATAGCCGATC AataCAACGA aGCAAGTtGg ATATATTTCC 850CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATGAGC 900GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKGAK GAWAAWATTT 1000ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA homology: blastx / nr

E value 7e-34 is with the homology 73/173 (42%) of registration number AC002332 (Arabidopis thaliana possible calcium combine EF chirality albumen), the rapid amplifying (RACE) of positive rate 107/173 (61%) cDNA end

With Phosphothion (MARATHON) cDNA amplification kit (Clontech), the rapid amplifying by the cDNA end obtains 5 of cDNA ' and 3 ' end.In order to produce double-stranded cDNA, merge respectively from inductive and non-inductive plant the isolating poly+RNA of (see above) as template.After joint connected, the joint primer one that provides with primer and the test kit of gene specific was reinstated the library as template amplification 5 ' with 3 ' hold.The rapid amplifying product of cDNA end is cloned among the pT-AdV, in the middle breeding of intestinal bacteria TOP10F ', and with the same sequence analysis of doing described above.The Northern engram analysis

In order to confirm that the difference formula expresses, separate total RNA of 5-10 microgram and transfer on the nylon membrane by (see above) on 1.5% formaldehyde agarose glue, carry out the northern engram analysis.Film is suppressing difference and is subtracting hybridization or terminal cDNA-probe rapid amplifying, digoxin-PCR-mark of cDNA in 60 ℃ of hybridization down with deriving under the tight condition, in Dig Easy Hyb (Boehringer).Film washs under room temperature 2 times with 2 times SSC/0.1%SDS, then with 0.1 times SSC/0.1%SDS in 60 ℃ of washings 3 times down.(Boehringer) detect according to " the digoxin system user guide of filter hybridization ".Material

Sequencing primer (at 5 ' end fluorescence dye IRD 800 modifieds):

M13 forward (21): 57 ℃ of 5 '-TGTAAAACGACGGCCAGT-3 ' Ta

M13 is (29): 57 ℃ of 5 '-CAGGAAACAGCTATGACC-3 ' Ta oppositely

T7-promotor: 56 ℃ of 5 '-TAATACGACTCACTATAGGG-3 ' Ta

The PCR primer:

Embedded primer 1:5 '-TCGAGCGGCCGCCCGGGCAGGT-3 '

Embedded primer 2 R:5 '-AGCGTGGTCGCGGCCGAGGT-3 '

Activator, (AP) composition of the sequencing gel of 1:5 '-CCATCCTAATACGACTCACTATAGGGC-3 ' 6%: 5 milliliters of Long Ranger of 10xTBE sol solution, (50%) 5 milliliter of urea 21 gram water adds 50 milliliters of TEMED, 25 microlitres, 10% adenosine-5'-phosphosulfate(APS), (APS) 250 microlitre 2xSSC:

Sodium-chlor 0.3M

Trisodium citrate: 30mM

PH 7.0RNA sample-loading buffer:

Formaldehyde (37%) 260 microlitre

Methane amide 720 microlitres

Glycerine 80 microlitres

Tetrabromophenol sulfonphthalein (water saturation solution) 80 microlitres

10 * MOPS, 180 microlitres

Ethidium bromide (10mg/ml) 100 microlitres

Water 100 microlitre stationary liquids

Methyl alcohol 45% (volume/volume)

Acetate 10% (volume/volume) dyeing solution A in water:

Yellow soda ash 5% (weight/volume) dyeing solution B in water:

Ammonium nitrate 0.2% (weight/volume)

Silver Nitrate 0.2% (weight/volume)

Tungstosilicic acid 1% (weight/volume)

Formaldehyde 1.4% (weight/volume)

Stop buffer in water:

Acetate 10% (volume/volume)

Glycerine 10% (volume/volume) 5x phosphonoacetic acid (PAA)-damping fluid:

Urea 7M

Tetrabromophenol sulfonphthalein 0.25% (weight/volume)

Dimethylbenzene nitrile indigo plant 0.25% (weight/volume)

EDTA 10mM

Glycerine 30% (volume/volume) sol solution:

Urea 7M

Acrylamide soln (37.5: 1 acrylamides: the methene acrylamide) 11.625 milliliters

7.5 milliliters of 10xTBE

Water adds 75 milliliters of 10xTBE:

Tris alkali 0.9M

Boric acid 0.9M

EDTA 25mM

Reference: Altschul, Stephen F., Thomas L.Madden, Alejandro A.Sch  ffer, Jinghui Zhang, Zheng Zhang, Webb Miller, the BLAST and the PSI-BLAST of and David J.Lipman (1997) breach: the Protein Data Bank search utility of a new generation.Nucleic acids research 25,3389-3402.Ausubel，F.M.，Brent，R.，Kingston，R.F.，Moore，D.D.，Seidman，J.G.，Smith，J.A.，Struhl，K。(editor).The modern molecular biology flow process.John Wiley and Sons company.2000。Diatchenko, L., Lau; Y.-F., Campbell, A.P., Chenchik, A., Moqadam, B.H., Lukyanov, S., Lukyanov, K., Gurskaya, N., Sverdlov, E.D.and Siebert, institute of P.D. (1996) NAS newspaper 93,6025-6030.Dudler, R., Hertig, C., Rebmann, G., Bull, J., and Mauch, the wheat cdna coding and the glutathione-S-transferase homologous protein of a kind of pathogenic bacterium inducing of F. (1991).MPMI?4，14-18。Faske, N., Backhausen, J.E., Sendker, M., SingerBayrle, M., Scheibe, R., von Schaewen, A. (1997) express the transgene tobacco of pea chloroplast justice and antisense Nmdh cDNA.To the effect of NADP-MDH level, the growth of transformant stability and plant.Plant physiology 115,705-715.Gottlieb, M.and Chavko, the silver dyeing of the nature on N. (1987) agarose gel and the eukaryotic DNA of sex change.Analytical biochemistry 165,33-37.Hinze，K.，Thompson，R.D.，Ritrer，E.，Salamini，F.，andSchulze-Lefert，P。(1991) target-seeking of barley (Horcleum vulgare) the molecule resistant gene seat of RFLP mediation.Institute of NAS newspaper 88,3691-3695.Mason，H.S，and?Mullet，J.E。(1990) two of soybean nutrition storage protein genes are being grown neutralization to the moisture shortage, the expression of wound and jasmonic reaction.Vegetable cell 2,569-579Mason, H.S., DeWald, D.B., and Mullet, the evaluation in the jasmonic reaction structure territory that methylates in J.E. (1993) the soybean vspB promotor.Vegetable cell 5,241-251.Berger, S., Bell, E., Sadka, A., and Mullet, the VspA and the VspB dna homolog of the coding nutrition storage protein acid phosphatase lipase of the Atvsp of J.E. (1995) Arabidopis thaliana and soybean, and be subjected to the methyl jasmonic, wound, sucrose, light and phosphoric acid are similarly regulated.Molecular biology of plants 1995,2,933-942.Metraux, J.P., Ahl-Goy, P., Staub, T., Speich, T., Steinemann, A., Ryals, J., and Ward, E. (1991) cucumber is to 2, the induction of resistance of 6-dichloro-isonicotinic acid and cause of disease reaction.Henneke, H., und Verma, D.P.S. waits editor, the development of molecular genetics of plant-microorganism interaction, the first roll.The Kluwer academic press, Dordrecht, Holland, 432-439 page or leaf.Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) molecular cloning: laboratory manual, second edition, cold spring harbor laboratory, cold spring port, New York.Taylor, C.B. (1997) understand inhibition altogether.Vegetable cell 9,1245-1249.Thompson J.D., Higgins D.G., Gibson T.J. (1994) CLUSTAL W: by the sequence weight, breach point penalty that the position is special and location matrix are selected, and improve the susceptibility that progressive multisequencing is arranged.Nucleic acids research 22,4673-4680.

Claims

A method selected from the sequence (A) to (H) a DNA molecule or derivative thereof: A: ACATGAAGAG CAGCGACGGC AAGGTCTACG ACTCCTTCAC CATCCACAGG 50 GATTACCGCG ACGTGCTCAG CTGCGTGCAC GACAGCTGCT TCCCCACCAC 100 GCTCGCTAGC TAGCTCATAT CGTCCGGCCG TCATGTCAAT GTAATGGAGG 150 GTCATCCATC CAATAAAATT GTGGGCATGT GTTGAGTAAT AAAATTGGTC 200 AGCTGCACAA TTTATATGTG CTAGTAAAAA GATCATGCAA GAGGTGGGTG 250 TATGCTCGTT ATATATGCTT TGTAACTCCT TCATGTCATA TTWTTATGGG 300 TTAATAAAAA CATCCTTTAT CAAAAAAAAA AAAAAAAAAA GCTTGT 350B: ACAGTATTGG TTGATATGAT TGCTAATCCG GCCCTAGCTC GCGCAGTAAG 50 GGCATCTCCA ATGATTGTAT GATCATCGTT GGTAATATTG CCACATAGAT 100 GATTTTGATG ACATGACGCC TAATAAAAGA AGAAAGAGAA TGAAATCGTA 150 TGAACTTGAA GCAACGGTTC ACGCACAAGC TCCGAGGCAA AGCATGGTTA 200 TTTCTTCCAT GTTTAGTGGA CCCGCAATGC ATGCATGGAG GTGTATCTTA 250 CGATTGATCG CAATTAATAA AGTGTTTCGG TACGATAGTA GCC: ACTTATCTTG AGCATGACAT TAGTCAGCAA ACATCCGGCG aTCATCAGAA 50 GaTCTTACTA GCCTATGTGG GCATTCCACG CTACGAAGGT CCAGAGGTTG 100 ATCCCACTAT AGTGACACAT GATGCGAAGG ACCTCTACAA AGCTGGTGAG 150 AAGAAGCTGG GCACAGATGA GAAAACCTTC ATCCGCATCT TCACTGAACG 200 CAGCTGGGCA CACATGGCAG CTGTTGCTTC TGCTTACCAG CACATGTATG 250 MTCGGTCATT ACAGAAGGTT GTGAAGAGTG AAACATCTGG AAACTTTGAA 300 GTTGCTCTGA TaACTATCCT CAGATGTGCT GAGAATCCAG CTAAGTaTTT 350 TGCTAAGGTG TTAAGGAAGT CCATGAAAGG TCTAGGTE: ACAGAAGCTT GGACAGTTCC ACTCGGAAGC TTCGGTTCGC GCTACCGTTC 50 CCGATGCTCG CCTACCCATT CTACTTGTGG TCAAGGAGTC CAGGGAAGTC 100 AGGCTCGCAT TTCCACCCGA GCAGCGATTT GTTCCAGCCG AACGAGAAGA 150 ACGACATACT GACGTCGACG ACATGCTGGC TTGCCATGGC TGGCCTGCTC 200 GCTGGGCTCA CTGCCGTGAT GGGCCCCCTT CAGATACTCA AGCTCTACGC 250 CGTCCCCTAC TGGATTTTTG TTATGTGGCT GGACTTTGTC ACCTACCTGC 300 ACCACCACGG CCACAACGAC AAGCTTCCCT GGTATCGCGG AAAGGCATGG 350 AGCTATCTGC GCGGGGGCCT GACAACGCTC GACAGGGACT ACGGGTGGCT 400 CAACAACATC CACCACGACA TCGGGACTCA CGTGATCCGC CATCTTTTCC 450 CGCAAATCCC GCATTACCAT CTAGTGGAGG CGACCGAGGC GGCGAACAGS 500 TGCTAGGGAA GTF: ACAAGCTTTT TTTTTTTTTT TTTTTTTTTT TTTTTGGTGG TAAACAACAA 150 CTGCTTCATA TGAACAACGG GCTTGACAAT CAAAATTCTT CCATATGTTG 200 TTATTATACA AAAAATTGCT TAGAGCCAGA GTGAAATTAC ATCAAAGGCC 250 TTTAAAACTT TGTTATAAAA TCTAGTCTCA AACTCCCCCT CGTCTACAGG 300 TGTTCTCCGA GATATTCTCC CCTTGCCTCC AGTGCGAAAT TTCTGCTATG 350 TCTATGCTCT ATTCACACGG ATGGTTTGTC CTTCCAATTC TGTGTTGTTG 400 AAAGTAGAAA CCACAGCTTC AACTTCCTCC TCTGAAGAAA ACGTGACAAA 450 ACCATATCCC TTGGACTTTG GGGTTCCCGG AATTCGGGAT ACCGTGGCAC 500 TGAGGACCTC CCCCTTTTCA GAGAAGAAGT TCTTGAGAAC TTCCGTCGTC 550 ACTTTCTTCG CAAGATTGCC AACATAAACC TTGTG: GTCAAGTTCA CGCCTGCCGA ATCGAGGATG ASTGCGCCGC CCCAAAAGCC 50 CCCGCCGGAG AGGAGAAGAA GACGTCATGG CCGGAGGTAG CGGGAAAGTC 100 CATCGAGGAG GCCAAGGAGA TCATCCTTAA GGACATGCCT GAAGCGGACA 150 TCGTCGTCCT CCCAGCCGGC TCGCCGGTGA CCCTCGACTT CAGGACCAAC 200 CGTGTCCGCA TCTTCGTCGA CACTGTCGCG TCCACTCCCC ACATTGGCTA 250 GCTAGCTTTG CAAGCAAAGG CAACATGGAT GCATTGTGGA TGCTGATGAA 300 TAAGTH: CAGGATCATA GCTACAGGCG ACAATGCCCG GTCATCGACA ATCGCTGGCA 50 GCGGCTTCTC TGAAGCTACC ATCTCTTCTG CTTCTGTGGA TCTTTAGCTG 100 GAACTGGGGG CATGCCGTGG CCAAGTTTGA TCCTGCAAAC ATGACGGAGC 150 TTCAGAAACA TGTCTCCTTT TTCGACCGGA ACAAGGATGG CTTCATCACT 200 CCTACAGAAA CCATCCAAGG GTTTGTTGCA ATCGGTTGCG AGTATGCATT 250 TGCTACTGCT GCCTCTGCCG CCATTCACGG TGCCCTTGCT CCTCAAACAA 300 CCCCGGCTGG TACACCACTG CCTCACTTGA CAATATACGT AGAGAATATC 350 CACAAAGCTA TGCATGGAAG TGATCCAGGT GTATATGATG CCAAAGGAAG 400 GTTTCTTCCC CAAAACTTTG AGGAATTATT CAAAACATAT GCAATACTCC 450 GACCAGATGC GTTGACTCTT GCGGAGATGC ATGTGATGCT CTTTGCAAAA 500 CGGGATCTAG ACCCTATATC ATGGGCACCA CCACAGGTTG AGTGGGGCCT 550 ATTATTCACG CTTGCAAGCG ATTGGCTTGG GTTCCTTCAC AAAGACAGTG 600 TTAGAGGTAT ATATGATGGA AGCCTGTTTA TCAAGTTGGA AAAGAAATGG 650 CACCCTTTTC AAAGTGCTAT GCGATGAACT TGGTGCTAGT TTAGAGTGAG 700 AGTTTGGATA TGGAAAGGTT TGTCCCGAAG AAGGTTTTCC TGCTATCTCC 750 AAATTCAACT AGAGTTTATT TTCTTTTCCT CCAAGTTGTA ATTGGTTTTA 800 TAAGACCTTC ATAGCCGATC AATACAACGA AGCAAGTTGG ATATATTTCC 850 CGACCTTGTA TTCTCTCTCA TGKGCCCCTT ATTATGTTTG CGCCATGAGC 900 GCCTACCCAA GAKGAGCCAT AAGCATAAGG CTCATCCACC TATTGGCCAC 950 GACTACTGTT GGAAATATTC CCTACAKGCA ATATTGKGAK GAWAAWATTT 1000 ATCTATAAAA AAAAAAAAAA AAAAAAAAAA AAAAA...
2. detection compound, composition or mixture are induced the method for the ability of enhanced resistance state (ERS) in plant, said method comprises: (a) plant or plant part are contacted with compound, this plant or plant part comprise expression can be by the gene fragment of the sequence (A) to (H) in the SAR inducing compounds inductive claim 1, (b) expression of the described gene fragment of analysis, the expression of wherein said gene fragment shows described compound, and composition or mixture have the ability of inducing ERS.
3. according to the method for claim 2, the expression of wherein said gene fragment is can not be by the phytopathogen inductive.
4. according to the method for claim 2 or 3, wherein the expression of said gene fragment can also be induced by the ISR inducing compounds.
5. according to the method for arbitrary claim of claim 2 to 4, wherein said gene fragment is given any nucleotide sequence in a kind of and the claim 1, has the gene fragment of substantial homology with the mutant of the gene fragment that limits in the claim 1 or with the gene fragment of the one or more parts that comprise above-mentioned gene fragment and mutant.
6. according to the method for arbitrary claim of claim 2 to 5, wherein said plant or plant part are selected from down group: plant protoplast, vegetable cell, plant tissue, callus, the plantlet of growth, jejune whole strain plant, sophisticated whole strain Plants and Seeds.
7. according to the method for arbitrary claim of claim 2 to 6, wherein said plant or plant part are the plants of cereal class, especially Hordeum or from the plant part in its source.
8. according to the method for arbitrary claim of claim 2 to 7, wherein (A) of claim 1 comprises a kind of nucleotide sequence of encode indicator protein or polypeptide to the gene fragment of (H), and the expression of wherein analyzing described gene fragment is undertaken by the expression of the nucleotide sequence of monitoring coding indicator protein or polypeptide.
9. the screening compound of in plant inducing ERS of having the ability, the method of composition or mixture, wherein use arbitrary method of claim 2 to 8, and wherein said screened compound, composition or mixture provide with the synthetic combinatorial libraries form of compound and/or with the library form of natural product.
10. one kind provides the method with compound of inducing ERS ability in plant, and this method may further comprise the steps:

(a) produce compound a kind of synthetic combinatorial libraries and

(b) method that requires with claim 9 is screened the compound in described library.
11. the new compound with inducing ERS ability, composition or mixture, they are obtained by the method for claim 10.
12. the compound of claim 11, composition or mixture be as agrochemicals, especially the purposes of the agrochemicals of inducing ERS in plant.
13. one kind as arbitrary dna molecular encoded polypeptide or protein that claim 1 limited.
A 14. identification specifically and in conjunction with polypeptide or proteic mono-clonal or polyclonal antibody that claim 13 limited.
15. transgenic plant that comprise recombinant DNA sequence, plant part or seed, described recombinant DNA sequence contains the dna molecular of claim 1.