Summary of the invention
The nucleic acid sequence that the object of the present invention is to provide a kind of for detecting herbicide tolerant corn plant DBN9888 andIts detection method, transgenic corn events DBN9888 have preferable tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide,And detection method can quickly and accurately identify in biological sample whether include specific transgenic corn events DBN9888 DNAMolecule.
To achieve the above object, the present invention provides in a kind of nucleic acid sequence, including SEQ ID NO:3 or its complementary seriesAt least 11 continuous nucleotide at least 11 continuous nucleotide, and/or SEQ ID NO:4 or its complementary series.
Preferably, the nucleic acid sequence includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or it is mutualComplementary series.
Further, the nucleic acid sequence include SEQ ID NO:3 or its complementary series, and/or SEQ ID NO:4 or itsComplementary series.
Further, the nucleic acid sequence includes SEQ ID NO:5 or its complementary series.
The SEQ ID NO:1 or its complementary series are 5 ' ends in transgenic corn events DBN9888 in insetion sequenceEnd is located at the sequence that a length near insertion junction is 22 nucleotide, the SEQ ID NO:1 or its complementary sequenceColumn span the DNA sequence dna of the flanking genomic DNA sequence of corn insertion point and 5 ' ends of insetion sequence, comprising describedSEQ ID NO:1 or its complementary series can be accredited as the presence of transgenic corn events DBN9888.The SEQ ID NO:2Or its complementary series is to be located near insertion junction in transgenic corn events DBN9888 in 3 ' ends of insetion sequenceOne length is the sequence of 22 nucleotide, and the SEQ ID NO:2 or its complementary series span 3 ' ends of insetion sequenceDNA sequence dna and corn insertion point flanking genomic DNA sequence, be comprising the SEQ ID NO:2 or its complementary seriesThe presence of transgenic corn events DBN9888 can be accredited as.
In the present invention, the nucleic acid sequence can be inserted into sequence for the SEQ ID NO:3 or its complementary series transgenicAny portion of at least 11 or more continuous polynucleotides (the first nucleic acid sequence) of column, or be the SEQ ID NO:3 or its complementary series in 5 ' flank corn gene group DNA regions any portion of at least 11 or more continuous multicore glycosidesSour (second nucleotide sequence).The nucleic acid sequence may further be derived from or be complementary to comprising the complete SEQ ID to be sameA part of the SEQ ID NO:3 of NO:1.When the first nucleic acid sequence is used together with second nucleotide sequence, these nucleic acidSequence includes DNA primer group in the DNA cloning method for generating amplified production.It is produced using DNA primer in DNA cloning methodRaw amplified production is that can diagnose transgenic corn events DBN9888 or thereafter when including the amplified production of SEQ ID NO:1The presence in generation.Well known to those skilled in the art, the first and second nucleic acid sequences need not be only made of DNA, may also compriseThe mixture or DNA, RNA of RNA, DNA and RNA or it is other not as the nucleotide of one or more polymerase templates or itsThe combination of analog.In addition, heretofore described probe or primer should be at least about 11,12,13,14,15,16,17,18, the length of 19,20,21 or 22 continuous nucleotides can be selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDNucleotide described in NO:3, SEQ ID NO:4 and SEQ ID NO:5.When selected from SEQ ID NO:3, SEQ ID NO:4 andWhen nucleotide shown in SEQ ID NO:5, the probe and primer can be for length at least about 21 to about 50 orMore continuous nucleotides.The SEQ ID NO:3 or its complementary series are in transgenic corn events DBN9888 in insertion sequenceColumn 5 ' ends be located at insertion junction near a length be 960 nucleotide sequence, the SEQ ID NO:3 orIts complementary series by 693 nucleotide corn flanking genomic DNA sequence (the nucleotide 1-693 of SEQ ID NO:3), 77The DBN10006 construct DNA sequence (the nucleotide 694-770 of SEQ ID NO:3) of nucleotide and 190 nucleotide5 ' end DNA sequence of pr35S promoter sequence (the nucleotide 771-960 of SEQ ID NO:3) composition includes the SEQ IDNO:3 or its complementary series can be accredited as the presence of transgenic corn events DBN9888.
The nucleic acid sequence can be any portion of the SEQ ID NO:4 or its complementary series transgenic insetion sequenceAt least 11 or more the continuous polynucleotides (third nucleic acid sequence) divided, or be the SEQ ID NO:4 or its complementationAny portion of at least 11 or more continuous polynucleotides (the 4th cores in 3 ' flank corn gene group DNA regions in sequenceAcid sequence).The nucleic acid sequence may further be derived from or be complementary to comprising the described of the complete SEQ ID NO:2 to be sameA part of SEQ ID NO:4.When third nucleic acid sequence is used together with the 4th nucleic acid sequence, these nucleic acid sequences are being generatedIt include DNA primer group in the DNA cloning method of amplified production.The amplification generated in DNA cloning method is produced using DNA primerObject is can to diagnose transgenic corn events DBN9888 or the presence of its offspring when including the amplified production of SEQ ID NO:2.The SEQ ID NO:4 or its complementary series are to be located to insert in 3 ' ends of insetion sequence in transgenic corn events DBN9888Enter the sequence that a length near junction is 753 nucleotide, the SEQ ID NO:4 or its complementary series are by 159The DBN10006 building of the tNos terminator sequence (the nucleotide 1-159 of SEQ ID NO:4) of a nucleotide, 113 nucleotideThe corn integration site flanking genomic dna of body DNA sequence dna (the nucleotide 160-272 of SEQ ID NO:4) and 481 nucleotideSequence (273-753 of SEQ ID NO:4) composition, can be accredited as comprising the SEQ ID NO:4 or its complementary series and turn baseBecause of the presence of corn event DBN9888.
The SEQ ID NO:5 or its complementary series are that the length of characterization transgenic corn events DBN9888 is 5952The sequence of nucleotide, the genome and genetic elements for specifically including are as shown in table 1.Comprising the SEQ ID NO:5 or its mutuallyComplementary series can be accredited as the presence of transgenic corn events DBN9888.
The genome and genetic elements that table 1, SEQ ID NO:5 include
The nucleic acid sequence or its complementary series can be used in DNA cloning method to generate amplicon, the inspection of the ampliconSurvey diagnosis biological sample transgenic corn event DBN9888 or the presence of its offspring；The nucleic acid sequence or its complementary seriesIt can be used in nucleotide detection method, to detect the presence of biological sample transgenic corn event DBN9888 or its offspring.
To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event DBN9888 a kind ofExisting method, comprising:
Contact sample to be tested in nucleic acid amplification reaction at least two primers；
Carry out nucleic acid amplification reaction；
Detect the presence of amplified production；
The amplified production includes at least 11 continuous nucleotide or SEQ in SEQ ID NO:3 or its complementary seriesAt least 11 continuous nucleotide in ID NO:4 or its complementary series.
Further, the amplified production includes 1-11 or 12-22 in SEQ ID NO:1 or its complementary series1-11 or 12-22 continuous nucleotides in continuous nucleotide or SEQ ID NO:2 or its complementary series.
Further, the amplified production include SEQ ID NO:1 or its complementary series, SEQ ID NO:2 or its mutuallyComplementary series, SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.
In the above-mentioned technical solutions, the primer includes at least one nucleic acid sequence.
Specifically, the primer includes the first primer and the second primer, the first primer be selected from SEQ ID NO:8 andSEQ ID NO:10；Second primer is selected from SEQ ID NO:9 and SEQ ID NO:11.
To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event DBN9888 a kind ofExisting method, comprising:
Contact sample to be tested with probe, the probe includes at least 11 in SEQ ID NO:3 or its complementary seriesAt least 11 continuous nucleotide in continuous nucleotide or SEQ ID NO:4 or its complementary series；
Hybridize the sample to be tested and the probe under stringent hybridization conditions；
Detect the hybridisation events of the sample to be tested and the probe.
The stringent condition can in 6 × SSC (sodium citrate), 0.5%SDS (lauryl sodium sulfate) solution,Hybridize at 65 DEG C, is then respectively washed film 1 time with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS.
Further, the probe include in SEQ ID NO:1 or its complementary series 1-11 or 12-22 it is continuous1-11 or 12-22 continuous nucleotides in nucleotide or SEQ ID NO:2 or its complementary series.
Further, the probe has SEQ ID NO:1 or its complementary series, SEQ ID NO:2 or its complementary sequenceColumn, SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.
Selectively, at least one fluorophor label of at least one described probe.
To achieve the above object, the present invention also provides a kind of test sample transgenic corn event DBN9888'sMethod existing for DNA, comprising:
Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:3 orIn its complementary series at least 11 continuous nucleotide or SEQ ID NO:4 or its complementary series at least 11 it is continuousNucleotide；
Hybridize the sample to be tested and the marker nucleic acid molecules under stringent hybridization conditions；
The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then are educated by marker auxiliaryKind analysis is to determine that glyphosate tolerant and/or glufosinate tolerant and marker nucleic acid molecules are chain on science of heredity.
Further, the marker nucleic acid molecules include 1-11 or in SEQ ID NO:1 or its complementary series1-11 or 12-22 continuous nucleotides in 12-22 continuous nucleotides or SEQ ID NO:2 or its complementary series.
Further, the marker nucleic acid molecules have SEQ ID NO:1 or its complementary series, SEQ ID NO:2Or its complementary series, SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.
To achieve the above object, the present invention also provides a kind of DNA detection kit, including at least one DNA molecular, institutesStating DNA molecular includes at least 11 continuous nucleotide or SEQ in the homologous sequence or its complementary series of SEQ ID NO:3At least 11 continuous nucleotide, can be used as transgenic corns in the homologous sequence of ID NO:4 or its complementary seriesEvent DBN9888 or its offspring have the DNA primer or probe of specificity.
Further, the DNA molecular includes 1-11 or 12-22 in SEQ ID NO:1 or its complementary series1-11 or 12-22 continuous nucleotides in continuous nucleotide or SEQ ID NO:2 or its complementary series.
Further, the DNA molecular has the homologous sequence or its complementary series, SEQ ID of SEQ ID NO:1The homologous sequence of NO:2 or its complementary series, the homologous sequence of SEQ ID NO:6 or its complementary series or SEQ ID NO:7Homologous sequence or its complementary series.
To achieve the above object, the present invention also provides a kind of plant cells, include coding glyphosate tolerant EPSPS eggWhite nucleic acid sequence, the nucleic acid sequence of coding glufosinate tolerant PAT albumen and the nucleic acid sequence of specific region, the given zoneThe nucleic acid sequence in domain includes SEQ ID NO:1, SEQ ID NO:2, sequence shown in SEQ ID NO:6 or SEQ ID NO:7.
To achieve the above object, the present invention also provides a kind of corn plants for generating and having tolerance to glyphosate herbicidalThe method of strain, the nucleic acid sequence including introducing coding glyphosate tolerant EPSPS albumen into the genome of the plantSEQ ID NO:1, SEQ ID NO:2, SEQ ID are selected from the nucleic acid sequence of the nucleic acid sequence of specific region, the specific regionAt least one of NO:3, SEQ ID NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequenceColumn.
Specifically, the method for generating the plant for having tolerance to glyphosate herbicidal includes:
By to glyphosate herbicidal have tolerance the first parental maize plant of transgenic corn events DBN9888 withThe the second parental maize plant sexual hybridization for lacking glyphosate tolerant, to generate a large amount of progeny plants；
The progeny plant is handled with glyphosate herbicidal；
The progeny plant of selection tolerance glyphosate.
To achieve the above object, the present invention also provides a kind of corn plants for generating and having tolerance to glufosinate-ammonium herbicideStrain method, including into the genome of the plant introduce coding glufosinate tolerant PAT albumen nucleic acid sequence andThe nucleic acid sequence of the nucleic acid sequence of specific region, the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ IDAt least one of NO:3, SEQ ID NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequenceColumn.
Specifically, the method for generating the plant for having tolerance to glufosinate-ammonium herbicide includes:
By first parental maize plant of transgenic corn events DBN9888 to glufosinate-ammonium herbicide with tolerance and lackSecond parental maize plant sexual hybridization of few glufosinate tolerant, to generate a large amount of progeny plants；
The progeny plant described in glufosinate-ammonium herbicide treatment；
The progeny plant of selection tolerance glyphosate.
To achieve the above object, the present invention also provides a kind of generations to have to glyphosate herbicidal and glufosinate-ammonium herbicideThe method of the plant of tolerance, including introducing coding glyphosate tolerant EPSPS into the genome of the plantThe nucleic acid sequence of the nucleic acid sequence of albumen, the nucleic acid sequence for encoding glufosinate tolerant PAT albumen and specific region, it is described specificThe nucleic acid sequence in region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, at least one of sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence.
Specifically, the method for generating the plant that there is tolerance to glyphosate herbicidal and glufosinate-ammonium herbicideInclude:
To there is the first parent of transgenic corn events DBN9888 of tolerance to glyphosate herbicidal and glufosinate-ammonium herbicideThis plant and the second parental maize plant sexual hybridization for lacking glyphosate and/or glufosinate tolerant, to generate bigMeasure progeny plant；
The progeny plant described in glyphosate herbicidal and glufosinate-ammonium herbicide treatment；
The progeny plant of selection tolerance glyphosate and glufosinate-ammonium.
To achieve the above object, the present invention also provides a kind of cultures has the corn plant of tolerance to glyphosate herbicidalThe method of object, comprising:
An at least corn seed is planted, includes coding glyphosate tolerant EPSPS in the genome of the corn seedNucleic acid sequence and specific region nucleic acid sequence；
The corn seed is set to grow up to plant；
The plant is sprayed with effective dose glyphosate herbicidal, harvest does not have the nucleic acid of specific region with otherThe plant of sequence compares the plant with the plant injury weakened；
The nucleic acid sequence of the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDAt least one of NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence.
To achieve the above object, the present invention also provides a kind of cultures has the corn plant of tolerance to glufosinate-ammonium herbicideThe method of object, comprising:
An at least corn seed is planted, includes coding glufosinate tolerant PAT egg in the genome of the corn seedThe nucleic acid sequence of white nucleic acid sequence and specific region；
The corn seed is set to grow up to plant；
The plant described in effective dose glufosinate-ammonium herbicide spray, harvest do not have the nucleic acid of specific region with otherThe plant of sequence compares the plant with the plant injury weakened；
The nucleic acid sequence of the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDAt least one of NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence.
To achieve the above object, the present invention also provides a kind of cultures to have to glyphosate herbicidal and glufosinate-ammonium herbicideThe method of the corn plant of tolerance, comprising:
An at least corn seed is planted, includes coding glyphosate tolerant EPSPS in the genome of the corn seedThe nucleic acid sequence of the nucleic acid sequence of albumen, the nucleic acid sequence for encoding glufosinate tolerant PAT albumen and specific region；
The corn seed is set to grow up to plant；
The plant described in effective dose glyphosate herbicidal and glufosinate-ammonium herbicide spray, harvest do not have with otherThe plant of the nucleic acid sequence of specific region compares the plant with the plant injury weakened；
The nucleic acid sequence of the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDAt least one of NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence.
To achieve the above object, the present invention also provides a kind of sides for protecting the plants from the damage as caused by herbicideMethod is planted including the herbicide containing effective dose glyphosate and/or glufosinate-ammonium is applied at least one transgenic corns of plantationThe big Tanaka of object, the rotaring gene corn plant include selected from SEQ ID NO:1, SEQ ID NO:2, SEQ in its genomeAt least one of ID NO:3, SEQ ID NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 coreAcid sequence, the rotaring gene corn plant have the tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide.
To achieve the above object, the present invention also provides a kind of methods for controlling weeds in field, including will contain effective agentAmount glyphosate and/or the herbicide of glufosinate-ammonium are applied to the big Tanaka for planting at least one rotaring gene corn plant, described to turn baseBecause corn plant includes selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO in its genome:4, at least one of sequence shown in SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence, the transgenosis are beautifulRice plant has the tolerance to glyphosate herbicidal and/or glufosinate-ammonium herbicide.
To achieve the above object, the present invention also provides a kind of crop field glyphosate for controlling glyphosate-tolerant plant is anti-Property weeds method, including the herbicide containing effective dose glufosinate-ammonium is applied at least one glyphosate tolerant of plantationThe big Tanaka of rotaring gene corn plant, the rotaring gene corn plant of the glyphosate tolerant include to be selected from its genomeSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQAt least one of sequence shown in ID NO:7 nucleic acid sequence, the rotaring gene corn plant of the glyphosate tolerant have pair simultaneouslyThe tolerance of glufosinate-ammonium herbicide.
To achieve the above object, the present invention also provides a kind of methods for delaying insect-resistant, are included in the pest-resistant jade of plantationThe big Tanaka of rice plant plants at least one rotaring gene corn plant with glyphosate and/or glufosinate tolerant, the grassThe rotaring gene corn plant of sweet phosphine tolerance includes selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID in its genomeAt least one of NO:3, SEQ ID NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequenceColumn.
To achieve the above object, the present invention also provides a kind of multicore glycosides comprising SEQ ID NO:1 or SEQ ID NO:2The agricultural product or commodity of acid, the agricultural product or commodity are corn flour, maize flour, corn oil, cornstarch, corn gluten, jadeRice cake, cosmetics or filler.
It is defined below and square in nucleic acid sequence and its detection method of the present invention for detecting antiweed corn plantMethod can preferably define the present invention and those skilled in the art is instructed to implement the present invention, unless otherwise mentioned, according toThe conventional usage of those of ordinary skill in the art understands term.
" corn " refers to maize (Zea mays), and all plant varieties including that can mate with corn,Including field corn kind.
Term "comprising" refers to " including but not limited to ".
Term " plant " includes that whole plant, plant cell, plant organ, plant protoplast, plant can therefrom againIt is complete in raw plant cell tissue cultures, plant callus, vegetation bed (plant clumps) and plant or plant partWhole plant cell, the plant part such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stalk, root, the tip of a root, flowerMedicine etc..The part for the genetically modified plants being interpreted as in the scope of the invention includes but is not limited to plant cell, protoplast, groupIt knits, callus, embryo and flower, stem, fruit, Ye Hegen, the above plant part are originated from advance with DNA molecular conversion of the inventionAnd the genetically modified plants being therefore at least partly made of transgenic cell or its filial generation.
Term " gene " refers to the nucleic acid fragment of expression specific protein, including adjusting sequence (5 ' the non-volumes before coded sequenceCode sequence) and coded sequence after adjusting sequence (3 ' non-coding sequence)." natural gene ", which refers to, is naturally found to have its ownAdjust the gene of sequence." mosaic gene " refer to be not natural gene any gene, it includes non-natural discovery adjusting andCoded sequence." endogenous gene " refers to natural gene, and the natural gene is located in organism genome its natural place." foreign gene " is the alien gene being not present in the existing genome for being biology and originally, also refers to and imports through Transgenic proceduresThe gene of recipient cell.Foreign gene may include the natural gene or mosaic gene of insertion non-native organism." transgenosis "It is the gene that genome is had been incorporated by Transformation Program.The site that recombinant DNA has been inserted into Plant Genome can claimFor " insertion point " or " target site ".
" flanking DNA " may include the genome being naturally present in the organism of such as plant or be drawn by conversion processExternal source (heterologous) DNA entered, such as segment relevant to transformation event.Therefore, flanking DNA may include natural and exogenous DNACombination.In the present invention, " flanking region " or " flanking sequence " or " genome frontier district " or " genome border sequence " refer toThe base-pair of at least 3,5,10,11,15,20,50,100,200,300,400,1000,1500,2000,2500 or 5000 is longerSequence, be located at initial external source insertion DNA molecular immediately upstream or downstream and with initial external source be inserted into DNA molecular phaseIt is adjacent.When the flanking region is located at downstream, it is referred to as " left margin flank " or " 3 ' flank " or " 3 ' genome frontier district "Or " 3 ' border sequence of genome " etc..When the flanking region is located at upstream, it is referred to as " right margin flank " or " 5 ' sidesThe wing " or " 5 ' genome frontier district " or " 5 ' border sequence of genome " etc..
The Transformation Program of the random integration of exogenous DNA is caused to will lead to the transformant containing different flanking regions, the differenceFlanking region is that each transformant institute specificity contains.When recombinant DNA is introduced into plant by conventional hybridization, flanking region is logicalChang Buhui changes.Transformant also can be containing between heterologous insertion DNA and the section of genomic DNA or between two sections of genomic DNAsOr the unique engagement between two sections of allogeneic dna sequence DNAs." engagement " is the point of two specific DNA fragmentation connections.For example, engagement existsIn the position of insert DNA connection flanking DNA.Junction is also present in the organism of conversion, and two of them DNA fragmentation is to repairAdorn linking together for the mode found from native organism." engagement DNA " refers to the DNA comprising junction.
The present invention provides the referred to as transgenic corn events of DBN9888 and its offspring, the transgenic corn eventsDBN9888 is corn plant DBN9888 comprising the Plants and Seeds of transgenic corn events DBN9888 and its plant are thinBorn of the same parents or its renewable part, the plant part of the transgenic corn events DBN9888, including but not limited to cell, pollen, embryoPearl, flower, bud, root, stem, silk, inflorescence, ear fringe, leaf and the product from corn plant DBN9888, such as corn flour, cornFace, corn oil, corn pulp, corn silk, cornstarch and the biomass for staying in corn crop field.
Transgenic corn events DBN9888 of the present invention contains a DNA construct, when it is expressed in plant cellWhen, the transgenic corn events DBN9888 obtains the tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide.The DNAConstruct includes two concatenated expression cassettes, and first expression cassette is comprising the suitable promoter for expressing in plant and fitsThe polyadenylation signal sequence of conjunction, the promoter, which is operably connected, encodes 5- enol pyruvylshikimate -3- phosphoric acidThe gene of synthase (EPSPS), the EPSPS have tolerance to glyphosate herbicidal.Second expression cassette includes for plantingThe suitable promoter expressed in object and suitable polyadenylation signal sequence, the promoter are operably connected codingThe nucleic acid sequence of the gene of phosphinothricin N-acetyl transferase (PAT), the PAT albumen has tolerance to glufosinate-ammonium herbicideProperty.Further, the promoter can be the suitable promoter separated from plant, including composing type, induction type and/or tissueSpecificity promoter, the suitable promoter include but is not limited to cauliflower mosaic virus (CaMV) 35S promoter, radix scrophulariae flowerMosaic virus (FMV) 35S promoter, Tsf1 promoter, ubiquitin protein (Ubiquitin) promoter, actin (Actin) startingSon, soil Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) promoter, octopine synthase(OCS) promoter, Cestrum (Cestrum) yellow leaf curl virus promoter, patatin (Patatin) openMover, ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase/oxygenase (RuBisCO) promoter, glutathione S-transferase (GST) startingSon, E9 promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes)RolD promoter and Arabidopsis (Arabidopsis) Suc2 promoter.The polyadenylation signal sequence can forThe suitable polyadenylation signal sequence to work in plant, the suitable polyadenylation signal sequence include but unlimitedIn from the polyadenosine of soil Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) genePolyadenylation signal sequence derives from cauliflower mosaic virus (CaMV) 35S terminator, derives from pea ribulose -1,5- diphosphonic acidCarboxylase/oxygenase E9 terminator, from protease-inhibitor Ⅱ (PIN II) gene polyadenylation signal sequence andFrom the polyadenylation signal sequence of alpha-tubulin (α-tubulin) gene.
In addition, the expression cassette can also include other genetic elements, the genetic elements include but is not limited to enhanceSon and signal peptide/transit peptides.The expression of gene can be enhanced in the enhancer, and the enhancer includes but is not limited to cigaretteCareless etch virus (TEV) translation activity factor, CaMV35S enhancer and FMV35S enhancer.Signal peptide/the transit peptides can be withGuide EPSPS albumen and/or PAT Protein transport to extracellular or intracellular specific organelle or compartment, for example, using compilingCode chloroplast transit peptide sequence targets chloroplaset, or utilizes ' KDEL ' to retain sequence and target endoplasmic reticulum.
5- enol pyruvylshikimate -3- phosphate synthase (EPSPS) gene can be from soil AgrobacteriumIt is isolated in (Agrobacterium tumefaciens sp.) CP4 bacterial strain, and can by optimization codon or withOther way changes the polynucleotides of coding EPSPS, to reach the stability and utilizability that increase transcript in transformed cellsPurpose.5- enol pyruvylshikimate -3- phosphate synthase (EPSPS) gene can also be used as selected marker.
" glyphosate " refers to the salt of N- phosphonomethylglycine and it, is handled with " glyphosate herbicidal " and refers to useAny one is handled containing the herbicide formulations of glyphosate.In order to reach ebd and to certain glyphosate systemThe selection of agent utilization rate is no more than the technical ability of common agronomic technique personnel.Herbicide formulations using any one containing glyphosateProcessing contains the field of the vegetable material from antiweed corn plant DBN9888, miscellaneous in the field by controllingGrass growth, and the growth or yield of the vegetable material from herbicide tolerant corn plant DBN9888 are not influenced.
The enzyme phosphinothricin N-acetyl transfer separated from streptomycete (Streptomyces viridochromogenes)Enzyme (phosphinothricin N-acetyltransferase, PAT) gene is catalyzed L-phosphinothricin conversion by acetylationFor its inactive form, to assign plant to the tolerance of glufosinate-ammonium herbicide.Phosphinothricin (PTC, 2- amino-4- methylphosphine-butyric acid) be glutamine synthelase inhibitor.PTC is antibiotic 2- amino -4- methylphosphine acyl-the-the third ammonia of alanylThe structural units of acid, this tripeptides (PTT) have resisting gram-positive and gramnegative bacterium and antimycotic Botrytis cinereaThe activity of (Botrytis cinerea).Phosphinothricin N-acetyl transferase (PAT) gene can also be used as selected markerGene.
" glufosinate-ammonium " also known as glufosinate refer to 2- amino -4- [hydroxyl (methyl) phosphono] butyric acid ammonium, with " careless ammoniumPhosphine herbicide " processing, which refers to, to be handled using any one containing the herbicide formulations of glufosinate-ammonium.In order to reach effective biologyLearn dosage and to certain glufosinate-ammonium preparation utilization rate selection be no more than common agronomic technique personnel technical ability.Use any oneHerbicide formulations processing containing glufosinate-ammonium contains the vegetable material from herbicide tolerant corn plant DBN9888Field will control the weed growth in the field, and not influence from herbicide tolerant corn plant DBN9888Vegetable material growth or yield.
The DNA construct is introduced in plant using method for transformation, and the method for transformation includes but is not limited to agriculture barBacterium (Agrobacterium) mediated transformation method, Gene Knock-out Mice and pollen tube channel conversion method.
The Agrobacterium_mediated method is the common method of Plant Transformation.The exogenous DNA gram that will be introduced into plantBetween the grand left and right boundary consensus sequence to carrier, i.e. the area T-DNA.The carrier is transformed into agrobatcerium cell, then,The agrobatcerium cell is organized for infection plant, and the area T-DNA of the carrier comprising exogenous DNA is inserted into plant geneIn group.
The Gene Knock-out Mice is with carrier bombardment plant cell (the biological bullet that particle mediates comprising exogenous DNAHit conversion).
The pollen tube channel conversion method is that natural pollen tube channel (also known as pollen is formed by after pollinating using plantPipe guides tissue), through megarchidium channel, exogenous DNA is carried into blastular.
After conversion, it is necessary to have from the plant tissue regenerating plants of conversion, and using suitable label selectionThe offspring of exogenous DNA.
DNA construct is the combination that DNA molecular is interconnected, and this combination provides one or more expression cassettes.DNAConstruct preferably can the self-replacation in bacterial cell, and contain different restriction endonuclease sites plasmid,Contained restriction endonuclease sites provide functioning gene element, i.e. promoter, introne, leader sequence, volume for importingThe DNA molecular of code sequence, 3 ' terminator regions and other sequences.Expression cassette contained in DNA construct includes providing courierGenetic elements necessary to the transcription of RNA, the expression cassette can be designed as expressing in prokaryotic cell or eukaryocyte.This hairBright expression cassette is designed to most preferably express in plant cell.
Transgenosis " event " is to include one as obtained from converting plant cell with heterologous DNA construct and containThe expression of nucleic acid box of target gene is inserted into the plant population in Plant Genome with generation by transgene method, regenerationThe plant population, and selection have the specific plant of insertion specific gene group site feature.Term " event " refers to including heterologousThe original transformant of DNA and the offspring of the transformant.Term " event " also refers to transformant and other kinds containing allogeneic dna sequence DNAOffspring obtained from sexual hybridization is carried out between body, even if after be returned repeatedly with backcross parent, from transformant parentThis insertion DNA and flanking genomic dna exists in the same chromosome location in filial generation.Term " event ", which also refers to, to be comeFrom the DNA sequence dna of original transformant, the DNA sequence dna include insertion DNA and with the close adjacent flanking genomes sequence of insertion DNAColumn, which, which is expected, is transferred in filial generation, the filial generation by containing insertion DNA parental department (such as original transformant and itsIt is selfed the filial generation generated) sexual hybridization is carried out with the parental department without containing insertion DNA and is generated, and the filial generation is received comprising meshMark the insertion DNA of gene.
" recombination " refers to the DNA that generally can not be found and therefore generate by manual intervention in nature in the present inventionAnd/or the form of albumen and/or organism.This manual intervention can produce recombinant DNA molecules and/or recombinant plant." the weightIt is that isolated sequence section obtains, such as passes through chemistry in other cases that group DNA molecular ", which is by two kinds of artificial combination,Synthesis operates isolated nucleic acid segment by genetic engineering technology.The technology for carrying out nucleic-acid manipulation is well-known.
Term " transgenosis " includes any cell, cell line, callus, tissue, plant part or plant, above baseBecause type due to heterologous nucleic acids there are due to change, " transgenosis " includes Transgenics initially changed in this way and by mostThe offspring individual that first Transgenics are generated by sexual hybridization or vegetative propagation.In the present invention, term " transgenosis " does not wrap(chromosome the or extrachromosomal) change by conventional plant breeding method or the natural genome that event occurs is included, it is describedIt is natural that for example random allogamy of event, non-recombinant virus infection, non-recombinant Bacterial Transformation, non-recombinant swivel base or spontaneous prominent occursBecome.
" heterologous " refers to that the first molecule is not found usually and the second molecular combinations in nature in the present invention.For example,Molecule can be originated from the first species and be inserted into the genome of the second species.Therefore this molecule for host be it is heterologous andIt is artificially introduced in the genome of host cell.
The transgenic corn events DBN9888 that there is tolerance to glyphosate herbicidal and glufosinate-ammonium herbicide is cultivated, is led toIt crosses following steps: making the first parental corn plants and the second parental corn plants sexual hybridization first, to produce multiplicityFirst generation progeny plant, first parental corn plants are by cultivation transgenic corn event DBN9888 and its jade of offspringRice plant composition, transgenic corn events DBN9888 and its offspring be by using it is of the invention to glyphosate herbicidal andObtained from there is glufosinate-ammonium herbicide the expression cassette of tolerance to be converted, the second parental corn plants shortage removes glyphosateThe tolerance of careless agent and/or glufosinate-ammonium herbicide；Then the application to glyphosate herbicidal and/or glufosinate-ammonium herbicide is selected to haveThere is the progeny plant of tolerance, can cultivate there is the corn of tolerance to plant glyphosate herbicidal and glufosinate-ammonium herbicideObject.These steps, which may further include, makes the application to glyphosate herbicidal and/or glufosinate-ammonium herbicide have toleranceProgeny plant is returned with the second parental corn plants or third parental corn plants, then passes through application Gyphosate herbiciceAgent, glufosinate-ammonium herbicide or by molecular marked compound relevant to character (such as comprising being inserted into transgenic corn events DBN9888The DNA molecular of bond site that the 5 ' ends and 3 ' ends of sequence identify) identification select filial generation, glyphosate is removed to generateCareless agent and glufosinate-ammonium herbicide have the corn plant of tolerance.
It will also be appreciated that two different genetically modified plants can also hybridize to generate containing there are two independent, separationThe offspring of the foreign gene of formula addition.It is all homozygous for the available foreign gene added to two of the selfing of appropriate offspringThe Progeny plants of son.It is also as previously described to be expected to the backcrossing of parental plant and with the cutcross of non-transgenic plant, vegetative propagation is also same.
The corn of trans Bt gene can kill insect/pest of such as Lepidoptera and coleoptera, but there is also under a small amount of survivalInsect/the pest come, after several generations is bred, it is possible to create resistant insects/pest of anti-Bt albumen.In order to solve insect/evilWorm generates resistance this problem, and Environmental Protection Agency USA gives following guidance for the use of genetically modified crops, need to provide oneSanctuary's corn of certainty ratio (requires have 5%, 10%, 20% etc. according to product difference about sanctuary's corn.And it can beThe corn of non-pest-resistant transgenic corns (such as herbicide tolerant transgenic corns) or anti-Non-target pests, not necessarily right and wrongTransgenic corns).After insect/pest of the overwhelming majority is killed on corresponding transgenic insect-resistant corn, someInsect/pest is on sanctuary's corn without extremely, ensure that insect/pest population of not resistance accounts for governance quantity.So i.e.Make have the resistant insects/pest to survive on a small quantity, with account for rule quantity non-resistance insect/pest post-coitum resistant gene also bySignificantly dilute.
Term " probe " is the nucleic acid molecules of one section of separation, is combined with conventional detectable label or report point aboveSon, for example, radioactive isotope, ligand, chemiluminescent agent or enzyme.This probe is complementary with a chain of target nucleic acid, in the present invention, probe is complementary with a DNA chain from transgenic corn events DBN9888 genome, no matter the geneGroup DNA be also be derived from from transgenic corn events DBN9888 or seed transgenic corn events DBN9888 plant orSeed or extract.Probe of the invention not only includes DNA or ribonucleic acid, further include specifically with targetDNA sequence dna combines and can be used for detecting the existing polyamide and other probe materials of the target dna sequence.
Term " primer " is the nucleic acid molecules of one section of separation, is hybridized by nucleic acid, annealed combination to complementary target dnaOn chain, heterozygote is formed between primer and target dna chain, then under the action of polymerase (such as archaeal dna polymerase), along meshDNA chain is marked to extend.Primer pair of the invention is related to its application in target nucleic acid sequence amplification, for example, passing through polymerase chainFormula reacts (PCR) or other conventional nucleic acid amplification methods.
The length of probe and primer is usually 11 polynucleotides or more, preferably 18 polynucleotides or more,More preferably 24 polynucleotides or more, most preferably 30 polynucleotides or more.This probe and primer are in heightSpecifically hybridize under degree stringent hybridization condition with target sequence.Although being different from target dna sequence and being protected to target dna sequenceThe probe for holding hybridization ability can design by conventional method, however, it is preferred to, probe and primer in the present inventionThere is complete DNA sequence dna identity with the continuous nucleic acid of target sequence.
It can be determined by conventional method based on the primer and probe of flanking genomic dna and insetion sequence of the invention,For example, by separating corresponding DNA molecular from from the vegetable material of transgenic corn events DBN9888, and determining shouldThe nucleic acid sequence of DNA molecular.The DNA molecular includes transgene insert sequence and Maize genome flank region, and the DNA dividesThe segment of son may be used as primer or probe.
Nucleic acid probe and primer of the invention hybridizes with target dna sequence under strict conditions.Any conventional nucleic acid is miscellaneousIt hands over or amplification method may be used to identify in sample from the presence of the DNA of transgenic corn events DBN9888.Nucleic acid pointSon or its segment can carry out specific hybrid with other nucleic acid molecules in any case.As the present invention uses, if twoA nucleic acid molecules can form antiparallel double-strandednucleic acid structure, so that it may say that the two nucleic acid molecules are able to carry out specifically to each otherProperty hybridization.If two nucleic acid molecules show complete complementarity, claiming one of nucleic acid molecules is another nucleic acid point" complement " of son.As the present invention uses, when each nucleotide and another nucleic acid molecules of a nucleic acid moleculesThe corresponding nucleotide mutual added time, then the two nucleic acid molecules is claimed to show " complete complementarity ".If two nucleic acid molecules can be withEnough stability phase mutual crosses then claim to make them anneal and be bonded to each other under the conditions of at least conventional " low stringent "The two nucleic acid molecules are " minimum level is complementary ".Similarly, if two nucleic acid molecules can be mutual with enough stabilityHybridization then claims the two nucleic acid molecules to have to make them anneal and be bonded to each other under the conditions of conventional " height is stringent "" complementarity ".Deviateing from complete complementarity can permit, as long as not exclusively to prevent two molecules from being formed double for this deviationChain structure.In order to enable a nucleic acid molecules as primer or probe, it is only necessary to it is adequately complementary to guarantee that it has in sequenceProperty, so that stable duplex structure can be formed under used specific solvent and salinity.
As the present invention uses, substantially homologous sequence is one section of nucleic acid molecules, and the nucleic acid molecules are in high stringencySpecific hybrid can occur with the complementary strand of another section of nucleic acid molecules to match down.Promote the suitable stringent of DNA hybridizationCondition is then used under the conditions of 50 DEG C for example, about being handled under the conditions of 45 DEG C with 6.0 × sodium chloride/sodium citrate (SSC)2.0 × SSC washing, these conditions are well known to those skilled in the art.For example, the salinity in washing step can be selectedAbout 2.0 × SSC, 50 DEG C to high stringency of about 0.2 × SSC, 50 DEG C from Low stringency conditions.In addition, washing stepIn temperature condition can be increased to about 65 DEG C of high stringency from about 22 DEG C of room temperature of Low stringency conditions.Temperature stripPart and salinity can all change, can also one of them remain unchanged and another variable changes.Preferably, originallyInvention a nucleic acid molecules can under moderate stringency, such as at about 2.0 × SSC and about 65 DEG C with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, in SEQ ID NO:6 and SEQ ID NO:7Specific hybrid occurs for any segment of one or more nucleic acid molecules or its complementary series or above-mentioned sequence.It is highly preferred thatA nucleic acid molecules of the invention under high stringency with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, one or more nucleic acid molecules or its complementary series in SEQ ID NO:6 and SEQ ID NO:7,Or specific hybrid occurs for any segment of above-mentioned sequence.In the present invention, preferred marker nucleic acid molecules have SEQ IDAny segment of NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ ID NO:7 or its complementary series or above-mentioned sequence.Another preferred marker nucleic acid molecules of the present invention and SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ IDAny segment of NO:7 or its complementary series or above-mentioned sequence is same with 80% to 100% or 90% to 100% sequenceProperty.SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 may be used as the mark in plant breeding methodObject is remembered to identify the offspring of genetic cross.Probe can be art technology by any one with hybridizing for target dna moleculeMethod known to personnel detects, these methods include but is not limited to fluorescent marker, radioactive label, antibody class labelAnd chemiluminescent labeling.
About the amplification (for example, passing through PCR) for using specific amplimer to carry out target nucleic acid sequence, " stringent itemPart " refers to the condition for only allowing primer pair target nucleic acid sequence to hybridize in the hot amplified reaction of DNA, has and target coreThe primer of the corresponding wild-type sequence of acid sequence (or its complementary series), can be and excellent in conjunction with the target nucleic acid sequenceChoosing generates unique amplified production, amplified production, that is, amplicon.
Term " specific binding (target sequence) " refers to probe under stringent hybridization conditions or primer only and comprising targetTarget sequence in the sample of sequence hybridizes.
As the present invention uses, " by the DNA of amplification " or " amplicon " refer to the target as nucleic acid-templated a partThe nucleic acid amplification product of nucleic acid sequence.For example, in order to determine corn plant whether by containing transgenic corn events of the present inventionDBN9888 is generated by sexual hybridization mode, or whether the corn sample acquired from field includes transgenic corn eventsWhether DBN9888 or corn extract, such as coarse powder, powder or oil include transgenic corn events DBN9888, from corn plantThe DNA that tissue sample or extract extract can be by using the nucleic acid amplification method of primer pair to generate for transgenic cornsThe presence of the DNA of event DBN9888 is diagnostic amplicon.The primer pair include one in the Plant Genome withThe first primer of the adjacent flanking sequence of the exogenous DNA insertion point of insertion, and second draw from the exogenous DNA of insertionObject.Amplicon has certain length and sequence, and the sequence is also diagnostic to the transgenic corn events DBN9888.The length range of amplicon can be the combination length of primer pair plus a nucleotide base pair, preferably add about 50 coresThuja acid base-pair more preferably adds about 250 nucleotide bases pair, most preferably adds about 450 nucleosides soda acidsBase to or more.
Optionally, primer pair can include entire insertion to generate from the flanking genomic sequence of the insertion two sides DNAThe amplicon of nucleotide sequence.One in the primer pair of plant genome sequences can be located at away from insertion DNA sequence dnaAt a certain distance from, which may range from a nucleotide base to about 20,000 nucleotide bases pair.Term " amplificationThe use of son " has been particularly intended to exclude the primer dimer formed in the hot amplified reaction of DNA.
Nucleic acid amplification reaction can be realized by any nucleic acid amplification reaction method known in the art, including polymerizationEnzyme chain reaction (PCR).Various nucleic acid amplification methods have been well-known to those skilled in the art.PCR amplification method has been sent outOpen up the phage DNA of the amplifiable up to genomic DNA of 22kb and up to 42kb.Other of these methods and this fieldDNA cloning method can be used for the present invention.The exogenous DNA array of insertion and flank from transgenic corn events DBN9888DNA sequence dna can be expanded by being expanded using genome of the provided primer sequence to transgenic corn events DBN9888The DNA sequencing of standard is carried out after increasing to the DNA of PCR amplification or clone.
DNA detection kit based on DNA cloning method contains DNA primer molecule, they are under reaction condition appropriateOn specific hybrid to target dna and expand diagnostic amplicon.Kit can provide the detection method based on Ago-GelOr many methods of checkout and diagnosis amplicon known in the art.Containing with SEQ ID NO:3 or SEQ ID NO:4'sAny part in Maize genome area is homologous or complementary and any part with the transgenosis insert district of SEQ ID NO:5The kit of homologous or complementary DNA primer is provided by the present invention.Particularly identify useful in DNA cloning method drawObject expands 5 ' transgenosis/genome with transgenic corn events DBN9888 to being SEQ ID NO:8 and SEQ ID NO:9A part of homologous diagnostic amplicon in area, wherein amplicon includes SEQ ID NO:1.Other DNA as DNA primer pointsSon can be selected from SEQ ID NO:5.
Amplicon caused by these methods can be detected by multiple technologies.One of method is GeneticBit Analysis, this method devise one across the DNA of insertion DNA sequence dna and adjacent flanking genomic DNA sequence widowNucleotide chain.The oligonucleotide chain is fixed in the micropore of a microwell plate, after carrying out PCR amplification to target area (A primer is respectively used in insetion sequence and in adjacent flanking genomic sequence), single stranded PCR products can be with fixed few nucleosidesSour chain is hybridized, and the template as single base extension, which has used archaeal dna polymerase and be nextThe ddNTPs of a expected base specific markers.Result can be obtained by fluorescence or ELISA class method.Signal represents slottingEnter/the presence of flanking sequence, illustrates that amplification, hybridization and single base extension are successful.
Another method is Pyrosequencing (pyrosequencing) technology.This method devises one across insertionThe oligonucleotide chain of DNA sequence dna and adjacent genomic DNA binding site.By the single-stranded of the oligonucleotide chain and target areaThen and DNA PCR product (primer is respectively used in insetion sequence and in adjacent flanking genomic sequence) is hybridized,Polymerase, ATP, sulfonyl enzyme, luciferase, apyrase, adenosine -5 '-phosphorus sulfate and luciferin are togetherIt is incubated.It is separately added into dNTPs, measures the optical signal of generation.Optical signal represents the presence of insertion/flanking sequence, saysBright amplification, hybridization and single base or polybase base extension are successful.
The Fluorescence polarization of Chen etc. (genome research (Genome Res.) 9:492-498,1999) description is also canIn a kind of method for detecting amplicon of the present invention.Need to design one in this way across insertion DNA sequence dna and phaseThe oligonucleotide chain of adjacent genomic DNA binding site.The single stranded PCR products of the oligonucleotide chain and target area (are being insertedEnter in sequence and respectively use in adjacent flanking genomic sequence a primer) hybridized, then with archaeal dna polymerase and oneThe ddNTP of kind fluorescent marker is incubated together.Single base extension will lead to insertion ddNTP.This insertion can use fluorescenceInstrument measures the change of its polarization.The change of polarization represents the presence of insertion/flanking sequence, illustrates amplification, hybridization and single alkaliBase extension is successful.
Taqman is described as a kind of detect and is mentioned with method existing for quantitative analysis DNA sequence dna, this method in manufacturerIt is discussed in detail in the operation instruction of confession.It is now briefly illustrated below, designs one across insertion DNA sequence dna and adjacent baseBecause of the FRET oligonucleotide probe of group flank binding site.The FRET probe and PCR primer are (in insetion sequence and adjacent sideA primer is respectively used in wing genome sequence) circular response is carried out in the presence of heat-stabilised poly synthase and dNTPs.FRET probeHybridization lead to the division of fluorescence part and quencher moieties and the release of fluorescence part on FRET probe.The generation of fluorescence signalThe presence of insertion/flanking sequence is represented, illustrates amplification and hybridization is successful.
Based on Hybridization principle, for detecting the plant material for deriving from herbicide tolerant transgenic corn events DBN9888The suitable technology of material can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization.Particularly, describedThe technology of being suitble to includes incubating probe and sample, is washed to remove whether unbonded probe and detection probe have hybridized.It is describedDetection method depend on the appended type marked of probe, for example, can detecte radioactive label by X-ray exposure and imagingProbe, or by substrate convert realize color change can detecte enzyme label probe.
Tyangi etc. (Nature Biotechnol (Nat.Biotech.) 14:303-308,1996) describes molecular labeling in sequenceApplication in column detection.It is briefly described as follows, designs one across insertion DNA sequence dna and adjacent flanking genomic binding siteFRET oligonucleotide probe.The unique texture of the FRET probe causes it to contain secondary structure, which can be closeApart from interior holding fluorescence part and quencher moieties.The FRET probe and PCR primer are (in insetion sequence and adjacent flanking geneA primer is respectively used in group sequence) circular response is carried out in the presence of heat-stabilised poly synthase and dNTPs.By successful PCRAmplification, the hybridization of FRET probe and target sequence leads to the forfeiture of probe secondary structure, to make fluorescence part and quencher moietiesIt spatially separates, generates fluorescence signal.The generation of fluorescence signal represents the presence of insertion/flanking sequence, explanationAmplification and hybridization are successful.
The method of other descriptions, such as the method that microfluid (microfluidics) provides separation and DNA amplification sampleAnd equipment.Photoinitiator dye is for detecting and measuring specific DNA molecular.Include the electronic sensor or knot for detecting DNA molecularClose specific DNA molecular receive pearl and thus can be detected receive test tube (nanotube) equipment for detecting DNA of the invention pointsSon is useful.
Method that composition and DNA detection field of the present invention describes or known can be used to develop DNA inspectionTest agent box.The kit is conducive to identify the DNA that whether there is transgenic corn events DBN9888 in sample, can be withFor cultivating the corn plant of the DNA containing transgenic corn events DBN9888.The kit can containing DNA primer orProbe at least part for being derived from or being complementary to SEQ ID NO:1,2,3,4 or 5, or contains other DNA primers or spyNeedle, with being derived from or being complementary to DNA contained in the genetically modified element of DNA, these DNA sequence dnas can be used for DNA cloningReaction, or as the probe in DNA hybridization method.It is containing in the corn genome and what is illustrated in Fig. 1 and table 1 turns baseBecause the DNA structure of insetion sequence and Maize genome binding site includes: the corn for being located at 5 ' end of transgene insert sequence is plantedObject DBN9888 flanking genomes region, a part of insetion sequence of the left boundary area (LB) from Agrobacterium, first tableUp to box by the cauliflower mosaic virus 35 S promoter (pr35S) of the tandem sequence repeats containing enhancer region, it is operably connected toOn the phosphinothricin N-acetyl transferase (cPAT) of the glufosinate tolerant of streptomycete, and it is operably connected to cauliflower flowerIt is formed on mosaic virus 35S terminator (t35S), second expression cassette, can by 1 promoter of rice actin (prOsAct1)It is operationally connected on the coded sequence (spAtCTP2) of arabidopsis EPSPS chloroplast transit peptides, is operably connected to agrobacteriumOn the 5- enol-pyrovyl shikimic acid -3- phosphate synthase (cEPSPS) for belonging to the glyphosate tolerant of CP4 bacterial strain, it is operatively connectedIt is formed on to the transcription terminator (tNos) of nopaline synthase, a part in the right side boundary region (RB) from Agrobacterium is insertedEnter sequence, and be located at 3 ' end of transgene insert sequence corn plant DBN9888 flanking genomes region (SEQ ID NO:5).In DNA cloning method, the DNA molecular as primer, which can be, is inserted into sequence from corn plant DBN9888 transgenicAny part of column is also possible to times from the region of DNA domain of flank Maize genome in transgenic corn events DBN9888What part.
Transgenic corn events DBN9888 can be combined with other transgenic maize varieties, such as herbicide tolerant is (such as2,4-D, dicamba etc.) corn, or carry the transgenic maize varieties of other anti insect genes (such as Cry1Ab, Vip3A).InstituteThere are the various combinations of these different transgenic events, the breeding together with transgenic corn events DBN9888 of the invention, Ke YitiFor resisting a variety of insect pests and being resistant to the improvement hybrid transgenic corn varieties of a variety of herbicides.These kinds are compared to non-transgenic productThe transformed variety of kind and unisexuality shape can show the superior features such as yield promotion.
The present invention provides a kind of for detecting the nucleic acid sequence and its inspection of herbicide tolerant corn plant DBN9888Survey method, the phytotoxicity that transgenic corn events DBN9888 is resistant to the agriculture herbicide containing glyphosate and/or glufosinate-ammonium are madeWith.The 5- enol pyruvylshikimate-of the glyphosate resistance of the plant expression Agrobacterium strains CP4 of the dual character3- phosphate synthase (EPSPS) albumen assigns plant to the tolerance of glyphosate, and expresses the phosphine of the glufosinate resistance of streptomyceteSilk rhzomorph N- acetyltransferase (PAT) albumen assigns plant to the tolerance of glufosinate-ammonium.Dual character corn has as followsAdvantage: 1) apply agriculture herbicide containing glyphosate and be used for the ability that broad-spectrum weeding controls to corn crop；2) glufosinate-ammonium is resistant toProperty character glufosinate-ammonium herbicide (mix or be used alternatingly with glyphosate herbicidal) be applied in combination can be used as a kind of effectively managementThe non-selective means of glyphosate-resistant weeds；3) herbicide tolerant transgenic corns are as non-pest-resistant transgenic corns, withTransgenic insect-resistant corn is planted together with certain proportion, and insect/pest can be delayed to generate resistance；4) corn yield does not dropIt is low.In addition, the gene linkage of coding glyphosate tolerant and glufosinate tolerant character and exists in same DNA sectionIn on the term single gene seat of transgenic corn events DBN9888 genome, this point provides the breeding efficiency of enhancing and makesThe transgenic insert in reproductive population and its filial generation can be tracked with molecular labeling.Simultaneously in detection methodSEQ ID NO:1 or its complementary series, SEQ ID NO:2 or its complementary series, SEQ ID NO:6 or its complementary series orSEQ ID NO:7 or its complementary series can be used as DNA primer or probe and be diagnosed as transgenic corn events DBN9888 to generateOr the amplified production of its offspring, and can be quick, accurate, stable identify from transgenic corn events DBN9888'sThe presence of vegetable material.
BRIEF DESCRIPTION OF THE SEQUENCES
The insertion point and Maize genome of 5 ' transgenic fragments in SEQ ID NO:1 transgenic corn events DBN988811 nucleotide of every side of DNA；
The insertion point and Maize genome of 3 ' transgenic fragments in SEQ ID NO:2 transgenic corn events DBN988811 nucleotide of every side of DNA；
It is located at insertion junction in 5 ' ends of insetion sequence in SEQ ID NO:3 transgenic corn events DBN9888A neighbouring length is the sequence of 960 nucleotide；
It is located at insertion junction in 3 ' ends of insetion sequence in SEQ ID NO:4 transgenic corn events DBN9888A neighbouring length is the sequence of 753 nucleotide；
The flank maize genomic sequence of the entire T-DNA sequence of SEQ ID NO:5,5 ' and 3 '；
SEQ ID NO:6 is located at the sequence inside SEQ ID NO:3, span DBN10006 construct DNA sequence dna andPr35S promoter sequence；
SEQ ID NO:7 is located at the sequence inside SEQ ID NO:4, span tNos transcription terminator andDBN10006 construct DNA sequence dna；
The first primer of SEQ ID NO:8 amplification SEQ ID NO:3；
The second primer of SEQ ID NO:9 amplification SEQ ID NO:3；
The first primer of SEQ ID NO:10 amplification SEQ ID NO:4；
The second primer of SEQ ID NO:11 amplification SEQ ID NO:4；
Primer on 5 ' flanking genomic sequence of SEQ ID NO:12；
The primer of SEQ ID NO:13 and SEQ ID NO:12 pairing being located on T-DNA；
Primer on 3 ' flanking genomic sequence of SEQ ID NO:14 can detecte with SEQ ID NO:12 pairing and turnGene is homozygote or heterozygote；
The primer of SEQ ID NO:15 and SEQ ID NO:14 pairing being located on T-DNA；
The primer 1 of SEQ ID NO:16 Taqman detection EPSPS；
The primer 2 of SEQ ID NO:17 Taqman detection EPSPS；
The probe 1 of SEQ ID NO:18 Taqman detection EPSPS；
The primer 3 of SEQ ID NO:19 Taqman detection PAT；
The primer 4 of SEQ ID NO:20 Taqman detection PAT；
The probe 2 of SEQ ID NO:21 Taqman detection PAT；
The first primer of SEQ ID NO:22 corn endogenous gene Ubiquitin；
The second primer of SEQ ID NO:23 corn endogenous gene Ubiquitin；
The probe of PAT in SEQ ID NO:24 Southern hybridization check；
The probe of EPSPS in SEQ ID NO:25 Southern hybridization check；
SEQ ID NO:26 is located at the primer on T-DNA, consistent with the direction ID NO:13 SEQ；
SEQ ID NO:27 is located at the primer on T-DNA, contrary with SEQ ID NO:13, is used as and obtains flank sequenceColumn；
SEQ ID NO:28 is located at the primer on T-DNA, contrary with SEQ ID NO:13, is used as and obtains flank sequenceColumn；
SEQ ID NO:29 is located at the primer on T-DNA, consistent with the direction ID NO:15 SEQ；
SEQ ID NO:30 is located at the primer on T-DNA, contrary with SEQ ID NO:15, is used as and obtains flank sequenceColumn；
SEQ ID NO:31 is located at the primer on T-DNA, contrary with SEQ ID NO:15, is used as and obtains flank sequenceColumn.
Below by drawings and examples, technical scheme of the present invention will be described in further detail.
Specific embodiment
Further illustrate the present invention for detecting herbicide tolerant corn plant DBN9888 below by specific embodimentNucleic acid sequence and its detection method technical solution.
First embodiment, clone and conversion
1.1, carrier cloning
Recombinant expression carrier DBN10006 (as shown in Figure 2) is constructed using the gene clone technology of standard.The carrierDBN10006 include two concatenated transgene expression cassettes, first expression cassette by the tandem sequence repeats containing enhancer region flowerCauliflower mosaic virus 35S promoter (pr35S) is operably connected to the phosphinothricin N- second of the glufosinate tolerant of streptomyceteOn acyltransferase (cPAT), and it is operably connected on cauliflower mosaic virus 35S terminator (t35S) and forms；SecondA expression cassette is operably connected to arabidopsis EPSPS chloroplast transit peptides by 1 promoter of rice actin (prOsAct1)On coded sequence (spAtCTP2), it is operably connected to 5- enol-acetone of the glyphosate tolerant of Agrobacterium CP4 bacterial strainOn acyl shikimic acid -3- phosphate synthase (cEPSPS), it is operably connected on the transcription terminator (tNos) of nopaline synthase and groupAt.
The carrier DBN10006 is transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA with liquid nitrogen method；Cat.No:18313-015 in), and with 5- enol pyruvylshikimate -3- phosphate synthase (EPSPS) be selected marker to turnChange cell to be screened.
1.2, Plant Transformation
It is converted using conventional Agrobacterium infestation method, by institute in the maize immature embryos of sterile culture and the present embodiment 1.1The Agrobacterium stated co-cultures, and the T-DNA in the recombinant expression carrier DBN10006 of building is transferred in maize chromosome group,To generate transgenic corn events DBN9888.
For the corn transformation of mediated by agriculture bacillus, briefly, immature rataria is separated from corn, is suspended with AgrobacteriumLiquid contacts rataria, and wherein the nucleotide sequence of the nucleotide sequence of EPSPS gene and pat gene can be transferred to children by AgrobacteriumAt least one cell (step 1: infecting step) of one of embryo, in this step, rataria preferably immerses agrobacterium suspension(OD660=0.4-0.6 infects culture medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, grapeSugared 36g/L, acetosyringone (AS) 40mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, pH 5.3)) in starting connectKind.Rataria and Agrobacterium co-culture one period (3 days) (step 2: co-culturing step).Preferably, rataria is after infecting stepIn solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetyl clovesKetone (AS) 100mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH 5.8) on cultivate.It co-cultures hereinAfter stage, there can be " recovery " step of a selectivity.In " recovery " step, recovery media (MS salt 4.3g/L, MS dimensionHe is life, casein 300mg/L, sucrose 30g/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH5.8) at least in the presence of one kind, oneself knows the antibiotic (cephalosporin) for inhibiting Agrobacterium growth in, does not add the selection of vegetable transformantAgent (step 3: recovering step).Preferably, rataria is cultivated on having antibiotic but the not solid medium of selective agent, to eliminateAgrobacterium simultaneously provides convalescence for infected cell.Then, the rataria of inoculation is cultivated on the culture medium containing selective agent (glyphosate)And select the transformed calli (step 4: selection step) grown.Preferably, rataria is in the screening solid training for having selective agentSupport base (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30 g/L, N- (phosphine carboxymerhyl) glycine 0.25mol/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH 5.8) on cultivate, cause conversion cell selectionProperty growth.Then, callus regeneration is at plant (step 5: regeneration step), it is preferable that raw on the culture medium containing selective agentLong callus is cultivated on solid medium (MS differential medium and MS root media) with aftergrowth.
It screens obtained resistant calli and is transferred to the MS differential medium (MS salt 4.3g/L, MS vitamin, cheesePlain 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, N- (phosphine carboxymerhyl) glycine 0.125mol/L, plant gel3g/L, pH 5.8) on, differentiation is cultivated at 25 DEG C.It differentiates the seedling come and is transferred to the MS root media (MS salt 2.15g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH 5.8) on, at 25 DEG CCulture moves to hot-house culture to solid to about 10cm high.In the greenhouse, it is cultivated 16 hours at 28 DEG C daily, at 20 DEG CCulture 8 hours.
1.3, the identification and screening of transgenic event
332 separate transgenic T are produced altogether0Plant.
Pass through TaqManTMIt analyzes (referring to second embodiment) and detects regenerated transgenic corn plant with the presence or absence of EPSPSAnd pat gene, and characterize the copy number of tolerance glyphosate and glufosinate-ammonium strain.By screening, the event DBN9888 of having selected is excellentDifferent, there is single copy transgenosis, good glyphosate herbicide tolerance, glufosinate-ammonium herbicide tolerant and economical characterPerformance (referring to the 5th embodiment).
Second embodiment carries out transgenic corn events DBN9888 detection with TaqMan
Take the blade about 100mg of transgenic corn events DBN9888 as sample, with the DNeasy Plant of QiagenMaxi Kit extracts its genomic DNA, detects EPSPS gene and pat gene by Taqman fluorescence probe quantitative PCR methodCopy number.Simultaneously using wild-type corn plant as control, tested and analyzed according to the method described above.Experiment sets 3 repetitions, takesAverage value.
The specific method is as follows:
Step 11, the blade 100mg for taking transgenic corn events DBN9888, are ground into homogenate with liquid nitrogen in mortar, eachSample takes 3 repetitions；
Step 12, the genomic DNA that above-mentioned sample is extracted using the DNeasy Plant Mini Kit of Qiagen, specificallyMethod refers to its product description；
Step 13, the genomic DNA concentration that above-mentioned sample is measured with NanoDrop 2000 (Thermo Scientific)；
Step 14, the genomic DNA concentration of the above-mentioned sample of adjustment to same concentration value, the range of the concentration value is 80-100ng/μl；
Step 15, the copy number that sample is identified using Taqman fluorescence probe quantitative PCR method, by being copied known to identificationThe sample of shellfish number is as standard items, and using the sample of wild-type corn plant as control, 3 repetitions of each sample take it averageValue；Fluorescence quantification PCR primer and probe sequence are respectively:
Following primer and probe is used to detect EPSPS gene order:
Primer 1:CTGGAAGGCGAGGACGTCATCAATA is as shown in SEQ ID NO:16 in sequence table；
Primer 2: TGGCGGCATTGCCGAAATCGAG is as shown in SEQ ID NO:17 in sequence table；
Probe 1:ATGCAGGCGATGGGCGCCCGCATCCGTA is as shown in SEQ ID NO:18 in sequence table；
Following primer and probe is used to detect pat gene sequence:
Primer 3:CAGTTGAGATTAGGCCAGCTACAG is as shown in SEQ ID NO:19 in sequence table；
Primer 4:TTCACTGTAGACGTCTCAATGTAATGG is as shown in SEQ ID NO:20 in sequence table；
Probe 2:CAGCTGATATGGCCGCGGTTTGTG is as shown in SEQ ID NO:21 in sequence table；
PCR reaction system are as follows:
50 × the primer/probe mixture includes each 45 μ L of every kind of primer, 50 μ of probe of 100 μM of concentration of 1mM concentrationL and 860 μ lL1 × TE buffers, and at 4 DEG C, it is housed in amber tube.
PCR reaction condition are as follows:
Data are analyzed using SDS2.3 software (Applied Biosystems), obtain the transgenic corn events singly copiedDBN9888。
3rd embodiment, transgenic corn events DBN9888 detection
3.1, extracting genome DNA
DNA is extracted according to CTAB (cetyl trimethylammonium bromide) method routinely used: taking 2 grams of tender transgenosis beautifulAfter the blade of rice event DBN9888 is pulverized in liquid nitrogen, the DNA that 0.5mL is preheated in 65 DEG C of temperature is added and extracts CTABBuffer (20g/L CTAB, 1.4M NaCl, 100mM Tris-HCl, 20mM EDTA (ethylenediamine tetra-acetic acid), with NaOH tune pHTo 8.0), after mixing well, extracted 90 minutes in 65 DEG C of temperature；0.5 times of volume of phenol is added, 0.5 times of volume of chloroform overturns mixedIt is even；It is centrifuged 10 minutes under 12000rpm (revolutions per minute) revolving speed；2 times of volume dehydrated alcohols are added in Aspirate supernatant, soft to shakeDynamic centrifuge tube stands 30 minutes in 4 DEG C of temperature；It is centrifuged again under 12000rpm revolving speed 10 minutes；DNA is collected to tube bottom；Abandon supernatantLiquid, the ethyl alcohol for being 70% with 1mL mass concentration, washing precipitating；It is centrifuged 5 minutes under 12000rpm revolving speed；Vacuum is drained or superNet platform drying；DNA is precipitated and dissolved in suitable TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), is stored inUnder the conditions of -20 DEG C of temperature.
3.2, the analysis of flanking DNA sequence
Concentration mensuration is carried out to the DNA sample of said extracted, is located at the concentration of sample to be tested between 80-100ng/ μ L.With restriction enzyme BamH I, Xma I, Kpn I, Sac II (5 ' end analysis) and Spe I, Pst I, the Eco57I selected(3 ' end analysis) difference digestion genomic DNA.It is added that 26.5 μ L genomic DNAs, 0.5 μ L is above-mentioned selects in each digestion systemRestriction enzyme and 3 μ L enzyme cutting buffering liquids, digestion 1 hour.After to digestion, 70 μ L are added into digestion systemDehydrated alcohol, ice bath 30 minutes, revolving speed 12000rpm was centrifuged 7 minutes, abandoned supernatant, and 8.5 μ L distilled water (dd are added in drying laterH2O)、1μL 10X T4Buffer and 0.5 μ L T4Ligase is stayed overnight in 4 DEG C of temperature connections.It is carried out with a series of nested primersPCR amplification separates 5 ' and 3 ' transgenosis/genomic DNA.Specifically, 5 ' transgenosis of separation/genomic DNA primer combination includesSEQ ID NO:13, SEQ ID NO:26 as the first primer, SEQ ID NO:27, SEQ ID NO:28 as the second primer,SEQ ID NO:13 is as sequencing primer.Separating 3 ' transgenosis/genomic DNA primer combination includes SEQ ID NO:15, SEQID NO:29 is as the first primer, and SEQ ID NO:30, SEQ ID NO:31 are as the second primer, and SEQ ID NO:15 is as surveySequence primer, PCR reaction condition are as shown in table 3.
Amplicon obtained electrophoresis on 2.0% Ago-Gel then uses QIAquick to separate PCR reactantGel extracts kit (catalogue #_28704, Qiagen Inc., Valencia, CA) separates target fragment from agarose matrix.So(for example, ABI PrismTM 377, PE Biosystems, Foster City, CA) is sequenced to the PCR product of purifying afterwards and is dividedIt analyses (for example, DNASTAR sequence analysis software, DNASTAR Inc., Madison, WI).
5 ' and 3 ' flanking sequences and junction sequences are confirmed using standard pcr.5 ' flanking sequences and junction sequences can makeConfirmed with SEQ ID NO:8 or SEQ ID NO:12, combination S EQ ID NO:9, SEQ ID NO:13 or SEQ ID NO:26.SEQ ID NO:11 or SEQ ID NO:14, combination S EQ ID NO:10, SEQ ID can be used in 3 ' flanking sequences and junction sequencesNO:15 or SEQ ID NO:29 confirms.PCR reaction system and amplification condition are as shown in table 2 and table 3.Those skilled in the artIt will be understood that other primer sequences can also be used for confirmation flanking sequence and junction sequences.
The DNA sequencing of PCR product provides the DNA that can be used for designing other DNA moleculars, other described DNA moleculars are madeThe identification of the corn plant or seed from transgenic corn events DBN9888 is used for for primer and probe.
It was found that 1-693, the nucleotide displays in SEQ ID NO:5 are maize genomic sequence in transgenic corns thingThe right margin flank (5 ' flanking sequence) of part DBN9888 insetion sequence, 5472-5952, nucleotide in SEQ ID NO:5 are aobviousShow the left margin flank (3 ' flanking sequence) for being maize genomic sequence in transgenic corn events DBN9888 insetion sequence.5 ' junction sequences are listed in SEQ ID NO:1, and 3 ' junction sequences are listed in SEQ ID NO:2.
3.3, PCR zygosity determination
Junction sequence is relatively short polynucleotide molecule, is new DNA sequence dna, examines when in polynucleotide tests and analyzesIt is diagnostic for the DNA of transgenic corn events DBN9888 when measuring.Connecing in SEQ ID NO:1 and SEQ ID NO:2Close every side of insertion point and corn gene group DNA that sequence is transgenic corn events DBN9888 transgenic segment11 polynucleotides.Longer or shorter polynucleotides junction sequence can be selected from SEQ ID NO:3 or SEQ ID NO:4It selects.Junction sequence (5 ' the join domain SEQ ID join domain SEQ ID of NO:1 and 3 ' NO:2) is used as DNA probe or conductDNA primer molecule is useful in DNA detection method.Junction sequence SEQ ID NO:6 and SEQ ID NO:7 is also transgenosisNew DNA sequence dna in corn event DBN9888 can also be used as DNA probe or beautiful as DNA primer Molecular Detection transgenosisThe presence of rice event DBN9888DNA.The SEQ ID NO:6 (694-960, the nucleotide of SEQ ID NO:3) spansDBN10006 construct DNA sequence dna and pr35S promoter sequence, the SEQ ID NO:7 (nucleotide of SEQ ID NO:41-272) span tNos transcription terminator and DBN10006 construct DNA sequence dna.
In addition, amplicon is generated by using at least one primer from SEQ ID NO:3 or SEQ ID NO:4,The diagnostic amplicon of transgenic corn events DBN9888 is generated when the primer is in PCR method.
Specifically, PCR product is generated from 5 ' ends of transgene insert sequence, which is to turn base comprising deriving fromBecause in the genome of the vegetable material of corn event DBN9888 flank in the genomic DNA of 5 ' ends of T-DNA insetion sequenceA part.This PCR product includes SEQ ID NO:3.In order to carry out PCR amplification, design and flank are in transgene insert sequence5 ' ends genomic dna sequence hybridization primer 5 (SEQ ID NO:8) and the paired transgenosis tNos that is located at turnRecord the primer 6 (SEQ ID NO:9) of termination sequence.
PCR product is generated from 3 ' ends of transgene insert sequence, which includes to derive from transgenic corn eventsFlank is in a part of the genomic DNA of 3 ' ends of T-DNA insetion sequence in the genome of the vegetable material of DBN9888.ThisA PCR product includes SEQ ID NO:4.In order to carry out PCR amplification, design and flank are in 3 ' ends of transgene insert sequenceThe pr35S of primer 8 (the SEQ ID NO:11) and the paired 3 ' ends positioned at insert of genomic dna sequence hybridizationThe primer 7 (SEQ IDNO:10) of promoter sequence.
The DNA cloning condition illustrated in table 2 and table 3 can be used for above-mentioned PCR zygosity test to generate transgenic cornsThe diagnostic amplicon of event DBN9888.The detection of amplicon can be by using Stratagene as shown in table 3Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradien thermal cycleInstrument etc. carries out, or carries out by methods known to those skilled in the art with equipment.
Table 2,5 ' transgenic insertions/genome engaging zones identification PCR for transgenic corn events DBN9888Step and reaction mixture condition
Table 3, Perkin-Elmer9700 thermal cycler condition
It lightly mixes, if not having hot top on thermal cycler, 1-2 drop mineral can be added above each reaction solutionOil.Using following loop parameter (table 3) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJEngine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) orPCR is carried out on Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermal cycler.MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run under the mode of calculating.Cooling rate (ramp speed) is set as maximum value when 9700 thermal cycler of Perkin-Elmer is run.
The results showed that primer 5 and 6 (SEQ ID NO:8 and 9), when it is used in transgenic corn events DBN9888 baseWhen because in the PCR reaction of group DNA, the amplified production of 960bp segment is generated, when it is used in unconverted corn gene group DNA and non-When in the PCR reaction of DBN9888 corn gene group DNA, no segment is amplified；Primer 7 and 8 (SEQ ID NO:10 and 11),When it is used in the PCR reaction of transgenic corn events DBN9888 genomic DNA, the amplified production of 753bp segment is generated,When in the PCR reaction that it is used in unconverted corn gene group DNA and non-DBN9888 corn gene group DNA, expanded without segmentIncrease.
PCR zygosity determination can also be used in identification from transgenic corn events DBN9888 material be homozygote orIt is heterozygote.Primer 9 (SEQ ID NO:12), primer 10 (SEQ ID NO:13) and primer 11 (SEQ ID NO:14) are used forAmplified reaction is to generate the diagnostic amplicon of transgenic corn events DBN9888.The DNA cloning condition illustrated in table 4 and table 5It can be used for above-mentioned zygosity test to generate the diagnostic amplicon of transgenic corn events DBN9888.
Table 4, zygosity determination reaction solution
Table 5, zygosity determination Perkin-Elmer9700 thermal cycler condition
Using following loop parameter (table 5) Stratagene Robocycler (Stratagene, La Jolla, CA),MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA)Or it is carried out on Eppendorf Mastercycler Gradient (Eppendorf, Hamburg, Germany) thermal cyclerPCR.MJ Engine or Eppendorf Mastercycler Gradient thermal cycler should be run under the mode of calculating.Cooling rate (ramp speed) is set as maximum value when 9700 thermal cycler of Perkin-Elmer is run.
In the amplified reaction, the biological sample containing template DNA, which contains, diagnoses the sample transgenic corn eventDBN9888 there are the DNA of situation.Or reaction will generate two by the biological sample containing the DNA from Maize genomeA different DNA cloning, the DNA from Maize genome in transgenic corn events DBN9888 relative to existingThe corresponding allele of insertion DNA be heterozygosis.The two different amplicons, which will correspond to, derives from wild-type corn baseBecause group locus the first amplicon and diagnosis transgenic corn events DBN9888DNA there are the second amplicons of situation.OnlyGenerate the maize dna sample for corresponding to the single amplicon of the second amplicon for the description of heterozygous genes group, diagnosable determinationThe presence of sample transgenic corn event DBN9888, and the sample in rotaring gene corn plant DBN9888 by relative to depositingThe corresponding allele of insertion DNA be produced by homozygous corn seed.
It should be noted that the primer pair of transgenic corn events DBN9888 is used to transgenic corn eventsDBN9888 genomic DNA is diagnostic amplicon.These primer pairs include but is not limited to (the SEQ ID NO:8 of primer 5 and 6With 9) and primer 7 and 8 (SEQ ID NO:10 and 11), in the DNA cloning method.In addition, for expanding in cornOne control primer 12 and 13 (SEQ ID NO:22 and 23) of source gene is included, as in one of reaction conditionStandard.DNA extracting sample analysis to transgenic corn events DBN9888 should include a transgenic corn eventsThe assaypositive tissue DNA extract of DBN9888 compares, and a negative DNA from non-transgenic corn event DBN9888 is extractedObject control and a negative control without containing template maize dna extract.Other than these primer pairs, it can also use andFrom any primer pair of SEQ ID NO:3 or SEQ ID NO:4 or its complementary series, when they are used for DNA amplification reactionGenerate respectively for the tissue from transgenic event corn plant DBN9888 be it is diagnostic comprising SEQ ID NO:1 orThe amplicon of SEQ ID NO:2.The DNA cloning condition illustrated in table 2- table 5 is used for suitable primer pair to generateThe diagnostic amplicon of transgenic corn events DBN9888.It generates when being tested in DNA cloning method to transgenic corns thingPart DBN9888 be diagnostic amplicon, presumption contain corn plant or kind comprising transgenic corn events DBN9888The extract of sub- DNA, or from the product of transgenic corn events DBN9888, it is used as the template of amplification, to determineWith the presence or absence of transgenic corn events DBN9888.
Fourth embodiment carries out transgenic corn events DBN9888 detection by Southern blot hybridization
4.1, it is extracted for the DNA of Southern blot hybridization
Southern engram analysis is carried out using T4, T5 generation homozygous transformation event.Using mortar and pestle, in liquid nitrogen10g plant tissue is arrived in grinding about 5.In 12.5mL Extraction buffer A (0.2M Tris pH8.0,50mM EDTA, 0.25MNaCl, 0.1%v/v β-dredges base ethyl alcohol, 2.5%w/v Polyvinyl-pyrrolidone) in resuspension plant tissue, with 4000rpm fromThe heart 10 minutes (2755g).After discarding supernatant, 2.5mL Extraction buffer B (0.2M Tris pH 8.0,50mM EDTA,0.5M NaCl, 1%v/v β-dredges base ethyl alcohol, 2.5%w/v Polyvinyl-pyrrolidone, 3% flesh aminoacyl, 20% ethyl alcohol) in be resuspendedIt drifts along shallow lake, and is incubated 30 minutes at 37 DEG C.It is primary with asepsis ring mixing sample during incubation.After incubation, addition is isometricChloroform/isoamyl alcohol (24:1), by be inverted be gently mixed, with 4000rpm centrifugation 20 minutes.Water-bearing layer is collected, and is being addedAdd after 0.54 volume isopropanol with 4000rpm centrifugation 5 minutes to precipitate DNA.Supernatant is discarded, and is resuspended in 500 μ LTEFloating DNA precipitating.DNA and 1 μ L 30mg/mlLRNAase A is incubated 30 minutes in order to degrade any existing RNA at 37 DEG C,With 4000rpm centrifugation 5 minutes, and in the presence of 0.5 volume 7.5M ammonium acetate and 0.54 volume isopropanol, by with14000rpm is centrifuged 10 minutes precipitating DNA.After discarding supernatant, precipitating is washed with the ethyl alcohol that 500 μ L mass fractions are 70%, andAfter making it dry in 100 μ L TE resuspension.
4.2, enzymic digestion is limited
Utilize spectrophotometer or fluorometric quantification detection DNA concentration (utilizing 1 × TNE and Hoechst dyestuff).
In 100 μ L reaction systems, 5 μ g DNA are digested every time.Disappeared respectively with restriction enzyme Sac I and Hind IIIChange genomic DNA, the partial sequence of the EPSPS using on T-DNA and PAT is as probe.It is warm at a proper temperature for every kind of enzymeIt educates and is digested overnight object.Using SpeedVac (speed vacuum) rotation sample to reduce volume to 30 μ L.
4.3, gel electrophoresis
Bromophenol blue Loading Dye is added to each sample in the present embodiment 4.2, and by each sample pipetting volumeIt onto 0.7% Ago-Gel containing ethidium bromide, is separated by electrophoresis in TBE electrophoretic buffer, the electrophoresis coagulating under 20 voltsGlue is stayed overnight.
Gel is washed in 0.25M HCl 15 minutes so that DNA depurination, is then washed with water.It is miscellaneous to set Southern traceIt hands over as follows: placing 20 thick drying trace paper in disk, place 4 thin drying trace paper again thereon.In 0.4M NaOH1 thin trace paper is moistened in advance, and is placed on the pile, and then placement 1 moistens in advance in 0.4M NaOHHybond-N+ transfer membrane (Amersham Pharmacia Biotech, #RPN303B).Gel is seated in top, it is ensured that solidifyingThere is no bubble between glue and film.3 trace paper in addition impregnated in advance are placed on gel top, and are filled out with 0.4M NaOHFull buffer disk.With the wick connection gel stack and buffer disk being immersed in 0.4M NaOH in advance, DNA is transferred to filmOn.DNA transfer in about 4 hours is carried out at room temperature.After transfer, rinsed Hybond film 10 seconds in 2 × SSC, DNA passes through UVCrosslinking is in conjunction with film.
4.4, hybridize
It is prepared with the DNA sequence dna that PCR amplification is suitble to for probe.The DNA probe is SEQ ID NO:24 and SEQ IDNO:25, or it is homologous or complementary with above-mentioned Sequence.25ng DNA probe is boiled 5 minutes in 45 μ LTE, is put on iceIt sets 7 minutes, is then transferred into Rediprime II (Amersham Pharmacia Biotech, #RPN1633) test tube.ToRediprime test tube adds 5 μ l32P label dCTP after, 37 DEG C incubation probe 15 minutes.According to the manufacturer's instructions,It is centrifuged by micro- centrifugation G-50 pillar (Amersham Pharmacia Biotech, #27-5330-01), is not incorporated into removalDNTPs purifies the probe.Probe activity is measured using scintillation counter.
By in 65 DEG C of Church prehybridization solution (500mM Na with 20mL pre-heating3P04, 1mM EDTA, 7%SDS,1%BSA) moisten the Hybond film 30 minutes, the prehybridization Hybond film.It boils the probe of label 5 minutes, and puts on iceIt sets 10 minutes.Appropriate probe (every 1mL pre-hybridization buffer 1,000,000 times countings) is added to pre-hybridization buffer, overnight at 65 DEG CHybridized.Second day, hybridization buffer is discarded, with (the 40mM Na of 20mLChurch rinse solution 13P04, 1mM EDTA, 5%SDS, 0.5%BSA) rinsing after, at 65 DEG C, wash film 20 minutes in 150mL Church rinse solution 1.It is rinsed with Church(the 40mM Na of solution 23P04, 1mM EDTA, 1%SDS) and it repeats the process 2 times.The film is exposed to phosphorus screen or X-ray to detectThe position that probe combines.
Include three kinds of control samples on each Southern: (1) DNA of the segregant from negative (unconverted),For identify it is any can be with element-specific probe hybridization endogenous corn sequence；(2) DNA from negative segregant, whereinThe DBN10006 of Hind III- digestion is introduced, amount is based on probe length and is equivalent to a copy number, beautiful in detection with explanationWhen individual gene in rice genome copies, the sensitivity of the experiment；(3) copy number is equivalent to based on probe lengthThe DBN10006 plasmid of Hind III- digestion, the sensitivity as the positive control hybridized and for illustrating experiment.
The evidence that hybridization data provides confirmation supports TaqManTMPCR analysis, i.e. corn plant DBN9888 contain EPSPSIt is copied with the list of pat gene.Using the EPSPS probe, Sac I and Hind III enzymatic hydrolysis generate respectively size about 7.8kb andThe single band of 12kb；Using the PAT probe, Sac I and Hind III enzymatic hydrolysis generate size about 5.5kb's and 4.8kb respectivelySingle band.This shows that each copy of EPSPS and PAT is present in corn transformation event DBN9888.
The herbicide tolerant detection of 5th embodiment, event
This test selects agriculture up to herbicide (41% glyphosate-isopropylammonium aqua) and protects examination up to herbicide (effective component18% glufosinate-ammonium) it is sprayed.Using RANDOMIZED BLOCK DESIGN, 3 repetitions.Plot area is 15m2(5m × 3m), line-spacing60cm, spacing in the rows 25cm, conventional cultivation management have the wide isolation strip of 1m between cell.Transgenic corn events DBN9888 is distinguishedIt carries out following 3 kinds of processing: 1) not spraying；2) agriculture is sprayed up to herbicide, then in V8 in the V3 leaf phase by 1680g a.e./ha dosagePhase is sprayed agriculture by same dose up to herbicide again；3) guarantor's examination is sprayed up to (Basta) in the V3 leaf phase by 800g a.i./ha dosageThen herbicide is sprayed guarantor's examination up to (Basta) herbicide again in the V8 phase by same dose.It should be noted that different contentThe form of equivalent glyphosate is converted into the glyphosate herbicidal of dosage form and the glufosinate-ammonium solution of various concentration is converted intoEquivalent effective component glufosinate-ammonium is stated to be suitable for draw a conclusion.
The 1 week and 2 weeks investigation symptom of chemical damage after medication respectively, and harvest when measure cell corn yield.Phytotoxicity diseaseShape classification is as shown in table 6.The index for using the aggrieved rate of herbicide as the herbicide tolerant of evaluation transformation event is specifically removedThe careless aggrieved rate of agent (%)=∑ (aggrieved strain number × number of levels at the same level)/(total strain number × highest level)；The wherein aggrieved rate of herbicideIncluding the aggrieved rate of glyphosate and the aggrieved rate of glufosinate-ammonium, the aggrieved rate of herbicide is the medicine according to 2 weeks after glyphosate or glufosinate-ammonium processingEvil investigation result and determination.The corn yield of each cell is the niblet total output (weight) for weighing 3 rows among each cell,Volume variance between different disposal is measured in the form of yield percentage, and yield percentage (%)=spraying yield/does not sprayApply yield.The results are shown in Table 7 for result and corn yield of the transgenic corn events DBN9888 to herbicide tolerant.
Table 6, herbicide are to the grade scale of corn phytotoxicity degree
| Phytotoxicity rank | Symptom description |
| 1 | Growth is normal, without any damage symptoms |
| 2 | Slight phytotoxicity, phytotoxicity are less than 10% |
| 3 | Medium phytotoxicity can restore later, not influence yield |
| 4 | Phytotoxicity is heavier, it is difficult to restore, cause the underproduction |
| 5 | Phytotoxicity is serious, cannot restore, and causes the obvious underproduction or total crop failure |
Table 7, transgenic corn events DBN9888 are to the result and corn yield result of herbicide tolerant
As a result illustrate, in terms of herbicide (glyphosate and glufosinate-ammonium) aggrieved rate: 1) transgenic corn events DBN9888 existsAggrieved rate is essentially 0 under glyphosate herbicidal (1680g a.e./ha) processing；Transgenic corn events DBN9888 is in glufosinate-ammoniumAggrieved rate is also essentially 0 under herbicide (800g a.i./ha) processing；Transgenic corn events DBN9888 has good as a result,Herbicide (glyphosate and glufosinate-ammonium) tolerance.
In terms of yield: transgenic corn events DBN9888 do not spray, glyphosate herbicidal (1680g a.e./ha)Handling lower yield with 3 kinds of glufosinate-ammonium herbicide (800g a.i./ha) does not have notable difference；After herbicide spraying, transgenosis is beautifulThe yield of rice event DBN9888 does not reduce substantially, and it is good to further demonstrate that transgenic corn events DBN9888 has as a result,Herbicide (glyphosate and glufosinate-ammonium) tolerance.
Sixth embodiment
Such as agricultural product or commodity can be produced by transgenic corn events DBN9888.If in the agricultural product or commodityIn detect enough expression quantity, the agricultural product or commodity are expected containing can diagnose transgenic corn events DBN9888 materialExpect the nucleotide sequence present in the agricultural product or commodity.The agricultural product or commodity include but is not limited to corn oil, jadeRice coarse powder, maize flour, corn gluten, corn-dodger, cornstarch and will be as food source for any other of animal consumptionFood or additionally as the ingredient in swelling agent or make-up composition for cosmetic use etc..Based on probe or primer pairNucleic acid detection method and/or kit can be developed to detect such as SEQ ID NO:1 or SEQ ID NO:2 in biological sampleShown in transgenic corn events DBN9888 nucleotide sequence, wherein probe sequence or primer sequence are selected from such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, sequence shown in SEQ ID NO:4 and SEQ ID NO:5, to diagnose transgenosis jadeThe presence of rice event DBN9888.
In conclusion transgenic corn events DBN9888 of the present invention has glyphosate herbicidal and glufosinate-ammonium herbicidePreferable tolerance, on yield without influence, and whether detection method can quickly and accurately be identified in biological sample comprising turning baseBecause of the DNA molecular of corn event DBN9888.
Seed corresponding to transgenic corn events DBN9888 is deposited in China Microbiological bacterium on December 24th, 2014Kind preservation administration committee common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, inInstitute of microbiology of the academy of sciences of state, postcode 100101), classification naming: corn (Zea mays), deposit number CGMCCNo.10216.Preserved material will be 30 years in depository's preservation.
It should be noted last that the above examples are only used to illustrate the technical scheme of the present invention and are not limiting, although ginsengIt is described the invention in detail according to preferred embodiment, those skilled in the art should understand that, it can be to the present inventionTechnical solution be modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.