CN104878091B

CN104878091B - For detecting the nucleic acid sequence and its detection method of corn plant DBN9978

Info

Publication number: CN104878091B
Application number: CN201510218637.2A
Authority: CN
Inventors: 丁德荣; 康越景; 张云珠; 刘海利; 庞洁; 王利君; 贾志伟; 黄金存; 郭函子; 王磊; 傅学乾; 周毅; 李风; 鲍晓明; 吕玉平; 张世平
Original assignee: Beijing Dabeinong Technology Group Co Ltd; Beijing Dabeinong Biotechnology Co Ltd
Current assignee: Beijing Dabeinong Biotechnology Co Ltd
Priority date: 2015-04-30
Filing date: 2015-04-30
Publication date: 2019-05-17
Anticipated expiration: 2035-04-30
Also published as: WO2016173362A1; AR104430A1; CN104878091A

Abstract

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of corn plant DBN9978, and the nucleic acid sequence of the corn plant includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series.Corn plant DBN9978 of the present invention with preferable resistance and has preferable tolerance to glyphosate herbicidal to lepidopterous insects, on yield without influence, and detection method can quickly and accurately identify in biological sample whether include transgenic corn events DBN9978 DNA molecular.

Description

For detecting the nucleic acid sequence and its detection method of corn plant DBN9978

Technical field

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of corn plant DBN9978, especially relates to And a kind of pair of insect it is resistant and be resistant to glyphosate herbicidal application transgenic corn events DBN9978 and for detecting life In object sample whether include specific transgenic corn events DBN9978 nucleic acid sequence and its detection method.

Background technique

Many areas are all main cereal crops to corn (Zea mays L.) in the world.Biotechnology has been applied In corn to improve its economical character and quality.Insect-resistant is an important economical character in maize production, especially To resistance of lepidopterous insects, such as corn borer, bollworm, armyworm etc..Corn can be by turning to the resistance of lepidopterous insects The method of gene makes the resistant gene of lepidopterous insects express and obtain in corn plant.Another important economical character is Herbicide tolerant, especially tolerance glyphosate herbicidal.Corn can pass through transgenosis to the tolerance of glyphosate herbicidal Method so that glyphosate herbicide tolerant type gene (such as EPSPS) is expressed and is obtained in corn plant.

Known foreign gene is influenced in the intracorporal expression of plant by their chromosome location, it may be possible to due to dyeing Matter structure (such as heterochromatin) or transcription regulatory element (such as enhancer) are close to integration site.Thus, it usually needs screening is a large amount of Event be possible to identify can be with commercialized event (event that the target gene imported obtains optimal expression).Example Such as, have been observed that the expression quantity of quiding gene there may be very big difference between event in plant and other organisms；In table On the space reached or time mode may there is also differences, such as between different plant tissues transgenosis relative expression exist it is poor Different, this species diversity shows that actual expression pattern may be pre- with the transcription regulatory element institute in the gene construct according to importing The expression pattern of phase is inconsistent.It is thus typically necessary to generate hundreds and thousands of different events and filter out from these events Single incident with transgene expression amount and expression pattern desired for the purpose of being commercialized.With expected transgenosis table Event up to amount and expression pattern can be used for that transgenosis is penetrated into other by sexual cutcross using conventional breeding methods In genetic background.The transgenic expression characteristics of original transformant are maintained by the offspring that this Crossing system generates.Using this Kind strategy pattern may insure there is reliable gene expression in many kinds, and these kinds can well adapt to locality Growth conditions.

It will be beneficial that the presence of particular event, which is able to detect, so that whether the offspring for determining sexual hybridization includes target gene. In addition, the method for detection particular event also will be helpful to abide by relevant laws and regulations, such as thrown from the food of recombination crops It needs to obtain official approval before entering market and is marked.Transgenosis is detected by any well known polynucleotides detection method Presence be all it is possible, such as polymerase chain reaction (PCR) or using polynucleotide probes DNA hybridization.These detections Method is usually focused on common genetic elements, such as promoter, terminator, marker gene etc..Therefore, unless with insertion turn The sequence of the adjacent chromosomal DNA of gene DNA (" flanking DNA ") be it is known, above-mentioned this method cannot be used to distinguish Different events, especially those events generated with identical DNA construct.Insertion is spanned so often utilizing at present The pair of primers of the junction of transgenosis and flanking DNA identifies transgenosis particular event by PCR, specifically includes The first primer in flanking sequence and the second primer comprising insetion sequence.

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 DBN9978, Transgenic corn events DBN9978 with preferable resistance and has preferable tolerance to glyphosate herbicidal to insect, and Detection method can quickly and accurately identify whether the DNA comprising specific transgenic corn events DBN9978 divides in biological sample Son.

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 least At 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 mutual Complementary series.

Further, the nucleic acid sequence include SEQ ID NO:3 or its complementary series, and/or SEQ ID NO:4 or its Complementary 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 DBN9978 in insetion sequence End is located at the sequence that a length near insertion junction is 22 nucleotide, the SEQ ID NO:1 or its complementary sequence Column span the DNA sequence dna of the flanking genomic DNA sequence of corn insertion point and 5 ' ends of insetion sequence, comprising described SEQ ID NO:1 or its complementary series can be accredited as the presence of transgenic corn events DBN9978.The SEQ ID NO:2 Or its complementary series is to be located near insertion junction in transgenic corn events DBN9978 in 3 ' ends of insetion sequence One length is the sequence of 22 nucleotide, and the SEQ ID NO:2 or its complementary series span 3 ' ends of insetion sequence DNA sequence dna and corn insertion point flanking genomic DNA sequence, be comprising the SEQ ID NO:2 or its complementary series The presence of transgenic corn events DBN9978 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 transgenic Any 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 glycosides Sour (second nucleotide sequence).The nucleic acid sequence may further be derived from or be complementary to comprising the complete SEQ ID to be same A 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 acid Sequence includes DNA primer group in the DNA cloning method for generating amplified production.Using DNA primer in DNA cloning method The amplified production of generation be when including the amplified production of SEQ ID NO:1 can diagnose transgenic corn events DBN9978 or its The 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 comprise The mixture or DNA, RNA of RNA, DNA and RNA or it is other not as the nucleotide of one or more polymerase templates or its The 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 ID Nucleotide described in NO:3, SEQ ID NO:4 and SEQ ID NO:5.When selected from SEQ ID NO:3, SEQ ID NO:4 and When nucleotide shown in SEQ ID NO:5, the probe and primer can be for length at least about 21 to about 50 or More continuous nucleotides.The SEQ ID NO:3 or its complementary series are in transgenic corn events DBN9978 in insertion sequence 5 ' ends of column are located at the sequence that a length near insertion junction is 1557 nucleotide, the SEQ ID NO:3 Or its complementary series by 1374 nucleotide corn flanking genomic DNA sequence (the nucleotide 1-1374 of SEQ ID NO:3), The DBN10124 construct DNA sequence dna (the nucleotide 1375-1458 of SEQ ID NO:3) of 84 nucleotide and 99 nucleotide 3 ' end DNA sequences (the nucleotide 1459-1557 of SEQ ID NO:3) group of tNos (rouge alkali synthetase) transcription terminator At the presence of transgenic corn events DBN9978 can be accredited as comprising the SEQ ID NO:3 or its complementary series.

The nucleic acid sequence can be any portion of the SEQ ID NO:4 or its complementary series transgenic insetion sequence At least 11 or more the continuous polynucleotides (third nucleic acid sequence) divided, or be the SEQ ID NO:4 or its complementation Any portion of at least 11 or more continuous polynucleotides (the 4th cores in 3 ' flank corn gene group DNA regions in sequence Acid 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 same A 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 generated It include DNA primer group in the DNA cloning method of amplified production.The amplification generated in DNA cloning method is produced using DNA primer Object is can to diagnose transgenic corn events DBN9978 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 DBN9978 Enter the sequence that a length near junction is 672 nucleotide, the SEQ ID NO:4 or its complementary series are by 51 The DBN10124 building of the t35s transcription terminator sequences (the nucleotide 1-51 of SEQ ID NO:4) of nucleotide, 141 nucleotide The corn integration site flanking genomic dna of body DNA sequence dna (the nucleotide 52-192 of SEQ ID NO:4) and 480 nucleotide Sequence (the nucleotide 193-672 of SEQ ID NO:4) composition, can identify comprising the SEQ ID NO:4 or its complementary series For the presence of transgenic corn events DBN9978.

The SEQ ID NO:5 or its complementary series are that the length of characterization transgenic corn events DBN9978 is 9219 The 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 mutually Complementary series can be accredited as the presence of transgenic corn events DBN9978.

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 amplicon Survey diagnosis biological sample transgenic corn event DBN9978 or the presence of its offspring；The nucleic acid sequence or its complementary series It can be used in nucleotide detection method, to detect the presence of biological sample transgenic corn event DBN9978 or its offspring.

To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event DBN9978 a kind of Existing 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 series At 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 series 1-11 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 mutually Complementary 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 and SEQ 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 DBN9978's Method 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 series At 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 continuous 1-11 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 sequence Column, 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.

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:3 or In its complementary series at least 11 continuous nucleotide or SEQ ID NO:4 or its complementary series at least 11 it is continuous Nucleotide；

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 auxiliary Kind 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 series 1-11 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:2 Or 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, institutes Stating DNA molecular includes at least 11 continuous nucleotide or SEQ in the homologous sequence or its complementary series of SEQ ID NO:3 At least 11 continuous nucleotide, can be used as transgenic corns in the homologous sequence of ID NO:4 or its complementary series Event DBN9978 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 series 1-11 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:1 The 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:7 Homologous sequence or its complementary series.To achieve the above object, the present invention also provides a kind of plant cells, include coding insect The nucleic acid sequence of resistance Cry1Ab albumen, the nucleic acid sequence for encoding glyphosate herbicide tolerance EPSPS albumen and specific region Nucleic acid sequence, the nucleic acid sequence of the specific region includes SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 or SEQ Sequence 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, including At least one transgenic corn plant cell is provided in the diet of target insect, the transgenic corn plant cell is in its gene All 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 group At 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 ingesting Insect 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 of Method 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 genome At 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 turn Gene 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 turn Gene corn plant includes selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID in its genome At 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 base Because 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 seed Nucleic 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 region The 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 ID At 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 herbicidal The method of plant, comprising:

An at least corn seed is planted, includes coding glyphosate herbicide tolerance in the genome of the corn seed The 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 other The plant of sequence compares the plant with the plant injury weakened；

To achieve the above object, a kind of resistant to insect the present invention also provides culture and tolerance Gyphosate herbicice The 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 seed Nucleic 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 other The plant of sequence compares the plant with the plant injury weakened, and the plant pair insect with the plant injury weakened is taken the photograph Food damage is also resistant；

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 introducing The 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 ID At 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 DBN9978 of insect and lacking insect-resistant The 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 weakened Progeny plant；

The transgenic corn events DBN9978 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:7 Kind nucleic acid sequence.

To achieve the above object, the present invention also provides a kind of corns for generating and having tolerance to glyphosate herbicidal The method of plant, the nucleic acid sequence including introducing coding glyphosate tolerant EPSPS albumen into the genome of the plant The 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, SEQ At 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 core Acid sequence.

Specifically, the method for generating the plant for having tolerance to glyphosate herbicidal includes:

By first parental maize plant of transgenic corn events DBN9978 to glyphosate herbicidal with tolerance and lack Second 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；

To achieve the above object, and tolerance glyphosate herbicidal resistant to insect is generated the present invention also provides a kind of The method of the plant of application, comprising:

By the first parental maize plant of transgenic corn events DBN9978 of glyphosate tolerance and insect-resistant and lack grass Second 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 glyphosate Also resistant；

To achieve the above object, the present invention also provides a kind of multicore glycosides comprising SEQ ID NO:1 or SEQ ID NO:2 The 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:2 The agricultural product or commodity of acid, the agricultural product or commodity are corn flour, maize flour, corn oil, cornstarch, corn gluten, jade Rice 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 more The 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 field Lead 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 again It is complete in raw plant cell tissue cultures, plant callus, vegetation bed (plant clumps) and plant or plant part Whole plant cell, the plant part such as embryo, pollen, ovule, seed, leaf, flower, branch, fruit, stalk, root, the tip of a root, flower Medicine 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, group It knits, callus, embryo and flower, stem, fruit, Ye Hegen, the above plant part are originated from advance with DNA molecular conversion of the invention And 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 sequence Code sequence) and coded sequence after adjusting sequence (3 ' non-coding sequence)." natural gene ", which refers to, is naturally found to have its own Adjust the gene of sequence." mosaic gene " refer to be not natural gene any gene, it includes non-natural discovery adjusting and Coded 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 procedures The 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 claim For " 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 process External source (heterologous) DNA entered, such as segment relevant to transformation event.Therefore, flanking DNA may include natural and exogenous DNA Combination.In the present invention, " flanking region " or " flanking sequence " or " genome frontier district " or " genome border sequence " refer to The base-pair of at least 3,5,10,11,15,20,50,100,200,300,400,1000,1500,2000,2500 or 5000 is longer Sequence, be located at initial external source insertion DNA molecular immediately upstream or downstream and with initial external source be inserted into DNA molecular phase It 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 ' sides The 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 difference Flanking region is that each transformant institute specificity contains.When recombinant DNA is introduced into plant by conventional hybridization, flanking region is logical Chang Buhui changes.Transformant also can be containing between heterologous insertion DNA and the section of genomic DNA or between two sections of genomic DNAs Or the unique engagement between two sections of allogeneic dna sequence DNAs." engagement " is the point of two specific DNA fragmentation connections.For example, engagement exists In 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 repair Adorn 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 DBN9978 and its offspring, the transgenic corn events DBN9978 is corn plant DBN9978 comprising the Plants and Seeds of transgenic corn events DBN9978 and its plant are thin Born of the same parents or its renewable part, the plant part of the transgenic corn events DBN9978, including but not limited to cell, pollen, embryo Pearl, flower, bud, root, stem, silk, inflorescence, ear fringe, leaf and the product from corn plant DBN9978, such as corn flour, corn Face, corn oil, corn pulp, corn silk, cornstarch and the biomass for staying in corn crop field.

Transgenic corn events DBN9978 of the present invention contains a DNA construct, when it is expressed in plant cell When, the transgenic corn events DBN9978 obtains the resistance to insect and the tolerance to glyphosate herbicidal.The DNA Construct includes two concatenated expression cassettes, and first expression cassette is comprising the suitable promoter for expressing in plant and fits The polyadenylation signal sequence of conjunction, the promoter are operably connected the nucleic acid sequence of Cry1Ab albumen, described The nucleic acid sequence of Cry1Ab albumen is mainly resistant to lepidopterous insects.Second expression cassette includes for expressing in plant Suitable 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 herbicidal There 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, soil Earth 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, E9 Promoter, GOS promoter, alcA/alcR promoter, Agrobacterium rhizogenes (Agrobacterium rhizogenes) RolD starting Son and Arabidopsis (Arabidopsis thaliana) Suc2 promoter.The polyadenylation signal sequence can for The suitable polyadenylation signal sequence to work in plant, the suitable polyadenylation signal sequence include but unlimited In from the polyadenosine of soil Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) gene Polyadenylation 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) gene Sequence.

In addition, the expression cassette can also include other genetic elements, the genetic elements include but is not limited to enhance Son and signal peptide/transit peptides.The expression of gene can be enhanced in the enhancer, and the enhancer includes but is not limited to cigarette Careless etch virus (TEV) translation activity factor, CaMV35S enhancer and FMV35S enhancer.Signal peptide/the transit peptides can be with Guide Cry1Ab albumen and/or EPSPS Protein transport to extracellular or intracellular specific organelle or compartment, for example, sharp Chloroplaset 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 reach The 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 most Mesh, 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 Agrobacterium It is isolated in (Agrobacterium tumefaciens sp.) CP4 bacterial strain, and can by optimization codon or In other ways change coding EPSPS gene polynucleotides, with reach increase transformed cells in transcript stability and can The purpose of usability.5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) gene can also be used as selective mark Remember gene.

" glyphosate " refers to the salt of N- phosphonomethylglycine and it, is handled with " glyphosate herbicidal " and refers to use Any one is handled containing the herbicide formulations of glyphosate.In order to reach ebd and to certain glyphosate system The selection of agent utilization rate is no more than the technical ability of common agronomic technique personnel.Herbicide formulations using any one containing glyphosate Processing contains the field of the vegetable material from transgenic corn events DBN9978, will control the weeds in the field Growth, and the growth or yield of the vegetable material from transgenic corn events DBN9978 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 bar Bacterium (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 plant Between 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 gene In group.

The Gene Knock-out Mice is with carrier bombardment plant cell (the biological bullet that particle mediates comprising exogenous DNA Hit conversion).

The pollen tube channel conversion method is that natural pollen tube channel (also known as pollen is formed by after pollinating using plant Pipe 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 selection The offspring of exogenous DNA.

DNA construct is the combination that DNA molecular is interconnected, and this combination provides one or more expression cassettes.DNA Construct 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 importing The DNA molecular of code sequence, 3 ' terminator regions and other sequences.Expression cassette contained in DNA construct includes providing courier Genetic elements necessary to the transcription of RNA, the expression cassette can be designed as expressing in prokaryotic cell or eukaryocyte.This hair Bright 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 one Expression of nucleic acid box containing target gene is inserted into Plant Genome to generate plant population by transgene method, then The raw plant population, and selection have the specific plant of insertion specific gene group site feature.Term " event " refers to including different The original transformant of source DNA and the offspring of the transformant.Term " event " also refers to transformant and other kinds containing allogeneic dna sequence DNA Offspring obtained from sexual hybridization is carried out between individual, even if after be returned repeatedly with backcross parent, from transformant The insertion DNA and flanking genomic dna of parent exists in the same chromosome location in filial generation.Term " event " also refers to DNA sequence dna from original transformant, the DNA sequence dna include insertion DNA and with the close adjacent flanking genomes of insertion DNA Sequence, which, which is expected, is transferred in filial generation, the filial generation by containing insertion DNA parental department (such as original transformant and It 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 includes The 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 invention And/or the form of albumen and/or organism.This manual intervention can produce recombinant DNA molecules and/or recombinant plant." the weight It 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 base Because type due to heterologous nucleic acids there are due to change, " transgenosis " includes Transgenics initially changed in this way and by most The 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 described It is natural that for example random allogamy of event, non-recombinant virus infection, non-recombinant Bacterial Transformation, non-recombinant swivel base or spontaneous prominent occurs Become.

" 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 and It is artificially introduced in the genome of host cell.

Cultivate transgenic corn events resistant to lepidopterous insects and that there is tolerance to glyphosate herbicidal DBN9978 passes through following steps: making the first parental corn plants and the second parental corn plants sexual hybridization first, to produce Given birth to the first generation progeny plant of multiplicity, first parental corn plants by cultivation transgenic corn event DBN9978 and The corn plant of its offspring forms, and transgenic corn events DBN9978 and its offspring are of the invention to squama wing by utilizing Mesh insect it is resistant and to glyphosate herbicidal have tolerance expression cassette convert obtained from, the second parental maize Plant lacks to the resistance of lepidopterous insects and/or has tolerance to glyphosate herbicidal；Then it selects to lepidopterous insects Invasion it is resistant and/or to glyphosate herbicidal have tolerance progeny plant, can cultivate to lepidopterous insects Corn plant resistant and that there is tolerance to glyphosate herbicidal.These steps, which may further include, makes Lepidoptera elder brother The progeny plant and the second parental corn plants or third parental corn plants of worm resistance and/or glyphosate tolerant are returned It hands over, then by being applied or by molecular marked compound relevant to character with lepidopteran insect infestation, glyphosate herbicidal (as wrapped The DNA molecular of bond site that the 5 ' ends and 3 ' ends of insetion sequence identify in DBN9978 containing transgenic corn events) identification Filial 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, separation The 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 offspring The 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 above Son, 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 DBN9978 genome, no matter the gene Group DNA be also be derived from from transgenic corn events DBN9978 or seed transgenic corn events DBN9978 plant or Seed or extract.Probe of the invention not only includes DNA or ribonucleic acid, further include specifically with target DNA 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 dna On chain, heterozygote is formed between primer and target dna chain, then under the action of polymerase (such as archaeal dna polymerase), along mesh DNA 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 chain Formula 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 height Specifically hybridize under degree stringent hybridization condition with target sequence.Although being different from target dna sequence and being protected to target dna sequence The probe for holding hybridization ability can design by conventional method, however, it is preferred to, probe and primer in the present invention There 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 DBN9978, and determining should The nucleic acid sequence of DNA molecular.The DNA molecular includes transgene insert sequence and Maize genome flank region, and the DNA divides The 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 miscellaneous It hands over or amplification method may be used to identify in sample from the presence of the DNA of transgenic corn events DBN9978.Nucleic acid point Son or its segment can carry out specific hybrid with other nucleic acid molecules in any case.As the present invention uses, if two A 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 other Property 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 molecules The corresponding nucleotide mutual added time, then the two nucleic acid molecules is claimed to show " complete complementarity ".If two nucleic acid molecules can be with Enough 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 stability Hybridization 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 deviation Chain 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 sequence Property, 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 stringency Specific hybrid can occur with the complementary strand of another section of nucleic acid molecules to match down.Promote the suitable stringent of DNA hybridization Condition 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 selected About 2.0 × SSC, 50 DEG C to high stringency of about 0.2 × SSC, 50 DEG C from Low stringency conditions.In addition, washing step In 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 strip Part and salinity can all change, can also one of them remain unchanged and another variable changes.Preferably, originally Invention 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:7 Specific hybrid occurs for any segment of one or more nucleic acid molecules or its complementary series or above-mentioned sequence.It is highly preferred that A nucleic acid molecules of the invention under high stringency with SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID 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 ID Any 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 ID Any segment of NO:7 or its complementary series or above-mentioned sequence is same with 80% to 100% or 90% to 100% sequence Property.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 method Object is remembered to identify the offspring of genetic cross.Probe can be art technology by any one with hybridizing for target dna molecule Method known to personnel detects, these methods include but is not limited to fluorescent marker, radioactive label, antibody class label And chemiluminescent labeling.

About the amplification (for example, passing through PCR) for using specific amplimer to carry out target nucleic acid sequence, " stringent item Part " 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 core The 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 sequence Choosing 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 target Target 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 part The 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 invention DBN9978 is generated by sexual hybridization mode, or whether the corn sample acquired from field includes transgenic corn events Whether DBN9978 or corn extract, such as coarse powder, powder or oil include transgenic corn events DBN9978, from corn plant The DNA that tissue sample or extract extract can be by using the nucleic acid amplification method of primer pair to generate for transgenic corns The presence of the DNA of event DBN9978 is diagnostic amplicon.The primer pair include one in the Plant Genome with The first primer of the adjacent flanking sequence of the exogenous DNA insertion point of insertion, and second draw from the exogenous DNA of insertion Object.Amplicon has certain length and sequence, and the sequence is also diagnostic to the transgenic corn events DBN9978. The length range of amplicon can be the combination length of primer pair plus a nucleotide base pair, preferably add about 50 cores Thuja acid base-pair more preferably adds about 250 nucleotide bases pair, most preferably adds about 450 nucleosides soda acids Base to or more.

Optionally, primer pair can include entire insertion to generate from the flanking genomic sequence of the insertion two sides DNA The amplicon of nucleotide sequence.One in the primer pair of plant genome sequences can be located at away from insertion DNA sequence dna At a certain distance from, which may range from a nucleotide base to about 20,000 nucleotide bases pair.Term " amplification The 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 polymerization Enzyme chain reaction (PCR).Various nucleic acid amplification methods have been well-known to those skilled in the art.PCR amplification method has been sent out Open up the genomic DNA of amplifiable 22kb and the phage DNA of 42kb.Other DNA cloning sides of these methods and this field Method can be used for the present invention.The exogenous DNA array of insertion and flanking DNA sequence from transgenic corn events DBN9978 can With by being expanded using genome of the provided primer sequence to transgenic corn events DBN9978, to PCR after amplification Amplicon 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 appropriate On specific hybrid to target dna and expand diagnostic amplicon.Kit can provide the detection method based on Ago-Gel Or many methods of checkout and diagnosis amplicon known in the art.Containing with SEQ ID NO:3 or SEQ ID NO:4's Any part in Maize genome area is homologous or complementary and any part with the transgenosis insert district of SEQ ID NO:5 The kit of homologous or complementary DNA primer is provided by the present invention.Particularly identify useful in DNA cloning method draw Object expands 5 ' transgenosis/genome with transgenic corn events DBN9978 to being SEQ ID NO:8 and SEQ ID NO:9 A part of homologous diagnostic amplicon in area, wherein amplicon includes SEQ ID NO:1.Other DNA as DNA primer points Son can be selected from SEQ ID NO:5.

Amplicon caused by these methods can be detected by multiple technologies.One of method is Genetic Bit Analysis, this method devise one across the DNA of insertion DNA sequence dna and adjacent flanking genomic DNA sequence widow Nucleotide 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 nucleosides Sour chain is hybridized, and the template as single base extension, which has used archaeal dna polymerase and be next The 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 insertion The oligonucleotide chain of DNA sequence dna and adjacent genomic DNA binding site.By the single-stranded of the oligonucleotide chain and target area PCR product (in insetion sequence and adjacent flanking genomic sequence in respectively use a primer) hybridized, then and Archaeal dna polymerase, ATP, sulfonyl enzyme, luciferase, apyrase, adenosine -5 '-phosphorus sulfate and luciferin one It 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 can In a kind of method for detecting amplicon of the present invention.Need to design one in this way across insertion DNA sequence dna and phase The oligonucleotide chain of adjacent genomic DNA binding site.The single stranded PCR products of the oligonucleotide chain and target area (are being inserted Enter in sequence and respectively use in adjacent flanking genomic sequence a primer) hybridized, then with archaeal dna polymerase and one The ddNTP of kind fluorescent marker is incubated together.Single base extension will lead to insertion ddNTP.This insertion can use fluorescence Instrument measures the change of its polarization.The change of polarization represents the presence of insertion/flanking sequence, illustrates amplification, hybridization and single alkali Base 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 manufacturer It 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 base Because of the FRET oligonucleotide probe of group flank binding site.The FRET probe and PCR primer are (in insetion sequence and adjacent side A primer is respectively used in wing genome sequence) circular response is carried out in the presence of heat-stabilised poly synthase and dNTPs.FRET probe Hybridization lead to the division of fluorescence part and quencher moieties and the release of fluorescence part on FRET probe.The generation of fluorescence signal The 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 DBN9978 It can also include Southern blot hybridization, Northern blot hybridization and in situ hybridization.Particularly, the suitable technology includes Probe and sample are incubated, is washed to remove whether unbonded probe and detection probe have hybridized.The detection method takes The certainly type of the label appended by probe, for example, can detecte radiolabeled probe by X-ray exposure and imaging, or logical It 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 sequence Application in column detection.It is briefly described as follows, designs one across insertion DNA sequence dna and adjacent flanking genomic binding site FRET oligonucleotide probe.The unique texture of the FRET probe causes it to contain secondary structure, which can be close Apart from interior holding fluorescence part and quencher moieties.The FRET probe and PCR primer are (in insetion sequence and adjacent flanking gene A primer is respectively used in group sequence) circular response is carried out in the presence of heat-stabilised poly synthase and dNTPs.By successful PCR Amplification, the hybridization of FRET probe and target sequence leads to the forfeiture of probe secondary structure, to make fluorescence part and quencher moieties It spatially separates, generates fluorescence signal.The generation of fluorescence signal represents the presence of insertion/flanking sequence, explanation Amplification and hybridization are successful.

The method of other descriptions, such as the method that microfluid (microfluidics) provides separation and DNA amplification sample And equipment.Photoinitiator dye is for detecting and measuring specific DNA molecular.Include the electronic sensor or knot for detecting DNA molecule Close specific DNA molecular receive pearl and thus can be detected receive test tube (nanotube) equipment for detecting DNA of the invention points Son is useful.

Method that composition and DNA detection field of the present invention describes or known can be used to develop DNA inspection Test agent box.The kit is conducive to identify the DNA that whether there is transgenic corn events DBN9978 in sample, can be with For cultivating the corn plant of the DNA containing transgenic corn events DBN9978.The kit can containing DNA primer or Probe 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 spy Needle, 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 cloning Reaction, 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 base Because the DNA structure of insetion sequence and Maize genome binding site includes: being located at the corn of 5 ' end of transgene insert sequence DBN9978 flanking genomes region, a part of insetion sequence of the left boundary area (LB) from Agrobacterium, first expression Box is operably connected to jade by the cauliflower mosaic virus 35 S promoter (pr35S) of the tandem sequence repeats containing enhancer region On rice heat shock 70kDa albumen introne (iZmHSP70), it is operably connected to the insect-resistant of bacillus thuringiensis On Cry1Ab albumen (cCry1Ab), and it is operably connected on the transcription terminator (tNos) of nopaline synthase and forms, the Two 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 big On oxalic acid -3- phosphate synthase (cEPSPS), and it is operably connected on cauliflower mosaic virus 35S terminator (t35S) and group At, a part of insetion sequence in the right side boundary region (RB) from Agrobacterium, and it is located at 3 ' end of transgene insert sequence Corn plant DBN9978 flanking genomes region (SEQ ID NO:5).In DNA cloning method, the DNA as primer divides Son can be any part from transgenic corn events DBN9978 transgenic insetion sequence, be also possible to derive from Any part in the region of DNA domain of flank Maize genome in transgenic corn events DBN9978.

Transgenic corn events DBN9978 can be combined with other transgenic maize varieties, such as herbicide is (such as careless ammonium Phosphine, dicamba etc.) tolerance corn, or carry the transgenic maize varieties of other anti insect genes.All these differences turn base Because of the various combinations of event, the breeding together with transgenic corn events DBN9978 of the invention can provide and resist a variety of insect pests simultaneously Resist the improvement hybrid transgenic corn variety of a variety of herbicides.These kinds turn base compared to non-transgenic kind and unisexuality shape Because 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 events DBN9978's is resistant to the feeding damage of lepidoptera pest, and is resistant to the plant of the agriculture herbicide containing glyphosate Toxic effect.The Cry1Ab albumen of the plant expression bacillus thuringiensis of the dual character, provides to Lepidoptera The resistance of pest (such as Ostrinia furnacalis) feeding damage, and express the 5- enol-of the glyphosate resistance of Agrobacterium strains CP4 Pyruvoyl shikimic acid -3- phosphate synthase (EPSPS) albumen assigns plant to the tolerance of glyphosate.Dual character corn tool It 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 remove Careless 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 grass The gene linkage of sweet phosphine tolerance trait is present in transgenic corn events DBN9978 genome in same DNA section Term single gene seat on, this point provide the breeding efficiency of enhancing and make it possible to be tracked with molecular labeling reproductive population and Transgenic insert in its filial generation.SEQ ID NO:1 or its complementary series, SEQ ID in detection method simultaneously NO: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 as DNA primer or probe to generate the amplified production for being diagnosed as transgenic corn events DBN9978 or its offspring, and can quickly, Accurately, the stable presence for identifying the vegetable material from transgenic corn events DBN9978.

BRIEF DESCRIPTION OF THE SEQUENCES

Below by drawings and examples, technical scheme of the present invention will be described in further detail.

Detailed description of the invention

Fig. 1 is the transgenosis insertion of nucleic acid sequence and its detection method of the present invention for detecting corn plant DBN9978 The structural schematic diagram of sequence and Maize genome binding site；

Fig. 2 is that recombinant expression of the present invention for the nucleic acid sequence and its detection method that detect corn plant DBN9978 carries The structural schematic diagram of body DBN10124；

Fig. 3 is the present invention for detecting the nucleic acid sequence of corn plant DBN9978 and its transgenic corns of detection method Field efficacy figure of the event DBN9978 in lobus cardiacus phase and spinning phase inoculation Ostrinia furnacalis；

Fig. 4 is the present invention for detecting the nucleic acid sequence of corn plant DBN9978 and its transgenic corns of detection method The field efficacy figure of event DBN9978 inoculation east armyworm；

Fig. 5 is the present invention for detecting the nucleic acid sequence of corn plant DBN9978 and its transgenic corns of detection method The field efficacy figure of event DBN9978 inoculation bollworm；

Fig. 6 is the present invention for detecting the nucleic acid sequence of corn plant DBN9978 and its transgenic corns of detection method Field efficacy figure of event DBN9978 under the conditions of dichocrocis punctiferalis naturally-occurring；

Fig. 7 is the present invention for detecting the nucleic acid sequence of corn plant DBN9978 and its transgenic corns of detection method Field efficacy figure of event DBN9978 under the conditions of beet armyworm naturally-occurring.

Specific embodiment

Below by specific embodiment further illustrate the present invention for detect corn plant DBN9978 nucleic acid sequence and The 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 carrier DBN10124 include two concatenated transgene expression cassettes, first expression cassette by the tandem sequence repeats containing enhancer region flower Cauliflower 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, and It is operably connected on the transcription terminator (tNos) of nopaline synthase and forms；Second expression cassette is by rice actin 1 promoter (prOsAct1) is operably connected on arabidopsis EPSPS chloroplast transit peptides (spAtCTP2), operationally It is connected to the 5- enol-pyrovyl shikimic acid -3- phosphate synthase (cEPSPS) of the glyphosate tolerant of Agrobacterium CP4 bacterial strain On, 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 turn Change 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 1.1 The 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 DBN9978.

For the corn transformation of mediated by agriculture bacillus, briefly, immature rataria is separated from corn, is suspended with Agrobacterium Liquid contacts rataria, and wherein Agrobacterium can transmit the nucleotide sequence of the nucleotide sequence of Cry1Ab gene and EPSPS gene To at least one cell (step 1: infecting step) of one of rataria, in this step, rataria preferably immerses Agrobacterium suspension Liquid (OD₆₆₀=0.4-0.6 infects culture medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, Portugal Grape sugar 36g/L, acetosyringone (AS) 40mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, pH 5.3)) in starting Inoculation.Rataria and Agrobacterium co-culture one period (3 days) (step 2: co-culturing step).Preferably, rataria is infecting step Afterwards in solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetyl fourth Ketone musk (AS) 100mg/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH 5.8) on cultivate.It trains altogether herein After the stage of supporting, there can be " recovery " step of a selectivity.In " recovery " step, recovery media (MS salt 4.3g/L, MS Vitamin, casein 300mg/L, sucrose 30g/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH 5.8) 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 transformant Agent (step 3: recovering step).Preferably, rataria is cultivated on having antibiotic but the not solid medium of selective agent, to eliminate Agrobacterium 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 agent Screening solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, N- (phosphine carboxymerhyl) sweet ammonia Sour 0.25mol/L, 2,4- dichlorphenoxyacetic acid (2,4-D) 1mg/L, plant gel 3g/L, pH 5.8) on cultivate, cause to convert Cell selective growth.Then, callus regeneration is at plant (step 5: regeneration step), it is preferable that containing selective agent The callus grown on culture medium is cultivated on solid medium (MS differential medium and MS root media) to regenerate and plant Object.

It screens obtained resistant calli and is transferred to the MS differential medium (MS salt 4.3g/L, MS vitamin, cheese Plain 300mg/L, sucrose 30g/L, 6-benzyladenine 2mg/L, N- (phosphine carboxymerhyl) glycine 0.125mol/L, plant gel 3g/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 C Culture 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 C Culture 8 hours.

1.3, the identification and screening of transgenic event

770 separate transgenic T are generated altogether₀Single plant.

Pass through TaqMan^TMIt analyzes (referring to second embodiment) and detects regenerated transgenic corn plant with the presence or absence of Cry1Ab With EPSPS gene, and the copy number of insect-resistant and glyphosate herbicide tolerance strain is characterized.According to the copy of target gene Several, good insect-resistant, glyphosate herbicide tolerance and Agronomic (are implemented referring to the 5th embodiment and the 6th Example), by screening, the event DBN9978 of having selected be it is excellent, have single copy transgenosis, good insect-resistant, grass sweet Phosphine herbicide tolerant and Agronomic (participating in the 5th embodiment and sixth embodiment).

Second embodiment carries out transgenic corn events DBN9978 detection with TaqMan

Take the blade about 100mg of transgenic corn events DBN9978 as sample, with the DNeasy Plant of Qiagen Maxi Kit extracts its genomic DNA, and the copy of Cry1Ab and EPSPS is detected by Taqman fluorescence probe quantitative PCR method Number.Simultaneously using wild-type corn plant as control, tested and analyzed according to the method described above.Experiment sets 3 repetitions, is averaged Value.

The specific method is as follows:

Step 11, the blade 100mg for taking transgenic corn events DBN9978, are ground into homogenate with liquid nitrogen in mortar, each Sample takes 3 repetitions；

Step 12, the genomic DNA that above-mentioned sample is extracted using the DNeasy Plant Mini Kit of Qiagen, specifically Method 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 identification The 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 average Value；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 concentration L 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 copied DBN9978。

3rd embodiment, transgenic corn events DBN9978 detection

3.1, extracting genome DNA

DNA is extracted according to CTAB (cetyl trimethylammonium bromide) method routinely used: taking 2 grams of tender transgenosis beautiful After the blade of rice event DBN9978 is pulverized in liquid nitrogen, the DNA that 0.5mL is preheated in 65 DEG C of temperature is added and extracts CTAB Buffer (20g/L CTAB, 1.4M NaCl, 100mMTris-HCl, 20mM EDTA (ethylenediamine tetra-acetic acid), with NaOH tune pH To 8.0), after mixing well, 65 DEG C of extracting 90min of Yu Wendu；0.5 times of volume of phenol is added, 0.5 times of volume of chloroform overturns mixed It 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 shake Dynamic centrifuge tube, 4 DEG C of standing 30min of Yu Wendu；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 bench Drying；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 selected Restriction enzyme and 3 μ L enzyme cutting buffering liquids, digestion 1 hour.After to digestion, be added into digestion system 70 μ L without Water-ethanol, ice bath 30min, revolving speed 12000rpm are centrifuged 7min, abandon supernatant, and 8.5 μ L distilled water (dd are added in drying later H₂O)、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 primers PCR amplification separates 5 ' and 3 ' transgenosis/genomic DNA.Specifically, 5 ' transgenosis of separation/genomic DNA primer combination includes SEQ 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, SEQ ID 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 survey Sequence primer, PCR reaction condition are as shown in table 3.

Amplicon obtained electrophoresis on 2.0% Ago-Gel then uses QIAquick to separate PCR reactant Gel 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 divided It 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 make Confirmed 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 sequences NO: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 art It 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 made The identification of the corn plant or seed from transgenic corn events DBN9978 is used for for primer and probe.

It was found that 1-1374, the nucleotide displays in SEQ ID NO:5 are maize genomic sequence in transgenic corns thing The right margin flank (5 ' flanking sequence) of part DBN9978 insetion sequence, 8740-9219, nucleotide in SEQ ID NO:5 are aobvious Show the left margin flank (3 ' flanking sequence) for being maize genomic sequence in transgenic corn events DBN9978 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 analyzes It is diagnostic for the DNA of transgenic corn events DBN9978 when measuring.Connecing in SEQ ID NO:1 and SEQ ID NO:2 Close every side of insertion point and corn gene group DNA that sequence is transgenic corn events DBN9978 transgenic segment 11 polynucleotides.Longer or shorter polynucleotides junction sequence can be selected from SEQ ID NO:3 or SEQ ID NO:4 It 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 conduct DNA primer molecule is useful in DNA detection method.Junction sequence SEQ ID NO:6 and SEQ ID NO:7 is also transgenosis New DNA sequence dna in corn event DBN9978 can also be used as DNA probe or beautiful as DNA primer Molecular Detection transgenosis The presence of rice event DBN9978DNA.The SEQ ID NO:6 (1375-1557, the nucleotide of SEQ ID NO:3) spans DBN10124 construct DNA sequence dna and tNos transcription terminator, SEQ ID NO:7 (the nucleotide 1- of SEQ ID NO:4 192) 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 DBN9978 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 from Because in the genome of the vegetable material of corn event DBN9978 flank in the genomic DNA of 5 ' ends of T-DNA insetion sequence A part.This PCR product includes SEQ ID NO:3.In order to carry out PCR amplification, design and flank are in transgene insert sequence 5 ' ends genomic dna sequence hybridization primer 5 (SEQ ID NO:8) and the paired transgenosis t35S that is located at turn Record 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 events Flank is in a part of the genomic DNA of 3 ' ends of T-DNA insetion sequence in the genome of the vegetable material of DBN9978.This A PCR product includes SEQ ID NO:4.In order to carry out PCR amplification, design and flank are in 3 ' ends of transgene insert sequence The primer 8 (SEQ ID NO:11) of genomic dna sequence hybridization and the tNos of the paired 3 ' ends positioned at insert turn Record 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 corns The diagnostic amplicon of event DBN9978.The detection of amplicon can be by using Stratagene as shown in table 3 Robocycler, MJ Engine, Perkin-Elmer 9700 or Eppendorf Mastercycler Gradien thermal cycle Instrument 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 DBN9978 Step 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 solution Oil.Using following loop parameter (table 3) in Stratagene Robocycler (Stratagene, La Jolla, CA), MJ Engine (MJ R-Biorad, Hercules, CA), Perkin-Elmer 9700 (Perkin Elmer, Boston, MA) or PCR 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 DBN9978 base When because in the PCR reaction of group DNA, the amplified production of 1557bp segment is generated, when it is used in unconverted corn gene group DNA and non- When in the PCR reaction of DBN9978 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 DBN9978 genomic DNA, the amplified production of 672bp segment is generated, When in the PCR reaction that it is used in unconverted corn gene group DNA and non-DBN9978 corn gene group DNA, expanded without segment Increase.

PCR zygosity determination can also be used in identification from transgenic corn events DBN9978 material be homozygote or It is heterozygote.Primer 9 (SEQ ID NO:12), primer 10 (SEQ ID NO:13) and primer 11 (SEQ ID NO:14) are used for Amplified reaction is to generate the diagnostic amplicon of transgenic corn events DBN9978.The DNA cloning condition illustrated in table 4 and table 5 It can be used for above-mentioned zygosity test to generate the diagnostic amplicon of transgenic corn events DBN9978.

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 cycler PCR.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 event DBN9978 there are the DNA of situation.Or reaction will generate two by the biological sample containing the DNA from Maize genome A different DNA cloning, the DNA from Maize genome in transgenic corn events DBN9978 relative to existing The corresponding allele of insertion DNA be heterozygosis.The two different amplicons, which will correspond to, derives from wild-type corn base Because group locus the first amplicon and diagnosis transgenic corn events DBN9978DNA there are the second amplicons of situation.Only Generate the maize dna sample for corresponding to the single amplicon of the second amplicon for the description of heterozygous genes group, diagnosable determination The presence of sample transgenic corn event DBN9978, and the sample in rotaring gene corn plant DBN9978 by relative to depositing The corresponding allele of insertion DNA be produced by homozygous corn seed.

It should be noted that the primer pair of transgenic corn events DBN9978 is used to transgenic corn events DBN9978 genomic DNA is diagnostic amplicon.These primer pairs include but is not limited to (the SEQ ID NO:8 of primer 5 and 6 With 9) and primer 7 and 8 (SEQ ID NO:10 and 11), in the DNA cloning method.In addition, for expanding in corn One control primer 12 and 13 (SEQ ID NO:22 and 23) of source gene is included, as in one of reaction condition Standard.Analysis to transgenic corn events DBN9978DNA extracting sample should include a transgenic corn events The assaypositive tissue DNA extract of DBN9978 compares, and a negative DNA from non-transgenic corn event DBN9978 is extracted Object control and a negative control without containing template maize dna extract.Other than these primer pairs, it can also use and From any primer pair of SEQ ID NO:3 or SEQ ID NO:4 or its complementary series, when they are used for DNA amplification reaction Generate respectively for the tissue from transgenic event corn plant DBN9978 be it is diagnostic comprising SEQ ID NO:1 or The amplicon of SEQ ID NO:2.The DNA cloning condition illustrated in table 2- table 5 is used for suitable primer pair to generate The diagnostic amplicon of transgenic corn events DBN9978.It generates when being tested in DNA cloning method to transgenic corns thing Part DBN9978 be diagnostic amplicon, presumption contain corn plant or seed comprising transgenic corn events DBN9978 The extract of DNA, or from the product of transgenic corn events DBN9978, it is used as the template of amplification, to determine With the presence or absence of transgenic corn events DBN9978.

Fourth embodiment carries out transgenic corn events DBN9978 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 nitrogen 10g plant tissue is arrived in grinding about 5.In 12.5mL Extraction buffer A (0.2M Tris pH8.0,50mM EDTA, 0.25M NaCl, 0.1%v/v β-dredges base ethyl alcohol, 2.5%w/v Polyvinyl-pyrrolidone) in resuspension plant tissue, with 4000rpm from The 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 resuspended It 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 isometric Chloroform/isoamyl alcohol (24:1), by be inverted be gently mixed, with 4000rpm centrifugation 20 minutes.Water-bearing layer is collected, and is being added Add after 0.54 volume isopropanol with 4000rpm centrifugation 5 minutes to precipitate DNA.Supernatant is discarded, and is resuspended in 500 μ L TE Floating 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 with 14000rpm is centrifuged 10 minutes precipitating DNA.After discarding supernatant, precipitating is washed with the ethyl alcohol that 500 μ L mass fractions are 70%, and After 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 III The partial sequence of digested genomic dna, the Cry1Ab using on T-DNA and EPSPS are as probe.For every kind of enzyme, in temperature appropriate Be incubated overnight digest under degree.Using SpeedVac (speed vacuum) rotation sample to reduce volume to 30 μL。

4.3, gel electrophoresis

Bromophenol blue Loading Dye is added to each sample in the present embodiment 4.2, and by each sample pipetting volume It onto 0.7% Ago-Gel containing ethidium bromide, is separated by electrophoresis in TBE electrophoretic buffer, the electrophoresis coagulating under 20 volts Glue 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 trace It hands over as follows: placing 20 thick drying trace paper in disk, place 4 thin drying trace paper again thereon.In 0.4M NaOH 1 thin trace paper is moistened in advance, and is placed on the pile, and then placement 1 moistens in advance in 0.4M NaOH Hybond-N+ transfer membrane (Amersham Pharmacia Biotech, #RPN303B).Gel is seated in top, it is ensured that solidifying There 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 NaOH Full buffer disk.With the wick connection gel stack and buffer disk being immersed in 0.4M NaOH in advance, DNA is transferred to film On.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 UV Crosslinking 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 ID NO: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 ice It sets 7 minutes, is then transferred into Rediprime II (Amersham Pharmacia Biotech, #RPN1633) test tube.To Rediprime 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 removal DNTPs 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 ice It 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 C Hybridized.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 detect The 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, wherein The DBN10124 of Hind III- digestion is introduced, amount is based on probe length and is equivalent to a copy number, beautiful in detection with explanation When individual gene in rice genome copies, the sensitivity of the experiment；(3) copy number is equivalent to based on probe length The 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 TaqMan^TMPCR analysis, i.e. corn plant DBN9978 contain Single copy of Cry1Ab and EPSPS gene., using the Cry1Ab probe, EcoR V and Hind III enzymatic hydrolysis generate size respectively The single band of about 11kb and 12kb；Using the EPSPS probe, EcoR V and Hind III enzymatic hydrolysis generate size about 10kb respectively With the single band of 4kb.This shows that each copy of Cry1Ab and EPSPS is present in corn transformation event DBN9978.

The insect-resistant detection of 5th embodiment, event

5.1, the bioassay of corn plant DBN9978

By transgenic corn events DBN9978 and wild-type corn plant (non-transgenic, NGM) 2 plants respectively to Asia Continent corn borer (Ostrinia furnacalis, ACB), dichocrocis punctiferalis (Conogethes punctiferalis, YPM), 2 points of committees Noctuid (Athetis lepigone, LPG), pink rice borer (Sesamia inferens, PSB), east armyworm (Mythimna Seperata, OAW), prodenia litura (Spodoptera litura, TCW), striped rice borer (Chilo suppressalis, SSB), Bollworm (Helicoverpa armigera, CBW) and beet armyworm (Spodoptera exigua, BAW) are as follows Carry out bioassay:

The new of transgenic corn events DBN9978 and wild-type corn plant (non-transgenic, NGM) 2 plants is taken respectively Fresh leaves (V3-V4 period), it is clean with aseptic water washing and blotted the water on blade with gauze, then maize leaf is removed Vein, 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 cut Blade 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 dish Work 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 water It is flat to be identified, corrected mortality (%)=(1- survival number/connect borer population-wild type control death rate)/(1- wild type control is dead Die rate) × 100%.Other insect statistical larvae development progresses, three Xiang Zhibiao of the death rate and blade injury rate 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 injury Rate).5 plants are selected to be tested respectively from transgenic corn events DBN9978 and wild-type corn plant (non-transgenic, NGM), often Strain 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 DBN9978

Pest-resistant bioassay results-resistance total score of table 7, transgenic corn events DBN9978

The result shows that: transgenic corn events DBN9978 is to Ostrinia furnacalis, dichocrocis punctiferalis, 2 committee noctuid insect, pink rice borer, east Square armyworm, prodenia litura, striped rice borer, bollworm and beet armyworm all have preferable resistance, and transgenic corn events The test worm death rate and resistance total score of DBN9978 is significantly higher than NGM.

5.2, the field effect of transgenic corn events DBN9978

The seed of transgenic corn events DBN9978 and wild-type corn plant (non-transgenic, NGM) 2 plants are set It is handled for 2, each processing is by pressing RANDOMIZED BLOCK DESIGN, 3 repetitions, plot area 30m²(5m × 6m), line-spacing 60cm, strain Away 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/filigree About 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 days Situation.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 as Effectively, 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 nothing Effect.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 worm Killed 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 place Reason blade is caused harm the average value of degree (food leaf level): averagely food leaf level=∑ (food leaf level × rank plant number)/adjust Look 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.Transgenosis The corn event DBN9978 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 deposited Quantity 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 DBN9978 spinning Phase is as shown in table 14 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 DBN9978 lobus cardiacus phase are to the resistance result of Ostrinia furnacalis

Table 13, transgenic corn events DBN9978 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 DBN9978 has Ostrinia furnacalis There is preferable resistance level；The food leaf level average value of lobus cardiacus phase, transgenic corn events DBN9978 are substantially less than NGM.Spinning Phase, it is significant that female fringe percentage of injury, larvae alive number, length of tunnel and the female fringe of transgenic corn events DBN9978 is killed rank Lower than NGM.Field efficacy such as Fig. 3 institute of the transgenic corn events DBN9978 in lobus cardiacus phase and spinning phase inoculation Ostrinia furnacalis Show.

(2) east armyworm

Experimental design and test method are substantially consistent with the evaluation resistance of Ostrinia furnacalis as described above.Different It 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 cardiacus Meet 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 days Afterwards, investigation maize leaf is caused harm degree by east armyworm.Caused harm degree according to maize leaf by east armyworm, is calculated each small Area 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 table Resistance level of the 15 judgment of standard corn to east armyworm.The transgenic corn events DBN9978 lobus cardiacus phase is to east armyworm Resistance 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 DBN9978 lobus cardiacus phase are to the resistance result of east armyworm

The result shows that: transgenic corn events DBN9978 has preferable resistance level to east armyworm, and transgenosis is beautiful Rice event DBN9978's incises ratio and food leaf level substantially less than NGM, transgenic corn events DBN9978 inoculation east The 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.Different It 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 about 20, 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 days Rate, 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 worm To 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 calculated For 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 18 Standard differentiates corn ear period to the resistance level of bollworm.Resistance knot of the transgenic corn events DBN9978 spinning phase to bollworm Fruit 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 DBN9978 spinning phase are to the resistance result of bollworm

The result shows that: transgenic corn events DBN9978 has preferable resistance level, and transgenic corns to bollworm The female fringe percentage of injury of event DBN9978, larvae alive number, female fringe are killed length and female fringe is killed rank and is substantially less than NGM, turn base Because the field efficacy of corn event DBN9978 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.Different It 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 events DBN9978 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 DBN9978 natural INFESTATION

The result shows that: under the conditions of dichocrocis punctiferalis naturally-occurring, compared with NGM, dichocrocis punctiferalis is to transgenic corn events The rate significant decrease of causing harm of DBN9978, thus illustrates that transgenic corn events DBN9978 has preferable resistance to dichocrocis punctiferalis, Field efficacy of transgenic corn events DBN9978 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.Different It is that corn only carries out natural INFESTATION in the more serious area of beet armyworm naturally-occurring.Occur insect pest 10-15 days first Afterwards, 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 corns Event DBN9978 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 DBN9978 natural INFESTATION

The result shows that: under the conditions of beet armyworm naturally-occurring, compared with NGM, beet armyworm is to transgenic corn events Thus it is preferable anti-to illustrate that transgenic corn events DBN9978 has beet armyworm for the rate significant decrease of causing harm of DBN9978 Property, field efficacy of transgenic corn events DBN9978 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 corns The field effect and its bioassay results to insect of event DBN9978, shows the application transgenic corn events DBN9978 The method and/or purposes of control pest are realized, specially 2 committee noctuid insect, dichocrocis punctiferalis, prodenia litura, pink rice borer and east is glutinous Worm；Namely control 2 committee noctuid insect, dichocrocis punctiferalis, twill may be implemented in the rotaring gene corn plant of any expression Cry1Ab albumen The 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 groups Meter, 3 repetitions.Plot area is 15m²(5m × 3m), line-spacing 60cm, spacing in the rows 25cm, conventional cultivation management have 1m between cell Wide isolation strip.Transgenic corn events DBN9978 is carried out to following 2 kinds of processing respectively: 1) not being sprayed；2) 1680g is pressed A.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 needs Illustrate, 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 point Grade 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 aggrieved Rate 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.Often The corn yield of a cell is the niblet total output (weight) for weighing 3 rows among each cell, the volume variance between different disposal It is measured in the form of yield percentage, yield percentage (%)=spraying yield/does not spray yield.Transgenic corn events DBN9978 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 DBN9978 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 DBN9978 is removed in glyphosate Aggrieved rate is essentially 0 under careless agent (1680g a.e./ha) processing, and transgenic corn events DBN9978 has good grass as a result, Sweet phosphine herbicide tolerant.

In terms of yield: transgenic corn events DBN9978 is not spraying and is spraying 2 kinds of 1680g a.e./ha glyphosate Handling lower yield does not have notable difference, and after spraying glyphosate herbicidal, the yield of transgenic corn events DBN9978 is omited instead There is increase, further demonstrates that transgenic corn events DBN9978 has good glyphosate herbicide tolerance as a result,.

7th embodiment

Such as agricultural product or commodity can be produced by transgenic corn events DBN9978.If in the agricultural product or commodity In detect enough expression quantity, the agricultural product or commodity are expected containing can diagnose transgenic corn events DBN9978 material Expect the nucleotide sequence present in the agricultural product or commodity.The agricultural product or commodity include but is not limited to corn oil, jade Rice coarse powder, maize flour, corn gluten, corn-dodger, cornstarch and will be as food source for any other of animal consumption Food or additionally as the ingredient in swelling agent or make-up composition for cosmetic use etc..Based on probe or primer pair Nucleic acid detection method and/or kit can be developed to detect such as SEQ ID NO:1 or SEQ ID NO:2 in biological sample Shown in transgenic corn events DBN9978 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 jade The presence of rice event DBN9978.

In conclusion transgenic corn events DBN9978 of the present invention has preferable resistance to lepidopterous insects, while right Glyphosate herbicidal tolerance with higher, on yield without influence, and detection method can quickly and accurately identify biological sample In product whether include transgenic corn events DBN9978 DNA molecular.

Seed corresponding to transgenic corn events DBN9978 is deposited in China Microbiological bacterium on December 24th, 2014 Kind preservation administration committee common micro-organisms center (abbreviation CGMCC, address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, in Institute of microbiology, the academy of sciences, state, postcode 100101), classification naming: corn (Zea mays), deposit number CGMCC No.10292.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 ginseng It is described the invention in detail according to preferred embodiment, those skilled in the art should understand that, it can be to the present invention Technical solution be modified or replaced equivalently, without departing from the spirit and scope of the technical solution of the present invention.

Claims

1. a kind of nucleic acid sequence, which is characterized in that including SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or Its complementary series, the nucleic acid sequence be originated from transgenic corn events DBN9978, the transgenic corn events DBN9978 with The form of seed and to be preserved in China Committee for Culture Collection of Microorganisms with deposit number CGMCC No.10292 commonly micro- Bio-Centers.

2. nucleic acid sequence according to claim 1, which is characterized in that the nucleic acid sequence include SEQ ID NO:3 or its Complementary series, and/or SEQ ID NO:4 or its complementary series.

3. nucleic acid sequence according to claim 2, which is characterized in that the nucleic acid sequence include SEQ ID NO:5 or its Complementary series.

4. a kind of method existing for DNA of test sample transgenic corn event DBN9978 characterized by comprising

Carry out nucleic acid amplification reaction；

Detect the presence of amplified production；

The amplified production includes SEQ ID NO:1 or its complementary series, and/or SEQ ID NO:2 or its complementary series；

The amplified production is originated from transgenic corn events DBN9978, and the transgenic corn events DBN9978 is with the shape of seed Formula and China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved in deposit number CGMCC No.10292.

5. method existing for the DNA of test sample transgenic corn event DBN9978 according to claim 4, feature It is, the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of test sample transgenic corn event DBN9978 according to claim 4 or 5, special Sign is that described two primers include SEQ ID NO:8 and SEQ ID NO:9 or SEQ ID NO:10 and SEQ ID NO: 11。

7. a kind of method existing for DNA of test sample transgenic corn event DBN9978 characterized by comprising

Contact sample to be tested with probe, the probe includes SEQ ID NO:1 or its complementary series or SEQ ID NO: 2 or its complementary series, the source probe transgenic corn event DBN9978, the transgenic corn events DBN9978 is to plant Son form and China Committee for Culture Collection of Microorganisms's commonly micro- life is preserved in deposit number CGMCC No.10292 Object center；

Detect the hybridisation events of the sample to be tested and the probe.

8. method existing for the DNA of test sample transgenic corn event DBN9978 according to claim 7, feature It is, the probe further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

9. special according to method existing for the DNA of the test sample transgenic corn event DBN9978 of claim 7 or 8 Sign is that at least one described probe is marked at least one fluorophor.

10. a kind of method existing for DNA of test sample transgenic corn event DBN9978 characterized by comprising

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or it is mutual Complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic corn events DBN9978, the transgenic corn events DBN9978 are preserved in the form of seed and with deposit number CGMCC No.10292 China Committee for Culture Collection of Microorganisms's common micro-organisms center；

The hybridisation events of the sample to be tested and the marker nucleic acid molecules are detected, and then pass through marker assistant breeding point Analysis is to determine that insect-resistant and/or herbicide tolerant and marker nucleic acid molecules are chain on science of heredity.

11. method existing for the DNA of test sample transgenic corn event DBN9978 according to claim 10, special Sign is that the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or it is mutual Complementary series.

12. a kind of DNA detection kit, which is characterized in that including at least one DNA molecular, the DNA molecular includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, can be used as transgenic corn events DBN9978 or its offspring have one of DNA primer of specificity or probe；The DNA molecular is originated from transgenic corn events DBN9978, the transgenic corn events DBN9978 are preserved in the form of seed and with deposit number CGMCC No.10292 China Committee for Culture Collection of Microorganisms's common micro-organisms center.

13. DNA detection kit according to claim 12, which is characterized in that further include when the DNA molecular is as probe SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

14. a kind of protection corn plant is from the method for insect infestations, which is characterized in that including being provided in the diet of target insect At least one transgenic corn plant cell, the transgenic corn plant cell successively include SEQ ID in its genome NO:1,1459-8598 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 or the transgenic corn plant cell Genome in include SEQ ID NO:5；Ingest the transgenic corn plant cell target insect be suppressed further ingest The corn plant.

15. a kind of method for protecting corn plant from the damage as caused by herbicide, which is characterized in that including that will contain effectively Dosage glyphosate herbicidal is applied to the big Tanaka for planting at least one rotaring gene corn plant, and the rotaring gene corn plant exists In its genome successively include SEQ ID NO:1,1459-8598 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, It or include SEQ ID NO:5 in the genome of the rotaring gene corn plant；The rotaring gene corn plant has sweet to grass The tolerance of phosphine herbicide.

16. a kind of method for the big Tanaka weeds for controlling maize planting plant, which is characterized in that including effective dose grass will be contained Sweet phosphine herbicide is applied to the big Tanaka for planting at least one rotaring gene corn plant, and the rotaring gene corn plant is in its gene Successively comprising SEQ ID NO:1,1459-8598 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2, Huo Zhesuo in group It states in the genome of rotaring gene corn plant comprising SEQ ID NO:5；The rotaring gene corn plant has to Gyphosate herbicice The tolerance of agent.

17. a kind of method for the corn plant that culture is resistant to insect characterized by comprising

An at least corn seed is planted, includes the nucleic acid sequence of specific region, the spy in the genome of the corn seed The nucleic acid sequence for determining region successively includes SEQ ID NO:1,1459-8598 nucleic acid sequences of SEQ ID NO:5 and SEQ ID The nucleic acid sequence of NO:2 or the specific region includes SEQ ID NO:5；

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 the specific region The plant of plant injury with decrease.

18. a kind of method that culture has the corn plant of tolerance to glyphosate herbicidal characterized by comprising

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 the specific region with other The plant of sequence compares the plant with the plant injury weakened.

19. a kind of method for cultivating corn plant that is resistant to insect and being resistant to glyphosate herbicidal, which is characterized in that Include:

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 the specific region with other The plant of sequence compares the plant with the plant injury weakened, and the plant pair insect with the plant injury weakened is taken the photograph Food damage is also resistant.

20. a kind of method for generating the plant resistant to insect, which is characterized in that including will successively be wrapped in genome The plant of ID containing SEQ NO:1, SEQ ID NO:5 1459-8598 nucleic acid sequences and SEQ ID NO:2, and it is another Kind plant hybridization, to generate a large amount of progeny plants；Selecting genome includes that the filial generation of the nucleic acid sequence of specific region is planted Strain, the nucleic acid sequence of the specific region successively includes 1459-8598 SEQ ID NO:1, SEQ ID NO:5 nucleic acid sequences It include SEQ ID NO:5 with the nucleic acid sequence of SEQ ID NO:2 or the specific region, and the progeny plant is to insect Ingest with weaken plant injury.

21. generating the method for the plant resistant to insect according to claim 20 characterized by comprising

By to resistant the first parental maize plant of transgenic corn events DBN9978 of insect with lack the of insect-resistant Two parental maize plant sexual hybridizations, 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 the specific region described in the plant injury weakened Progeny plant；

The transgenic corn events DBN9978 is preserved in China in the form of seed and with deposit number CGMCC No.10292 Microbiological Culture Collection administration committee common micro-organisms center.

22. a kind of method for generating the plant that there is tolerance to glyphosate herbicidal, which is characterized in that including by gene Successively the corn comprising SEQ ID NO:1,1459-8598 nucleic acid sequences of SEQ ID NO:5 and SEQ ID NO:2 is planted in group Strain, hybridizes with another plant, to generate a large amount of progeny plants；Selecting genome includes the nucleic acid sequence of specific region Progeny plant, the nucleic acid sequence of the specific region successively includes SEQ ID NO:1, SEQ ID NO:5 1459-8598 The nucleic acid sequence of nucleic acid sequence and SEQ ID NO:2 or the specific region includes SEQ ID NO:5, and the filial generation is planted Strain has glyphosate tolerant.

23. the generation according to claim 22 has the method for the plant of tolerance, feature to glyphosate herbicidal It is, comprising:

By first parental maize plant of transgenic corn events DBN9978 to glyphosate herbicidal with tolerance and lack grass Second parental maize plant sexual hybridization of sweet phosphine tolerance, to generate a large amount of progeny plants；

The progeny plant is handled with glyphosate herbicidal；

The progeny plant of selection tolerance glyphosate；

24. a kind of method for generating plant that is resistant to insect and being resistant to glyphosate herbicidal application, feature exist In, comprising:

By the first parental maize plant of transgenic corn events DBN9978 of glyphosate tolerance and insect-resistant and lack glyphosate Second parental maize plant sexual hybridization of 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, the progeny plant for being resistant to glyphosate also have the feeding damage of insect Resistance；

25. a kind of composition for being produced from transgenic corn events DBN9978, which is characterized in that the composition be corn flour, Maize flour, corn oil, corn silk or cornstarch；The transgenic corn events DBN9978 is in the form of seed and to protect Hiding number CGMCC No.10292 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center.

26. a kind of agricultural product or commodity for being produced from transgenic corn events DBN9978, which is characterized in that the agricultural product or Commodity are corn flour, maize flour, corn oil, cornstarch, corn gluten, corn-dodger, cosmetics or filler；The transgenosis Corn event DBN9978 is preserved in Chinese microorganism strain preservation in the form of seed and with deposit number CGMCC No.10292 Administration committee's common micro-organisms center.