Summary of the invention
The object of the present invention is to provide a kind of for detecting the nucleic acid sequence and its detection method of corn plant DBN9936,Transgenic corn events DBN9936 with preferable resistance and has preferable tolerance to glyphosate herbicidal to insect, andDetection method can quickly and accurately identify whether the DNA comprising specific transgenic corn events DBN9936 divides in biological sampleSon.
To achieve the above object, the present invention provides a kind of nucleic acid sequence, in SEQ ID NO:3 or its complementary series at leastAt least 11 continuous nucleotide in 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 DBN9936 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 DBN9936.The SEQ ID NO:2Or its complementary series is to be located near insertion junction in transgenic corn events DBN9936 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 DBN9936 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.Using DNA primer in DNA cloning methodThe amplified production of generation be when including the amplified production of SEQ ID NO:1 can diagnose transgenic corn events DBN9936 or itsThe presence of offspring.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 DBN9936 in insertion sequence5 ' ends of column are located at the sequence that a length near insertion junction is 1001 nucleotide, the SEQ ID NO:3Or its complementary series by 832 nucleotide corn flanking genomic DNA sequence (the nucleotide 1-832 of SEQ ID NO:3), 77The tNos of DBN10124 construct DNA sequence dna (the nucleotide 833-909 of SEQ ID NO:3) and 92 nucleotide of a nucleotide3 ' the end DNA sequences (the nucleotide 910-1001 of SEQ ID NO:3) of terminator form, comprising the SEQ ID NO:3 orIts complementary series can be accredited as the presence of transgenic corn events DBN9936.
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 DBN9936 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 DBN9936Enter the sequence that a length near junction is 1204 nucleotide, the SEQ ID NO:4 or its complementary series are by 38The DBN10124 structure of the t35S transcription terminator sequences (the nucleotide 1-38 of SEQ ID NO:4) of a nucleotide, 152 nucleotideBuild the corn integration site flanking genomes of body DNA sequence dna (the nucleotide 39-190 of SEQ ID NO:4) and 1014 nucleotideDNA sequence dna (the nucleotide 191-1204 of SEQ ID NO:4) composition includes the SEQ ID NO:4 or its complementary seriesIt is accredited as the presence of transgenic corn events DBN9936.
The SEQ ID NO:5 or its complementary series are that the length of characterization transgenic corn events DBN9936 is 9215The 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 DBN9936.
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 DBN9936 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 DBN9936 or its offspring.
To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event DBN9936 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 a kind of test sample transgenic corn event DBN9936'sMethod existing for DNA, 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 includes 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 the DNA of test sample transgenic corn event DBN9936 a kind ofExisting method, 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 insect-resistant and/or herbicide 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 include 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 DBN9936 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 includes 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 insectThe nucleic acid sequence of resistance Cry1Ab albumen, the nucleic acid sequence for encoding glyphosate herbicide tolerance EPSPS albumen and specific regionNucleic acid sequence, the nucleic acid sequence of the specific region includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQSequence shown in ID NO:7.
To achieve the above object, the present invention also provides a kind of protection corn plants from the method for insect infestations, includingAt least one transgenic corn plant cell is provided in the diet of target insect, the transgenic corn plant cell is in its geneAll comprising being selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ in groupAt least one of sequence shown in ID NO:6 and SEQ ID NO:7 nucleic acid sequence, the target for the transgenic corn plant cell of ingestingInsect is suppressed the corn plant of further ingesting.
To achieve the above object, protect corn plant from the damage as caused by herbicide the present invention also provides a kind ofMethod plants the big Tanaka of at least one rotaring gene corn plant including that will be applied to containing effective dose glyphosate herbicidal,The rotaring gene corn plant includes selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ in its genomeAt least one of ID NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence, described turnGene corn plant has the tolerance to glyphosate herbicidal.
To achieve the above object, the present invention also provides it is a kind of control maize planting plant big Tanaka weeds method,Plant the big Tanaka of at least one rotaring gene corn plant including that will be applied to containing effective dose glyphosate herbicidal, described turnGene corn plant includes selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID in its genomeAt least one of NO:4, SEQ ID NO:5, sequence shown in SEQ ID NO:6 and SEQ ID NO:7 nucleic acid sequence, it is described to turn baseBecause corn plant has the tolerance to glyphosate herbicidal.
To achieve the above object, the present invention also provides a kind of method of culture corn plant resistant to insect,Include:
An at least corn seed is planted, includes coding insect-resistant Cry1Ab albumen 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 described in target insect infestations harvests compared with the plant of other nucleic acid sequences for not having specific regionThe plant of plant injury with decrease；
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 the corns that a kind of culture has tolerance to glyphosate herbicidalThe method of plant, comprising:
An at least corn seed is planted, includes coding glyphosate herbicide tolerance in the genome of the corn seedThe nucleic acid sequence of EPSPS albumen and the nucleic acid sequence of specific region；
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, a kind of resistant to insect the present invention also provides culture and tolerance Gyphosate herbiciceThe method of the corn plant of agent, comprising:
An at least corn seed is planted, includes coding insect-resistant Cry1Ab albumen in the genome of the corn seedNucleic acid sequence, encode glyphosate herbicide tolerance EPSPS albumen nucleic 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, and the plant pair insect with the plant injury weakened is taken the photographFood damage is also resistant；
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 method for generating the plant resistant to insect,Nucleic acid sequence from coding insect-resistant Cry1Ab albumen to the genome of the plant and specific region including introducingThe nucleic acid sequence of nucleic acid sequence, 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.
Specifically, the method for generating the plant resistant to insect includes:
It will be to resistant the first parental maize plant of transgenic corn events DBN9936 of insect and lacking insect-resistantThe second parental maize plant sexual hybridization, to generate a large amount of progeny plants；
The progeny plant described in target insect infestations；
It selects to have compared with the plant of other nucleic acid sequences for not having specific region described in the plant injury weakenedProgeny plant；
The transgenic corn events DBN9936 include in its genome 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 in sequence shown in SEQ ID NO:6 and SEQ ID NO:7Kind nucleic acid sequence.
To achieve the above object, the present invention also provides a kind of corns for generating and having tolerance to glyphosate herbicidalThe method of plant, the nucleic acid sequence including introducing coding glyphosate tolerant EPSPS albumen into the genome of the plantThe nucleic acid sequence of the nucleic acid sequence of column and specific region, the specific region is selected from SEQ ID NO:1, SEQ ID NO:2, SEQAt 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.
Specifically, the method for generating the plant for having tolerance to glyphosate herbicidal includes:
By first parental maize plant of transgenic corn events DBN9936 to glyphosate herbicidal with tolerance and lackSecond parental maize plant sexual hybridization of few 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；
The transgenic corn events DBN9936 include in its genome 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 in sequence shown in SEQ ID NO:6 and SEQ ID NO:7Kind nucleic acid sequence.
To achieve the above object, and tolerance glyphosate herbicidal resistant to insect is generated the present invention also provides a kind ofThe method of the plant of application, comprising:
By the first parental maize plant of transgenic corn events DBN9936 of glyphosate tolerance and insect-resistant and lack grassSecond parental maize plant sexual hybridization of sweet phosphine tolerance and/or insect-resistant, to generate a large amount of progeny plants；
The progeny plant is handled with glyphosate；
The progeny plant of selection tolerance glyphosate, is resistant to feeding damage of the progeny plant to insect of glyphosateAlso resistant；
The transgenic corn events DBN9936 include in its genome 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 in sequence shown in SEQ ID NO:6 and SEQ ID NO:7Kind nucleic acid sequence.
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 composition of acid, the composition are corn flour, maize flour, corn oil, corn silk or cornstarch.
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.
In nucleic acid sequence and its detection method of the present invention for detecting corn plant, defined below and method can be moreThe present invention is defined well and those skilled in the art is instructed to implement the present invention, it is unless otherwise mentioned, general according to this fieldLead to the conventional usage of technical staff to understand term.
" corn " refers to maize (Zea mays), and all plant varieties including that can mate with corn,Including field corn kind.
The "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 DBN9936 and its offspring, the transgenic corn eventsDBN9936 is corn plant DBN9936 comprising the Plants and Seeds of transgenic corn events DBN9936 and its plant are thinBorn of the same parents or its renewable part, the plant part of the transgenic corn events DBN9936, including but not limited to cell, pollen, embryoPearl, flower, bud, root, stem, silk, inflorescence, ear fringe, leaf and the product from corn plant DBN9936, such as corn flour, cornFace, corn oil, corn pulp, corn silk, cornstarch and the biomass for staying in corn crop field.
Transgenic corn events DBN9936 of the present invention contains a DNA construct, when it is expressed in plant cellWhen, the transgenic corn events DBN9936 obtains the resistance to insect and the tolerance to glyphosate herbicidal.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 are operably connected the nucleic acid sequence of Cry1Ab albumen, describedThe nucleic acid sequence of Cry1Ab albumen is mainly resistant to lepidopterous insects.Second expression cassette includes for expressing in plantSuitable promoter and suitable polyadenylation signal sequence, the promoter, which is operably connected, encodes 5- enol-The nucleic acid sequence of the gene of pyruvoyl shikimic acid -3- phosphate synthase (EPSPS), the EPSPS albumen has glyphosate herbicidalThere is tolerance.Further, the promoter can be the suitable promoter that separates from plant, including composing type, induction type and/Or tissue-specific promoter, the suitable promoter include but is not limited to, cauliflower mosaic virus (CaMV) 35S promoter,Figwort mosaic virus (FMV) 35S promoter, ubiquitin protein (Ubiquitin) promoter, actin (Actin) promoter, soilEarth Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) promoter, octopine synthase (OCS)Promoter, Cestrum (Cestrum) yellow leaf curl virus promoter, patatin (Patatin) promoter,Ribulose-1,5-bisphosphate, 5- diphosphonic acid carboxylase/oxygenase (RuBisCO) promoter, glutathione S-transferase (GST) promoter, E9Promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD startingSon and Arabidopsis (Arabidopsis thaliana) 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 protease-inhibitor Ⅱ (PIN II)The polyadenylation signal sequence of gene and the polyadenylation signal for deriving from alpha-tubulin (α-tubulin) geneSequence.
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 Cry1Ab albumen and/or EPSPS Protein transport to extracellular or intracellular specific organelle or compartment, for example, sharpChloroplaset is targeted with encoding chloroplast transit peptide sequence, or utilizes ' KDEL ' to retain sequence and targets endoplasmic reticulum.
The Cry1Ab gene can be from thuringiensis (Bacillus thuringiensis, abbreviation Bt)Isolated, and the nucleotide sequence of Cry1Ab gene can be changed by optimization codon or in other ways, to reachThe stability of transcript and the purpose of utilizability into increase transformed cells.
" Lepidoptera ", scientific name Lepidoptera, including moth, two class insect of butterfly are one of agriculture and forestry injurious insect at mostMesh, such as corn borer, bollworm, east armyworm, 2 committee noctuid insect, dichocrocis punctiferalis.
5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) gene can be from soil AgrobacteriumIt is isolated in (Agrobacterium tumefaciens sp.) CP4 bacterial strain, and can by optimization codon orIn other ways change coding EPSPS gene polynucleotides, with reach increase transformed cells in transcript stability and canThe purpose of usability.5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) gene can also be used as selective markRemember gene.
" 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 transgenic corn events DBN9936, will control the weeds in the fieldGrowth, and the growth or yield of the vegetable material from transgenic corn events DBN9936 are not influenced.
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 as obtained from converting plant cell with heterologous DNA construct, that is, includes at least oneExpression of nucleic acid box containing target gene is inserted into Plant Genome to generate plant population by transgene method, thenThe raw plant population, and selection have the specific plant of insertion specific gene group site feature.Term " event " refers to including differentThe original transformant of source 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 individual, even if after be returned repeatedly with backcross parent, from transformantThe insertion DNA and flanking genomic dna of parent exists in the same chromosome location in filial generation.Term " event " also refers toDNA sequence dna from original transformant, the DNA sequence dna include insertion DNA and with the close adjacent flanking genomes of insertion DNASequence, which, which is expected, is transferred in filial generation, the filial generation by containing insertion DNA parental department (such as original transformant andIt 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 receives and includesThe insertion DNA of target 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.
Cultivate transgenic corn events resistant to lepidopterous insects and that there is tolerance to glyphosate herbicidalDBN9936 passes through following steps: making the first parental corn plants and the second parental corn plants sexual hybridization first, to produceGiven birth to the first generation progeny plant of multiplicity, first parental corn plants by cultivation transgenic corn event DBN9936 andThe corn plant of its offspring forms, and transgenic corn events DBN9936 and its offspring are of the invention to squama wing by utilizingMesh insect it is resistant and to glyphosate herbicidal have tolerance expression cassette convert obtained from, the second parental maizePlant lacks to the resistance of lepidopterous insects and/or has tolerance to glyphosate herbicidal；Then it selects to lepidopterous insectsInvasion it is resistant and/or to glyphosate herbicidal have tolerance progeny plant, can cultivate to lepidopterous insectsCorn plant resistant and that there is tolerance to glyphosate herbicidal.These steps, which may further include, makes Lepidoptera elder brotherThe progeny plant and the second parental corn plants or third parental corn plants of worm resistance and/or glyphosate tolerant are returnedIt hands over, then by being applied or by molecular marked compound relevant to character with lepidopteran insect infestation, glyphosate herbicidal (as wrappedThe DNA molecular of bond site that the 5 ' ends and 3 ' ends of insetion sequence identify in DBN9936 containing transgenic corn events) identificationFilial generation is selected, to generate corn plant to lepidopterous insects resistant and that there is tolerance to glyphosate herbicidal.
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.
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 DBN9936 genome, no matter the geneGroup DNA be also be derived from from transgenic corn events DBN9936 or seed transgenic corn events DBN9936 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 DBN9936, 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 DBN9936.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 inventionDBN9936 is generated by sexual hybridization mode, or whether the corn sample acquired from field includes transgenic corn eventsWhether DBN9936 or corn extract, such as coarse powder, powder or oil include transgenic corn events DBN9936, 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 DBN9936 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 DBN9936.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 genomic DNA of amplifiable 22kb and the phage DNA of 42kb.Other DNA cloning sides of these methods and this fieldMethod can be used for the present invention.The exogenous DNA array of insertion and flanking DNA sequence from transgenic corn events DBN9936 canWith by being expanded using genome of the provided primer sequence to transgenic corn events DBN9936, to PCR after amplificationAmplicon or the DNA of clone carry out the DNA sequencing of standard.
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 DBN9936 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 expected base specific markers.Result can be obtained by fluorescence or ELISA class method.Signal represent insertion/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 areaPCR product (in insetion sequence and adjacent flanking genomic sequence in respectively use a primer) hybridized, then andArchaeal dna polymerase, ATP, sulfonyl enzyme, luciferase, apyrase, adenosine -5 '-phosphorus sulfate and luciferin oneIt rises and is incubated.It is separately added into dNTPs, measures the optical signal of generation.Optical signal represents the presence of insertion/flanking sequence,Illustrate amplification, hybridization and single base or polybase base extension is 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 suitable technology for deriving from the vegetable material of transgenic corn events DBN9936It can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization.Particularly, the suitable technology includesProbe and sample are incubated, is washed to remove whether unbonded probe and detection probe have hybridized.The detection method takesThe certainly type of the label appended by probe, for example, can detecte radiolabeled probe by X-ray exposure and imaging, or logicalIt crosses substrate conversion and realizes that color change can detecte the probe of enzyme label.
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 moleculeClose 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 DBN9936 in sample, can be withFor cultivating the corn plant of the DNA containing transgenic corn events DBN9936.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: being located at the corn of 5 ' end of transgene insert sequenceDBN9936 flanking genomes region, a part of insetion sequence of the left boundary area (LB) from Agrobacterium, first expressionBox is operably connected to jade by the cauliflower mosaic virus 35 S promoter (pr35S) of the tandem sequence repeats containing enhancer regionOn rice heat shock 70k Da albumen introne (iZmHSP70), it is operably connected to the insect-resistant of bacillus thuringiensisOn Cry1Ab albumen (cCry1Ab), and it is operably connected on the transcription terminator (tNos) of nopaline synthase and forms, theTwo expression cassettes are operably connected to arabidopsis EPSPS chloroplast transit by 1 promoter of rice actin (prOsAct1)On peptide (spAtCTP2), the 5- enol-pyrovyl for being operably connected to the glyphosate tolerant of Agrobacterium CP4 bacterial strain is bigOn oxalic acid -3- phosphate synthase (cEPSPS), and it is operably connected on cauliflower mosaic virus 35S terminator (t35S) and groupAt, a part of insetion sequence in the right side boundary region (RB) from Agrobacterium, and it is located at 3 ' end of transgene insert sequenceCorn plant DBN9936 flanking genomes region (SEQ ID NO:5).In DNA cloning method, the DNA as primer dividesSon can be any part from transgenic corn events DBN9936 transgenic insetion sequence, be also possible to derive fromAny part in the region of DNA domain of flank Maize genome in transgenic corn events DBN9936.
Transgenic corn events DBN9936 can be combined with other transgenic maize varieties, such as herbicide is (such as careless ammoniumPhosphine, dicamba etc.) tolerance corn, or carry the transgenic maize varieties of other anti insect genes.All these differences turn baseBecause of the various combinations of event, the breeding together with transgenic corn events DBN9936 of the invention can provide and resist a variety of insect pests simultaneouslyResist the improvement hybrid transgenic corn variety of a variety of herbicides.These kinds turn base compared to non-transgenic kind and unisexuality shapeBecause kind can show the superior features such as yield promotion.
The present invention provides a kind of for detecting the nucleic acid sequence and its detection method of corn plant, transgenic corn eventsDBN9936's is resistant to the feeding damage of lepidoptera pest, and is resistant to the plant of the agriculture herbicide containing glyphosateToxic effect.The Cry1Ab albumen of the plant expression bacillus thuringiensis of the dual character, provides to LepidopteraThe resistance of pest (such as Ostrinia furnacalis) feeding damage, and express the 5- enol-of the glyphosate resistance of Agrobacterium strains CP4Pyruvoyl shikimic acid -3- phosphate synthase (EPSPS) albumen assigns plant to the tolerance of glyphosate.Dual character corn toolIt has the following advantages: 1) being damaged from the economy as caused by lepidoptera pest (such as Ostrinia furnacalis, east armyworm and dichocrocis punctiferalis etc.)It loses, Ostrinia furnacalis, east armyworm and dichocrocis punctiferalis etc. are the primary pests of corn-growing regions；2) apply the agricultural containing glyphosate to removeCareless agent is used for the ability that broad-spectrum weeding controls to corn crop；3) corn yield does not reduce.In addition, coding insect-resistant and grassThe gene linkage of sweet phosphine tolerance trait is present in transgenic corn events DBN9936 genome in same DNA sectionTerm single gene seat on, this point provide the breeding efficiency of enhancing and make it possible to be tracked with molecular labeling reproductive population andTransgenic insert in its filial generation.SEQ ID NO:1 or its complementary series, SEQ ID in detection method simultaneouslyNO:2 or its complementary series, SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series can be used asDNA primer or probe to generate the amplified production for being diagnosed as transgenic corn events DBN9936 or its offspring, and can quickly,Accurately, the stable presence for identifying the vegetable material from transgenic corn events DBN9936.
BRIEF DESCRIPTION OF THE SEQUENCES
The insertion point and Maize genome of 5 ' transgenic fragments in SEQ ID NO:1 transgenic corn events DBN993611 nucleotide of every side of DNA；
The insertion point and Maize genome of 3 ' transgenic fragments in SEQ ID NO:2 transgenic corn events DBN993611 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 DBN9936A neighbouring length is the sequence of 1001 nucleotide；
It is located at insertion junction in 3 ' ends of insetion sequence in SEQ ID NO:4 transgenic corn events DBN9936A neighbouring length is the sequence of 1204 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 DBN10124 construct DNA sequence dna andTNos transcription terminator；
SEQ ID NO:7 is located at the sequence inside SEQ ID NO:4, spans t35S transcription terminator and DBN10124Construct 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 SEQ ID NO:125 ' flanking genomic sequence；
The primer of SEQ ID NO:13 and SEQ ID NO:12 pairing being located on T-DNA；
Primer on SEQ ID NO:143 ' flanking genomic sequence can detecte with SEQ ID NO:12 pairing and turn baseBecause being 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:16Taqman detection Cry1Ab；
The primer 2 of SEQ ID NO:17Taqman detection Cry1Ab；
The probe 1 of SEQ ID NO:18Taqman detection Cry1Ab；
The primer 3 of SEQ ID NO:19Taqman detection EPSPS；
The primer 4 of SEQ ID NO:20Taqman detection EPSPS；
The probe 2 of SEQ ID NO:21Taqman detection EPSPS；
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 Cry1Ab in SEQ ID NO:24Southern hybridization check；
The probe of EPSPS in SEQ ID NO:25Southern 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
Below by specific embodiment further illustrate the present invention for detect corn plant DBN9936 nucleic acid sequence andThe technical solution of its detection method.
First embodiment, clone and conversion
1.1, carrier cloning
Recombinant expression carrier DBN10124 (as shown in Figure 2) is constructed using the gene clone technology of standard.The carrierDBN10124 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 maize Heat Shock 70kDa albumen introne(iZmHSP70) it on, is operably connected on the Cry1Ab albumen (cCry1Ab) of the insect-resistant of bacillus thuringiensis, andIt is operably connected on the transcription terminator (tNos) of nopaline synthase and forms；Second expression cassette is by rice actin1 promoter (prOsAct1) is operably connected on arabidopsis EPSPS chloroplast transit peptides (spAtCTP2), operationallyIt is connected to the 5- enol-pyrovyl shikimic acid -3- phosphate synthase (cEPSPS) of the glyphosate tolerant of Agrobacterium CP4 bacterial strainOn, and be operably connected on cauliflower mosaic virus 35S terminator (t35S) and form.
The carrier DBN10124 is transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA with liquid nitrogen method；Cat.No:18313-015 in), and with 5- enol-pyrovyl shikimic acid -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 DBN10124 of building is transferred in maize chromosome group,To generate transgenic corn events DBN9936.
For the corn transformation of mediated by agriculture bacillus, briefly, immature rataria is separated from corn, is suspended with AgrobacteriumLiquid contacts rataria, and wherein Agrobacterium can transmit the nucleotide sequence of the nucleotide sequence of Cry1Ab gene and EPSPS geneTo at least one cell (step 1: infecting step) of one of rataria, in this step, rataria preferably immerses Agrobacterium suspensionLiquid (OD660=0.4-0.6 infects culture medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, PortugalGrape sugar 36g/L, acetosyringone (AS) 40mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, pH 5.3)) in startingInoculation.Rataria and Agrobacterium co-culture one period (3 days) (step 2: co-culturing step).Preferably, rataria is infecting stepAfterwards in solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetyl fourthKetone musk (AS) 100mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH 5.8) on cultivate.It trains altogether hereinAfter the stage of supporting, there can be " recovery " step of a selectivity.In " recovery " step, recovery media (MS salt 4.3g/L, MSVitamin, 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 containing selective agent (N- (phosphine carboxymerhyl) glycine)It is cultivated on culture medium and selects the transformed calli (step 4: selection step) grown.Preferably, rataria is having selective agentScreening solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, N- (phosphine carboxymerhyl) sweet ammoniaSour 0.25mol/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH 5.8) on cultivate, cause to convertCell selective growth.Then, callus regeneration is at plant (step 5: regeneration step), it is preferable that containing selective agentThe callus grown on culture medium is cultivated on solid medium (MS differential medium and MS root media) to regenerate and plantObject.
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
770 separate transgenic T are generated altogether0Single plant.
Pass through TaqManTMIt analyzes (referring to second embodiment) and detects regenerated transgenic corn plant with the presence or absence of Cry1AbWith EPSPS gene, and the copy number of insect-resistant and glyphosate herbicide tolerance strain is characterized.According to the copy of target geneSeveral, good insect-resistant, glyphosate herbicide tolerance and Agronomic (are implemented referring to the 5th embodiment and the 6thExample), by screening, the event DBN9936 of having selected be it is excellent, have single copy transgenosis, good insect-resistant, grass sweetPhosphine herbicide tolerant and Agronomic (participating in the 5th embodiment and sixth embodiment).
Second embodiment carries out transgenic corn events DBN9936 detection with TaqMan
Take the blade about 100mg of transgenic corn events DBN9936 as sample, with the DNeasy Plant of QiagenMaxi Kit extracts its genomic DNA, and the copy of Cry1Ab and EPSPS is detected by Taqman fluorescence probe quantitative PCR methodNumber.Simultaneously using wild-type corn plant as control, tested and analyzed according to the method described above.Experiment sets 3 repetitions, is averagedValue.
The specific method is as follows:
Step 11, the blade 100mg for taking transgenic corn events DBN9936, 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 Cry1Ab gene order:
Primer 1:CGAACTACGACTCCCGCAC is as shown in SEQ ID NO:16 in sequence table；
Primer 2: GTAGATTTCGCGGGTCAGTTG is as shown in SEQ ID NO:17 in sequence table；
Probe 1:CTACCCGATCCGCACCGTGTCC is as shown in SEQ ID NO:18 in sequence table；
Following primer and probe is used to detect EPSPS gene order:
Primer 3:CTGGAAGGCGAGGACGTCATCAATA is as shown in SEQ ID NO:19 in sequence table；
Primer 4:TGGCGGCATTGCCGAAATCGAG is as shown in SEQ ID NO:20 in sequence table；
Probe 2:ATGCAGGCGATGGGCGCCCGCATCCGTA 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 L 1 × 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 copiedDBN9936。
3rd embodiment, transgenic corn events DBN9936 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 DBN9936 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, in 65 DEG C of extracting 90min of temperature；0.5 times of volume of phenol is added, 0.5 times of volume of chloroform overturns mixedIt is even；10min is centrifuged under 12000rpm (revolutions per minute) revolving speed；2 times of volume dehydrated alcohols are added in Aspirate supernatant, soft to shakeDynamic centrifuge tube, in 4 DEG C of standing 30min of temperature；It is centrifuged 10min again under 12000rpm revolving speed；DNA is collected to tube bottom；Supernatant is abandoned,The ethyl alcohol for being 70% with 1mL mass concentration, washing precipitating；5min is centrifuged under 12000rpm revolving speed；Vacuum is drained or in super-clean benchDrying；DNA is precipitated and dissolved in suitable TE buffer (10mM Tris-HCl, 1mM EDTA, pH 8.0), is stored in temperature-Under the conditions of 20 DEG C.
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 Sac I, Kpn I, Xma I, Nhe I (5 ' end analysis) and Spe I, Pst I, the BssH II selected(3 ' end analysis) difference digestion genomic DNA.26.5 μ L genomic DNAs are added in each digestion system, 0.5 μ L is above-mentioned to be selectedRestriction enzyme and 3 μ L enzyme cutting buffering liquids, digestion 1 hour.After to digestion, be added into digestion system 70 μ L withoutWater-ethanol, ice bath 30min, revolving speed 12000rpm are centrifuged 7min, abandon 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 DBN9936 is used for for primer and probe.
It was found that 1-832, the nucleotide displays in SEQ ID NO:5 are maize genomic sequence in transgenic corns thingThe right margin flank (5 ' flanking sequence) of part DBN9936 insetion sequence, 8202-9215, nucleotide in SEQ ID NO:5 are aobviousShow the left margin flank (3 ' flanking sequence) for being maize genomic sequence in transgenic corn events DBN9936 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 DBN9936 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 DBN9936 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 DBN9936 can also be used as DNA probe or beautiful as DNA primer Molecular Detection transgenosisThe presence of rice event DBN9936DNA.The SEQ ID NO:6 (833-1001, the nucleotide of SEQ ID NO:3) spansDBN10124 construct DNA sequence dna and tNos transcription terminator, SEQ ID NO:7 (the nucleotide 1- of SEQ ID NO:4190) span t35S transcription terminator and DBN10124 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 DBN9936 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 DBN9936 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 t35S 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 DBN9936.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 primer 8 (SEQ ID NO:11) of genomic dna sequence hybridization and the tNos of the paired 3 ' ends positioned at insert turnRecord the primer 7 (SEQ IDNO:10) of termination 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 DBN9936.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 DBN9936Step 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 DBN9936 baseWhen because in the PCR reaction of group DNA, the amplified production of 1001bp segment is generated, when it is used in unconverted corn gene group DNA and non-When in the PCR reaction of DBN9936 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 DBN9936 genomic DNA, the amplified production of 1204bp segment is generated,When in the PCR reaction that it is used in unconverted corn gene group DNA and non-DBN9936 corn gene group DNA, expanded without segmentIncrease.
PCR zygosity determination can also be used in identification from transgenic corn events DBN9936 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 DBN9936.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 DBN9936.
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 eventDBN9936 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 DBN9936 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 DBN9936DNA 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 DBN9936, and the sample in rotaring gene corn plant DBN9936 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 DBN9936 is used to transgenic corn eventsDBN9936 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.Analysis to transgenic corn events DBN9936DNA extracting sample should include a transgenic corn eventsThe assaypositive tissue DNA extract of DBN9936 compares, and a negative DNA from non-transgenic corn event DBN9936 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 DBN9936 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 DBN9936.It generates when being tested in DNA cloning method to transgenic corns thingPart DBN9936 be diagnostic amplicon, presumption contain corn plant or seed comprising transgenic corn events DBN9936The extract of DNA, or from the product of transgenic corn events DBN9936, it is used as the template of amplification, to determineWith the presence or absence of transgenic corn events DBN9936.
Fourth embodiment carries out transgenic corn events DBN9936 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 L TEFloating DNA precipitating.DNA and 1 μ L 30mg/mL RNAase 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.Distinguished with restriction enzyme EcoR V and Hind IIIThe partial sequence of digested genomic dna, the Cry1Ab using on T-DNA and EPSPS are as probe.For every kind of enzyme, in temperature appropriateBe incubated overnight digest under degree.Using SpeedVac (speed vacuum) rotation sample to reduce volume to 30μL。
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 L TE, 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 1 (40mM Na of 20mL Church rinse solution3P04, 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 DBN10124 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 DBN10124 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 DBN9936 containSingle copy of Cry1Ab and EPSPS gene., using the Cry1Ab probe, EcoR V and Hind III enzymatic hydrolysis generate size respectivelyThe single band of about 10kb and 9kb；Using the EPSPS probe, EcoR V and Hind III enzymatic hydrolysis generate respectively size about 8kb andThe single band of 15kb.This shows that each copy of Cry1Ab and EPSPS is present in corn transformation event DBN9936.
The insect-resistant detection of 5th embodiment, event
5.1, the bioassay of corn plant DBN9936
By transgenic corn events DBN9936 and wild-type corn plant (non-transgenic, NGM) 2 plants respectively to AsiaContinent corn borer (Ostrinia furnacalis, ACB), dichocrocis punctiferalis (Conogethes punctiferalis, YPM), 2 points of committeesNoctuid (Athetis lepigone, LPG), pink rice borer (Sesamia inferens, PSB), east armyworm (MythimnaSeperata, OAW), prodenia litura (Spodoptera litura, TCW), striped rice borer (Chilo suppressalis, SSB),Bollworm (Helicoverpa armigera, CBW) and beet armyworm (Spodoptera exigua, BAW) are as followsCarry out bioassay:
The new of transgenic corn events DBN9936 and wild-type corn plant (non-transgenic, NGM) 2 plants is taken respectivelyFresh leaves (V3-V4 period), it is clean with aseptic water washing and blotted the water on blade with gauze, then maize leaf is removedVein, while it being cut into the strip of about 1cm × 3cm, the strip after taking 1-3 piece (determining blade quantity according to insect appetite) to cutBlade is put on the filter paper of round plastic culture dish bottom, and the filter paper is soaked with distilled water, and 10 tribal chief are put in each culture dishWork raising newly hatched larvae, worm try culture dish cover after, 26-28 DEG C of temperature, relative humidity 70%-80%, the photoperiod (light/Statistical result after secretly) being placed 3 days under conditions of 16:8.Ostrinia furnacalis counts the death rate, passes through corrected mortality antagonism waterIt is flat to be identified, corrected mortality (%)=(1- survival number/connect borer population-wild type control death rate)/(1- wild type control is deadDie rate) × 100%.Three other insect statistical larvae development progresses, the death rate and blade injury rate indexs obtain resistance total score(full marks 300 divide): resistance total score=100 × corrected mortality+[100 × death rate+90 × (just incubate borer population/connect worm sum)+60× (just incubate-negative control borer population/and connect worm sum)+10 × and (negative control borer population/connect worm sum)]+100 × (1- blade injuryRate).5 plants are selected to be tested respectively from transgenic corn events DBN9936 and wild-type corn plant (non-transgenic, NGM), oftenStrain is repeated 6 times.As a result as shown in table 6 and table 7.
Pest-resistant bioassay results-death rate (%) of table 6, transgenic corn events DBN9936
Pest-resistant bioassay results-resistance total score of table 7, transgenic corn events DBN9936
The result shows that: transgenic corn events DBN9936 is to Ostrinia furnacalis, dichocrocis punctiferalis, 2 committee noctuid insect, pink rice borer, eastSquare armyworm, prodenia litura, striped rice borer, bollworm and beet armyworm all have preferable resistance, and transgenic corn eventsThe test worm death rate and resistance total score of DBN9936 is significantly higher than NGM.
5.2, the field effect of transgenic corn events DBN9936
The seed of transgenic corn events DBN9936 and wild-type corn plant (non-transgenic, NGM) 2 plants are setIt is handled for 2, each processing is by pressing RANDOMIZED BLOCK DESIGN, 3 repetitions, plot area 30m2(5m × 6m), line-spacing 60cm, strainAway from 25cm, conventional cultivation management, the time of infertility does not spray insecticide.Different insects connect the interval for having 2m between worm experimental plot,Avoid diffusion of the insect between different community.(1) Ostrinia furnacalis
Respectively the corn lobus cardiacus phase (the toy trumpet mouth phase, plant be developed to exhibition the 6-8 leaf phase) and spin phase Artificial Inoculation of Anoplophora glabripennis,It respectively connects worm 2 times.Every cell Artificial Inoculation of Anoplophora glabripennis is no less than 40 plants, and the newly hatched larvae of artificial feeding is connect in every plant of corn lobus cardiacus/filigreeAbout 60, after connecing worm 3 days, worm is connect for the second time, connects borer population amount with for the first time.It is killed by strain investigation corn after connecing worm 14-21 daysSituation.It institutes an inquiry within 14 days after usually connecing worm, if the rank that causes harm of negative control material (NGM) reaches sense or high sense, is considered asEffectively, it if investigation can suitably be postponed by not reaching, but connects 21 days after worm and is still not up to appropriate level, then this connects worm and is considered as nothingEffect.The lobus cardiacus phase connects worm investigation plant middle and upper part blade by Ostrinia furnacalis feeding situation；The spinning phase investigates female fringe after connecing wormKilled degree and plant are killed situation.Each processing randomly selects 15-20 plants/row.
The lobus cardiacus phase: Ostrinia furnacalis is recorded by the description in table 8 by strain and eats leaf level.Ostrinia furnacalis is calculated to each placeReason blade is caused harm the average value of degree (food leaf level): averagely food leaf level=∑ (food leaf level × rank plant number)/adjustLook into total strain number.According to the average value of food leaf level, each processing is divided to the resistance level of Ostrinia furnacalis, such as table 9.TransgenosisThe corn event DBN9936 lobus cardiacus phase is as shown in table 12 to the resistance result of Ostrinia furnacalis.
The spinning phase: situation, channel quantity, channel length of tunnel (cm) and survival instar larvae are killed according to female fringe and depositedQuantity living calculates each cell ear period Ostrinia furnacalis and is killed rank average value to the resistance of female fringe, and judgment criteria is as shown in table 10,Then by the judgment of standard corn ear period of table 11 to the resistance level of Ostrinia furnacalis.Transgenic corn events DBN9936 spinningPhase is as shown in table 13 to the resistance result of Ostrinia furnacalis.
Table 8, Ostrinia furnacalis cause harm the grade scale of degree to corn lobus cardiacus
| Eat leaf level | Symptom description |
| 1 | Only there is 1-2 aperture≤1mm worm channel on individual blades |
| 2 | Only there is 3-6 aperture≤1mm worm channel on individual blades |
| 3 | A small number of blades have 7 or more apertures≤1mm worm channel |
| 4 | There is 1-2 aperture≤2mm worm channel on individual blades |
| 5 | There is 3-6 aperture≤2mm worm channel on a small number of blades |
| 6 | Partial blade has 7 or more apertures≤2mm worm channel |
| 7 | There is 1-2 aperture to be greater than the worm channel of 2mm on a small number of blades |
| 8 | There is 3-6 aperture to be greater than the worm channel of 2mm on partial blade |
| 9 | There are 7 or more apertures to be greater than the worm channel of 2mm on most of blade |
Table 9, corn are to the evaluation criterion of Ostrinia furnacalis resistance
| The lobus cardiacus phase eats leaf level average value | Resistance level |
| 1.0-2.9 | Highly resistance (HR) |
| 3.0-4.9 | Anti- (R) |
| 5.0-6.9 | In resist (MR) |
| 7.0-8.9 | Feel (S) |
| 9.0 | Height sense (HS) |
Table 10, corn ear period are caused harm the grade scale of degree by Ostrinia furnacalis
Table 11, corn ear period are to the Evaluation standard of resistance of Ostrinia furnacalis
| Female fringe is killed rank average value | Resistance level |
| 1.0-2.0 | Highly resistance (HR) |
| 2.1-3.0 | Anti- (R) |
| 3.1-5.0 | In resist (MR) |
| 5.1-7.0 | Feel (S) |
| ≥7.1 | Height sense (HS) |
Table 12, transgenic corn events DBN9936 lobus cardiacus phase are to the resistance result of Ostrinia furnacalis
Table 13, transgenic corn events DBN9936 spinning phase are to the resistance result of Ostrinia furnacalis
The result shows that: either the lobus cardiacus phase still spins the phase, and transgenic corn events DBN9936 has Ostrinia furnacalisThere is preferable resistance level；The food leaf level average value of lobus cardiacus phase, transgenic corn events DBN9936 are substantially less than NGM.SpinningPhase, it is significant that female fringe percentage of injury, larvae alive number, length of tunnel and the female fringe of transgenic corn events DBN9936 is killed rankLower than NGM.Field efficacy such as Fig. 3 institute of the transgenic corn events DBN9936 in lobus cardiacus phase and spinning phase inoculation Ostrinia furnacalisShow.
(2) east armyworm
Experimental design and test method are substantially consistent with the evaluation resistance of Ostrinia furnacalis as described above.DifferentIt is only to carry out Artificial Inoculation of Anoplophora glabripennis in the corn lobus cardiacus phase (plant is developed to the exhibition 4-6 leaf phase), connect worm 2 times, in every plant of corn lobus cardiacusMeet second instar larvae about 20 of artificial feeding.After connecing worm 3 days, worm is connect for the second time, connects borer population amount with for the first time.Connecing worm 14 daysAfterwards, investigation maize leaf is caused harm degree by east armyworm.Caused harm degree according to maize leaf by east armyworm, is calculated each smallArea east armyworm causes harm the average value of rank (food leaf level) to maize leaf, and judgment criteria is as shown in table 14, then presses tableResistance level of the 15 judgment of standard corn to east armyworm.The transgenic corn events DBN9936 lobus cardiacus phase is to east armywormResistance result is as shown in table 16.
Table 14, maize leaf are caused harm the grade scale of degree by east armyworm
| Eat leaf level | Symptom description |
| 1 | Blade is without killed, or only has needle prick shape (≤1mm) worm channel on blade |
| 2 | Only there is a small amount of shell hole size (≤5mm) worm channel on individual blades |
| 3 | A small number of blades have shell hole size (≤5mm) worm channel |
| 4 | (≤10mm) is incised on individual blades |
| 5 | (≤10mm) is incised on a small number of blades |
| 6 | (≤10mm) is incised on partial blade |
| 7 | Individual blade-sections have sheet to incise (≤10mm) by feeding on a small number of blades |
| 8 | A small number of blades have sheet to incise (≤10mm) by feeding on partial blade |
| 9 | Most of blade is by feeding |
Table 15, corn are to the Evaluation standard of resistance of east armyworm
| The lobus cardiacus phase eats leaf level average value | Resistance level |
| 1.0-2.0 | Highly resistance (HR) |
| 2.1-4.0 | Anti- (R) |
| 4.1-6.0 | In resist (MR) |
| 6.1-8.0 | Feel (S) |
| 8.1-9.0 | Height sense (HS) |
Table 16, transgenic corn events DBN9936 lobus cardiacus phase are to the resistance result of east armyworm
The result shows that: transgenic corn events DBN9936 has preferable resistance level to east armyworm, and transgenosis is beautifulRice event DBN9936's incises ratio and food leaf level substantially less than NGM, transgenic corn events DBN9936 inoculation eastThe field efficacy of armyworm is as shown in Figure 4.
(3) bollworm
Experimental design and test method are substantially consistent with the evaluation resistance of Ostrinia furnacalis as described above.DifferentIt is only to carry out Artificial Inoculation of Anoplophora glabripennis in the corn silking phase, connect worm 2 times, the newly hatched larvae of artificial feeding is connect in every plant of corn capillament about20, after connecing worm 3 days, worm is connect for the second time, connects borer population amount with for the first time.It is killed by strain investigation female fringe after connecing worm 14-21 daysRate, each female fringe survival larva number, female fringe are killed length.It is instituted an inquiry within 14 days after usually connecing worm, if negative control material (NGM)The rank that causes harm reach sense or high sense, then be considered as effectively, if investigation can suitably be postponed by not reaching, but do not reached yet within 21 days after connecing wormTo appropriate level, then this connects worm and is considered as in vain.It is killed length (cm) according to female fringe percentage of injury, survival larva number, female fringe, is calculatedFor each cell corn ear period bollworm to the rank average value of causing harm of female fringe, judgment criteria is as shown in table 17, then presses the mark of table 18Standard differentiates corn ear period to the resistance level of bollworm.Resistance knot of the transgenic corn events DBN9936 spinning phase to bollwormFruit is as shown in table 19.
Table 17, maize ear are caused harm the grade scale of degree by bollworm
| Female fringe is killed rank | Symptom description |
| 0 | Female fringe is not aggrieved |
| 1 | Only filigree is killed |
| 2 | Fringe top is killed 1cm |
| 3+ | Every increase 1cm is killed under fringe top, killed rank increases by 1 grade accordingly |
| …N | |
Table 18, maize ear are to the Evaluation standard of resistance of bollworm
| Female fringe is killed rank average value | Resistance level |
| 0-1.0 | Highly resistance (HR) |
| 1.1-3.0 | Anti- (R) |
| 3.1-5.0 | In resist (MR) |
| 5.1-7.0 | Feel (S) |
| ≥7.1 | Height sense (HS) |
Table 19, transgenic corn events DBN9936 spinning phase are to the resistance result of bollworm
The result shows that: transgenic corn events DBN9936 has preferable resistance level, and transgenic corns to bollwormThe female fringe percentage of injury of event DBN9936, larvae alive number, female fringe are killed length and female fringe is killed rank and is substantially less than NGM, turn baseBecause the field efficacy of corn event DBN9936 inoculation bollworm is as shown in Figure 5.
(4) dichocrocis punctiferalis
Experimental design and test method are substantially consistent with the evaluation resistance of Ostrinia furnacalis as described above.DifferentIt is that corn only carries out natural INFESTATION in the more serious area of dichocrocis punctiferalis naturally-occurring.After first occur insect pest 14-21 days,And NGM is when being mostly that 4-5 age high instar larvae endangers, by strain investigation dichocrocis punctiferalis to the rate that causes harm of plant.Transgenic corn eventsDBN9936 is as shown in table 20 to the resistance result of dichocrocis punctiferalis.
To the resistance result of dichocrocis punctiferalis under the conditions of table 20, transgenic corn events DBN9936 natural INFESTATION
The result shows that: under the conditions of dichocrocis punctiferalis naturally-occurring, compared with NGM, dichocrocis punctiferalis is to transgenic corn eventsThe rate significant decrease of causing harm of DBN9936, thus illustrates that transgenic corn events DBN9936 has preferable resistance to dichocrocis punctiferalis,Field efficacy of transgenic corn events DBN9936 under the conditions of dichocrocis punctiferalis naturally-occurring is as shown in Figure 6.
(5) beet armyworm
Experimental design and test method are substantially consistent with the evaluation resistance of Ostrinia furnacalis as described above.DifferentIt is that corn only carries out natural INFESTATION in the more serious area of beet armyworm naturally-occurring.Occur insect pest 10-15 days firstAfterwards, when and NGM is mostly that 4-6 age high instar larvae endangers, by strain investigation beet armyworm to the rate that causes harm of plant.Transgenic cornsEvent DBN9936 is as shown in table 21 to the resistance result of beet armyworm.
To the resistance result of beet armyworm under the conditions of table 21, transgenic corn events DBN9936 natural INFESTATION
The result shows that: under the conditions of beet armyworm naturally-occurring, compared with NGM, beet armyworm is to transgenic corn eventsThus it is preferable anti-to illustrate that transgenic corn events DBN9936 has beet armyworm for the rate significant decrease of causing harm of DBN9936Property, field efficacy of transgenic corn events DBN9936 under the conditions of beet armyworm naturally-occurring is as shown in Figure 7.
What is particularly worth mentioning is that according to Chinese patent (application) number be 201210509817.2,201210511214.6,201310576970.1, the content recorded in 201310578129.6 and 201310681139.2 and the application transgenic cornsThe field effect and its bioassay results to insect of event DBN9936, shows the application transgenic corn events DBN9936The method and/or purposes of control pest are realized, specially 2 committee noctuid insect, dichocrocis punctiferalis, prodenia litura, pink rice borer and east is glutinousWorm；Namely control 2 committee noctuid insect, dichocrocis punctiferalis, twill may be implemented in the rotaring gene corn plant of any expression Cry1Ab albumenThe method and/or purposes of noctuid, pink rice borer and/or east armyworm pest.
The herbicide tolerant detection of sixth embodiment, event
This test selects agriculture to be sprayed up to herbicide (41% glyphosate-isopropylammonium aqua).It is set using random district's groupsMeter, 3 repetitions.Plot area is 15m2(5m × 3m), line-spacing 60cm, spacing in the rows 25cm, conventional cultivation management have 1m between cellWide isolation strip.Transgenic corn events DBN9936 is carried out to following 2 kinds of processing respectively: 1) not being sprayed；2) 1680g is pressedA.e./ha dosage sprays agriculture up to herbicide in the V3 leaf phase, then sprays agriculture again by same dose in the V8 phase up to herbicide.It needsIllustrate, the form that the glyphosate herbicidal of different content and dosage form is converted into equivalent glyphosate is suitable for draw a conclusion.
The 1 week and 2 weeks investigation symptom of chemical damage after medication respectively, and harvest when measure cell yield.Symptom of chemical damage pointGrade is as shown in table 22.Use the aggrieved rate of herbicide as the index of evaluation index assessment transformation event herbicide tolerant, specifically,The aggrieved rate of herbicide (%)=∑ (aggrieved strain number × number of levels at the same level)/(total strain number × highest level)；Wherein herbicide is aggrievedRate refers to that the aggrieved rate of glyphosate, the aggrieved rate of glyphosate are 2 weeks after handling according to glyphosate phytotoxicity investigation results and determination.OftenThe corn yield of a cell is the niblet total output (weight) for weighing 3 rows among each cell, the volume variance between different disposalIt is measured in the form of yield percentage, yield percentage (%)=spraying yield/does not spray yield.Transgenic corn eventsDBN9936 is as shown in table 23 to the result and corn yield result of herbicide tolerant.
Table 22, glyphosate herbicidal 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 23, transgenic corn events DBN9936 are to the result and corn yield result of glyphosate herbicide tolerance
As a result illustrate, in terms of herbicide (glyphosate) aggrieved rate: 1) transgenic corn events DBN9936 is removed in glyphosateAggrieved rate is essentially 0 under careless agent (1680g a.e./ha) processing, and transgenic corn events DBN9936 has good grass as a result,Sweet phosphine herbicide tolerant.
In terms of yield: transgenic corn events DBN9936 is not spraying and is spraying 2 kinds of 1680g a.e./ha glyphosateHandling lower yield does not have notable difference, and after spraying glyphosate herbicidal, the yield of transgenic corn events DBN9936 is omited insteadThere is increase, further demonstrates that transgenic corn events DBN9936 has good glyphosate herbicide tolerance as a result,.
7th embodiment
Such as agricultural product or commodity can be produced by transgenic corn events DBN9936.If in the agricultural product or commodityIn detect enough expression quantity, the agricultural product or commodity are expected containing can diagnose transgenic corn events DBN9936 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 DBN9936 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 DBN9936.
In conclusion transgenic corn events DBN9936 of the present invention has preferable resistance to lepidopterous insects, while rightGlyphosate herbicidal tolerance with higher, on yield without influence, and detection method can quickly and accurately identify biological sampleIn product whether include transgenic corn events DBN9936 DNA molecular.
Seed corresponding to transgenic corn events DBN9936 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.10219.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.