CN110195122A - It is a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4 - Google Patents

Abstract

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4, 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.The detection method can quickly and accurately identify in biological sample whether include transgenic corn events T anti-4 DNA molecular.

Description

It is a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4

Technical field

The present invention relates to a kind of for detecting the nucleic acid sequence and its detection method of corn plant T anti-4, more particularly to It is a kind of for detect in biological sample whether include specific transgenic corn events T anti-4 nucleic acid sequence and its detection method.

Background technique

Weeds in field and crop competition water, fertilizer, light and growing space, directly affect crop yield and quality.Permitted simultaneously More weeds are the vector of crop pathogens and pest again, are one of important biomolecule restriction factors of crop yield.According to joint Food and Agriculture Organization, state statistics, annual up to 95,000,000,000 dollars because of grain-production loss caused by weeds of the whole world, is equivalent to loss 3.8 hundred million tons of wheats are roughly equal to more than half of global wheat yield in 2009.It is poor in 95,000,000,000 dollars of economic loss Developing country bear about 70,000,000,000 dollars (FAO.The lurking menace of weeds [J/OL] (http: // Www.fao.org/news/story/en/item/29402/icode/), 2009-08-11.).Therefore, field is efficiently controlled Weeds are one of the important measures for promoting increases in grain production.In China, the weeds type for endangering corn has more than 40 kinds, wherein endanger compared with Big weeds have more than 10 kinds.It can make corn underproduction 10-20% in general time weeds, more up to 30-50% when serious.In addition, As China's people in the countryside are toward the quickening of urban migration speed, the scale and mechanization of corn planting be one it is foreseeable become Gesture, this makes traditional artificial weeding mode become unrealistic.Currently, widely applied selective herbicide amount of application in the market Greatly, residual life, is long, is easy to influence the normal growth of second stubble crop.The steriland herbicides such as glyphosate have efficient, low toxicity, easily drop The features such as solution, noresidue.But their weedings cannot be used directly in the growth period of crop without selectivity.Pass through transgenic technology The corn for cultivating such resistance to steriland herbicide can overcome this problem.Spraying 1-2 times in corn growing season can effectively solve Certainly weed problem reduces the dosage and input cost of herbicide.Therefore, herbicide-resistant transgenic corns have boundless Application value and market potential.

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 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 T anti-4 or the presence of its offspring；The nucleic acid sequence or its complementary series can For in nucleotide detection method, to detect the presence of biological sample transgenic corn event T anti-4 or its offspring.

To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event T anti-4 a kind of to deposit 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 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 T anti-4.

In the above-mentioned technical solutions, the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

Specifically, the primer includes the first primer and the second primer, the first primer be selected from SEQ ID NO:1 or its Complementary series, SEQ ID NO:8 and SEQ ID NO:10；Second primer be selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 and SEQ ID NO:11.

To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event T anti-4 a kind of to deposit Method, 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 T anti-4；

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 further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or it is mutual Complementary series.

Selectively, at least one fluorophor label of at least one described probe.

To achieve the above object, the present invention also provides the DNA of test sample transgenic corn event T anti-4 a kind of to deposit Method, comprising:

Contact sample to be tested with marker nucleic acid molecules, the marker nucleic acid molecules include SEQ ID NO:1 or Its complementary series or SEQ ID NO:2 or its complementary series, the marker nucleic acid molecules are originated from transgenic corn events T Anti- 4；

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 herbicide tolerant and marker nucleic acid molecules are chain on science of heredity.

Further, the marker nucleic acid molecules further include 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 SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series, be can be used as pair There is one of DNA primer of specificity or probe in transgenic corn events T anti-4 or its offspring；The DNA molecular, which is originated from, to be turned Gene corn event T anti-4.

It further, further include SEQ ID NO:6 or its complementary series or SEQ when the DNA molecular is as probe ID NO:7 or its complementary series.

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 upstream, it is referred to as " left margin flank " or " 5 ' flank " or " 5 ' flanking genomic area " Or " 5 ' flanking sequence of genome " etc..When the flanking region is located at downstream, it is referred to as " right margin flank " or " 3 ' sides The wing " or " 3 ' flanking genomic area " or " 3 ' flanking 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 transgenic corn events of referred to as T anti-4 and its offspring, the transgenic corn events T anti-4 As corn plant T anti-4 comprising the Plants and Seeds and its plant cell of transgenic corn events T anti-4 or its is renewable Part, the plant part of the transgenic corn events T anti-4, including but not limited to cell, pollen, ovule, flower, bud, root, Stem, silk, inflorescence, ear fringe, leaf and the product from corn plant T anti-4, such as corn flour, maize flour, corn oil, corn Slurry, corn silk, cornstarch and the biomass for staying in corn crop field.

Transgenic corn events T anti-4 of the present invention contains a DNA construct, when it is expressed in plant cell, The transgenic corn events T anti-4 obtains the tolerance to glyphosate herbicidal.The DNA construct includes an expression Box, expression cassette includes the suitable promoter and suitable polyadenylation signal sequence for expressing in plant, described Promoter, which is operably connected, encodes the gene of 5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS), the EPSPS egg White nucleic acid sequence has tolerance to glyphosate herbicidal.Further, the promoter can be suitable for what is separated from plant Promoter is closed, including composing type, induction type and/or tissue-specific promoter, the suitable promoter include but is not limited to, flower Cauliflower mosaic virus (CaMV) 35S promoter, figwort mosaic virus (FMV) 35S promoter, ubiquitin protein (Ubiquitin) open Mover, actin (Actin) promoter, soil Agrobacterium (Agrobacterium tumefaciens) rouge alkali synthetase (NOS) promoter, octopine synthase (OCS) promoter, Cestrum (Cestrum) yellow leaf curl virus promoter, Ma Ling Potato wedge stem storage protein (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 promoter and Arabidopsis (Arabidopsis thaliana) Suc2 starting Son.The polyadenylation signal sequence can be the suitable polyadenylation signal sequence to work in plant, institute Stating suitable polyadenylation signal sequence includes but is not limited to, and derives from soil Agrobacterium (Agrobacterium Tumefaciens) polyadenylation signal sequence of rouge alkali synthetase (NOS) gene, derive from cauliflower mosaic virus (CaMV) 35S terminator, the polyadenylation signal sequence from protease inhibitors II (PIN II) gene and source In the polyadenylation signal sequence of alpha-tubulin (α-tubulin) gene.

In addition, the expression cassette can also include other genetic elements, the genetic elements include but is not limited to 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 EPSPS Protein transport to extracellular or intracellular specific organelle or compartment, for example, utilizing encoding chloroplast transit Peptide sequence targets chloroplaset, or utilizes ' KDEL ' to retain sequence and target endoplasmic reticulum.

5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) gene can be from soil Agrobacterium In (Agrobacterium tumefaciens sp.) CP4 bacterial strain or from Pseudomonas Pseudomonas (pseudomonas Fluorescens) isolated in G2 bacterial strain, and coding can be changed by optimization codon or in other ways The polynucleotides of EPSPS gene, to achieve the purpose that increase the stability and utilizability of transcript in transformed cells.The 5- Enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) gene can also be used as selected marker.

" glyphosate " refers to the salt of N- phosphonomethylglycine and it, is handled with " glyphosate herbicidal " and refers to 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 T anti-4, raw by the weeds in the field are controlled It is long, and the growth or yield of the vegetable material from transgenic corn events T anti-4 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 sequence of insertion DNA Column, which, which is expected, is transferred in filial generation, the filial generation by containing insertion DNA parental department (such as original transformant and its 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 is received comprising mesh Mark the insertion DNA of gene.

" recombination " refers to the DNA that generally can not be found and therefore generate by manual intervention in nature in the present 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.

The transgenic corn events T anti-4 that there is tolerance to glyphosate herbicidal is cultivated, passes through following steps: making first First parental corn plants and the second parental corn plants sexual hybridization, to produce the first generation progeny plant of multiplicity, institute It states the first parental corn plants to be made of the corn plant of cultivation transgenic corn event T anti-4 and its offspring, the transgenosis Corn event T anti-4 and its offspring are by there is the expression cassette of tolerance to turn glyphosate herbicidal using of the invention Obtained from change, the second parental corn plants, which lack, has tolerance to glyphosate herbicidal；Then it selects to Gyphosate herbicice Agent has the progeny plant of tolerance, can cultivate the corn plant for having tolerance to glyphosate herbicidal.These steps May further include the progeny plant for making glyphosate tolerant and the second parental corn plants or third parental corn plants into Row backcrossing, then by being applied with glyphosate herbicidal or by molecular marked compound relevant to character (such as comprising transgenosis jade The DNA molecular of the bond site that the 5 ' ends and 3 ' ends of insetion sequence identify in rice event T anti-4) identification select filial generation, To generate the corn plant 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 anti-4 genome of transgenic corn events T, no matter the genome DNA be also be derived from from transgenic corn events T anti-4 or seed transgenic corn events T anti-4 plant or seed or Extract.Probe of the invention not only includes DNA or ribonucleic acid, further include specifically with target dna sequence In conjunction with 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 T anti-4, and determine the DNA The nucleic acid sequence of molecule.The DNA molecular includes transgene insert sequence and Maize genome flank region, the DNA molecular Segment 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 T anti-4.Nucleic acid molecules Or its segment can carry out specific hybrid with other nucleic acid molecules in any case.As the present invention uses, if two 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 specificity to each other Hybridization.If two nucleic acid molecules show complete complementarity, claiming one of nucleic acid molecules is another nucleic acid molecules " complement ".As the present invention uses, when pair of each nucleotide and another nucleic acid molecules of a nucleic acid molecules The nucleotide mutual added time is answered, then the two nucleic acid molecules is claimed to show " complete complementarity ".If two nucleic acid molecules can be with foot Enough stability phase mutual crosses then claim this to make them anneal and be bonded to each other under the conditions of at least conventional " low stringent " Two nucleic acid molecules are " minimum level is complementary ".Similarly, if two nucleic acid molecules can be mutually miscellaneous with enough stability It hands over that them is made to anneal and be bonded to each other under the conditions of conventional " height is stringent ", then the two nucleic acid molecules is claimed to have " mutually Benefit property ".Deviateing from complete complementarity can permit, as long as this deviation not exclusively prevents two molecules from forming double-strand knot 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, with 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 Any segment of SEQ ID NO:7 or its complementary series or above-mentioned sequence is with 80% to 100% or 90% to 100% Sequence identity.SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:6 and SEQ ID NO:7 may be used as plant breeding side Marker in method is to identify the offspring of genetic cross.Probe can be this by any one with hybridizing for target dna molecule Method known to the technical staff of field detects, these methods include but is not limited to that fluorescent marker, resists radioactive label Body 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 T of the present invention Anti- 4 are generated by sexual hybridization mode, or whether the corn sample acquired from field includes transgenic corn events T anti-4, or Whether corn extract, such as coarse powder, powder or oil include transgenic corn events T anti-4, from corn plant tissue sample or are mentioned The DNA for taking object to extract can generate anti-for transgenic corn events T 4 by using the nucleic acid amplification method of primer pair The presence of DNA is diagnostic amplicon.The primer pair includes an exogenous DNA in Plant Genome with insertion The first primer of the adjacent flanking sequence of insertion point, and the second primer of the exogenous DNA from insertion.Amplicon has one Measured length and sequence, the sequence are also diagnostic to the transgenic corn events T anti-4.

The length range of amplicon can be the combination length of primer pair plus a nucleotide base pair, preferably plus about 50 nucleotide bases pair more preferably add about 250 nucleotide bases pair, most preferably add about 450 Nucleotide 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 T anti-4 can be with It is expanded by the genome using provided primer sequence anti-to transgenic corn events T 4, to PCR amplification after amplification Son 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/genomic region with transgenic corn events T anti-4 to being SEQ ID NO:8 and SEQ ID NO:9 A part of homologous diagnostic amplicon, wherein amplicon includes SEQ ID NO:1.Other DNA moleculars as DNA primer It 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 Then and DNA PCR product (primer is respectively used in insetion sequence and in adjacent flanking genomic sequence) is hybridized, Polymerase, ATP, sulfonyl enzyme, luciferase, apyrase, adenosine -5 '-phosphorus sulfate and luciferin are together It is incubated.It is separately added into dNTPs, measures the optical signal of generation.Optical signal represents the presence of insertion/flanking sequence, says Bright amplification, hybridization and single base or polybase base extension are successful.

Fluorescence polarization also can be used for detecting amplicon of the present invention a kind of method (Chen X, Levine L, and Kwok PY.Fluorescence polarization in homogeneous nucleic acid analysis [J] .Genome Res, 1999,9 (5): 492-8.).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 of the vegetable material from transgenic corn events T anti-4 also It may include Southern blot hybridization, Northern blot hybridization and in situ hybridization.Particularly, the suitable technology includes temperature Probe and sample are educated, is washed to remove whether unbonded probe and detection probe have hybridized.The detection method depends on The type of the label appended by probe for example, can detecte radiolabeled probe by X-ray exposure and imaging, or passes through Substrate conversion realizes that color change can detecte the probe of enzyme label.

(Tyagi S and Kramer F R.Molecular can also be detected to sequence using molecular labeling Beacons:probes that fluoresce upon hybridization [J] .Nat Biotechnol, 1996,14 (3): 303-8.).One is designed to visit across the FRET oligonucleotides of insertion DNA sequence dna and adjacent flanking genomic binding site Needle.The unique texture of the FRET probe causes it to contain secondary structure, which can keep fluorescence portion in short distance Point and quencher moieties.The FRET probe and PCR primer (respectively use one in insetion sequence and in adjacent flanking genomic sequence A primer) in the presence of heat-stabilised poly synthase and dNTPs carry out circular response.By successful PCR amplification, FRET probe and mesh The hybridization of mark sequence leads to the forfeiture of probe secondary structure, thus separate fluorescence part and quencher moieties spatially, Generate fluorescence signal.The generation of fluorescence signal represents the presence of insertion/flanking sequence, illustrates amplification and hybridization is success 's.

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 molecular 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 T anti-4 in sample, can also use In the corn plant for cultivating the DNA containing transgenic corn events T anti-4.The kit can contain DNA primer or probe, It is 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 probe, same It is derived from or is complementary to DNA contained in the genetically modified element of DNA, these DNA sequence dnas can be used for DNA amplification reaction, or As the probe in DNA hybridization method.Transgenosis insertion that is containing in the corn genome and illustrating in Fig. 1 and table 1 Sequence and the DNA structure of Maize genome binding site include: being located at anti-4 left side corn T of 5 ' end of transgene insert sequence Wing genome area, a part of insetion sequence of the left boundary area (LB) from Agrobacterium, first expression cassette is by corn Ubiquitin protein gene promoter (ubiquitin promoter) is operably connected to pea rbcS chloroplast transit peptide sequence (SP) on, it is operably connected to the 5- enol-pyrovyl thick grass that Pseudomonas fluorescence belongs to the glyphosate tolerant of G2 bacterial strain On acid -3- phosphate synthase gene (2mG2-epsps), and it is operably connected to rouge alkali synthetase gene terminator (no Terminator it is formed on), a part of insetion sequence in the right side boundary region (RB) from Agrobacterium, and positioned at turning base Because of the anti-4 right side flap genome area of the corn plant T of 3 ' end of insetion sequence (SEQ ID NO:5).In DNA cloning method, DNA molecular as primer can be any part from the anti-4 transgenic insetion sequence of transgenic corn events T, It can be any part from the region of DNA domain of flank Maize genome in transgenic corn events T anti-4.

Transgenic corn events T anti-4 can be combined with other transgenic maize varieties, such as herbicide (such as glufosinate-ammonium, Dicamba etc.) tolerance corn, or carry anti insect gene transgenic maize varieties.All these difference transgenic events Various combinations can provide pest-resistant and resist a variety of herbicides with anti-4 breeding together of transgenic corn events T of the invention Improve hybrid transgenic corn variety.These kinds can be showed compared to the transformed variety of non-transgenic kind and unisexuality shape 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 T anti-4 is the phytotoxic effects for being resistant to the agriculture herbicide containing glyphosate.The plant of the character expresses Pseudomonas 5- enol-pyrovyl shikimic acid -3- phosphate synthase (EPSPS) albumen of the glyphosate resistance of Bacillus genus strain G2 is assigned and being planted Tolerance of the object to glyphosate.Simultaneously SEQ ID NO:1 or its complementary series in detection method, 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 can be used as DNA primer Or probe to be to generate the amplified production for being diagnosed as transgenic corn events T anti-4 or its offspring, and can quickly, it is accurate, stablize The presence for identifying the vegetable material from transgenic corn events T anti-4.

BRIEF DESCRIPTION OF THE SEQUENCES:

In SEQ ID NO:1 transgenic corn events T anti-4 5 ' transgenic insert locus left side flap corn gene group DNAs and Each 11 nucleotide sequences of transgenic fragment left margin；

3 ' transgenic insert locus transgenic fragment right margins and the right side in SEQ ID NO:2 transgenic corn events T anti-4 Each 11 nucleotide sequences of flank corn gene group DNA；

It is attached positioned at insertion junction in 5 ' ends of insetion sequence in SEQ ID NO:3 transgenic corn events T anti-4 A close length is the sequence of 448 nucleotide；

It is attached positioned at insertion junction in 3 ' ends of insetion sequence in SEQ ID NO:4 transgenic corn events T anti-4 A close length is the sequence of 611 nucleotide；

SEQ ID NO:55 ' left side flap maize genomic sequence, entire T-DNA sequence and 3 ' right side flap Maize genome sequences Column；

SEQ ID NO:6 is located at the sequence inside SEQ ID NO:3, span 5 ' left side flap maize genomic sequences, 2mG2-epsps-pC3301 construct left margin DNA sequence dna and ubiquitin promoter sequence；

SEQ ID NO:7 is located at the sequence inside SEQ ID NO:4, spans no transcription terminator, 2mG2- Epsps-pC3301 construct right margin DNA sequence dna and 3 ' right side flap maize genomic sequences；

The first primer of SEQ ID NO:8 amplification SEQ ID NO:6；

The second primer of SEQ ID NO:9 amplification SEQ ID NO:6；

The first primer of SEQ ID NO:10 amplification SEQ ID NO:7；

The second primer of SEQ ID NO:11 amplification SEQ ID NO:7；

The first primer of SEQ ID NO:12PCR detection 2mG2-epsps；

The second primer of SEQ ID NO:13PCR detection 2mG2-epsps；

The primer of SEQ ID NO:14 acquisition right margin flanking sequence；

The primer of SEQ ID NO:15 acquisition right margin flanking sequence；

The primer of SEQ ID NO:16 acquisition right margin flanking sequence；

The primer of SEQ ID NO:17 acquisition right margin flanking sequence；

Probe in SEQ ID NO:18Southern hybridization check；

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

Detailed description of the invention

The structural schematic diagram that 1 transgene insert sequence of attached drawing and Maize genome engaging portion are stood.

The physical map of 2 recombinant expression carrier 2mG2-epsps-pC3301 of attached drawing.Each element English and abbreviation meaning are enumerated It is as follows:

The T-DNA left margin sequence of T-Border (left) Agrobacterium C58.

The promoter of ubiquitin promoter Maize Ubiquitin gene.

RbcS chloroplast transit peptides in SP pea.

2mG2-epsps derives from the glyphosate tolerance gene of G2 bacterial strain.

The terminator of no terminator rouge alkali synthetase gene.

The T-DNA right border sequence of T-Border (right) Agrobacterium C58.

The plasmid stabilisation site of PVS1 sta pVS1 plasmid.

The replication origin of PVS1 rep pVS1 plasmid.

The site bom of PBR322 bom pBR322 plasmid.

The replication origin of PBR322 ori pBR322 plasmid.

Kanamycin (R) encodes aminoglycoside phosphotransferase albumen, assigns bacterium kalamycin resistance.

The Southern marking of the anti-4 foreign gene insertion copy number of 3 T of attached drawing hybridizes figure.A:SacI digestion；B:BstEII Digestion；The area C:T-DNA restriction enzyme site and probe location schematic diagram, the stripe size after digital representation digestion, unit kb are used Probe location is labeled in lower part.Swimming lane 1:DNA Marker III, DIG-labeled (Roche), stripe size is labeled in side, Unit kb；Swimming lane 2: blank；599 genomic DNA pools of swimming lane 3:2mG2-epsps-pC3301 plasmid and receptor 18；Swimming lane 4: 599 genomic DNA of receptor 18；Anti- 4 T of swimming lane 5:T₆For genomic DNA；Anti- 4 T of swimming lane 6:T₇For genomic DNA.

Attached drawing 4 sprays the variable rate technology of transformation event T anti-4 after glyphosate 4 weeks.A: receptor 18 599 does not spray glyphosate； B: receptor 18 599 sprays glyphosate；C: transformation event T anti-4 does not spray glyphosate；D: transformation event T anti-4 sprays glyphosate.

The testing result of 5 transformation event T anti-4 of attached drawing.A: left margin detection, the expected size 353bp of target stripe；B: right Border detection, the expected size 414bp of target stripe；M: molecular weight standard, be followed successively by from top to bottom 2kb, 1kb, 750bp, 500bp,250bp,100bp；1: water；2: corn material 0151；3: corn material is at single 30；4: corn material T anti-4-0151； 5: corn material 16-1.

Specific embodiment

This application involves transformation event T anti-4 refer to corn inbred line 18599 be receptor obtained by genetic transformation The plant of foreign gene insert (T-DNA insert) is inserted between specific gene group sequence.In a particular embodiment, Transgenosis expression carrier used thereof has attached physical map shown in FIG. 1, and obtained T-DNA insert has SEQ ID NO:5 293-4277 nucleotide shown in sequence.Transformation event T anti-4 can refer to this transgenic protocol, can also refer to by this The combination of T-DNA insert or T-DNA insert and flanking sequence in the obtained genome of process, or can refer to by this The plant that one transgenic protocol obtains.In specific example, which is also applied for same expression vector and converts other Receptor kind, thus the plant that T-DNA insert is inserted into same genomic locations and is obtained.Transformation event T anti-4 may be used also With refer to as above-mentioned plant carry out vegetative propagation, sexual propagation, it is double-diminished or double breeding or above combination obtained from offspring plant Object.

The building of 1 conversion carrier of embodiment

(1) according to the needs of the expressing gene function in plant, one is increased at the end of the gene coding region 2mG2-epsps 5 ' Section chloroplaset leads peptide SP sequence, to guarantee that the EPSPS albumen translated is transported to the synthesizing site chloroplaset of aromatic amino acid It is interior.(2) 2mG2-epsps sequence is synthesized, and introduces BamHI and SacI restriction enzyme site respectively at sequence both ends；SP sequence is cloned, And PstI and BamHI restriction enzyme site is introduced respectively at sequence both ends.

(3) BamHI+SacI handles 2mG2-epsps sequence and PUC19 carrier and connects, and obtains 2mG2-epsps-PUC19 Carrier.PstI+BamHI processing SP sequence and 2mG2-epsps-PUC19 carrier simultaneously connect, and obtain SP-2mG2-epsps-PUC19 Carrier.

(4) HindIII+PstI digestion PAH17 carrier, which obtains ubiquitin promoter and is connected into, uses HindIII+ The SP-2mG2-epsps-PUC19 carrier of PstI processing, obtains Pubi-SP-2mG2-epsps-PUC19 carrier.

(5) AseI+BstEII handle pCABIA3301 carrier and left end be AseI+HindIII, right end SacI+ The DNA fragmentation of BstEII obtains improved pCAMBIA3301 carrier after connection.

(6) HindIII+SacI digestion Pubi-SP-2mG2-epsps-PUC19 carrier and improved pCAMBIA3301 Pubi-SP-am79 epsps is accessed pCAMBIA3301 carrier, obtains the load for being named as 2mG2-epsps-pC3301 by carrier Body.

Resulting vehicle size is 10.261kb, and carrier physical map is shown in attached drawing 1.

The conversion of 2 maize genetic of embodiment

Method used in maize transformation is agrobacterium-mediated transformation, and operation sequence is as follows:

(1) maize ear pollinated 10-13 days is chosen, the rataria (1.5-2.0mm) being of moderate size therefrom is removed:

(2) picking Agrobacterium (containing expression vector) on three days YEP plates is grown from 19 DEG C with oese, is suspended in and infects In culture solution, room temperature, 75rpm, 2-4h, until OD550=0.3-0.5, infects the rataria of removing；

(3) rataria infected, which is transferred to, to be co-cultured on base, and 20 DEG C are cultivated 3 days in the dark；

Rataria is transferred in recovery media after (4) 3 days, 28 DEG C are cultivated 7 days in the dark；

(5) rataria is transferred in screening and culturing medium after renewal cultivation, conversion is primary every two weeks, and therefrom selection growth is rapid Callus；

(6) it is sprouted under the callus light chosen, Molecular Detection determines transgenic plant after growing up to regeneration plant；

The screening of 3 transformant of embodiment

(1) target gene Molecular Detection is carried out for transformation seedlings to 810 T0 that conversion obtains.It is designed according to gene order PCR primer pair, primer sequence are respectively SEQ ID NO:12 and SEQ ID NO:13.Conversion seedling leaf is taken to extract genomic DNA, It is expanded according to following PCR parameter:

Reaction system:

Response procedures:

The amplified fragments that the target gene amplified fragments size of positive transformant compares PCR with positive plasmid are in the same size, It is 499bp, harvests the T1 seed of 13 positive single plants.

(2) in the greenhouse to T1-T3 for plant through herbicide screening, the screenings such as segregation ratio is investigated obtain number be T it is anti-1, T is anti-2, T is anti-3, T is anti-4, T is anti-5,6 strains of T anti-6.

(3) in the greenhouse to T₄-T₆Economical character is further investigated for plant to screen to obtain transformant T anti-4.

In terms of yield traits, T anti-4 has tapered fringe, Hard grain type, yellow grain color and white axis, these are all consistent with 18599. T anti-4 and 18599 is in spike length, prominent point, fringe are thick, in terms of tassel row number, row grain number, 100-grain weight and yield without significant difference (p > 0.05) (table 2).

Anti- 4 yield of 2 T of table and species test character investigation result

LSD method testing significance of difference (α=0.05) is used with column data.

The flanking sequence of anti-4 exogenous array of 4 transformation event T of embodiment and Maize genome insertion position

Design primer (SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, the SEQ ID of Flanking sequence isolation NO:17), the method for expanding and being sequenced using Tail-PCR obtains right side flap sequence (the SEQ ID NO:5 of transformant T anti-4 4278-4601), and by PCR amplification sequencing, (primer sequence is SEQ ID NO:8, SEQ ID according to genome sequence NO:9 left side flap sequence (SEQ ID NO:5 1-292)) is obtained.PlantGDB database (http: // Www.plantgdb.org/ZmGDB/cgi-bin/blastGDB.p1 use BLASTN tool by flanking sequence and corn gene in) Group sequence carries out homologous comparison analysis, using B73 RefGen_V2 as reference sequences.The anti-4 insertion corn of transformant T known to analysis At the position genome C hr01:227594470.

The operating procedure of Tail-PCR is as follows:

1) corn gene group DNA is extracted.

2) template reacted using plant genome DNA as first round PCR, reaction system are as follows:

Response procedures are as follows: 94 DEG C, 5min；(94 DEG C, 30sec；62 DEG C, 2min；72 DEG C, 2.5min) × 5cycles；94 DEG C, 30sec；25 DEG C, 3min；72 DEG C (32%ramp), 3min；(94 DEG C, 30sec；62 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；62 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；45 DEG C, 1min；72 DEG C, 2.5min) × 15cycles；72 DEG C, 7min；20 DEG C, 10min.

It 3) is that template carries out the second wheel PCR amplification with PCR product (mother liquor) corresponding in step 2).Reaction system is as follows:

Response procedures are as follows: 94 DEG C, 5min；(94 DEG C, 30sec；65 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；65 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；45 DEG C, 1min；72 DEG C, 2.5min) × 20cycles；72 DEG C, 7min；20 DEG C, 10min.

It 4) is that template carries out third round PCR amplification with PCR product corresponding in step 3) (30 letter of dilution).Reaction system is such as Under:

Response procedures are as follows: 94 DEG C, 5min；(94 DEG C, 30sec；65 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；65 DEG C, 1min；72 DEG C, 2.5min；94 DEG C, 30sec；45 DEG C, 1min；72 DEG C, 2.5min) × 20cycles；72 DEG C, 7min；20 DEG C, 10min.

5) product of third round PCR in step 4) electrophoresis detection in 1% (w/v) 1 × TAE Ago-Gel is taken, is recycled Meet the DNA fragmentation of purpose size.

6) segment of recycling is connected into carrier T, 16 DEG C of connections overnight.

7) connection product of conversion 6).

8) converted product in amplification 7), and picking positive colony shakes bacterium upgrading grain.

9) plasmid is sent to be sequenced.

The overall length insetion sequence for expanding and being sequenced T anti-4 is further segmented by over-lap PCR.Transformant T anti-4 is practical to be inserted Enter sequence and left and right flank maize genomic sequence as shown in SEQ ID NO:5.

3 Flanking sequence isolation the primer information of table

N=A/T/C/G；V=G/C/A；1: unit bp.

The copy number of 5 transformation event T anti-4 of embodiment detects

The copy number of foreign gene insertion is determined using the method that the Southern marking hybridizes.2mG2-epsps genetic fragment Insertion copy number hybridization check choose two kinds of restriction enzymes of SacI and BstEII and distinguish digestion positive control 2mG2- The mixture of epsps-pC3301 plasmid and 18599 genomic DNA of wild type, 18599 genomic DNA of negative control wild type and T₆、T₇For the anti-4 transformant genomic DNA of T.2mG2-epsps Probe labelling is used after running glue transferring film.Results of hybridization is shown in attached drawing Shown in 4A, B.The probe location of target gene 2mG2-epsps and the restriction enzyme site of restriction enzyme SacI and BstEII are for example attached Shown in Fig. 4 C.

SacI the area T-DNA restriction enzyme site only one, positioned at the right side of 2mG2-epsps gene probe, by digestion Anti- 4 genomic DNA of T and specific probe hybridization after the slug band that obtains should T-DNA sequence including 3.6kb and its The unknown sequence of size on the genome of left side, whole fragment length are greater than 3.6kb, mark single hybridising band in experiment, Size is about 15kb (attached drawing 4A swimming lane 5,6), meets expection.

There are two restriction enzyme sites in the area T-DNA by BstEII, the right side of 2mG2-epsps gene probe are all located at, by digestion The anti-4 transformant genomic DNA of T and specific probe hybridization after the slug band that obtains should include 2.6kb T-DNA sequence The unknown sequence of size on column and its left side genome, whole fragment length are greater than 2.6kb, mark single hybridization in experiment Band, size are about 2.7kb (attached drawing 4B swimming lane 5,6), meet expection.

Meanwhile blank control and the two kinds of digestion with restriction enzyme marks of SacI and BstEII of negative control receptor 18 599 Band (swimming lane 2,4) is not all seen after note；599 genomic DNA of positive control 2mG2-epsps-pC3301 plasmid and receptor 18 Hybridize after mixture SacI and BstEII two kinds of digestion with restriction enzyme label band out and plasmid size 10.3kb or 10.1kb coincide (swimming lane 3), these all show the specificity of hybridization probe.

The experimental results showed that, the 2mG2-epsps genetic fragment singly copied, and T-DNA are contained in the area T-DNA of T anti-4 above Area's unit point is inserted into Maize genome.External source Insert Fragment can stablize heredity by zoogamy between different generations.

The tolerance of the anti-4 pairs of target herbicides of 6 transformation event T of embodiment

Under the natural environment of field, it is sweet to herbicide grass that anti-4 transformant of T is measured by the method for artificial spraying's herbicide The tolerance of phosphine, with the validity of clear transformant herbicide-resistant objective trait.Herbicide used reaches for agriculture, by the Monsanto Far East Company's production, the content of active constituent glyphosate isopropyl amine salt are 41%, and dosage form is aqua.Its field recommended dose is 150- 250mL/ mus, 200mL/ mus (active component content is 82g/ mus) are taken, 30L/ mus is watered and sprays.

4 processing are arranged in test process:

(1) transgenic corns not herbicide spraying；

(2) transgenic corns spray target herbicide；

(3) corresponding non-transgenic corn not herbicide spraying；

(4) corresponding non-transgenic corn sprays target herbicide；

Herbicide sprayed dose be respectively spray measured in clear water (0 ×) and field recommended dose 1 times (1 ×, active constituent 82g/ mus of content), 2 times (2 ×, 164g/ mus of active component content) and 4 times (4 ×, 328g/ mus of active component content).

Character investigation: investigation in 1 week, 2 weeks and 4 weeks and record planting percent, plant height (are chosen highest after medication respectively 5 plants), symptom of chemical damage (choose symptom of chemical damage most light 5 plants).

(1) it if phytotoxicity can be counted or measure, is indicated with absolute figure, such as plant number (spray strain number, dead strain Number) or plant height (choosing highest 5 plants).

(2) it after medication 1 week, defines the level by table 4 to each processing phytotoxicity.

4 herbicide damage Syndrome Scale table of table

The aggrieved rate of herbicide is calculated by formula (1).

In formula:

The aggrieved rate of X-, unit are percentage (%)；

N- aggrieved strain number at the same level；

S- number of levels；

T- total strain number；

M- highest level.

After spraying 0 times of glyphosate solution 1 week, transformant and the control of non-transgenic receptor can grow survival, but miscellaneous Thick grass is raw, and corn growing way is very poor (attached drawing 4A, C).And the glyphosate solution measured in spraying 1 times of field recommended dose is after 1 week, it is non- Transgene receptor compare corn it is all withered together with weeds, dead (attached drawing 4B), the plant of transformant can normal growth, and Since weeds all kill, growing way is more preferable (attached drawing 4D) when corn is than not herbicide spraying.Over time, grass is being sprayed Sweet phosphine solution is after 4 weeks, spray the transformant plant of various dose glyphosate still can normal growth, and under various dose Appearance is the same.

Phytotoxicity investigation result is as shown in table 5, sprays lower plant death in 1 multiple dose of herbicide in control receptor 18 599, does not have There is survival seedling.Transformation event T anti-4 investigates no plant after spraying 1 week and all survives, and aggrieved rate is 0%, and plant height is clear Without significant difference between water process and herbicide treatment；Occur without phytotoxicity and the plant phenomena of mortality within 2 weeks and 4 weeks after medicine, Plant strain growth is not suppressed.

The tolerance sex expression of the anti-4 pairs of glyphosates of 5 T of table

Dosage is indicated with the multiple measured in recommended dose.Numerical value is with the duplicate mean+SD of 3 secondary pollutants It indicates, data use LSD method testing significance of difference (α=0.05) between same column difference sprayed dose.

These results indicate that transformation event T anti-4 has stronger glyphosate tolerant.It is measured at 4 times in recommended dose When reason, phytotoxicity rate is still 0, and tolerance grade reaches outstanding.Therefore, transformation event T anti-4 can protect corn from by weeding Damage caused by agent.Pass through plantation transformation event T anti-4 and spray suitable dosage herbicide can control it is miscellaneous in corn field Grass.

The method that embodiment 7 generates the plant for having tolerance to glyphosate herbicidal using transformation event T anti-4

It finds that the resistance of T anti-4 and other economical characters are all very excellent by pre-stage test, therefore is improved with the event transformation Southwest produces the backbone parent 0151 being widely used, 08-641, G533 etc., to improve the weeding of these backbone parents Agent resistance.Start within 2010 to do first-filial generation, be then returned for 3 generations with receptor parent, then it is more than generation to be selfed 4, material is just substantially steady It is fixed.It is annual to plant for two generations by Sichuan Province, Hainan Province two places shuttle breeding.Every generation is with regard to objective trait --- antiweed Glyphosate is selected under high concentration (4 times of glyphosate recommended dose), selects fine individual plant in conjunction with other economical characters It reserves seed for planting with head progeny row, until character stablizes purifying.

In addition, the stable self-mating system of anti-4 transformation of T can also assemble cross combination.Transgenic corns Combination nova 16-1 be with Inbred Lines P9325 be it is maternal with using backbone parent 0151 be background and parent comprising transformation event T anti-4 as male parent in It matches within 2015；Corn Combination nova 16-1 participates in corn Combination nova variety comparative test in 2016, average product 570.68kg/ Mu, relatively control are at single 30 volume increase 9.2%.

It not only can 4 progress anti-to T during Parent improvement and cross combination assemble using method provided by the invention Whether detection, provides molecule supplementary means with the breeding for kind, can also be used in identification corn variety containing anti-4 conversion of T Event.Following methods are the specific example for identifying T anti-4.

Self-mating system the T anti-4-0151 and transgenic corns Combination nova 16-1 obtained is improved to parent 0151 carries out molecule inspection It surveys, to confirm in the two materials comprising transformation event T anti-4.PCR primer pair is designed according to gene order, is converted by detection The presence on two boundaries of event or so is to determine the presence of transformation event T anti-4.The wherein primer sequence difference of left margin detection Primer sequence for SEQ ID NO:8 and SEQ ID NO:9, left margin detection is respectively SEQ ID NO:10 and SEQ ID NO: 11.It takes plant blade to extract genomic DNA, is expanded according to following PCR parameter:

PCR reaction system:

Response procedures are as follows:

94 DEG C, 5min；(94 DEG C, 30sec；55 DEG C, 30sec；72 DEG C, 1.0min) × 35 circulations；72 DEG C, 7min；4 DEG C, 5min。

Take PCR product electrophoresis detection in 1% (w/v) 1 × TAE Ago-Gel.

The results are shown in attached figure 5.The sample of T anti-4-0151 and 16-1 can amplify corresponding target stripe, and have same Background but band cannot be amplified without containing the corn sample of anti-4 event of T, this proves that anti-4 event of T succeed transformation to close It also include anti-4 event of T with its cross combination 16-1 prepared in sheet 0151.

The herbicide tolerant of 4-0151 anti-to T and 16-1 identifies that identification method is referring to embodiment 7, qualification result It is shown in Table 6.Herbicide will not 4-0151 and 16-1 material anti-to T damage, but will lead to the control material without T anti-4 0151 and dead at single 30, it can be shown that and can produce the jade that there is tolerance to glyphosate herbicidal using transformation event T anti-4 Rice plant.

Tolerance sex expression of 6 T of the table anti-4-0151 and 16-1 to glyphosate

Dosage is 4 times measured in recommended dose.Numerical value indicates with the duplicate mean+SD of 3 secondary pollutants, LSD method testing significance of difference (α=0.05) is used with column data.

The detection method of 8 transformation event T anti-4 of embodiment

It can be by transgenic corn events T anti-4 production such as agricultural product or commodity.If in the agricultural product or commodity Detect enough expression quantity, agricultural product or commodity are expected exists containing can diagnose anti-4 material of transgenic corn events T Nucleotide sequence present in the agricultural product or commodity.The agricultural product or commodity include but is not limited to corn oil, Corn Crude Powder, maize flour, corn gluten, corn-dodger, cornstarch and will be as food source for any other food of animal consumption Product or additionally as the ingredient in swelling agent or make-up composition for cosmetic use etc..Core based on probe or primer pair Sour detection method and/or kit can be developed to detect such as SEQ ID NO:1 or SEQ ID NO:2 institute in biological sample Anti- 4 nucleotide sequence of the transgenic corn events T shown, wherein probe sequence or primer sequence are selected from such as SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, shown in SEQ ID NO:6 and SEQ ID NO:7 Sequence, to diagnose the presence of transgenic corn events T anti-4.

In conclusion the anti-4 pairs of glyphosate herbicidals of transgenic corn events T of the present invention tolerance with higher, and examine Survey method can quickly and accurately identify in biological sample whether include transgenic corn events T anti-4 DNA molecular.

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 (10)

1. a kind of method existing for DNA of test sample transgenic corn event T anti-4 characterized by 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 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 T anti-4.

2. method existing for the DNA of test sample transgenic corn event T anti-4, feature exist according to claim 1 In the amplified production further includes SEQ ID NO:6 or its complementary series, and/or SEQ ID NO:7 or its complementary series.

3. method existing for the DNA of test sample transgenic corn event T anti-4 according to claim 1 or claim 2, feature Be, the primer includes the first primer and the second primer, the first primer be selected from SEQ ID NO:1 or its complementary series, SEQ ID NO:8 and SEQ ID NO:10；Second primer is selected from SEQ ID NO:2 or its complementary series, SEQ ID NO:9 With SEQ ID NO:11.

4. a kind of method existing for DNA of test sample transgenic corn event T anti-4 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 T anti-4；

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.

5. method existing for the DNA of test sample transgenic corn event T anti-4, feature exist according to claim 4 In the probe further includes SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary series.

6. method existing for the DNA of test sample transgenic corn event T anti-4 according to claim 4 or 5, feature It is, at least one fluorophor label of at least one described probe.

7. a kind of method existing for DNA of test sample transgenic corn event T anti-4 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 T anti-4；

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 pass through marker assistant breeding point Analysis is to determine that herbicide tolerant and marker nucleic acid molecules are chain on science of heredity.

8. method existing for the DNA of test sample transgenic corn event T anti-4, feature exist according to claim 7 In the marker nucleic acid molecules further include SEQ ID NO:6 or its complementary series or SEQ ID NO:7 or its complementary sequence Column.

9. 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 anti-for transgenic corn events T 4 or its offspring there is one of DNA primer or probe of specificity；The DNA molecular is originated from transgenic corn events T anti-4.

10. DNA detection kit according to claim 9, 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.