CN104762279A - Fixed-point knockout system for Bel gene of paddy rice and application thereof - Google Patents

Abstract

The invention discloses a fixed-point knockout system for the Bel gene of paddy rice and application thereof, specifically a fixed-point knockout method for the bentazone gene of paddy rice and application thereof, belonging to the field of plant genetic engineering. The invention provides a TALEN site-specific mutagenesis system preferably directed at plant genomic DNA. The TALEN site-specific mutagenesis system includes four recognition modules. Compared with conventional bentazone-sensitive plant creating technology, the TALEN site-specific mutagenesis system provided by the invention has the advantages of convenience and rapidness; with the TALEN site-specific mutagenesis system, one scientific research personnel can acquire a plurality of homozygous mutant plants with different mutation sites in one and a half years, so labor and material resources are substantially saved; obtained mutants do not contain transgenic components, so misgivings of people about transgene are eliminated; and the TALEN site-specific mutagenesis system is of great significance to creation of novel germplasm resources through plant genetic engineering.

Description

A kind of fixed point of paddy rice Bel gene knocks out system and application thereof

Technical field

The invention belongs to plant genetic engineering field, be specifically related to a kind of fixed point knockout technique and application thereof of paddy rice bentazone gene.

Background technology

The selective herbicide that wild rice is applied mainly contains two classes, one class is diazosulfide class (benzothiadiazole) weedicide, as bentazone, its effective constituent absorbs by the root of crop, leaf, it kills except effect to the most dicotyledons beyond pulse family and sedge weed, harmless to grass.The mechanisms of bentazone suppresses the Hill reaction in photosynthesis of plant.

TALENs (transcriptional activation increment effector nuclease) is the gene site-directed modification new technology occurred in recent years, has investigation and application widely in fields such as animal, plant and the mankind.Transcriptional activation increment effector (TranscriptionActivator-Like Protein Effector, TALE) be a kind of toxic protein that xanthomonas pathogenic bacteria is secreted in host plant cell, can DNA of plants be identified, drive genetic expression.TALENs is exactly the nuclease utilizing the DNA binding domains of TALE and the DNA cutting structure territory synthetic of Fok I.Two TALEN monomers are combined on DNA double chain according to certain way, DNA cuts off by the dimer being formed with nicking activity, produce DNA double splitting of chain (DSB), cell starts repair mechanism, can site-directed point mutation be produced by this coarse repair mode of non-homologous end joining, accurate gene site-directed insertion or gene replacement can be realized by homologous recombination (HR) mode reparation.The advantages such as compare Zinc finger nuclease (ZFNs) and Meganucleases technology, TALENs technology has simplicity of design, and the high and cytotoxicity of mutation efficiency is low.The superiority of TALENs depends on the DNA evident characteristics of TALE uniqueness.The DNA binding domains of TALEN contains one and does not wait (the tandem repeat domains that the repeating unit of (1.5-33.5) forms by quantity.Each repeating unit is made up of 33-35 amino acid usually, the amino acid high conservative of repeating unit, but the 12nd of unit the and 13 two adjacent amino acids are variable, these two amino acid are commonly called and repeat variable pair of residue (Repeat-variable diresidue, RVD).There is simple corresponding relation in each RVD and Nucleotide A, T, C, G, modal recognition code is that NI identification identifies in conjunction with C, NG identification in conjunction with T in conjunction with A, HD, and NN identifies in conjunction with G (Moscou and Bogdanove., 2009).

General cultivation Resistant Herbicide Crops is mainly through following two kinds of methods: one is by physics and chemistry mutagenesis, obtains the mutant of Resistant Herbicide Crops; Two is by DNA recombinant technology, is imported by anti-herbicide gene in existing species, creates the novel material of antiweed.Because the method targeting of physics and chemistry mutagenesis is not strong, suddenly change a gene, needs process great many of experiments material, for ensureing the mutant obtaining goal gene, needing to do a large amount of screening operation, and easily carrying oneself unwanted mutator gene; There is the situation of foreign transgenes in DNA recombinant technology, in breeding time or Biosafety evaluation, there is certain problem.TALEN technology has accurately, fast, convenient and can not with the mutant of foreign gene, and mutant specy enriches, and adopting TALEN technology to obtain antiweed material will become a kind of technology trends.

Summary of the invention

The invention provides a kind of TALEN rite-directed mutagenesis system preferring to plant genome DNA, the nucleotide sequence of four identification modules that described TALEN rite-directed mutagenesis system comprises is as follows respectively: identification in conjunction with the nucleotide sequence of the NI module of base A as shown in SEQ ID NO:13; Identify in conjunction with the nucleotide sequence of the HD module of base C as shown in SEQ ID NO:14; Identify in conjunction with the nucleotide sequence of the NG module of base T as shown in SEQ ID NO:15; Identify in conjunction with the nucleotide sequence of the NN module of bases G as shown in SEQ ID NO:16.

Present invention also offers a kind of method of acquisition bentazone sensitive plant newly, be to provide a kind of in monocotyledons more specifically, in paddy rice, preferably formulate the method to the responsive plant of bentazone.Present method is using the nucleotide sequence of first exon initiator codon back of paddy rice Bel gene as target sequence, the nucleotide sequence of described target sequence is as shown in SEQ ID NO:10, wherein the sequence of the 17th to the 34th of SEQ ID NO:10 is intervening sequence, and both sides are the Module recognition sequence (respectively called after L and R) of TALENs.In the present invention, the L for this target sequence holds the TALEN-L designed to comprise TALE gene and Fok1 fusion rotein, and its nucleotide sequence is as shown in SEQ ID NO:6; R for this target sequence holds the TALEN-R designed to comprise TALE gene and Fok1 fusion rotein, and its nucleotide sequence is as shown in SEQ ID NO:8.The nucleotide sequence of this TALEN-L and TALEN-R is building up to same expression vector, under the driving of composition type expression promoter, is transferred to after in paddy rice, effectively can obtain Bel gene by the responsive plant of bentazone suddenlyd change.Achieve the actual demand that paddy rice Bel gene is practiced shooting accurately and efficiently.

Present invention also offers a kind of plant expression constructs, described construct contains SEQ ID NO:6 and/or the nucleotide sequence shown in SEQ ID NO:8.More specifically, the TALEN-L of described construct starts expression by Ubiquitin promotor, and TALEN-R is expressed by 35s promoters driven.

The mutant material screening means that current TALEN method obtains mainly contain: Phenotypic Observation, gene sequencing checking etc.Material for the bad observation of phenotype can only carry out gene sequencing, because progeny material is more, causes workload huge, cost up.Specific to bentazone sensitive mutant, phenotype is easily observed, but has phenotype plant can be dead gradually and can not receive seed, so can not carry out the screening of bentazone susceptibility in T0 generation, and order-checking exists mosaic and is not easy order-checking, and program is loaded down with trivial details, high in cost of production problem.

For solving the problem, present invention also offers a kind of method of screening this type of mutant, particularly for transforming the transfer-gen plant obtained, screening mutant plants by the method for high resolving power solubility curve (HRM).HRM (HighResolution Melt), Chinese is translated into " high resolving power melting " is the up-to-date SNP and mutation research instrument that rose in the world in recent years.This detection method is not subject to the limitation of mutating alkali yl site and type, without the need to sequence-specific probes, directly runs high resolving power and melts, can complete the analysis to sample genotype after PCR terminates.The invention provides a species specific HRM primer Ben-F for detecting paddy rice Bel transgenation situation and Ben-R, its nucleotide sequence is as shown in SEQ ID NO:11 and SEQ ID NO:12.

The present invention realizes successively through the following steps:

(1) Preference of monocotyledons codon and the nucleotide sequence of paddy rice Bel gene order to TALEN are transformed, this sequence, as shown in SEQ ID NO:6 in sequence table and SEQ ID NO:8, hands over precious biotechnology (Dalian) company limited to synthesize by improved nucleotide sequence;

(2) the driving expression of Pubi and P35S promotor downstream is connected to by synthesizing a pair TALEN nucleotide fragments obtained; Pubi and P35S promotor nucleotide sequence is respectively as shown in SEQ ID NO:5 in sequence table and SEQ ID NO:2.

(3) conversion of plant, particularly, preferred rice transformation;

(4) with the T0 obtained for transgenic paddy rice genome for template, carry out HRM analysis.Design one couple of PCR primers particularly, product is made to comprise Bel gene target sequence fragment, PCR primer length is about 120bp, solubility curve is obtained with Lightscanner (USIdaho) scanning, by comparing transfer-gen plant and WT lines PCR primer melting temperature difference screens possible mutant plant, utilize this method can a large amount of screening transgenic plant of fast and easy;

(5) by plant Bel transgenation situation that sequence verification screens.Screen for checking the mutant plants Bel gene obtained whether to suddenly change and how to suddenly change, by the DNA fragmentation of pcr amplification with target sequence, its real sequence of sequence verification.

(6) Bentazon herbicide sensitivity experiments is carried out to the mutant screened.Screening the Bel transgenation strain that obtains and WT lines is planted in same flowerpot simultaneously, it is new that plant to be planted grows to 3 leaves one, sprays with the bentazone of 1200mg/L, and one week " Invest, Then Investigate " plant leaves dries up situation, and take pictures (Fig. 4).

" promotor " of the present invention is one section of DNA sequence dna being positioned at that structure gene 5' holds upstream, can activate RNA polymerase, make it combine exactly with template DNA and have the specificity of transcription initiation.

" nucleotide sequence " of the present invention is putting in order of nucleic acid (DNA and RNA) nucleotide.In many situations, nucleotide sequence determines higher structure and the biological function of nucleic acid, and namely different sequence has different higher structures and different biological functions.

" plant expression vector " of the present invention refer to well known in the prior art, can in vegetable cell any one carrier of constant expression alien gene, as pCAMBIA1300, pBI121 etc.

" conversion " of the present invention refer to well known in the prior art, can by any one methods for plant transformation of exogenous gene transfered plant cell or plant tissue, as agrobacterium-mediated transformation and particle gun etc.

" transgenic plant " of the present invention refer to the plant individual being integrated with foreign gene obtained by gene transfer technique.In usual conversion of plant or transgenic plant genome, the stable nucleotide sequence with foreign gene, stably can entail the next generation by this exogenous nucleotide sequence.

The present invention is compared with the technology of existing establishment bentazone sensitive plant, it is advantageous that easily and fast, scientific research personnel the Mutants homozygous plant in multiple different mutational site can be obtained within the time of a year and a half by this technology, greatly save human and material resources, and the mutant obtained is not with transgene component, eliminate people to genetically modified misgivings, new germ plasm resource formulated to plant genetic engineering significant.

Accompanying drawing explanation

The TALEN target sequence location that Fig. 1 paddy rice Bel gene order and this patent relate to, Exon 1 and Exon 2 be respectively paddy rice Bel gene two exons 1s and 2, target sequence L and target sequence R be respectively TALEN albumen in conjunction with target site.

Fig. 2 is used for the carrier pOsBel schematic diagram of rice transformation.LB and RB is respectively left margin and the right margin of T-DNA; P35S is the constitutive promoter 35S promoter coming from tobacco mosaic virus (TMV), and Hyg is the hygromycin gene of synthetic; T35S is certainly in the 35S gene terminator of tobacco mosaic virus (TMV); Pubi represents the promotor of Maize Ubiquitin gene, and TALEN-L is the TALEN-L gene coding region from bacterium of synthetic, and Trbcs is the terminator coming from Arabidopis thaliana Rbcs gene; TALEN-R is the TALEN-R gene coding region coming from bacterium of synthetic, and Tnos represents the terminator of rouge alkali synthetase (no) gene.

Fig. 3 is that HRM detects transgenic paddy rice and wild rice Bel gene melting temperature(Tm) peak figure comparison diagram; That WT indicates is the peak figure that unwinds of wild rice Bel gene, 24 and Mutants indicates is the peak figure that unwinds after Bel gene is undergone mutation.

The mutant plants that Fig. 4 obtains and WT lines are analyzed bentazone class sensitivity to herbicides, left side is WT lines, grows normal, and right side is the plant that HRM screens the Bel gene that obtains and undergos mutation, show as bentazone sensitivity (blade dries up, and growth slows down).

Embodiment

The sequence of embodiment 1, paddy gene Bel and analysis

The sequence of paddy gene Bel is as shown in sequence 1.Sequential analysis shows, and this gene comprises 2 exons altogether, is respectively the 41-964 position (First Exon) of sequence 1,1691-2308 position (Second Exon).A kind of cytochrome p450 protein of this genes encoding.

The present invention is respectively with the target sequence of the sequence on First Exon for transcriptional activation increment effector nuclease (TALENs).(Fig. 1)

The structure of embodiment 2, TALENs design and recombinant expression vector thereof

One, the selection of TALENs target sequence

The rear edge sequence of the initiator codon of first exon of target sequence target paddy gene Bel, sequence is as follows: 5'-CAGCTTAGCCATGGAT aacgcctacattattgcc ATTCTCTCTGTAGCT-3'(is specifically as shown in SEQ ID NO:10) lowercase is wherein intervening sequence, both sides capitalization is TALENs Module recognition sequence (respectively called after L and R).

Two, the Design and synthesis of TALENs encoding gene

By each element of assembling talen expression cassette, carry out the pOsBel carrier shown in design of graphics 1.This carrier contains 3 expression cassettes, wherein, first expression cassette is hygromycin gene expression cassette, consist of the constitutive promoter 35S promoter P35S from tobacco mosaic virus (TMV), its nucleotide sequence is as shown in SEQ ID NO:2 in sequence table, the hygromycin gene Hyp of synthetic, its nucleotide sequence is as shown in SEQ ID NO:3 in sequence table, and from the terminator T35S of tobacco mosaic virus (TMV), its nucleotide sequence, as shown in SEQ ID NO:4 in sequence table, forms the first expression cassette 35S::Hyp::T35S; Second expression cassette is Talen-L expression cassette, consist of the constitutive promoter ubiquitin promotor Pubi coming from Maize Ubiquitin gene, its nucleotide sequence is as shown in SEQ ID NO:5 in sequence table, the talen-l gene of synthetic and Fok1 fusion rotein TALEN-L, its nucleotide sequence is as shown in SEQ ID NO:6 in sequence table, and from the rbcs terminator Trbcs of Arabidopis thaliana, its nucleotide sequence, as shown in SEQ ID NO:7 in sequence table, forms the second expression cassette Ubi::TALEN-L::Trbcs; 3rd expression cassette is Talen-R expression cassette, consist of the constitutive promoter 35S promoter P35S from tobacco mosaic virus (TMV), its nucleotide sequence is as shown in SEQ ID NO:2 in sequence table, the talen-R gene of synthetic and Fok1 fusion rotein TALEN-R, its nucleotide sequence is as shown in SEQ ID NO:8 in sequence table, and the Nos terminator Tnos of bacterial origin, its nucleotide sequence, as shown in SEQ ID NO:9 in sequence table, forms the second expression cassette 35S::TALEN-R::Tnos.

After the element sequence verification of above-mentioned each expression cassette, in pCAMBIA1301 carrier, be connected into above-mentioned fragment successively, finally obtain plant expression vector pOsBel, specifically as shown in Figure 2.

Embodiment 3, turn pOsBel carrier rice plant Bel genetic analysis

Recombinant vectors pOsBel thermal shock transformation Agrobacterium bacterial strain EHA105 embodiment 2 obtained, obtains the recombinational agrobacterium containing recombinant vectors pOsBel, called after EHA105/pOsBel, the method infecting callus by Agrobacterium obtains 100 strains and independently singly copies transfer-gen plant, with the genomic dna of transfer-gen plant for template, with primer Ben-F:5'-ACAGAAACACATCACACATTCG-3'(SEQ ID NO:11) and Ben-R:5'-TAGTAGTGGAGCAAGAAGAGGATAG-3'(SEQ ID NO:12) carry out pcr amplification, amplified production is analyzed through HRM, find 4, 10, 18, 19, 24, 36, 41, 44, 56, 66, 67, 76, the Bel gene solubility curve of 78 and No. 81 plant changes, concrete outcome as shown in Figure 3, that WT indicates is the peak figure that unwinds of wild rice Bel gene, its material has WT lines, also transgenosis is had but the Bel gene plant of not undergoing mutation, 24 and Mutants indicates is the peak figure that unwinds after Bel gene is undergone mutation.

HRM (High Resolution Melt), Chinese is translated into " high resolving power melting " is the up-to-date SNP and mutation research instrument that rose in the world in recent years.This detection method is not subject to the limitation of mutating alkali yl site and type, without the need to sequence-specific probes, directly runs high resolving power and melts, can complete the analysis to sample genotype after PCR terminates.The present invention devises according to target gene and severally to compare by experiment detection primer, and the specificity detecting primer Ben-F and Ben-R is best, and its nucleotide sequence is respectively as shown in SEQ ID NO:11 and SEQ ID NO:12.The operation steps that described HRM analyzes is as follows:

1. prepare PCR damping fluid.Be generally 10 μ l systems, comprise: 10*Buffer 1 μ l, dNTP 0.1 μ l, Primer 0.2 μ l, Taq enzyme 0.1 μ l, LCGreen 0.1 μ l, mark DNA1 μ l, genomic dna 0.2 μ l, water 7 μ l in high/low temperature.

2. carry out PCR reaction.

3. detect PCR primer with LightScanner instrument, and analytical results.

Embodiment 4, turn the sensitivity analysis of pOsBel carrier rice plant bentazone

When transfer-gen plant grows to 3-4 leaf, spray concentration is the bentazone of 1200mg/L, after one week, observe plant phenotype, and take pictures, its result as shown in Figure 4, left side WT lines growth is normal, the plant that the Bel gene that right side obtains for HRM screens is undergone mutation, shows as bentazone sensitivity (blade dries up, and growth slows down).Wherein, the plant after fractional mutations withers as offspring's plant part of the plant such as 4,18,20 and No. 24, illustrate that these plant strengthen bentazone susceptibility, and WT lines growth is normal.

Embodiment 5, the application of bentazone sensitive paddy plant in hybrid seeding

Bentazone sensitive rice transformation restorer, hybridisation rice producing method for seed can be reformed: do male parent by the restorer with bentazone sensitive gene, after free pollination, just male parent restorer can be killed by spraying bentazone, therefore can realize Parent mixed seeding miscegenation in breeding of hybrid rice, be conducive to improving outcrossing rate, reduce labor intensity, realize mechanized operation.Gone if forward in two-line sterile line, what can solve that the two-line hybrid rice production of hybrid seeds worries causes purity inadequate because of temperature variation, give and produce damnous great difficult problem.

The present invention with the responsive plant of bentazone (military fortune No. 7, round-grained rice) for male parent, the male sterile plant of Huang Huazhan sudden change is maternal, adjustment date of seeding, makes its flower synchronization, two row are maternal, a line male parent, and 20 meters of row are long, plant 30 row altogether, August 28 sprayed the bentazone of 1200mg/L, and after the week, male parent is all wilted death, September 29 mechanical harvesting.Get the seed seedling-raising that 1kg is dried, 3 leaf phases sprayed bentazone, did not find dead plant of wilting after one week.The seed not mixing male parent selfing and produce is described in gathered in the crops seed.

SEQUENCE LISTING

 

<110> Unnamed Xingwang System Crop Design Front Laboratory (Beijing) Co., Ltd.

Shenzhen Crop Molecular Design Breeding Institute

Shenzhen Xingwang Biological Seed Industry Co., Ltd.

Xingwang Investment Pty Ltd.

The fixed point of a <120> paddy rice Bel gene knocks out system and application thereof

<130>

<160> 16

<170> PatentIn version 3.3

 

<210> 1

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<213> paddy rice (Oryza Sativa)

 

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cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca ccctctttcc 900

 

ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca aatccacccg 960

 

tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc tctaccttct 1020

 

ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt tcatgtttgt 1080

 

gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga tgcgacctgt 1140

 

acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa tcctgggatg 1200

 

gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg ttgcataggg 1260

 

tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt cgggtcatct 1320

 

tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg tcgttctaga 1380

 

tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga tctgtatgtg 1440

 

tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat cgatctagga 1500

 

taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt tttgttcgct 1560

 

tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc ggagtagaat 1620

 

actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg tgtcatacat 1680

 

cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt atacatgttg 1740

 

atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc atatgctcta 1800

 

accttgagta cctatctatt ataataaaca agtatgtttt ataattattt tgatcttgat 1860

 

atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc tgccttcata 1920

 

cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg tttggtgtta 1980

 

cttggtac 1988

 

 

<210> 6

<211> 2778

<212> DNA

<213> synthetic

 

<400> 6

atggctccaa agaagaagcg taaggtagac tacaaagacc atgacggtga ttataaagat 60

 

catgacatcg attacaagga tgacgatgac aagggatctg tggatctacg cacgctcggc 120

 

tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 180

 

cacgaggcac tggtcggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 240

 

ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 300

 

gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 360

 

gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 420

 

cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 480

 

aatgcactga cgggtgcccc cctgaacctg accccggagc aggtggtggc catcgctagt 540

 

aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 600

 

gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaacaatgg tggcaaacag 660

 

gctcttgaaa ccgtacagcg cctactgcca gttctctgtc aagcccacgg tctgactccg 720

 

gagcaagttg tagcgattgc tagtcatgac ggtggcaaac aggctcttga aacagtccaa 780

 

cgactacttc cagttctctg tcaagcccac ggccttactc ctgagcaagt tgtagcgatt 840

 

gctagtaatg ggggtggcaa acaggctctt gagactgttc agcgccttct accagttctc 900

 

tgtcaagccc acggcctgac gcccgagcaa gttgtagcga ttgctagtaa tattggtggc 960

 

aaacaggctc ttgagacggt tcagcgcctc cttccagttc tctgtcaagc ccacggactc 1020

 

accccagatc aagttgtagc gattgctagt catgacggtg gcaaacaggc tcttgaaaca 1080

 

gtccaacgac tacttccagt tctctgtcaa gcccacggcc ttactcctga gcaagttgta 1140

 

gcgattgcta gtaatattgg tggcaaacag gctcttgaga cggttcagcg cctccttcca 1200

 

gttctctgtc aagcccacgg actcacccca gatcaagttg tagcgattgc tagtaacaat 1260

 

ggtggcaaac aggctcttga aaccgtacag cgcctactgc cagttctctg tcaagcccac 1320

 

ggtctgactc cggagcaagt tgtagcgatt gctagtaata ttggtggcaa acaggctctt 1380

 

gagacggttc agcgcctcct tccagttctc tgtcaagccc acggactcac cccagatcaa 1440

 

gttgtagcga ttgctagtaa caatggtggc aaacaggctc ttgaaaccgt acagcgccta 1500

 

ctgccagttc tctgtcaagc ccacggtctg actccggagc aagttgtagc gattgctagt 1560

 

aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 1620

 

gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaacaatgg tggcaaacag 1680

 

gctcttgaaa ccgtacagcg cctactgcca gttctctgtc aagcccacgg tctgactccg 1740

 

gagcaagttg tagcgattgc tagtaatatt ggtggcaaac aggctcttga gacggttcag 1800

 

cgcctccttc cagttctctg tcaagcccac ggactcaccc cagatcaagt tgtagcgatt 1860

 

gctagtaata ttggtggcaa acaggctctt gagacggttc agcgcctcct tccagttctc 1920

 

tgtcaagccc acggactcac cccagatcaa gttgtagcga ttgctagcaa tggcggcggc 1980

 

aggccggcgc tggagagcat tgttgcccag ttatctcgcc ctgatccggc gttggccgcg 2040

 

ttgaccaacg accacctcgt cgccttggcc tgcctcggcg gacgtcctgc gctggatgca 2100

 

gtgaaaaagg gattgccgca cgcgccggcc ttgatcaaaa gaaccaatcg ccgtattccc 2160

 

gaacgcacat cccatcgcgt tgccggatcc caactagtca aaagtgaact ggaggagaag 2220

 

aaatctgaac ttcgtcataa attgaaatat gtgcctcatg aatatattga attaattgaa 2280

 

attgccagaa atcccactca ggatagaatt cttgaaatga aggtaatgga attttttatg 2340

 

aaagtttatg gatatagagg tgagcatttg ggtggatcaa ggaaaccgga cggagcaatt 2400

 

tatactgtcg gatctcctat tgattacggt gtgatcgtgg atactaaagc ttatagcgga 2460

 

ggttataatc tgccaattgg ccaagcagat gccatgcaaa gctatgtcga agaaaatcaa 2520

 

acacgaaaca aacatatcaa ccctaatgaa tggtggaaag tctatccatc ttctgtaacg 2580

 

gaatttaagt ttttatttgt gagtggtcac tttaaaggaa actacaaagc tcagcttaca 2640

 

cgattaaatc atatcactaa ttgtaatgga gctgttctta gtgtagaaga gcttttaatt 2700

 

ggtggagaaa tgattaaagc cggcacatta accttagagg aagtgagacg gaaatttaat 2760

 

aacggcgaga taaacttt 2778

 

 

<210> 7

<211> 482

<212> DNA

<213> Arabidopis thaliana (Arabidopsis thaliana)

 

<400> 7

aggcctccca gctttcgtcc gtatcatcgg tttcgacaac gttcgtcaag ttcaatgcat 60

 

cagtttcatt gcccacacac cagaatccta ctaagtttga gtattatggc attggaaaag 120

 

ctgttttctt ctatcatttg ttctgcttgt aatttactgt gttctttcag tttttgtttt 180

 

cggacatcaa aatgcaaatg gatggataag agttaataaa tgatatggtc cttttgttca 240

 

ttctcaaatt attattatct gttgttttta ctttaatggg ttgaatttaa gtaagaaagg 300

 

aactaacagt gtgatattaa ggtgcaatgt tagacatata aaacagtctt tcacctctct 360

 

ttggttatgt cttgaattgg tttgtttctt cacttatctg tgtaatcaag tttactatga 420

 

gtctatgatc aagtaattat gcaatcaagt taagtacagt ataggctttt tgtgtcgagg 480

 

gg 482

 

 

<210> 8

<211> 2880

<212> DNA

<213> synthetic

 

<400> 8

atggctccaa agaagaagcg taaggtagac tacaaagacc atgacggtga ttataaagat 60

 

catgacatcg attacaagga tgacgatgac aagggatctg tggatctacg cacgctcggc 120

 

tacagccagc agcaacagga gaagatcaaa ccgaaggttc gttcgacagt ggcgcagcac 180

 

cacgaggcac tggtcggcca tgggtttaca cacgcgcaca tcgttgcgct cagccaacac 240

 

ccggcagcgt tagggaccgt cgctgtcaag tatcaggaca tgatcgcagc gttgccagag 300

 

gcgacacacg aagcgatcgt tggcgtcggc aaacagtggt ccggcgcacg cgctctggag 360

 

gccttgctca cggtggcggg agagttgaga ggtccaccgt tacagttgga cacaggccaa 420

 

cttctcaaga ttgcaaaacg tggcggcgtg accgcagtgg aggcagtgca tgcatggcgc 480

 

aatgcactga cgggtgcccc cctgaacctg accccggagc aggtggtggc catcgctagt 540

 

catgacggtg gcaaacaggc tcttgagacc gtccaacgcc ttctaccagt tctctgtcaa 600

 

gcccacggac taaccccagc gcaagttgta gcgattgcta gtaatattgg tggcaaacag 660

 

gctcttgaga cggttcagcg cctccttcca gttctctgtc aagcccacgg actcacccca 720

 

gatcaagttg tagcgattgc tagtaacaat ggtggcaaac aggctcttga aaccgtacag 780

 

cgcctactgc cagttctctg tcaagcccac ggtctgactc cggagcaagt tgtagcgatt 840

 

gctagtcatg acggtggcaa acaggctctt gaaacagtcc aacgactact tccagttctc 900

 

tgtcaagccc acggccttac tcctgagcaa gttgtagcga ttgctagtaa tgggggtggc 960

 

aaacaggctc ttgagactgt tcagcgcctt ctaccagttc tctgtcaagc ccacggcctg 1020

 

acgcccgagc aagttgtagc gattgctagt aatgggggtg gcaaacaggc tcttgagact 1080

 

gttcagcgcc ttctaccagt tctctgtcaa gcccacggcc tgacgcccga gcaagttgta 1140

 

gcgattgcta gtaatattgg tggcaaacag gctcttgaga cggttcagcg cctccttcca 1200

 

gttctctgtc aagcccacgg actcacccca gatcaagttg tagcgattgc tagtaacaat 1260

 

ggtggcaaac aggctcttga aaccgtacag cgcctactgc cagttctctg tcaagcccac 1320

 

ggtctgactc cggagcaagt tgtagcgatt gctagtcatg acggtggcaa acaggctctt 1380

 

gagaccgtcc aacgccttct accagttctc tgtcaagccc acggactaac cccagcgcaa 1440

 

gttgtagcga ttgctagtca tgacggtggc aaacaggctc ttgagaccgt ccaacgcctt 1500

 

ctaccagttc tctgtcaagc ccacggacta accccagcgc aagttgtagc gattgctagt 1560

 

aatattggtg gcaaacaggc tcttgagacg gttcagcgcc tccttccagt tctctgtcaa 1620

 

gcccacggac tcaccccaga tcaagttgta gcgattgcta gtaatggggg tggcaaacag 1680

 

gctcttgaga ctgttcagcg ccttctacca gttctctgtc aagcccacgg cctgacgccc 1740

 

gagcaagttg tagcgattgc tagtaacaat ggtggcaaac aggctcttga aaccgtacag 1800

 

cgcctactgc cagttctctg tcaagcccac ggtctgactc cggagcaagt tgtagcgatt 1860

 

gctagtaaca atggtggcaa acaggctctt gaaaccgtac agcgcctact gccagttctc 1920

 

tgtcaagccc acggtctgac tccggagcaa gttgtagcga ttgctagtaa caatggtggc 1980

 

aaacaggctc ttgaaaccgt acagcgccta ctgccagttc tctgtcaagc ccacggtctg 2040

 

actccggagc aagttgtagc gattgctagc aatggcggcg gcaggccggc gctggagagc 2100

 

attgttgccc agttatctcg ccctgatccg gcgttggccg cgttgaccaa cgaccacctc 2160

 

gtcgccttgg cctgcctcgg cggacgtcct gcgctggatg cagtgaaaaa gggattgccg 2220

 

cacgcgccgg ccttgatcaa aagaaccaat cgccgtattc ccgaacgcac atcccatcgc 2280

 

gttgccggat cccaactagt caaaagtgaa ctggaggaga agaaatctga acttcgtcat 2340

 

aaattgaaat atgtgcctca tgaatatatt gaattaattg aaattgccag aaatcccact 2400

 

caggatagaa ttcttgaaat gaaggtaatg gaatttttta tgaaagttta tggatataga 2460

 

ggtgagcatt tgggtggatc aaggaaaccg gacggagcaa tttatactgt cggatctcct 2520

 

attgattacg gtgtgatcgt ggatactaaa gcttatagcg gaggttataa tctgccaatt 2580

 

ggccaagcac gagaaatgca acgatatgtc gaagaaaatc aaacacgaaa caaacatatc 2640

 

aaccctaatg aatggtggaa agtctatcca tcttctgtaa cggaatttaa gtttttattt 2700

 

gtgagtggtc actttaaagg aaactacaaa gctcagctta cacgattaaa tcatatcact 2760

 

aattgtaatg gagctgttct tagtgtagaa gagcttttaa ttggtggaga aatgattaaa 2820

 

gccggcacat taaccttaga ggaagtgaga cggaaattta ataacggcga gataaacttt 2880

 

 

<210> 9

<211> 231

<212> DNA

<213> synthetic

 

<400> 9

agtttcttaa gattgaatcc tgttgccggt cttgcgatga ttatcatata atttctgttg 60

 

aattacgtta agcatgtaat aattaacatg taatgcatga cgttatttat gagatgggtt 120

 

tttatgatta gagtcccgca attatacatt taatacgcga tagaaaacaa aatatagcgc 180

 

gcaaactagg ataaattatc gcgcgcggtg tcatctatgt tactagatcg g 231

 

 

<210> 10

<211> 49

<212> DNA

<213> paddy rice (Oryza Sativa)

 

<400> 10

cagcttagcc atggataacg cctacattat tgccattctc tctgtagct 49

 

 

<210> 11

<211> 22

<212> DNA

<213> synthetic

 

<400> 11

acagaaacac atcacacatt cg 22

 

 

<210> 12

<211> 25

<212> DNA

<213> synthetic

 

<400> 12

tagtagtgga gcaagaagag gatag 25

  

<210> 13

<211> 102

<212> DNA

<213> synthetic

 

<400> 13

cttactcctg agcaagttgt agcgattgct agtaatattg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

  

<210> 14

<211> 102

<212> DNA

<213> synthetic

 

<400> 14

cttactcctg agcaagttgt agcgattgct agtcatgacg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

 

<210> 15

<211> 102

<212> DNA

<213> synthetic

 

<400> 15

cttactcctg agcaagttgt agcgattgct agtaatgggg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

  

<210> 16

<211> 102

<212> DNA

<213> synthetic

 

<400> 16

cttactcctg agcaagttgt agcgattgct agtaacaatg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

 

Claims (8)

1. a TALENs fixed point knocks out system, comprise the DNA binding domains of TALE and the DNA cutting structure territory of Fok I, it is characterized in that comprising in the DNA binding domains of described TALE the base identification module that quantity does not wait, wherein identify that the nucleotide sequence of the NI module of base A is as shown in SEQ ID NO:13, identify in conjunction with the nucleotide sequence of the HD module of base C as shown in SEQ ID NO:14, identify in conjunction with the nucleotide sequence of the NG module of base T as shown in SEQ ID NO:15, identify in conjunction with the nucleotide sequence of the NN module of bases G as shown in SEQ ID NO:16.

2. TALENs fixed point according to claim 1 knocks out the application of system in plant gene fixed point knocks out.

3. the fixed point of a paddy rice bentazone gene knocks out system, comprise the DNA binding domains of TALE and the DNA cutting structure territory of Fok I, it is characterized in that the DNA binding domains target of described TALE is attached to first exon of paddy rice bentazone gene, the nucleotide sequence of described target sequence is as shown in SEQ ID NO:10.

4. fixed point according to claim 3 knocks out system, the DNA binding domains of wherein said TALE comprises TALEN-L and TALEN-R, the nucleotide sequence of described TALEN-L is as shown in SEQ ID NO:6, and the nucleotide sequence of described TALEN-R is as shown in SEQ ID NO:8.

5. the fixed point described in claim 3 or 4 knocks out system and is obtaining the application in the responsive plant of paddy rice bentazone.

6. an expression vector, comprise the DNA binding domains of TALE and the DNA cutting structure territory of Fok I, it is characterized in that the DNA binding domains of described TALE comprises an a TALEN-L and TALEN-R structure, the nucleotide sequence of described TALEN-L is as shown in SEQ ID NO:6, and the nucleotide sequence of described TALEN-R is as shown in SEQ ID NO:8.

7. expression vector according to claim 6, a Ubiquitin promotor is connected with before it is characterized in that described TALEN-L, its nucleotide sequence, as shown in SEQ ID NO:5, is connected with a 35S promoter before described TALEN-R, its nucleotide sequence is as shown in SEQ ID NO:2.

8. a HRM method for Screening of Rice bentazone transgenation, is characterized in that the primer sequence of described HRM method is as shown in SEQ ID NO:11 and SEQ ID NO:12.