CN101578360A - tflA gene which can degrade toxoflavin and its chemical derivatives and transgenic organisms expressing tflA gene - Google Patents

Abstract

The present invention relates to a microorganism which can degrade toxoflavin and its derivatives, a protein which can degrade toxoflavin and its derivatives, a use of the protein as a selection marker for plant transformation, a gene which encodes the protein, a recombinant expression vector comprising the gene, a transgenic organism which is transformed with the vector, an expression cassette of a selection marker comprising tflA gene for plant transformation, a recombinant vector comprising the expression cassette, a plant which is transformed with the vector, a method of selecting transgenic plants using tflA gene, and a method of preparing transgenic plants using tflA gene. According to the present invention, transgenic plants which express tflA are given with the resistant character to toxoflavin. Particularly for rice cultivation, more grains can be harvested and quality of grains can be improved, thanks to a resistance to bacterial grain rot. In addition, instead of using expensive antibiotics, transgenic plants can be easily selected by using rather cheap toxoflavin.

Description

Can degrade toxoflavin and the tflA gene of chemical derivative and the genetically modified organism of expressing the tflA gene

Technical field

The present invention relates to the microorganism of energy can degrade toxoflavin and derivative thereof, the protein of energy can degrade toxoflavin and derivative thereof, this protein is as the purposes of the selected marker of Plant Transformation, the gene of code for said proteins, the recombinant expression vector that contains described gene, genetically modified organism with this carrier conversion, the expression cassette that contains the selected marker of the tflA gene that is useful on Plant Transformation, the recombinant vectors that contains this expression cassette, with described carrier plant transformed, use the screening method of the transgenic plant of tflA gene, with the method for using tflA genes produce transgenic plant.

Background technology

Rotting of rice grain is that gram negative bacterium by being called bacillary glume blight bacterium (Burkholderia glumae) causes, and rice grain rot very responsive to the variation of weather.Recently, in the country that the paddy rice of the country that comprises Korea S, Japan, South East Asia and the U.S. is planted, the cereal that takes much count of paddy rice rots.It is reported that the rotting of rice grain spread in the season that hot and humid paddy rice blooms, and caused 34% the underproduction.Report that in addition bacillary glume blight bacterium can produce the necessary virulence factor that causes the rotten of rice grain and bacterial leaf spot (bacterial blight) outburst, as toxoflavin, reumycin (reumycin), planomycin (fervenulin).Wherein, toxoflavin is most important virulence factor.

" Paenibacillus polymyxa (Paenibacillus polymyxa) " usually near plant roots growth vigorous, it can promote the growth of plant, and by with soil in other microbial interaction prevent the generation of plant disease.In addition, it can also produce multiple microbiotic and lytic enzyme, is considered to useful microorganism.In addition, the gram positive bacterium that it still can fixed nitrogen, recently, its importance more and more receives publicity.

Expression vector contains at least a genetic marker, and this genetic marker can be used for cell transformed is screened by the cell growth that suppresses not contain the selected marker.It is isolating from bacterium that the overwhelming majority is used for screening the selected marker who transforms plant, their a kind of enzymes that can make the selective chemical material carry out metabolic degradation of encoding, and described selective chemical material can be microbiotic or weedicide.

The most popular selected marker who is used for screening Plant Transformation is from the isolating neomycin phosphotransferase II of Tn5 (nptII).Other selected marker comprises hygromycin phosphotransferase gene, and a kind of antibiotic hygromycin of this gene pairs has resistance.

Many selected markers have been used to screen the transgenic plant tissue.Yet, some defectives or restriction are arranged based on the screen body cording of poisonous chemical substance.It is very difficult at first using the direct recovery of the transgenic plant that can survive normally of chemical sieve method.Secondly, not all selected marker system can both be applied to each tissue and each plant.The 3rd, some chemical substances that add in order successfully to screen are microbiotic.Therefore, for fear of making any virulence factor produce resistance, because of preventing from microbiotic and weedicide are had the propagation of the gene of resistance as much as possible.The 4th, some chemical substances that add in order successfully to screen are microbiotic, and these microbiotic are very expensive, therefore, need the cheap selected marker of exploitation.

The objective of the invention is tflA protein and this proteinic gene of coding for Paenibacillus polymyxa JH2 is provided, described protein relates to the septic resistance reaction to rice grain; Identify the proteinic feature of described tflA; Described gene is produced transgenic plant by recombinating; High quality and nontoxic paddy rice are provided, and this paddy rice has the resistance of improvement to septic blight and the deleterious insect that causes rice grain.In addition, the invention provides tflA enzyme that use can degrade toxoflavin and can be easily and the selected marker of easily transgenic plant being screened.

Summary of the invention

The protein of the tflA of the present inventor by expressing Paenibacillus polymyxa JH2 has prepared the genetically modified organism that rots to have resistance to rice grain, and rotting of described tflA protein and anti-rice grain is relevant.The present inventor is understanding under biological and the interactional situation of Paenibacillus polymyxa JH2, and the present inventor can provide a kind of new system that is used for the controlling plant disease.As a result, finished the present invention.

Therefore, an object of the present invention is to provide the microorganism of a kind of energy can degrade toxoflavin and derivative thereof.

Another object of the present invention provides the tflA protein of a kind of energy can degrade toxoflavin and derivative thereof.

Another object of the present invention provides the application of tflA protein as the selected marker of Plant Transformation.

Another object of the present invention provides the proteinic gene of tflA of coding energy can degrade toxoflavin and derivative thereof.

Another object of the present invention provides the recombinant expression vector that contains the tflA gene.

Another object of the present invention provides the tflA protein of the reorganization of being expressed by the recombinant expression vector of the described tflA of containing gene.

Another object of the present invention provides the genetically modified organism of the recombinant expression vector conversion of using the described tflA of containing gene.

Another object of the present invention provides the expression cassette of the selected marker that contains the tflA gene that is useful on Plant Transformation.

Another object of the present invention provides the recombinant vectors that contains described expression cassette.

Another object of the present invention provides uses described carrier plant transformed.

Another object of the present invention provides the method for using tflA genescreen transgenic plant.

Another object of the present invention provides uses the tflA gene to prepare the method for transgenic plant.

In order to realize purpose of the present invention, the invention provides the microorganism of energy can degrade toxoflavin and derivative thereof.Preferably, described microorganism is a bacterial origin.More preferably, described microbe-derived in series bacillus genus (genus Paenibacillus), more preferably derive from Paenibacillus polymyxa, more preferably derive from Paenibacillus polymyxa JH2, this Paenibacillus polymyxa JH2 carried out preservation (deposit number is KCTC 10959BP) by Korea S bio-engineering research institute (Korean Bioengineering Institute) on June 13rd, 2006.Described toxoflavin is to cause rice grain to rot and the necessary virulence factor of the bacterial leaf spots of farm crop.According to the present invention, the derivative of described toxoflavin comprises having the active any derivative identical with toxoflavin.Described derivative includes but not limited to 3-methyl toxoflavin, 4,8-dihydro toxoflavin and 3-methyl reumycin etc.

Paenibacillus polymyxa usually near plant roots the growth vigorous, can promote plant growth and by with soil in other microbial interaction prevent plant disease.And, owing to can produce multiple microbiotic and lytic enzyme, therefore be included into the row of useful microorganism.The present inventor finds can degrade toxoflavin from the tflA protein of Paenibacillus polymyxa JH2, and toxoflavin is a kind of septic material that can cause rice grain.

In addition, the invention provides the tflA protein of energy can degrade toxoflavin and derivative thereof.The variant of the gene of code for said proteins also belongs to scope of the present invention.Described variant can have different aminoacid sequences, but has similar function and immune characteristic to aminoacid sequence shown in SEQ ID NO:2.Different variants can comprise such sequence: this sequence and aminoacid sequence shown in SEQ ID NO:2 have at least 50%, preferably have at least 70%, more preferably have at least 80%, further preferably have at least 90%, further preferably have at least 95% sequence homology.Described variant can also comprise such protein sequence: after one or more amino-acid residue in the described protein sequence is substituted, inserts or deletes, can also keep the ability of can degrade toxoflavin.The method of described replacement, insertion or deletion amino-acid residue can be the known any method of various equivalent modifications.

According to an embodiment of the invention, above-mentioned tflA protein can be as the selected marker of Plant Transformation.TflA endonuclease capable can degrade toxoflavin of the present invention, and make plant have resistance to the toxoflavin chemical substance.Toxoflavin is to cause the septic most important virulence factor of rice grain.Genetically modified organism of the present invention can be a plant.Be preferably paddy rice or Arabidopis thaliana (Arabidopsis thaliana).

The present invention also provides coding tflA proteinic gene.Preferably, this gene is the gene that comprises the nucleotide sequence shown in SEQID NO:1.This gene can comprise with nucleotide sequence shown in SEQ ID NO:1 having at least 50% preferred at least 70%, more preferably at least 80%, the further nucleotide sequence of preferred at least 90%, further preferred at least 95% sequence homology.Gene with described sequence homology can prepare by the nucleotide sequence shown in SEQ ID NO:1 is replaced, inserts or deletes.The method of described replacement, insertion or deleted nucleotide can be those skilled in the relevant art various any method.Yet, should keep the ability of can degrade toxoflavin by the described genetic mutation encoded protein matter of the acquisition by described replacement, insertion or deleted nucleotide.

" per-cent of sequence homology (%) " can compare by the sequence to two researchs and determine, described two sequences has best comparison mutually at comparison domain.The polynucleotide sequence of described comparison domain and the part of peptide sequence can be included in interpolation or the deletion (that is breach) in the two sequences (not having increases or deletion) that relates to best comparison.Described per-cent be based on following calculate determine: determine to be present in the identical Nucleotide in the two sequences or the number of amino acid whose position; Obtain the number of the position of mating in them; The number of position of coupling divided by the total position number that exists in comparison domain, will be obtained institute's numerical value then and multiply by 100, obtain the per-cent (%) of sequence homology.Can use known working method by using computer (at Wisconsin heredity software package (Wisconsin GeneticsSoftware Package), heredity computer set (Genetics Computer Group) (GCG), 575Science Dr., Madison) in GAP, BESTFIT, FASTA and TFAST; WI or BlastN and BlastX that can (National Center for Biotechnology Information) obtains from national biotechnology information science center) or, compare the best comparison of sequence by determining.

Term " same basically " or " similar basically " mean the polypeptide with this feature be included under the stringent condition can with the sequence of target polypeptide hybridization.Herein, stringent condition is meant under 2 times SSC solution neutral temperature is 65 ℃ condition.

The polypeptide of " similar basically " has above-mentioned sequence, except because the difference that the position of different residues may exist in the two sequences that causes of amino acid whose conservative variation.Described amino acid whose conservative variation is meant in the exchange that has between the amino-acid residue of similar side chain.For example, the amino acid group with alkyl group side chain comprises glycine, L-Ala, Xie Ansuan, leucine and Isoleucine; Amino acid group with hydroxyl side chain comprises Serine and Threonine; Amino acid group with amide side chains comprises aspartic acid and glutamine; Amino acid group with aryl side chain comprises phenylalanine, tyrosine and tryptophane; Amino acid group with basic side chain comprises Methionin, arginine and Histidine; Amino acid group with sulfur-containing side chain comprises halfcystine and methionine(Met).

Basically same polynucleotide sequence is meant that the polynucleotide of research comprise having at least 70% identity, preferred at least 80% identity, more preferably at least 90% identity, the most preferably sequence of at least 95% identity.Basically same another kind of meaning is meant that then their sequence is same mutually basically when two nucleic acid molecule phase mutual crosses specifically under stringent condition.Described stringent condition changes with the sequence variation of Nucleotide.Therefore, described stringent condition may be different under different situations.Usually, under certain ionic strength and pH, low about 10 ℃ of the solvent temperature (Tm) of the selected temperature bit of stringent condition sequencing row.Tm is defined as 50% the target sequence and the temperature (under certain ionic strength and pH situation) of complementary probe hybridization fully.Tm is determined with forming by the length of the nucleotide base of probe, and can by document (referring to, Sambrook, T.et al., (1989) Molecular Cloning-A Laboratory Manual (second edition), Volume 1-3, ColdSpring Harbor Laboratory, Cold Spring) in the method described calculate.Usually, the stringent condition that carries out the southern blotting technique analysis is included in 65 ℃ of SSC with 0.2 times and washs.For suitable oligonucleotide probe, can wash at 42 ℃ of SSC with 6 times.

The present invention also provides the recombinant expression vector that contains above-mentioned tflA gene.Preferably, this carrier is the carrier that can express in intestinal bacteria, virus, plant or animal.In one embodiment, the invention provides the tflA expression vector, this expression vector is to prepare by being inserted in the pCamLA carrier from the tflA gene of Paenibacillus polymyxa JH2, comprises the hygromix phosphotransferase Hyg that is arranged in T-DNA in the pCamLA carrier ^RWith the kalamycin resistance gene that is positioned at outside the T-DNA.

" carrier " is meant the vehicle that is used for nucleic acid is transferred to host cell.Described carrier can be a replicon, other dna fragmentation attached on this replicon so that the fragment of adhering to can be replicated." replicon " function of the amplicon dna of performance single cell in the organism that lives, and corresponding to genetic elements (for example, plasmid, phage, clay, karyomit(e) and virus) that can self-replacation." carrier " is defined as importing in the body nucleic acid or the instrument in the external cell.It comprises viral carrier or non-viral carrier.Described viral carrier comprises retrovirus carrier, adeno associated virus (adeno-realted virus) carrier, baculovirus vector, herpes simplex carrier, cowpox carrier, Epstein-Barr virus carrier and adenovirus carrier etc.Described non-viral carrier comprises plasmid, liposome, charged grease (electrically charged lipids) (cytofectin), DNA-protein complex and biological polymer etc.Except that comprising nucleic acid, described carrier also comprises at least one regulation domain and/or selected marker, and this selected marker is used to screen, detect and monitor the result that nucleic acid transforms (for example, be transformed in certain tissue and continue expression etc.).

The present invention also provides the reorganization tflA protein of being expressed by recombinant expression vector of the present invention.At the reorganization tflA of expression in escherichia coli protein is not glycosylated, and the reorganization tflA albumen of expressing in plant or zooblast is glycosylated.Therefore, based on described proteinic application target, can select and use suitable reorganization tflA protein.

The present invention also provides the genetically modified organism that uses recombinant expression vector of the present invention to transform.Described genetically modified organism can be microorganism, viral plant or animal etc.Preferably, described genetically modified organism is plant, more preferably paddy rice.

The present invention also provides the method for preparing transgenic paddy rice, this method can be carried out vegetative propagation by tissue culture, wherein, the tflA gene gives expression to from the tflA expression vector, and this expression vector is to be inserted in the pCamLA carrier and to prepare by coming from tflA gene among the Paenibacillus polymyxa JH2; Described tflA gene can make the septic toxoflavin degraded that causes rice grain, comprises the hygromix phosphotransferase Hyg that is arranged in T-DNA in the pCamLA carrier ^RWith the kalamycin resistance gene that is positioned at beyond the T-DNA.

The present invention also provides by tissue culture can carry out vegetative transgenic paddy rice, wherein, the tflA gene is from Paenibacillus polymyxa JH2, and this gene can make the septic toxoflavin degraded that causes rice grain, thus the render transgenic plant to rice grain rot have resistance.

The present invention also provides the expression cassette of the selected marker that is used for Plant Transformation, and this expression cassette comprises the following sequence that connects according to 5 ' to 3 ' direction operability:

(i) promoter sequence;

The sequence of enzyme that (ii) can degrade toxoflavin; With

(iii) 3 ' untranslated terminator sequence.

For albumen mass-energy being expressed in host cell and being made host cell have resistance to having the toxic preparation of selection, the encoding sequence of enzyme that can degrade toxoflavin provides with the form of expression cassette with regulatory element usually, described regulatory element can come the recognition coding sequence by the biochemical mechanism in the host cell, and can regulate transcribing and translating of open reading-frame (ORF) in the host cell.Described expression cassette not only comprises the initiator that is used to transcribe that derives from any gene that can express in host cell usually, but also comprises in order to discern and to be connected ribosomal other initiator that is used to transcribe.In the eukaryote cell, described expression cassette also comprises the terminator zone that is positioned at described open reading-frame (ORF) downstream that is used to transcribe usually, to realize transcribing the termination with the polyadenous glycosidation of primary transcript.In addition, the usage quantity of codon can with the usage quantity fit of the codon that in host cell, is allowed.Be used for determining the fundamental principle of expression of the hybrid dna construct of the host cell selected, usually can be understood by those skilled in the art, and the preparation method of hybrid dna construct to be expressed is identical to any host cell that comprises prokaryotic cell prokaryocyte and eukaryotic cell.

For the expression cassette according to an embodiment of the invention, above-mentioned promotor can be but be not limited to CaMV 35S promoter, actin promoter, ubiquitin promoter, pEMU promotor, MAS promotor or histone promotor.Term " promotor " is meant the DNA zone that is positioned at the structure sequence upstream, and is meant and combines the dna molecular of transcribing with initiation with RNA polymerase." plant promoter " is meant and can causes the promotor of transcribing in vegetable cell." constitutive promoter " is meant to have active promotor under most environment and under the developmental condition and under the condition in cytodifferentiation.Because the screening of genetically modified organism can be carried out in being in the different tissues of different times, therefore, in the present invention, preferably uses constitutive promoter.Therefore, described constitutive promoter does not limit the possibility of screening.

For the expression cassette of one embodiment of the present invention, above-mentioned terminator can be but be not limited to: the terminator of rouge alkali synthetase (nopaline synthase (NOS)) or the terminator of paddy rice αDian Fenmei RAmy1 A.About the necessity of terminator, it has been generally acknowledged that to exist under the situation of terminator that the reliability and the validity of transcribing in the plant increase to some extent.Therefore, according to context of the present invention, very preferably use this terminator.

For the expression cassette of one embodiment of the present invention, the encoding sequence of the enzyme of above-mentioned energy can degrade toxoflavin can comprise the nucleotide sequence shown in SEQ ID NO:1.In addition, described encoding sequence can also comprise the nucleotide sequence that has at least 70%, preferred at least 80%, more preferably at least 90%, most preferably at least 95% sequence identity with nucleotide sequence shown in SEQ ID NO:1.

In the present invention, term " is operably connected " and is meant and can plays a role with expressing heterologous protein expression box element as a unit.For example, be operably connected to the promotor of allogeneic dna sequence DNA, coding can promote the protein with the generation of the corresponding function mRNA of allogeneic dna sequence DNA.

For the expression cassette according to an embodiment of the invention, the expression cassette of target protein comprises (i) promoter sequence, (ii) the encoding sequence of target protein and (iii) can also comprise 3 '-untranslated terminator sequence.Described target protein includes but not limited to: commercially available treatment protein and polypeptide, for example, promoting erythrocyte is produced plain (EPO), is organized fibrinogen matter N,O-Diacetylmuramidase activator (t-PA), urokinase and uPA (prourokinase), tethelin, cytokine, Factor IX, Epoetin Alfa (epoetin-α), granulocyte colony-stimulating factor and vaccine etc.

For the expression cassette according to an embodiment of the invention, the expression cassette of target protein can be built into one expression cassette, wherein, and the expression cassette of described target protein and the expression cassette of above-mentioned selected marker polyphone.In other words, the expression cassette of the expression cassette of target protein and selected marker can be one by one on sequence.In addition, also can make the expression cassette of target protein be arranged in different expression cassettes with the expression cassette of above-mentioned selected marker.

The present invention also provides recombinant vectors, and this recombinant vectors comprises expression cassette of the present invention.For open reading-frame (ORF) is remained in the host cell, described recombinant vectors provides with the form of replicon and selecteed host cell is duplicated; Described replicon comprises the open reading-frame (ORF) of the present invention that is connected on the DNA to be identified.Therefore, selected host cell is depended in the selection of replicon to a great extent.Selecting the replicon of suitable selected host cell is that those skilled in the relevant art are known.

Special replicon can be transferred to himself in other cell (as vegetable cell) in whole or in part.As a result, open reading-frame (ORF) of the present invention can automatically be transferred in the vegetable cell.Have this active replicon and be called as " carrier " of the present invention.The example of this carrier comprises the Ti-plasmid vector, and when this carrier was present in the appropriate host cell (as agrobacterium tumefaciens), this carrier can be transferred to the part (so-called T-zone) of himself in the vegetable cell.Also use another kind of Ti-plasmid vector, so that the DNA of existing vegetable cell or its assorted and DNA are transferred in the protoplastis, wherein, described dna sequence dna is suitable for importing in the genome of plant, to produce new floristics (referring to, EP0 116 718 B1).Particularly preferred Ti-plasmid vector is as at EP 0 120 516 B1 and USP No.4, the so-called binary vector of describing in 940,838.The another kind of carrier that can be used for DNA of the present invention is imported to plant host cell can be selected from the virus vector that derives from double-stranded plant virus (as CaMV), strand virus or geminivirus infection group etc., for example incomplete plant viral vector.When suitable conversion is difficult for carrying out in plant host cell, use this carrier especially useful.The example of this kind of plant can comprise that wood belongs to (lignum sp), especially tree and trailing plant.

In order to realize another object of the present invention, provide and used recombinant vectors of the present invention to carry out transformed host cells.Preferably, described host cell can belong to Agrobacterium.More preferably, described host cell can be an agrobacterium tumefaciens.

In order to realize another object of the present invention, provide and used recombinant vectors of the present invention to carry out plant transformed.Described plant can be paddy rice or Arabidopis thaliana.

The method of Plant Transformation comprises any method that DNA can be transferred in the plant.The method of described conversion must not need to comprise be used for the tissue culture box/or the breeding period.At present, the method for transformation of plant not only can be used for dicotyledons usually, can also be used for monocotyledons.In principle, any method that is used for Plant Transformation may be used to the DNA of hybridization of the present invention is imported to suitable ancester cell.Can suitably use the following method that is selected from: calcium/polyoxyethylene glycol method _ (Krens, F.A.et al., 1982, Nature 296,72-74; Negrutiu I.et al., June 1987, Plant Mol.Biol.8,363-373), the electroporation method of protoplastis (Shillito R.D.et al., 1985 Bio/Technol.3,1099-1102), method (the Crossway A.et al. of microinjection in the plant element, 1986, Mol.Gen.Genet.202,179-185), partickle bombardment method (DNA or RNA-coatedparticles) (the Klein T.M.et al. of different plant elements, 1987, Nature 327,70), utilize the method (EP 0 301 316) etc. of (incomplete) virus infection that is used for transgenosis that conversion mediated of the infiltration of plant or mature pollen or pollen granule by agrobacterium tumefaciens.In the present invention, preferable methods comprises by agriculture bacillus mediated DNA transfer.Particularly preferred method is to use the method for so-called binary vector as carrier, and this is described in EPA 120 516 and USP No.4, in 940,838.

In order to realize another object of the present invention, provide the transgenic seed that uses recombinant vectors of the present invention to transform.

In order to realize another object of the present invention, provide the screening method of the transgenic plant that comprise the following steps:

-with recombinant vectors of the present invention plant, plant organ or vegetable cell are transformed; With

-genetically modified organism that obtains is bred.Method of the present invention comprises the step of using recombinant vectors of the present invention that plant, plant organ or vegetable cell are transformed, and wherein, described conversion can mediate by agrobacterium tumefaciens.In addition, method of the present invention is included in the step that makes the genetically modified organism propagation that obtains in the developing medium that contains toxoflavin.Described genetically modified organism can be survived in containing the developing medium of toxoflavin, and not genetically modified biology can not be survived in containing the developing medium of toxoflavin.As a result, transgenic plant can be gone out by easily screened.

In order to realize another object of the present invention, provide the method that is used to produce transgenic plant that comprises the following steps:

-with recombinant vectors of the present invention vegetable cell is transformed;

-in containing the developing medium of toxoflavin, make the plant transformed cell proliferation that obtains and

-from the plant transformed cell that obtains, differentiate transgenic plant again.Method of the present invention comprises the step of using recombinant vectors of the present invention that vegetable cell is transformed, and wherein, described conversion can be by Agrobacterium tumefaciens mediated.In addition, method of the present invention is included in the step that makes the transformed plant cells propagation that obtains in the developing medium that contains toxoflavin.Described genetically modified organism can be survived in containing the developing medium of toxoflavin, and not genetically modified biology can not be survived in containing the developing medium of toxoflavin.As a result, transgenic plant can be gone out by easily screened.In addition, method of the present invention comprises be divided into the step of transgenic plant again from resulting transformed plant cells.From resulting transformed plant cells, the method that is divided into transgenic plant again can be the known any method of association area.

Description of drawings

Fig. 1 has shown the separation that is used for illustrating the Paenibacillus polymyxa JH2 that is derived from intermountain soil, rice field or field soil, rice paddy seed etc. and the synoptic diagram of feature.

Fig. 2 has shown that toxoflavin is carried the clay clone's of Paenibacillus polymyxa JH2 (all clones have the 1.5kbEcoRI fragment) intestinal bacteria degraded.

Fig. 3 has shown the outer dioxygenase (ring-cleavage extradioldioxygenase of Exiguobacterium sp.255-15) of open loop glycol of small Bacillaceae and the result of following proteic similarity analysis: (the agnoprotein matter of (2) salt tolerant genus bacillus C-125; (3) the agnoprotein matter of salt tolerant genus bacillus C-125; (4) NahC of the JF8 of Bacillaceae (1,2-dihydroxy naphthlene dioxygenase); (5) NahH of the JF8 of Bacillaceae; (6) ThnC of Sphingopyxis macrogoltabida TFA; (7) BphC of Rhodopseudomonas LB400; (8) the conservative putative protein matter of the pathogenic mutation (X.axonopodis pv.citri st) 306 of carpetweed Xanthomonas campestris citrus; (9) the conservative protein matter between two kinds of peptides that are marked as *).

Fig. 4 has shown the TflA protein of purifying, and wherein (A) carries out the painted result of coomassie to TflA protein, (B) is in order to determine Mn ⁺⁺And the influence of the degraded of dithiothreitol (DTT) (DTT) toxoflavin that TflA is caused and result's (toxoflavin that in each swimming lane, all contains 100 μ M: swimming lane 1, the MnCl of 1mM of the TLC plate analysis that carries out ₂ Swimming lane 2, the His-TflA of purifying and the MnCl of 1mM ₂Swimming lane 3, the DTT of 5mM; Swimming lane 4, the His-TflA of purifying and the DTT of 5mM; Swimming lane 5, the MnCl of the DTT/1mM of 5mM ₂Swimming lane 6, the His-TflA of purifying and the MnCl of DTT/1mM ₂).

Fig. 5 (A) has shown the optimum temps of the His-TflA can degrade toxoflavin of purifying; Fig. 5 (B) has shown the variation that amount that toxoflavin is degraded by His-TflA is passed in time.

Fig. 6 has shown the best pH of the His-TflA can degrade toxoflavin of purifying.

Fig. 7 has shown the chemical structure (the different functional group of circular expression) of toxoflavin and derivative thereof.

Fig. 8 has shown the toxoflavin that caused by His-TflA and the degraded of derivative thereof.

Fig. 9 is the L-B figure (Lineweaver-Burk) of the degraded of the toxoflavin that caused by His-TflA of expression and derivative thereof.

Figure 10 (A) is the figure of the genetic construction of expression pCamLA gene; Figure 10 (B) is figure (MCS, the multiple clone site of the genetic construction of expression pJ904 (pCamLA::tflA); LB, left boundary; RB, border, the right; 35S, 35S promoter; P, PstI; Sm, SmaI; S, SacI).

Figure 11 has comprised the image of preparation transgenic paddy rice.

Figure 12 (A) is the figure of the genetic construction of expression pJ904 gene (pCamLA::tflA); Figure 12 (B) has shown result (the T-DNA zone of (A) pJ904 to the DNA analysis of transgenic paddy rice; LB, left margin; RB, right margin; Hyg ^R, hygromix phosphotransferase; 35S, the CaMV 35S promoter; (B) DNA analysis of transgenic paddy rice T2; M, the molecular size mark; 1, Cv6-2; 2, Dt1-1; 3, Dt1-2; 4, Dt3-6; 5, Dt27-1; 6, Dt27-2; 7, Dt27-3; 8, Dt27-4; 9, Dt34-1; 10, Dt34-3; 11, Ct36-3; 12, pJ904; 13, Cv10-1; 14, Dt4-1; 15, Dt4-3; 16, Dt7-7; 17, Dt19-5; 18, Dt40-5; 19, Ct18-2; 20, Ct18-4; 21, Ct18-5; 22, Ct18-6; 23, Ct9-1; 24, pJ904; 25, Cv6-4; 26, Dt2-1; 27, Dt2-5; 28, Dt7-3; 29, Dt7-5; 30, Dt7-9; 31, Dt7-10; 32, Dt9-6; 33, Dt16-1; 34, Dt19-3; 35, Dt38-3; 36, pJ904).

Figure 13 has shown result (M, the mark to the DNA analysis of wild-type paddy rice and transgenosis T2 plant; WT, paddy rice (Dongjinbyeo) wild-type plant; Dt, paddy rice (Dongjinbyeo) the transgenosis T2 plant of expression tflA; TflA, the TflA of the His6 mark of purifying).

Figure 14 has comprised photo (A, the Dongjinbyeo wild-type plant of the rice leaf of handling with toxoflavin; B and C, different transgenic lines (B, Dt40-6; C, Dt19-5); D, Dongjinbyeo wild-type plant in the dark);

Figure 15 is the synoptic diagram that is included in the T-DNA among the pCamLA.

Figure 16 is the synoptic diagram that is included in the T-DNA among the pTflA.

Figure 17 has comprised with pCamLA (left side) or pTflA (the right) carrier and transforms the back and carry out the photo of the rice callus tissue behind the programmed screening with Totomycin (30 μ g/ml).

Figure 18 comprises the photo of using pCamLA (left side) or pTflA (the right) carrier to transform the back and using the rice callus tissue after toxoflavin (5 μ g/ml) screens, and wherein, described screening is organized at described rice callus again and carried out before the differentiation.

Figure 19 comprised with pCamLA and transformed the back and with the photo of the rice callus tissue after toxoflavin (the 7.5 μ g/ml) screening, and wherein, described screening is inserted and carried out after all around after breaking up in the developing medium again will stating rice callus tissue.

Figure 20 comprised with pTflA and transformed the back and with the photo of the rice callus tissue after toxoflavin (the 7.5 μ g/ml) screening, and wherein, described screening is inserted and carried out after all around after breaking up in the developing medium again will stating rice callus tissue.

Figure 21 has shown the result of the agarose electrophoresis of PCR product, and described PCR product is to obtain from the pcr amplification from the genomic dna of the genetically modified organism that is filtered out.

Figure 22 has shown transgenic plant, has inserted following carrier: pCamLA:tflA, pCamLA (Δ hpt) respectively in this plant: tflA, pMBP1:tflA and pMBP1 carrier.

Figure 23 has shown the result of the agarose electrophoresis of PCR product, and described PCR product is to obtain from the pcr amplification from the genomic dna of the genetically modified organism that is screened.

Figure 24 has shown the effect that transgenic plant are bleached.

Embodiment

Embodiment has hereinafter described the present invention in detail, their illustrative purposes, rather than in order to limit the present invention.Should be understood that scope of the present invention is not limited to these embodiment, can comprise other embodiment that belongs in aim of the present invention and the scope.

Embodiment

EXPERIMENTAL EXAMPLE 1: the condition of culturing bacterium cell

With the Paenibacillus polymyxa cell cultures in 28 ℃ liquid or solid LB substratum.All intestinal bacteria are cultivated in 37 ℃ liquid or solid LB substratum.Employed antibiotic concentration is as follows: Rifampin 50 μ g/ml, tsiklomitsin 10 μ g/ml, kantlex 30 μ g/ml, penbritin 100 μ g/ml, paraxin 25 μ g/ml.

The enzyme of EXPERIMENTAL EXAMPLE 2:DNA is handled

1, the preparation of chromosomal DNA

(Leach, J.E.et al. 1990.MPMI.3:238-246), extract chromosomal DNA from Paenibacillus polymyxa with method that improved N,O-Diacetylmuramidase-sodium laurylsulfonate (SDS) decomposes.In containing suitable antibiotic LB substratum (500ml), rotating speed is 230rpm with microbial culture, and temperature is 28 ℃.By centrifugal collection bacterial cell.With the 0.9%NaCl solution washing bacterial precipitation of 1ml, and with the dissolving of the GTE solution of 330 μ l (50mM glucose, 25mM Tris-HCl[pH 8.0], 10mM EDTA[pH8.0]).Subsequently, add 3 μ l N,O-Diacetylmuramidases (50mg/ml), and reacted 30 minutes down at 37 ℃.Use 10% the SDS smudge cells of 17 μ l then, be reflected at and carried out under 37 ℃ 10 minutes.Add the RNaseA (10mg/ml) of 10 μ l, and under 37 ℃, carried out 1 hour.The 0.5M EDTA that adds 17 μ l is reflected at and carried out under 37 ℃ 10 minutes.Add Proteinase K (20mg/ml) solution of 2.5 μ l, be reflected at and carried out under 37 ℃ 6 hours.Add phenol: chloroform: the ratio of primary isoamyl alcohol is 25: 24: 1 (volumes: volume: mixture volume), and with the mixture vigorous stirring that obtains 5 minutes.After under 16,816 * g centrifugal 5 minutes, supernatant is transferred in the new pipe, and added phenol, extract twice.Add the chloroform that has equal volume with described supernatant: the ratio of primary isoamyl alcohol is 24: 1 (volumes: mixture volume).Add the sodium acetate [pH 7.0] of the 3M of a volume and the ethanol of 2 volumes 95%.With mixture under 16,816 * g centrifugal 15 minutes.After centrifugal, pour out supernatant lightly, washing with alcohol precipitation with 70%.After all ethanol volatilizations, with the TE[pH 8.0 of 0.2ml] dissolution precipitation, and be kept at-20 ℃.

2, the preparation of plasmid DNA

Method (Sambrook, J.et.al., 1989.Molecular Cloning:ALaboratory Manula.2nd ed.Cold Spring Harbor Laboratory, Cold Sprong Harbor, NY) preparation e. coli plasmid dna with alkaline lysis.Bacillus coli cells is cultivated in containing of 2mL antibiotic LB substratum, and rotating speed is 200rpm, and temperature is 37 ℃.By centrifugal 1 minute collection bacterial cell under 16,816 * g.Shift out supernatant, and bacterial precipitation is mixed, add 5 μ lRNaseA solution (20 μ g/ml) then with the ice-cold solution I of 100 μ l (50mM glucose, 25mMTris-HCl[pH 8.0], 10mM EDTA[pH 8.0]).(0.2N NaOH 1%SDS), shakes the mixture that obtains gently to add the solution II of 200 μ l.Add the ice-cold solution III (the 5M potassium acetate of 60ml, 11.5ml Glacial acetic acid, 28.5ml sterile distilled water) of 150 μ l and mix.Is centrifugal bacterial lysate 10 minutes under 16,816 * g at 4 ℃ down and at rotating speed.Handle with phenol subsequently, supernatant is transferred in the new pipe, and add 95% the ethanol of 1ml, mix.By 4 ℃ be under the condition of 16,816 * g down and at rotating speed, obtained the DNA precipitation in centrifugal 15 minutes.With 70% washing with alcohol precipitation, and with the TE[pH 8.0 of 30 μ l] dissolution precipitation, be kept at 4 ℃ then.

3, use the agarose gel electrophoresis enzyme

Restriction enzyme, calf intestine alkaline phosphatase, T4DNA ligase enzyme and other related reagent are available from Takara (Japan), Boehringer Mannheim (climing extra large nurse, Germany), Stratagene (La Jolla, CA), GibcoBLR (Gaithersburg, MD) and Sigma (St.Louis, MO).Analysis condition carries out according to the described condition of the operation instruction of manufacturer.With different endonuclease bacterial digestion DNA, use sepharose (Sigma) (45mM Tris-borate, 1mM EDTA) (Ausubel, F.M.1991.Current Protocols in Molecular Biology.Wiley Interscience.New York) in 0.5 times tbe buffer system of 0.7% (weight/volume) to separate then.Especially, DNA is mixed, and use gel loading buffer (contain 0.25% tetrabromophenol sulfonphthalein, 0.25% dimethylbenzene aniline F, exist 15% in the water phenanthrene can) sample is joined on the gel.To obtain with the ethidium bromide solution of 0.5 μ g/ml gel-colored 30 minutes, under transilluminator, observe then.

4, with the separation of dna fragmentation in sepharose

Use QIAEX II gel extraction kit (150) (QIAGEN, Germany), dna fragmentation is separated in sepharose.

EXPERIMENTAL EXAMPLE 3: use the conversion of calcium chloride

As described (Maniatis, T.et al., 1982.Molecular Cloning such as Maniatis; ALaboratory Manual, Cold Spring Harbor Lab Press, New York), use calcium chloride to come transformed into escherichia coli.In order to prepare competent cell, Bacillus coli cells cultivated 12 hours after, be that 37 ℃, rotating speed are under the condition of 230rpm in temperature, cultivate exponential phase of growth (A600=0.6).Collect substratum then, and keep 20 minutes on ice.By being that 4 ℃, rotating speed are centrifugal under the condition of 2,700 * g in temperature, collecting precipitation from substratum.With the CaCl that places on ice ₂Solution adds (sterilized 10mM calcium chloride and 10% glycerine) and mixes.After ice bath reaction 20 minutes, with mixture under 4 ℃, in 2,700 * g centrifugal 10 minutes.With the CaCl that places on ice ₂Solution joins in the precipitation, mixes.Mixture is divided into equal portions (every part of 0.1ml), and places the centrifuge tube of precooling, be kept at-70 ℃, up to further use.In order to transform, the TE damping fluid of 85 μ l is joined in the connection mixture of 15 μ l.In the competent cell that slowly thaws on ice, add the connection mixture of described precooling lentamente, and mix.Reaction was carried out 20 minutes.By under 42 ℃, in the LB of 1ml, carry out after heat-shocked handles, with cell 37 ℃ of static cultivations 1 hour down.The cell that obtains is inoculated into equably contains in the antibiotic solid LB substratum.

EXPERIMENTAL EXAMPLE 4: the separation of the bacterium of can degrade toxoflavin

The minimal medium of 2mL is joined in the agricultural land soil sample of 1mg.After cultivating 48 hours under 37 ℃, they are inoculated in the LB nutrient agar of the toxoflavin that contains 40 μ g/ml equably.After cultivating 1 to 2 day, prepare single bacterium colony.With the rice paddy seed sterilization, under 37 ℃, in minimal medium, cultivated 24 hours.Then, add the toxoflavin of 40 μ g/ml, and cultivated again 48 hours.The culture that obtains is inoculated in the LB nutrient agar of the toxoflavin that contains 40 μ g/ml.After cultivating 2 to 3 days, prepare single bacterium colony, and be inoculated into equably again in the LB nutrient agar, to obtain pure single bacterium colony.

EXPERIMENTAL EXAMPLE 5: bacterial cell qualitative

For the isolated bacterial cell is carried out qualitative analysis, use Biolog programanalysis, GC-FAME (gas chromatographic analysis of fatty acid methyl ester), the physiology and the cultural characters of pair cell are analyzed, and 16S rDNA sequencing analysis.

By Biolog micro plate (Biolog GN MicroPlate; Biolog, Hayward CA), according to the operation instruction that manufacturer provides, carries out three times to the characteristic of utilizing carbon source of isolated cells and detects.After cultivating 24 hours and 48 hours respectively, use MicroLog 3-Automated Microstation system (Biolog) to read plate.Database (Version 4.0) with reference to the Microlog gram-positive microorganism carries out qualitative to bacterium.

For fatty acid methyl ester is analyzed, with above-mentioned trypticase soya broth (Becton Dickinson and Co., Franklin Lakes, NJ) agar plate last 48 hour of bacterial cell cultivation under 28 ℃ that is separated to of 9 kinds of expections.(Newark DE), extracts fatty acid methyl ester for Sasser, ofbacteria by gas chromatography of cellular fatty acids.Technical note#101.MDMI M.1997.Identification according to the method for standard.(MIDI Inc., Newark DE), analyze lipid acid to use Sherlock Microbial IdentificationSystem Version 2.11.Isolating fatty acid methyl ester is analyzed three times.

Order-checking to the 16S rDNA of isolated bacterial cell, undertaken by PCR, 10 times PCR damping fluid (the Takara Bio Inc. that contains 5 μ l in the total reaction volume of 50 μ l, Otsu, Japan), the various dNTP (2.5mM of 5 μ l, Takara), the primer of 1 μ l (100pmol, 27mF:5 ' AGAGTTTGATCMTGGCTCAG3 ' (SEQ ID NO:3), 1492mR:5 ' GGYTACCTTGTTACGACTT-3 ' (SEQ ID NO:4)), the Taq polysaccharase of 0.5 μ l (250U/ μ l, Takara) and the bacterium floating substance (A of 2 μ l _600nm=0.1).(Norwalk carries out in CT) pcr amplification for model PTC-150, Perkin-Elmer Cetus at automatic heating cycle machine.At first the condition of sex change be 94 ℃ following 5 minutes, carry out 29 circulations then under the following conditions: 94 ℃ of following sex change 1 minute, 55 ℃ down 1 minute, 72 ℃ of annealing prolong 1.5 minutes down (when amplification step finishes increase by one time 2 ℃ following 10 minutes).

Method (the Sambrook that use is described by people such as Sambrook, J.et al., 1989.MolecularCloning:A Laboratory Manula.2nd ed.Cold Spring Harbor Laboratory, ColdSprong Harbor, NY), at pBluescript II (SK+) (Stratagene, Cedat Creek, TX) clone the DNA that increases in SmaI site.

(Applied Biosystems Ins., Foster City CA), carry out dna sequencing to use dna sequencing ABI3700 automated DNA sequenator.The result that will obtain from dna sequencing, use the blast program (Altschul of biotechnology research institute's national center (National Center for Biotechnology Institute), S.F.et al., 1990.Basic local alignment search tool.J.Mol.Biol.215:403-410) analyze.

EXPERIMENTAL EXAMPLE 6: the structure of Paenibacillus polymyxa JH2 cosmid library

From the culture of the Paenibacillus polymyxa JH2 of 500ml, prepare chromosomal DNA, carry out the digestion of part then with Sau3A.By rotating speed at room temperature is 24, and the sucrose gradient centrifugation of 000rpm (10-40%[weight/volume]) 24 hours is isolated the fragment that length is 20-30kb.Use pLAFR3 (Tra subsequently ^-, Mob ⁺, RK2 replicon, Tet ^r, Staskawicz, B.et al., 1987.Molecular characterization of cloned avirulence genes from race 0 and race 1 ofPseudomonas syringar pv.glycinea.J.Bacteriol 169:5789-5794), they are connected.

According to the operation instruction of manufacturer, use phage that the DNA that connects is packed (Promega, Madison, the U.S.).Use described phage, to intestinal bacteria HB101 (F ^-McrB mrr hsdS20 (rB ^-MB ^-) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20 (S _m ^R) suoE44 λ ^-, Gibco BRL) transform.

EXPERIMENTAL EXAMPLE 7: screening library

By being 230rpm with cell cultures in the LB liquid nutrient medium and at rotating speed, temperature is under 37 ℃ the condition, the screening library.Cell cultivated 3 hours in the liquid LB of the toxoflavin that contains 40 μ g/ml substratum after, culturing cell is 12 hours again.Liquid nutrient medium is assigned to equably in the solid LB substratum of the toxoflavin that is added with 40 μ g/ml.After cultivating 1 day under 37 ℃, in this solid medium, observe bacterium colony.

EXPERIMENTAL EXAMPLE 8: order-checking

1, sequencing reaction

Working instructions according to QIAprep Spin Miniprep Kit (QIAGEN, Germany) manufacturer use template plasmid DNA.To contain the BigDye Terminat of 1 μ g or the reagent of ready-made reaction mixture, the T7 promoter primer of 2pmol, the template plasmid DNA of 5 μ l (100-200ng) mixes.With reactant transfer in the PCR test tube of 0.2ml, heated 5 minutes down at 95 ℃, (MinicyclerTM PTC-150 (MJ Research in thermo cycler then, Watertown, MA)) carry out amplification procedure, amplification condition comprises: heating is 20 seconds under 95 ℃, and 55 ℃ were heated 10 seconds down, and 60 ℃ were heated 4 minutes down; Circulation is 25 times under this amplification condition.

2, the purifying of PCR product

After sequencing reaction finished, (10 μ l) transferred in the centrifuge tube of 1.5ml with the PCR product.17 kinds of reagent (distilled water of 26 μ l and 64 μ l 95% ethanol) are joined in the reactor product of 10 μ l.After mixing, at room temperature kept 15 minutes.At room temperature centrifugal 20 minutes with 16,816 * g.After removing supernatant, with 70% the washing with alcohol precipitation of 250 μ l, and at air drying.Finished product is kept at-20 ℃.

Dna sequencing and data analysis

Insert the pJ9 (plasmid, wherein, clone's cosmid library) of DNA with the digestion of suitable restriction enzyme, and before order-checking with its subclone in pBluescript II SK (+).Use universal primer (SEQ IDNO:5) and reverse primer (SEQ ID NO:6) to carry out basic reaction, adopt synthetic primer (SEQ IDNO:7 and SEQ ID NO:8) that complete two strands is checked order.Use blast program (Gish, W.etal., 1993.Identification of protein coding regions by database similarity search.Nat.Genet.3:266-272), MEGALIGN software (DNASTAR) and GENETYX-WIN software (Software Development, Tokyo, Japan), the data of dna sequencing are analyzed.

Crossing of EXPERIMENTAL EXAMPLE 9:His-TflA expressed and partial purification

Use has the tflA gene of the primer amplification Paenibacillus polymyxa JH2 of the sequence shown in SEQ ID NO:9 or SEQ ID NO:10, use tflA gene clone that NdeI/BamHI makes Paenibacillus polymyxa JH2 (Novagen in the pET14b carrier then, Madison, WI, USA).There is e. coli bl21 (DE3) bacterial cell (pLysS) of pET14b to cultivate in containing the liquid LB substratum of penbritin and paraxin, importing at 37 ℃ of following wave and culture.Work as OD _600nmBe 0.8 o'clock, joining IPTG in the substratum and making its final concentration is 1mM.After cultivating 2 hours under 37 ℃, pass through centrifugal collecting cell.The sodium phosphate (pH 6.5) that adds 50mM, and mix with cell precipitation, then mixture is carried out supersound process.Partially purified proteinic concentration by centrifugal acquisition adopts Bradford (Bradford) method to measure, and adopt bovine serum albumin (BSA) as standard substance (Bradford, M.M.1976.A rapid and sensitive method for the quantitation ofmicrogram quantities of protein utilizing the principle of protein-dye binding.Anal.Biochem.72:248-254).Use the SDS-PAGE analysing protein, and use Coomassie blue stain.

The purifying of the His-TflA of EXPERIMENTAL EXAMPLE 10:N-end

In order to carry out purifying better, adopt the TflA of the His-mark of N-end.(Novagen, Madison, WI, e. coli bl21 USA) (DE3) (pLysS) cultivate in the LB substratum with carrying the pET14b carrier of cloning tflA.Use crossing of IPTG induced protein to express.Behind collecting cell, the cell in the sodium phosphate (pH6.5) of 50mM is carried out ultrasonication, under 4 ℃ centrifugal 20 minutes then with the rotating speed of 10,000 * g.Supernatant is joined Ni-NTA outstanding rotary column (QIAGEN, Valencia, CA, top USA).With the protein of His-mark under 4 ℃ centrifugal 2 minutes, be attached to then on the Ni-NTA matrix with the rotating speed of 1,000 * g.Use lavation buffer solution (imidazoles of 20mM, 50mM sodium phosphate (pH 6.5)) washing matrix twice, remove unconjugated protein.Elution buffer 1 (10mM imidazoles, 50mM sodium phosphate (pH 6.5)) by using 0.1ml in turn and the elution buffer 2 (10mM imidazoles, 50mM sodium phosphate (pH 6.5)) of 0.1ml with the protein of dissolving His-mark, and elute it.With 50mM sodium phosphate (pH 6.5) protein that obtains is dialysed, to remove imidazolium compounds.Adopt Bradford (Bradford) method and adopt standard substance (Bradford, M.M.1976.A rapid and sensitive method for the quantitation of microgram quantitiesof protein utilizing the principle of protein-dye binding.Anal.Biochem.72:248-254), the proteinic concentration of purifying is measured.

The enzyme characteristic of EXPERIMENTAL EXAMPLE 11:His-TflA

The analysis of enzymic activity

Use thin-layer chromatography chromatogram (TLC) plate to measure the activity of TflA, the thin-layer chromatography chromatosheet is followed the product that reacts between the reaction mixture of toxoflavin and enzyme basically and is changed.With the TflA protein of 5 μ M, the MnCl of 10 μ M ₂Join (50mM sodium phosphate (pH 6.5)) in the analysis buffer with the DTT (DTT) of 5mM, obtain the mixture of 200 μ l, be reflected under 25 ℃ and carry out.In reaction when just having begun, the concentration of toxoflavin is adjusted to 100 μ M.After reaction 10 minutes, the chloroform that adds 200 μ l is with termination reaction.After with the chloroform layer complete drying, add 100% the methyl alcohol of 10 μ l.Use transfer pipet to take out a reaction mixture that contains methyl alcohol, and join on the TLC plate.Then, at room temperature this plate is placed contain chloroform and methyl alcohol (95: 5, volume: in the TLC chamber of mixed solvent volume).Under UV light-emitting appearance (254nm and 365nm), described TLC plate is observed.

Metal ion is to the influence of enzymic activity

Studied the influence of metal ion to enzymic activity.Under study for action, the concentration of all metal ions is 1mM.

3, pH and temperature are to the influence of enzymic activity

Under different pH conditions, under 25 ℃, use the activity of sodium phosphate buffer (pH 4.0-8.0) the detection tflA of 50mM.In temperature is under 10-40 ℃, uses the sodium phosphate buffer (pH 6.5) of 50mM to detect enzymic activity.

4, enzymic activity kinetics

Determine kinetic parameter (Km and V from L-B (Lineweaver-Burk) figure _Max), described L-B figure is that the data that the toxoflavin (80-200 μ M) according to different concns is obtained are set up.Each test point is to detect the mean value that calculates the data that obtained from minimum three times.

Embodiment 1: the separation of the gene of can degrade toxoflavin

Obtain 500 kinds of different bacteriums from field soil, farm soil and rice paddy seed etc., and they are cultivated in being added with the minimal medium of toxoflavin.As a result, thus can separate and obtain the bacterial cell that can degrade toxoflavin can survive.In isolated bacterial from rice paddy seed, isolate the bacterial cell of pure energy specificity can degrade toxoflavin.Analyze by 16s dna sequencing, fatty acid analysis and Biolog, determine their characteristic.The result.These cells are confirmed as Paenibacillus polymyxa, and are called the JH2 (see figure 1).From the genomic DNA of Paenibacillus polymyxa JH2 library, be built among the intestinal bacteria HB101.From the library clone that makes up, filter out the bacterium colony that in containing the substratum of toxin, to survive.Adopt restriction enzyme to digest the clone that described separated DNA clone prepares, be used to determine the dna fragmentation (see figure 2) of the needed minimum of can degrade toxoflavin.The clone of the needed minimum of described toxin of degrading carries out the dna sequencing analysis with this fragment, with the open reading-frame (ORF) (SEQ ID NO:1) of determining to have 666bp for the long EcoRI fragment of 1.5kb.Analyze by NCBI BLAST, find that the outer dioxygenase (ring-cleavage extradioldioxygenase of Exiguobacterium sp) of open loop glycol of described fragment and small Bacillaceae has 67.3% similarity (see figure 3).The tflA gene of having found to relate to can degrade toxoflavin is a kind of new useful gene of never reporting.

Embodiment 2: the degraded of the expression of gene of the tflA of degraded toxin and the described toxin that caused by the protein of purifying

Use pET14-b (T7 promoter expression vector, Amp ^r, Novagen) the tflA gene of expression energy can degrade toxoflavin.By the described gene of pcr amplification, be cloned into pBluescript IISK (+) (ColEI, MCS-lacZ α, Amp with ^rCloning vector, Ampr, Stratagene) in, and analyze its sequence.To detect no problem clone by PCR, and be cloned into once more among the pET14-b (being pH904), and use described pH904 transformed into escherichia coli BL21 (DE3/pLysS), and adding IPTG (1mM) induces.Use the Ni-post to separate His-TflA protein (26.7kDa) (seeing Fig. 4 A) then.Then, detect the ability of the TflA protein degradation toxoflavin of purifying, and when finding TflA protein degradation toxoflavin, need have DTT and Mn simultaneously ²⁺(seeing Fig. 4 B).

For the temperature profile of the degraded that obtains the toxoflavin that TflA protein causes, detect under differing temps (20,25,30,35 and 40 ℃) by the amount of the toxoflavin of described protein degradation.Found that TflA protein shows the highest activity (referring to, Fig. 5 A) in the time of 30 ℃, best pH is 6.5 (referring to Fig. 6).In order to detect the proteinic activity specific of TflA, the amount of the toxoflavin that detection in per 10 minutes once is degraded.The result shows that the proteinic activity specific of TflA is 0.0413 μ mole/min/mg (referring to 5B).

Embodiment 3: the toxoflavin that is caused by TflA protein and the degraded of derivative thereof

1) toxoflavin and derivative thereof is synthetic

In order to study the reaction mechanism of the toxin degraded that TflA protein causes, the present inventor obtains toxoflavin and derivative thereof from Tomohisa doctor Nagamatsu, Tomohisa doctor Nagamatsu is the professor of Japanese Okayama Univ. (Okayama University JAPAN), and it also is the co-worker (referring to Fig. 7) of this project.Used the derivative of following toxoflavin, to determine the proteinic degradation capability of TflA, the derivative of described toxoflavin comprises: in carbon atoms numbered be have methyl on 3 the position 3-methyl toxoflavin (except as otherwise noted, for the numbering of carbon atom and nitrogen-atoms, with the toxoflavin of primary unmodified as n-compound); 4 and 8 the position of being numbered at nitrogen-atoms has 4 of hydrogen, 8-dihydro toxoflavin; In carbon atoms numbered be have on 3 the position methyl and nitrogen-atoms be numbered 4 and 8 position have 3 of hydrogen '-methyl 4,8-dihydro toxoflavin; The planomycin that has methyl on 1 the position that is numbered that 8 position replaces nitrogen-atoms that is numbered at nitrogen-atoms; It in carbon atoms numbered the 3-phenyl toxoflavin that has phenyl on 3 the position; 5-denitrogenation flavine (deazaflavin) with extra loop structure; The reumycin that 1 position does not have methyl that is numbered at nitrogen-atoms; It in the carbon atoms numbered of reumycin the 3-methyl reumycin that has methyl on 3 the position; It in the reumycin carbon atoms numbered 3-phenyl reumycin that has phenyl on 3 the position.

2) toxoflavin that causes by TflA protein and the degraded of derivative thereof

In order to detect the toxoflavin that causes by TflA protein and the degraded of derivative thereof, carried out a series of test, test-results shows toxoflavin, 3-methyl toxoflavin, 4,8-dihydro toxoflavin and 3-methyl reumycin are degraded in the given time fully.And 3-phenyl toxoflavin, 3-phenyl reumycin and 5-denitrogenation flavine are not degraded; Reumycin, 3-methyl 4,8-dihydro toxoflavin and planomycin are partly degraded.Generally speaking owing to be not degraded at the derivative that 3 position has phenyl that is numbered of carbon atom, therefore, think TflA protein identification toxoflavin carbon atom be numbered near 3 specific ring structure (referring to, Fig. 8)

The proteinic kinetics of embodiment 4:TflA

Detected the proteinic Michaelis-Menton constant (K of His-TflA of energy can degrade toxoflavin _m), maximum speed of reaction (V _Max) and activity specific.Confirm the activity of can degrade toxoflavin toxin with the TLC method.Found that the proteinic K of His-TflA _mAnd V _MaxValue be respectively 69.72 μ M and-0.45U/mg.Activity specific is 0.4 μ mol/mg (referring to Fig. 9).

Embodiment 5: the preparation of transgenic plant

The carrier that is used to transform is pCamLA, wherein, and hygromix phosphotransferase (Hyg ^R) being arranged in T-DNA, the gene of anti-kantlex is positioned at T-DNA outer (referring to Figure 10).The bacterial cell that is used to transform is an agrobacterium tumefaciens lba4404.Agrobacterium is cultivated in the AB minimal medium.(3,5-dimethoxy-4 '-hydroxy acetophenone, AA liquid nutrient medium Aldrich) recover and the dilution cultured cells with containing Syringylethanone.To in described cell mixture, soak 3-5 minute from the rice callus tissue of various paddy rice (Donjinbyeo, Chucheongbyeo and Nipponbare).The callus that obtains with aseptic filter paper drying, is placed substratum then, with Agrobacterium at 28 ℃ in dark place co-cultivation 3 days.Agrobacterium is removed from cultivated 3 days callus, place N6 to select substratum the callus that obtains.Under 25 ℃, cultivated about 30 days having under the condition of light, only select the callus of propagation, these callus are placed substratum again, to break up again.After obtaining again differentiated plants once more, they are transferred to do not contain any substratum that is used for the material of control growing, make its can freely take root (referring to Figure 11).Pick out the single plant that in containing the substratum of Totomycin, can normally germinate and take root, and tame.The transgenic plant that obtain are like this transferred in the jar, placed the greenhouse then.From the transgenic plant that repetition test obtains, pick out the plant that is in the normal growth state according to its outward appearance.The result has set up T1 generation and T2 for the transgenic paddy rice plant, and the result is summarised in the table 1.

The tabulation of table 1, transgenic paddy rice strain system

The rice cropping plant	Gene	Strain system numbering	Existing plant
				Donjinbyeo	tflA	Dt	46	T1 is for plant
Nipponbare	tflA	Nt	41						T1 is for plant
				Chucheongbyeo	tflA	Ct	43	T1 is for plant

Embodiment 6: the analysis of transgenic plant (T2 is for plant)

1) southern blotting technique analysis

The method of employing standard is extracted genomic dna from the leaf of 1 gram transgenic paddy rice plant.Handle genomic dna (15-20 μ g) with limiting enzyme EcoRI, and separate with sepharose (0.7%).With the separated DNA trace after lead on the film, use probe to carry out hybridization.The probe that uses is for containing the tflA fragment (2.5kb) of 3 ' NOS end.From the DNA of leaf texture and the tflA probe hybridization in T2 generations of transgenic plant, the DNA analysis result of acquisition is shown in Figure 12 B and table 2.For Donjinbyeo and two kinds of rice cropping plants of Chucheongbyeo, find to exist multiple integration mode and multiple copy number.

2) western blot analysis

Collect the leaf texture (300mg) in T2 generation of transgenic paddy rice, use the liquid nitrogen fragmentation.Add broken damping fluid (50mM Tris-HCl pH7.5,150mM NaCl, 1mM EDTA, 10% glycerine, 1mM PMSF, 0.05%Tween 20, protease inhibitor cocktail), then with centrifugal 15 minutes of mixture (4 ℃, 15,000rpm).Take out supernatant, recentrifuge is to extract total soluble protein.Total soluble protein (15 μ l) joined in 2 times the sample buffer, the mixture that obtains was heated 5 minutes in 100 ℃ boiling water.Then protein is carried out SDS-PAGE and analyzes, and subsequently trace to pvdf membrane.After handling film with lock solution (TBS-T that contains 5% skim-milk), with the antibody treatment of anti--TflA, antibody be immune purity (

).With NBT/BCIP detection kit (Amersham, England) detection signal.As shown in figure 13, TflA protein is expressed in generation at the T2 of transgenic paddy rice usually.

Embodiment 7: screening transgenic paddy rice plant (T3 is for plant)

Pick out the plant that in containing the substratum of Totomycin, can normally germinate and take root, and tame.The transgenic plant that obtain are like this transferred in the jar, placed the greenhouse then.From the transgenic plant that repetition test obtains, select the plant that is in the normal growth state by its outward appearance.From described plant, select rice paddy seed (T1 and T2), and as the test that detects the moisture resistance mycin.From sophisticated seed, remove exosper, the seed that obtains was soaked 1 minute in 100% ethanol.The result is summarised in the table 1.Under agitation, the chlorine bleach liquor with 2% handled seed 20 minutes, so that the seed outside surface is sterilized.Obtain aseptic seed three times like this with sterilized water washing, then this seed is placed 1/2 the MS substratum that is added with Totomycin (50mg/L).Under temperature is 26 ℃ and under the continuous illumination (3000 lux), accompany foster.After cultivating 10 days,, determine resistance to Totomycin by the stem of observation rice plants and the growing state of root.From each rice cropping plant, take out seed, determine to comprise the gene isolation ratio of the gene of moisture resistance mycin.As shown in table 2 below, the T3 of transgenic paddy rice that has set up two kinds of cultivated plants is for plant, and as further research.

The tabulation of table 2, transgenic paddy rice plant

Gene	The rice cropping plant	T1 system	T2 system	T3 system
					tflA	Dongjinbyeo
	25	21	16
					Chucheongbyeo	15	12	8

Embodiment 8: the determining of transgenic plant phenotype (T3 is for plant)

Detect always from the performance of transgenosis T3 for the antitoxin flavine of the blade of rice plants, described rice plants has grown to has 4-5 sheet leaf.Pre-research shows that the activation of toxoflavin needs light.Therefore, test of the present invention is carried out having under the optical condition.In the culture dish that is of a size of 60 * 15mm, add the sterilized water of 5ml, and add the toxoflavin (0,25,50 or 100 μ M) of various concentration.From grow to transgenosis T3 rice plants, get leaf, be cut into then and be of a size of 3 * 4mm, handled 48 hours with toxoflavin again, in described processing, be included in and grew 26 hours under 25 ℃ of illumination conditions and grew 8 hours 25 ℃ dark place with 4-5 sheet leaf.As shown in figure 14, after handling 40 hours, on the rice leaf of wild-type, begun to occur because the variable color that toxin causes with toxoflavin.Yet, after handling, on the blade of the transgenic plant that imported the tflA gene, do not observe variable color with toxoflavin.

Embodiment 9: the preparation of conversion carrier

Adopt pCamLA and pTflA as the carrier that transforms in the present invention.PCamLA carries the hygromycin phosphotransferase gene (Hyg that is arranged in T-DNA ^R) and be positioned at the gene (referring to Figure 15) of the anti-kantlex outside the T-DNA.PTflA carries tflA gene (that is, expressing the gene that the back produces the enzyme of can degrade toxoflavin), 35S promoter and the NOS terminator sequence (referring to Figure 16) that is arranged in T-DNA.

The carrier that is used for the Totomycin selection is the pCamLA carrier, and this carrier derives from binary vector pCAMBIA1300.In pCAMBIA1300, hygromycin phosphotransferase gene (Hyg ^R) be arranged in T-DNA, and the gene of anti-kantlex is positioned at outside the T-DNA.PCamLA, and breeds in this host as the host with bacillus coli DH 5 alpha.From bacterium, extract plasmid DNA.Method by electroporation is transformed into the pCamLA that extracts in the agrobacterium tumefaciens.

The pTflA carrier that is used to screen toxoflavin is made by pCamLA, deletes Hyg with the XhoI restrictive diges-tion ^RGene, and will have the tflA gene of XhoI joint in its initiator codon and terminator codon, be inserted into the position of being deleted.

The preparation method of tflA gene with XhoI joint is as follows: use pJ90 (1.2kb HindIII fragment, described fragment contains the tfl that is inserted in pBluscript II SK (+)) as template DNA, with J90XhoI-F (5 '-CTCGAGATGACTTCGATTAAACAGCTTAC-3 '; SEQ ID NO:11) and J90XhoI-R (5 '-CTCGAGTTAGATCACCAGTTCACC-3 '; SEQ ID NO:12) carry out the pcr amplification of tflA gene as primer, the PCR product cloning that amplification is obtained is to the XhoI position of pBluscriptII SK (+).Order-checking, the product of acquisition is called pJX90-6.To be inserted into the dna fragmentation that restriction enzyme XhoI digestion pJX90-6 obtains and remove Hyg ^RPCamLA in.The result obtains pTflA.

Embodiment 10: the conversion of rice plants

1) callus induces

The rice paddy seed (rice grain) of time results carries out test of the present invention before using.The concrete steps of the callus of inducing paddy rice are as follows:

1, selects high-quality rice paddy seed (noting not damaging plumule).

2, above-mentioned seed is placed hawk (Falcon) pipe, be immersed in 100% the ethanol, shook 30 seconds.

3, the chlorisis agent (Chlorox) of adding 1/2 was hatched this mixture 20 minutes then in the incubator that shakes.

4, wash above-mentioned seed 4-5 time with sterilized water.

5, seed is transferred in the substratum that does not contain 2N6, and the plumule of guaranteeing seed up, described substratum can be induced the formation callus.

Found that inducing the appropriate condition that produces callus is to induce 3-4 week at 28 ℃.Especially, induction time all around is best, can not surpass for five weeks.The present invention tests the culture dish (that is, use the culture dish that be of a size of 100/20mm) of the culture dish of employing greater than routine.

Table 3

Table 4, stoste (X 1000)

2), the conversion of callus

(1) hatches (dark place condition) altogether

Before cultivating altogether 3 days place new 2N6 substratum with the inductive callus, then second day and Agrobacterium co-cultivation.

1, Agrobacterium was cultivated 48 hours.

2, under 3000rpm, made cell precipitation in centrifugal 5 minutes.

3, with AAM substratum diluting cells, up to OD ₆₆₀=0.1.

4, the callus with appropriate amount places pipe, and soaks 30 seconds in described AAM substratum.

5, after 30 minutes supernatant is poured out carefully, be sprayed onto on the filter paper drying then.

6,, filter paper is placed culture dish, and add a spot of AAM substratum (that is, 1-2ml) in the time of dry 20-30 minute at callus with wetting filter paper.

7, the exsiccant callus is placed described filter paper above, and wrap culture dish with aluminium foil.Is to hatch under 24 ℃ 3 days this culture dish in temperature.

(2), screening (in the dark) for the first time

1, preparation contains 2N6, the Totomycin (selected marker of plant; 10mg/L (200 μ l-50mg/ml)) and match good fortune mycin (cephatoxin) substratum (1ml-250mg/ml) of 250mg/L.

2, the callus that will cultivate 3 days places aseptic pipe, with the sterilized water washing once.

3, with the solution of the match good fortune mycin that contains the sterilized water of 50ml and 50 μ l, washing callus three times, each 20 minutes.

4, callus is sprayed onto carries out drying on the filter paper.

5, the callus that obtains is placed screening culture medium for the first time.

6, wrap with aluminium foil, under 28 ℃, hatch callus.Observed 7 days.

(3), programmed screening (in the dark)

1, preparation contains the substratum of the match good fortune mycin (1ml-250mg/ml) of the Totomycin of 2N6,30mg/L (600 μ l-50mg/ml) and 250mg/L.

2, dark areas lacks resistance corresponding to contratoxin.Therefore, remove dark areas, only with the zone-transfer of white in the programmed screening substratum.

3, observed for three weeks.

(4), cultivated for three weeks (condition that light is arranged) with division culture medium for the first time.

(5), cultivated for three weeks (condition that light is arranged) with division culture medium for the second time.

(6) use bottled 1/2MS.

Table 5, AAM substratum

Table 6,10 * MSAA substratum

Be used for screening for the first time the substratum of callus

2N6 substratum-autoclaving of 1L

The Totomycin of 200 μ l (50mg/ml) (final concentration 150mg/L)

The match good fortune mycin (250mg/ml) (final concentration 1650mg/L) of 1ml

The substratum that is used for the programmed screening callus

2N6 substratum-autoclaving of 1L

The Totomycin of 600 μ l (50mg/ml)

The match good fortune mycin (250mg/ml) of 1ml

Table 7, be used for the substratum of the first time differentiation of callus

Table 8, be used for the substratum of the second time differentiation of callus

Table 9, final substratum (formation of callus-1/2MS substratum (using bottle))

	1L	500ml	250ml
				MS	2.15g	1.07g	0.54g
Sucrose	15g	7.5g	3.75g
				Gel resin (propolis (phyta gel))	4(3)g	2(1.5)g	1(0.75)g

Figure 17 has shown with the programmed screening result of Totomycin (30 μ g/ml) to the rice callus tissue that transforms respectively with pCamLA or pTflA carrier.The culture dish on the right has shown that the rice callus tissue beginning of using pTflA carrier of the present invention to transform is dead.

Figure 18 has shown the callus of the survival of screening with toxoflavin (5 μ g/ml) before will breaking up again with the rice plants that pCamLA or pTflA carrier transform.Because the rice callus tissue (being positioned at the substratum on the left side) that transforms with the pCamLA carrier does not have the enzyme of energy can degrade toxoflavin, therefore, the overwhelming majority's callus death.Yet, for the rice callus tissue that transforms with the pTflA carrier (being positioned at the substratum on the right), the only callus death of some.

Figure 19 has shown the callus of survival in the time will placing substratum to screen for 4 weeks to break up the back again with toxoflavin (7.5 μ g/ml) with the rice plants that the pCamLA carrier transforms.As shown in figure 19, owing to organize enzyme with energy can degrade toxoflavin with the rice callus of pCamLA carrier conversion, therefore, most callus death.

Figure 20 has shown the callus of survival in the time will placing substratum to screen for 4 weeks to break up the back again with toxoflavin (7.5 μ g/ml) with the rice plants that the pTflA carrier transforms.As shown in figure 20, because the rice callus tissue that transforms with the pTflA carrier has the enzyme of energy can degrade toxoflavin, therefore, callus normally breaks up again.

Result according to the callus of using toxoflavin (7.5 μ g/ml) Screening of Rice, calculate and break up ratio again, the callus of described paddy rice transforms with pCamLA or pTflA carrier, and the result of described callus with toxoflavin (7.5 μ g/ml) Screening of Rice is by placing rice plants the redifferential medium screening definite during 4 weeks.The result is summarized in following table 10.

Table 10, the ratio of breaking up again

	pCamLA(hpt)	pTflA
			Transform for the second time	2/11(18.18％)	28/84(33.3％)
Transform for the third time	11/109(10.09％)	26/192(13.54％)

In last table 10, Fen Hua ratio is meant the ratio of the number of the number of differentiation and total callus again again.Result by table 10 can clearly be seen that, compares with the callus that inserts the pCamLA carrier, and the differentiation ratio again of the callus of insertion pTflA carrier is higher.The plant that filters out is transferred in the jar, analyzed the tissue samples of these plants, to determine whether changed the tflA gene in the plant by PCR.Sequences Design PCR primer according to the tflA gene.Annealing temperature is 55 ℃.Use tfla140-U (5 '-TGCAGCTGCTGATGGAACAAA-3 '; SEQ ID NO:13) and TFLA370-L (5 '-TTATCCAGTACAGGTGCAGCT-3 '; SEQ ID NO:14) as the PCR primer.

Figure 21 has shown the result of agarose electrophoresis PCR product, the PCR product that described PCR product is an isolating genomic dna from the transgenic plant that filter out.In Figure 21, swimming lane 12,16,17,18,21 and 36 expressions do not have the PCR product.As shown in figure 21, the PCR product that transgenic plant have produced in desired location, therefore, this show can degrade toxoflavin of the present invention stable gene be incorporated in the genome of rice plants.

Embodiment 11: the conversion of Arabidopis thaliana

1) based on the comparative analysis of the existing conversion carrier of tflA and new conversion carrier

Carry out comparative analysis based on the existing conversion carrier of tflA and new conversion carrier.Specifically, the pMBP1 carrier that compares pMBP1:tflA (this carrier contains the gene of antitoxin flavine and Totomycin), pCamLA (Δ hpt): tflA (gene of disappearance moisture resistance mycin in this carrier), pMBP1:tflA (being inserted with the binary vector pMBP1 of tflA gene) and be generally used for transforming.

2) test method

With the Agrobacterium-mediated Transformation Arabidopis thaliana Col-0 that contains each construct.The detailed method that is used to transform is as follows.

In jar, cultivate Arabidopis thaliana Col-0 vigorous.When scape begins growth and blooming, cut scape with scissors.

Agrobacterium overnight incubation in the LB broth culture that will contain gene to be transformed.

The Agrobacterium of cultivating is centrifugal, and with 5% sucrose solution suspension, up to O.D.=0.8.Agricultural chemicals with 0.03% (Silwet) joins in the suspension.

After one week, cut scape, the scape of growth newly is immersed in 2-3 second in described 5% the sucrose solution.

Use the plastic bag packaging plant.Remove packing bag two days later.

After collecting all seeds, the construct that will contain the tflA resistant gene is distributed to and contains on the MS plate of toxoflavin that concentration is 20 μ M (3.84 μ g/ml).For the pMB1 that contains anti-kanamycin gene, seed is distributed to contains on the MS plate of kantlex that concentration is 50 μ g/ml.After ten days, select genetically modified organism from described plate.On each plate, disperse about 1,000 seeds, on each piece plate, obtain about 10 strain genetically modified organisms.

Figure 22 comprises the photo of the shooting of transgenic plant, and described transgenic plant transform pCamLA:tflA, pCamLA (Δ hpt): tflA, pMBP1:tflA or pMBP1 carrier.Be integrated with the transgenic plant of the gene of antitoxin flavine, in the substratum of the toxoflavin that contains 20 μ M, can normally grow.Root is well-grown also.Yet most of non-transgenic biologies can not rudiment, and, both having enabled rudiment, root can not normal growth.Use the level of conversion of the level of conversion of carrier of the present invention and widely used pMBP1 carrier similar.

The PCR of the genetically modified organism that 3) filters out detects and the photobleaching test

The PCR of the genetically modified organism that (1) filters out detects

Select some transgenic plant, and transfer in the jar.Carry out PCR, to determine conversion with tflA gene pairs plant.According to the sequence of tflA gene, be designed for the PCR primer of reaction.Annealing temperature is 55 ℃.Two PCR primers that are used to react are:

Tfla140-U (5 '-TGCAGCTGCTGATGGAACAAA-3 '; SEQ ID NO:13) and TFLA370-L (5 '-TTATCCAGTACAGGTGCAGCT-3 '; SEQ ID NO:14).Figure 23 has shown the result of the agarose gel electrophoresis that the PCR product is carried out, and described PCR product is the PCR product of isolating genomic dna from the genetically modified organism that filters out.(((the pcr analysis result of the genetically modified organism of 3-1～3-11) shows for 2-1～2-12) or pMBP1:tflA for 1-1～1-10), pCamLA (Δ hpt): tflA to contain construct pCamLA:tflA, transgenic plant can successfully germinate in containing the substratum of toxoflavin, and can take root well, reach the rank of contrast (being the tflA gene).On the other hand, for those roots can not normal growth plant, do not observe this rank.Therefore, can determine to use the selected marker of toxoflavin as transgenic plant.

(2) according to the evaluation of the genetically modified organism of photobleaching test

Toxoflavin is a kind of component that causes the septic virulence factor of rice grain, and when toxoflavin was used for plant, it can rely on light and produce bleaching action, and this phenomenon is present in all plants usually.In the present embodiment, detect of the effect of the toxoflavin of different concns to Arabidopis thaliana Col-O.Found that even under low concentration, toxoflavin also has bleaching action to plant.According to the bleaching effect of toxoflavin, detect genetically modified organism prepared in accordance with the present invention.Figure 24 has shown the result who genetically modified organism and non-transgenic biology is carried out bleaching test.For the genetically modified organism of selecting that is used to test, do not observe the photobleaching phenomenon.Yet, the photobleaching phenomenon has appearred for the non-transgenic biology.This is corresponding with above-mentioned PCR result.Therefore, compare according to the antibiotic tag selection system of use with previously used, the present invention utilizes toxoflavin to be as the usefulness of the system of selection of selected marker, does not need to use any microbiotic just can realize that the transgenic plant that preparation had needed gene screen.

Sequence table

＜110〉Seoul National University's property of the institution is learned industry financial group

＜120〉can degrade toxoflavin and the tflA gene of chemical derivative and the genetically modified organism of expressing the tflA gene

<160>14

<170>KopatentIn 1.71

<210>1

<211>666

<212>DNA

＜213〉Paenibacillus polymyxa

<400>1

atgacttcga ttaaacagct tacattgtat acggccgagc ttgaccggat gctagcattt 60

tatacgaata tgcttggtgc gcagcatgtg catgagcaag cagatgcgtt tacgatccag 120

ctaggagtat cacagattca atttcgtgca gctgctgatg gaacaaagcc cttttaccat 180

attgctatca atatcgcggc aaaccatttt caagagggaa aagcctggct cagcggcttt 240

ggtgaattgc taacggaaaa tgatgaagat caggcatact ttcccttctt taacgcgtac 300

tcctgttatg tagaagaccc gtctggtaat attattgaac tcatctcgcg tcagcaagct 360

gcacctgtac tggataagcc cttctcagcg gatcagctac taagcatcgg tgagattaat 420

ataacaacca gcgatgtaga gcaagctgca acacgattaa agcaagcaga actgcctgta 480

aagctagacc agattgagcc agcaggctta aattttatcg gtgatcagga tttgttcctg 540

ctgctgggtc ctccaggacg acgctggtta ttttcagaac gcgtagccgt gatctatccg 600

ttacagatgg agctggataa cggcgtcagt ctggcgatta cagagacagg tgaactggtg 660

atctaa 666

<210>2

<211>221

<212>PRT

＜213〉Paenibacillus polymyxa

<400>2

Met Thr Ser Ile Lys Gln Leu Thr Leu Tyr Thr Ala Glu Leu Asp Arg

1 5 10 15

Met Leu Ala Phe Tyr Thr Asn Met Leu Gly Ala Gln His Val His Glu

20 25 30

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

35 40 45

Arg Ala Ala Ala Asp Gly Thr Lys Pro Phe Tyr His Ile Ala Ile Asn

50 55 60

Ile Ala Ala Asn His Phe Gln Glu Gly Lys Ala Trp Leu Ser Gly Phe

65 70 75 80

Gly Glu Leu Leu Thr Glu Asn Asp Glu Asp Gln Ala Tyr Phe Pro Phe

85 90 95

Phe Asn Ala Tyr Ser Cys Tyr Val Glu Asp Pro Ser Gly Asn Ile Ile

100 105 110

Glu Leu Ile Ser Arg Gln Gln Ala Ala Pro Val Leu Asp Lys Pro Phe

115 120 125

Ser Ala Asp Gln Leu Leu Ser Ile Gly Glu Ile Asn Ile Thr Thr Ser

130 135 140

Asp Val Glu Gln Ala Ala Thr Arg Leu Lys Gln Ala Glu Leu Pro Val

145 150 155 160

Lys Leu Asp Gln Ile Glu Pro Ala Gly Leu Asn Phe Ile Gly Asp Gln

165 170 175

Asp Leu Phe Leu Leu Leu Gly Pro Pro Gly Arg Arg Trp Leu Phe Ser

180 185 190

Glu Arg Val Ala Val Ile Tyr Pro Leu Gln Met Glu Leu Asp Asn Gly

195 200 205

Val Ser Leu Ala Ile Thr Glu Thr Gly Glu Leu Val Ile

210 215 220

<210>3

<211>20

<212>DNA

＜213〉general 16s rDNA sequence

<400>3

agagtttgat cmtggctcag 20

<210>4

<211>19

<212>DNA

＜213〉general 16s rDNA sequence

<400>4

ggytaccttg ttacgactt 19

<210>5

<211>20

<212>DNA

<213>pBluescript II SK(+)

<400>5

aattaaccct cactaaaggg 20

<210>6

<211>22

<212>DNA

<213>pBluescript II SK(+)

<400>6

gtaatacgac tcactatagg gc 22

<210>7

<211>19

<212>DNA

＜213〉Paenibacillus polymyxa

<400>7

gaactcatct cgcgtcagc 19

<210>8

<211>19

<212>DNA

＜213〉Paenibacillus polymyxa

<400>8

gcagcttgct ctacatcgc 19

<210>9

<211>26

<212>DNA

＜213〉Paenibacillus polymyxa

<400>9

catatgactt cgattaaaca gcttac 26

<210>10

<211>24

<212>DNA

＜213〉Paenibacillus polymyxa

<400>10

ggatccttag atcaccagtt cacc 24

<210>11

<211>29

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>11

ctcgagatga cttcgattaa acagcttac 29

<210>12

<211>24

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>12

ctcgagttag atcaccagtt cacc 24

<210>13

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>13

tgcagctgct gatggaacaa a 21

<210>14

<211>21

<212>DNA

＜213〉artificial sequence

<220>

＜223〉primer

<400>14

ttatccagta caggtgcagc t 21

Claims (37)

1, a kind of microorganism, this microorganism can degrade toxoflavins or the derivative of toxoflavin.

2, microorganism according to claim 1 is characterized in that, this microorganism is a bacterium.

3, microorganism according to claim 2 is characterized in that, this bacterium belongs to series bacillus and belongs to.

4, microorganism according to claim 3 is characterized in that, it is Paenibacillus polymyxa that described series bacillus belongs to.

5, microorganism according to claim 4 is characterized in that, this microorganism is Paenibacillus polymyxa JH2, and the deposit number of this Paenibacillus polymyxa JH2 is KCTC10959BP.

6, a kind of tflA protein, the derivative of this tflA albumen mass-energy can degrade toxoflavin or toxoflavin.

7, tflA protein according to claim 6, wherein, this tflA protein comprises the aminoacid sequence shown in SEQID NO:2.

8, tflA protein according to claim 6, wherein, this tflA protein comprises the aminoacid sequence that has at least 80% sequence homology with aminoacid sequence shown in SEQ ID NO:2.

9, tflA protein according to claim 6, wherein, after inserting at least one amino-acid residue in deletion or after replacing at least one amino-acid residue in the proteinic aminoacid sequence of described tflA or in the proteinic aminoacid sequence of described tflA, described tflA protein still keeps the ability of the derivative of can degrade toxoflavin or toxoflavin.

10, tflA protein according to claim 6, wherein, this tflA protein is as the selected marker of Plant Transformation.

11, tflA protein according to claim 10 is characterized in that, described plant is paddy rice or Arabidopis thaliana.

12, a kind of gene, any described tflA protein among this genes encoding claim 6-11.

13, gene according to claim 12, wherein, this gene comprises the described nucleotide sequence as SEQ ID NO:1.

14, gene according to claim 13, this wherein gene comprise and the sequence that has at least 90% sequence homology as the described nucleotide sequence of SEQ ID NO:1.

15, a kind of recombinant expression vector, this recombinant expression vector contain the described tflA gene of claim 12.

16, recombinant expression vector according to claim 15 is characterized in that, this recombinant expression vector is to be used for colibacillary expression vector.

17, recombinant expression vector according to claim 15 is characterized in that, this recombinant expression vector is the expression vector that is used for virus.

18, recombinant expression vector according to claim 15 is characterized in that, this recombinant expression vector is the expression vector that is used for plant.

19, a kind of tflA protein of reorganization, the tflA protein of this reorganization are to be expressed by any described recombinant expression vector among the claim 15-18.

20, a kind of genetically modified organism, this genetically modified organism are to transform with any described recombinant expression vector among the claim 15-18.

21, genetically modified organism according to claim 20 is characterized in that, this genetically modified organism is microorganism, plant, virus or animal.

22, a kind of expression cassette that is used for the selected marker of Plant Transformation, this expression cassette contain the following sequence that connects according to 5 ' to 3 ' direction operability:

(i) promoter sequence;

The encoding sequence of enzyme that (ii) can degrade toxoflavin; With

(iii) 3 '-untranslated terminator sequence.

23, expression cassette according to claim 22 is characterized in that, this expression cassette also comprises the expression cassette of target protein, and the expression cassette of this target protein contains following sequence:

(i) promoter sequence;

The (ii) encoding sequence of target protein; With

(iii) 3 '-untranslated terminator sequence.

24, expression cassette according to claim 23 is characterized in that, the expression cassette of described target protein and the expression cassette of described selected marker polyphone make up, to form single expression cassette.

25, expression cassette according to claim 22 is characterized in that, described promotor is CaMV35S promotor, actin promoter, ubiquitin promoter, pEMU promotor, MAS promotor or histone promotor.

26, expression cassette according to claim 22 is characterized in that, described terminator is the terminator of rouge alkali synthetase or the terminator of RAmy1 A.

27, expression cassette according to claim 22 is characterized in that, the encoding sequence of described enzyme that can degrade toxoflavin comprises the nucleotide sequence shown in SEQ ID NO:1.

28, expression cassette according to claim 22 is characterized in that, the encoding sequence of described enzyme that can degrade toxoflavin comprises that the nucleotide sequence with SEQ ID NO:1 has the nucleotide sequence of at least 90% sequence homology.

29, a kind of recombinant vectors, this recombinant vectors comprise any described expression cassette among the claim 22-28.

30, a kind of host cell, this host cell transforms with the described carrier of claim 29.

31, host cell according to claim 20 is characterized in that, this host cell belongs to Agrobacterium.

32, a kind of plant, this plant transforms with the described carrier of claim 29.

33, plant according to claim 32 is characterized in that, described plant is paddy rice or Arabidopis thaliana.

34, a kind of transgenic seed, this transgenic seed are the seed of the described plant of claim 33.

35, the screening method of a kind of transgenic plant, this method comprises:

-with the described carrier of claim 29 plant, plant organ or vegetable cell are transformed and

-biological the propagation of render transgenic in containing the developing medium of toxoflavin.

36, method according to claim 35 is characterized in that, described conversion is by Agrobacterium tumefaciens mediated.

37, a kind of method of producing transgenic plant, this method may further comprise the steps:

-with the described carrier of claim 29 vegetable cell is transformed,

-in containing the developing medium of toxoflavin render transgenic vegetable cell propagation and

-from described genetically modified vegetable cell, differentiate transgenic plant again.

Images