CN110184276A - A kind of method for creating of Transgenic Resistant Herbicide corn event - Google Patents
A kind of method for creating of Transgenic Resistant Herbicide corn event Download PDFInfo
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- CN110184276A CN110184276A CN201910382625.1A CN201910382625A CN110184276A CN 110184276 A CN110184276 A CN 110184276A CN 201910382625 A CN201910382625 A CN 201910382625A CN 110184276 A CN110184276 A CN 110184276A
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G22/00—Cultivation of specific crops or plants not otherwise provided for
- A01G22/20—Cereals
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G7/00—Botany in general
- A01G7/06—Treatment of growing trees or plants, e.g. for preventing decay of wood, for tingeing flowers or wood, for prolonging the life of plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8274—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for herbicide resistance
- C12N15/8275—Glyphosate
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- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- Environmental Sciences (AREA)
- Forests & Forestry (AREA)
- Ecology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biodiversity & Conservation Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Cell Biology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The present invention relates to a kind of method for creating of Transgenic Resistant Herbicide corn event, the nucleic acid sequence of the transgenic corn events includes SEQ ID NO:1 or its complementary series or SEQ ID NO:2 or its complementary series.The anti-4 pairs of glyphosate herbicidals of corn event T in the present invention have preferable tolerance.
Description
Technical field
The present invention relates to plant biotechnology fields.Specifically, it is related to a kind of initiative of Transgenic Resistant Herbicide corn
Method and its nucleotide sequence.
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.
Summary of the invention
The object of the present invention is to provide a kind of Transgenic Resistant Herbicide corn event, the transgenic corn events T anti-4
There is preferable tolerance to glyphosate herbicidal.
To achieve the above object, the present invention provides a kind of nucleic acid sequence, the nucleic acid sequence include SEQ ID NO:1 or
Its complementary series, and/or SEQ ID NO:2 or its complementary series, the nucleic acid sequence are originated from transgenic corn events T anti-4.
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 in transgenic corn events T anti-4 in 5 ' ends of insetion sequence
A length near insertion junction is the sequence of 22 nucleotide, the SEQ ID NO:1 or its complementary series
The DNA sequence dna for spanning the left side flap genomic dna sequence of corn insertion point and the 5 ' end of left margin of insetion sequence includes
The SEQ ID NO:1 or its complementary series can be accredited as the presence of transgenic corn events T anti-4.The SEQ ID NO:
2 or its complementary series be transgenic corn events T anti-4 in 3 ' ends of insetion sequence be located at insertion junction near
One length is the sequence of 22 nucleotide, and the SEQ ID NO:2 or its complementary series span the right margin of insetion sequence
The DNA sequence dna of 3 ' ends and the right side flap genomic dna sequence of corn insertion point, include the SEQ ID NO:2 or it is mutual
Complementary series can be accredited as the presence of transgenic corn events T anti-4.
The SEQ ID NO:5 or its complementary series are that the length of characterization transgenic corn events T anti-4 is 4601 cores
The sequence of thuja acid, the genome and genetic elements for specifically including are as shown in table 1.Include the SEQ ID NO:5 or its complementation
Sequence can be accredited as the presence of transgenic corn events T anti-4.
The genome and genetic elements that 1 SEQ ID NO:5 of table includes
1: unit bp.
The present invention also provides a kind of preparation methods of transgenic corns antiweed corn, and its step are as follows:
A) conversion carrier containing sequence shown in the 3994244th nucleotide of SEQ ID NO:5 is constructed；
B) carrier is transformed into maize immature embryos using agrobcterium-mediated transformation, is selected after recovered culture
Callus grows up to new transgenic corn plant；
C) target gene Molecular Detection.PCR primer pair is designed according to gene order, primer sequence is respectively SEQ ID NO:
12 and SEQ ID NO:13 chooses transgenic corns blade and carries out target gene amplification, and retains the plant of the testing result positive；
D) transgenic corn plant screens herbicide tolerant；
E) transformant target gene copy number detects, and retains single copy plant；
F) separation of transformant flank and verifying.
In the method for present invention initiative transgenic corns, defined below and method can preferably define the present invention and refer to
It leads those skilled in the art and implements the present invention, unless otherwise mentioned, according to the routine of those of ordinary skill in the art
Usage understands 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.
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.
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.
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 P Y.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.
The present invention provides a kind of method for creating of Transgenic Resistant Herbicide corn, the BRIEF DESCRIPTION OF THE SEQUENCES being directed to is as follows:
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；
5 ' left side flap maize genomic sequence of SEQ ID NO:5, entire T-DNA sequence and 3 ' right side flap Maize genomes
Sequence；
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:12 PCR detection 2mG2-epsps；
The second primer of SEQ ID NO:13 PCR 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:18 Southern 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 of attached drawing 1 transgene insert sequence and Maize genome binding site.
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.pl use BLASTN tool by flanking sequence and corn gene in)
Group sequence carries out homologous comparison analysis, using B73RefGen_V2 as reference sequences.The anti-4 insertion corn-based of transformant T known to analysis
At group position Chr01:227594470.
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 times 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 detection method of 7 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 (4)
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 are originated from transgenic corn events T anti-4.
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 preparation method of transgenic corns antiweed corn event, its step are as follows:
A) conversion carrier containing sequence shown in SEQ ID 399-4244 nucleotide of NO:5 is constructed；
B) carrier is transformed into maize immature embryos using agrobcterium-mediated transformation, selects callus after recovered culture
Grow up to new transgenic corn plant；
C) target gene Molecular Detection.According to gene order design PCR primer pair, primer sequence be respectively SEQ ID NO:12 and
SEQ ID NO:13 chooses transgenic corns blade and carries out target gene amplification, and retains the plant of the testing result positive；
D) transgenic corn plant screens herbicide tolerant；
E) transformant target gene copy number detects, and retains single copy plant；
F) separation of transformant flank and verifying.
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| CN114015682B (en) * | 2021-11-16 | 2022-10-21 | 四川省农业科学院生物技术核技术研究所 | Specific probe, primer, kit and method for identifying nucleic acid sample |
Also Published As
| Publication number | Publication date |
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| CN110195122A (en) | 2019-09-03 |
| CN110881367A (en) | 2020-03-17 |
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