CN104762279B - Rice Bel gene site-directed knockout system and application thereof - Google Patents

Abstract

The invention discloses a rice Bel gene site-directed knockout system and application thereof, belongs to the field of plant genetic engineering, and particularly relates to a rice bentazon gene site-directed knockout method and application thereof. The invention provides a TALEN site-directed mutagenesis system which is better than plant genome DNA, and the TALEN site-directed mutagenesis system comprises four recognition modules. Compared with the prior art for creating bentazon sensitive plants, the method has the advantages that convenience and rapidness are realized, a scientific research worker can obtain homozygous mutant plants with a plurality of different mutation sites within one year and a half, manpower and material resources are greatly saved, the obtained mutant does not contain transgenic components, the consideration of people on transgenosis is eliminated, and the method has important significance for creating new germplasm resources for plant genetic engineering.

Description

Rice Bel gene site-directed knockout system and application thereof

Technical Field

The invention belongs to the field of plant genetic engineering, and particularly relates to a fixed-point knockout method of a rice bentazon gene and application thereof.

Background

The selective herbicide applied to wild rice mainly has two types, one type is benzothiadiazole (benzothiadiazole) herbicide, such as bentazon, the active ingredients of which can be absorbed by the roots and leaves of crops, and the selective herbicide has killing effect on most dicotyledons and cyperaceae weeds except leguminous plants and is harmless to gramineous plants. The herbicidal mechanism of bentazon is to inhibit the hill reaction in plant photosynthesis.

TALENs (transcription activator-like effector nucleases) are new techniques for gene site-directed modification that have appeared in recent years, and have been widely studied and applied in the fields of animals, plants, humans, and the like. Transcription activator-Like Effector (TALE) is a toxic Protein secreted by Xanthomonas pathogenic bacteria into host plant cells, and can recognize plant DNA and drive gene expression. TALENs are nucleases artificially synthesized using the DNA binding domain of TALE and the DNA cleavage domain of Fok I. Two TALEN monomers are combined on a DNA double strand according to a certain mode, a dimer with cutting activity is formed to cut off the DNA, a DNA Double Strand Break (DSB) is generated, a cell starts a repair mechanism, the inexact repair mode of connecting non-homologous ends can generate gene site-directed mutation, and the repair can realize accurate gene site-directed insertion or gene replacement through a Homologous Recombination (HR) mode. Compared with Zinc Finger Nucleases (ZFNs) and Meganucleas technologies, the TALENs technology has the advantages of simple design, high mutation efficiency, low cytotoxicity and the like. The superiority of TALENs depends on the unique DNA recognition properties of TALEs. The DNA binding domain of TALENs comprises a tandem Repeat domain of unequal numbers of Repeat units ((1.5-33.5). each Repeat unit is typically composed of 33-35 amino acids, the amino acids of the Repeat units are highly conserved, but the two adjacent amino acids at positions 12 and 13 of each unit are variable, and these two amino acids are commonly referred to as Repeat-variable diresives (RVDs). each RVD has a simple correspondence to nucleotide A, T, C, G, with the most common recognition codes being NI-recognition binding a, HD-recognition binding C, NG-recognition binding T, and NN-recognition binding G (Moscou and bogdanov, 2009).

Herbicide-resistant crops are generally cultivated mainly by the following two methods: firstly, a mutant of herbicide-resistant crops is obtained through physicochemical mutagenesis; secondly, by DNA recombination technology, the herbicide-resistant gene is introduced into the existing species to create a new herbicide-resistant material. Because the method of the physical and chemical mutagenesis has weak targeting property, a gene is mutated, a large amount of experimental materials need to be processed, a large amount of screening work needs to be carried out to ensure that the mutant of the target gene is obtained, and the mutant gene which is not needed by the user is easy to carry; DNA recombination techniques involve exogenous transgenes, which have certain problems in breeding time or biosafety evaluation. The TALEN technology is accurate, rapid and convenient, mutants without exogenous genes can be obtained, the mutants are rich in variety, and the method for obtaining the herbicide-resistant material by adopting the TALEN technology becomes a technical development trend.

Disclosure of Invention

The invention provides a TALEN site-directed mutagenesis system which is better than plant genome DNA, and nucleotide sequences of four recognition modules contained in the TALEN site-directed mutagenesis system are respectively as follows: the nucleotide sequence that recognizes the NI module that binds base a is set forth in SEQ ID NO: 13 is shown in the figure; the nucleotide sequence that recognizes the HD module that binds to base C is set forth in SEQ ID NO: 14 is shown in the figure; the nucleotide sequence of the NG module recognizing the binding base T is shown as SEQ ID NO: 15 is shown in the figure; the nucleotide sequence that recognizes the NN module that binds to base G is set forth in SEQ ID NO: shown at 16.

The invention also provides a novel method for obtaining the bentazon sensitive plant, in particular to a method for creating the bentazon sensitive plant in monocotyledons, preferably rice. The method takes a nucleotide sequence behind a first exon initiation codon of a rice Bel gene as a target sequence, wherein the nucleotide sequence of the target sequence is shown as SEQ ID NO. 10, a sequence from 17 th to 34 th of the SEQ ID NO. 10 is a spacer sequence, and module recognition sequences (named as L and R respectively) of TALENs are arranged on two sides of the spacer sequence. In the invention, the TALEN-L designed aiming at the L end of the target sequence comprises a TALE gene and a Fok1 fusion protein, and the nucleotide sequence of the TALEN-L is shown as SEQ ID NO. 6; the TALEN-R designed aiming at the R end of the target sequence comprises a TALE gene and a Fok1 fusion protein, and the nucleotide sequence of the TALEN-R is shown as SEQ ID NO. 8. The nucleotide sequences of the TALEN-L and the TALEN-R are constructed into the same expression vector, and are transferred into rice under the drive of a constitutive expression promoter, so that a bentazon sensitive plant with a mutant Bel gene can be effectively obtained. The practical requirement of accurately and efficiently targeting the Bel gene of the rice is met.

The invention also provides a plant expression construct, which comprises the nucleotide sequence shown in SEQ ID NO. 6 and/or SEQ ID NO. 8. More specifically, TALEN-L of the construct is initiated by a Ubiquitin promoter to express, and TALEN-R is driven by a 35s promoter to express.

The screening means of the mutant materials obtained by the TALEN method at present mainly comprises the following steps: phenotypic observation, gene sequencing verification, etc. For materials with bad phenotype observation, gene sequencing can be performed only, and the quantity of progeny materials is large, so that the workload is huge and the cost is increased. Particularly, the phenotype of the bentazon sensitive mutant is easy to observe, but the phenotype plant gradually dies and cannot receive seeds, so that bentazon sensitive screening cannot be carried out in the T0 generation, and the problems of difficult chimera sequencing, complex procedure, high cost and the like exist in sequencing.

In order to solve the problems, the invention also provides a method for screening the mutant, in particular to a method for screening the mutant by a high resolution melting curve (HRM) method for the transgenic plant obtained by transformation. HRM (high resolution melt), the translation of Chinese into "high resolution melting" is the latest SNP and mutation research tool that has emerged internationally in recent years. The detection method is not limited by mutation base sites and types, does not need sequence specific probes, and can finish the analysis of the genotype of the sample by directly running high-resolution melting after the PCR is finished. The invention provides specific HRM primers Ben-F and Ben-R for detecting the Bel gene mutation condition of rice, wherein the nucleotide sequences of the primers are shown as SEQ ID NO. 11 and SEQ ID NO. 12.

The invention is realized by the following steps in sequence:

(1) the nucleotide sequence of the TALEN is modified by the preference of the monocotyledon codon and the rice Bel gene sequence, and the modified nucleotide sequence is shown as SEQ ID NO:6 and SEQ ID NO:8, synthesizing the sequence by Jiebao bioengineering (Dalian) Co., Ltd;

(2) respectively connecting a pair of TALEN nucleotide fragments obtained by synthesis to the downstream of Pubi and P35S promoters to drive expression; the nucleotide sequences of the Pubi and P35S promoters are respectively shown in SEQ ID NO:5 and SEQ ID NO:2, respectively.

(3) Transformed plants, particularly, preferably transformed rice;

(4) HRM analysis was performed using the obtained T0 transgenic rice genome as a template. Specifically, a pair of PCR primers is designed, so that a product comprises a Bel gene target sequence fragment, the length of the PCR product is about 120bp, a dissolving curve is obtained by using Lightscanner (USIdaho) scanning, and possible mutant plants are screened by comparing the difference of melting temperatures of PCR products of a transgenic plant and a wild plant, and the method can be used for conveniently and rapidly screening a large number of transgenic plants;

(5) and verifying the Bel gene mutation condition of the screened plants by sequencing. In order to verify whether and how the Bel gene of the screened mutant plant is mutated, a DNA fragment with a target sequence is amplified by PCR, and the true sequence of the DNA fragment is verified by sequencing.

(6) The screened mutants were subjected to a bentazon herbicide sensitivity test. The Bel gene mutant strain obtained by screening and a wild plant are planted in the same flowerpot at the same time, 1200mg/L bentazon is used for spraying when the plant grows to have 3 new leaves, the dry and dry condition of the plant leaves is investigated after one week, and a picture is taken (figure 4).

The ' promoter ' of the present invention is one DNA sequence located in the 5' upstream region of the structural gene and can activate RNA polymerase to combine with template DNA precisely and to have transcription initiation specificity.

The "nucleotide sequence" of the present invention is the sequence of nucleotides in nucleic acids (DNA and RNA). In many cases, the nucleotide sequence determines the higher order structure and biological function of a nucleic acid, i.e., different sequences have different higher order structures and different biological functions.

The "plant expression vector" of the present invention refers to any vector known in the prior art, such as pCAMBIA1300, pBI121, etc., which can constantly express a foreign gene in a plant cell.

The term "transformation" as used herein refers to any plant transformation method known in the art, such as Agrobacterium-mediated method, gene gun, etc., which is capable of introducing a foreign gene into a plant cell or plant tissue.

The "transgenic plant" of the invention refers to a plant individual integrated with an exogenous gene obtained by a gene transfer technology. Usually, the nucleotide sequence stably carrying the foreign gene in the genome of the transformed plant or transgenic plant can be stably inherited to the next generation.

Compared with the prior art for creating bentazon sensitive plants, the method has the advantages that convenience and rapidness are realized, a scientific research worker can obtain homozygous mutant plants with a plurality of different mutation sites within one year and a half, manpower and material resources are greatly saved, the obtained mutant does not contain transgenic components, the consideration of people on transgenosis is eliminated, and the method has important significance for creating new germplasm resources for plant genetic engineering.

Drawings

FIG. 1 shows the rice Bel gene sequence and the TALEN target sequence position related to the patent, Exon 1 and Exon 2 are two exons 1 and 2 of the rice Bel gene respectively, and the target sequence L and the target sequence R are the binding target sites of TALEN protein respectively.

FIG. 2 schematic representation of the vector pOsBel used for transformation of rice. LB and RB are the left and right borders of the T-DNA, respectively; P35S is a constitutive promoter 35S promoter from tobacco mosaic virus, and Hyg is an artificially synthesized hygromycin resistance gene; T35S is a 35S gene terminator derived from tobacco mosaic virus; pubi represents a promoter of a maize ubiquitin gene, TALEN-L is an artificially synthesized TALEN-L gene coding region from bacteria, and Trbcs is a terminator from an Arabidopsis Rbcs gene; TALEN-R is the artificial TALEN-R gene coding region from bacteria, Tnos represents the terminator of nopaline synthase (nos) gene.

FIG. 3 is a graph comparing the melting temperature peaks of Bel gene of HRM-detected transgenic rice and wild rice; WT is a melting peak plot of the Bel gene of wild-type rice, 24 and Mutants are melting peak plots of the Bel gene after mutation.

The mutant plants and wild plants obtained in FIG. 4 were analyzed for sensitivity to bentazon herbicides, the wild plants on the left side showed a substantially normal growth, and the plants on the right side showed sensitivity to bentazon (dry leaves, slow growth) with mutated Bel gene obtained by HRM screening.

Detailed Description

Example 1 sequence and analysis of Rice Gene Bel

The sequence of the rice gene Bel is shown as the sequence 1 in the sequence table. Sequence analysis shows that the gene comprises 2 exons, namely, 41 th to 964 th (first exon) and 1691 th and 2308 th (second exon) of the sequence 1 in the sequence table. The gene encodes a cytochrome P450 protein.

The invention uses the sequence on the first exon as the target sequence of Transcription Activator Like Effector Nucleases (TALENs). (FIG. 1)

Example 2 design of TALENs and construction of recombinant expression vectors thereof

Selection of TALENs target sequences

The target sequence targets the rear sequence of the initiation codon of the first exon of the Bel of the rice gene, and the sequence is shown in 5'-CAGCTTAGCCATGGAT aacgcctacattattgcc ATTCTCTCTGTAGCT-3' (shown in SEQ ID NO:10 specifically), wherein the lower case letters are interval sequences, and the upper case letters on both sides are TALENs module recognition sequences (named L and R respectively).

Design and synthesis of coding gene of two, TALENs

The pOsBel vector shown in FIG. 1 was constructed by assembling the individual elements of the talen expression cassette. The vector contains 3 expression frames, wherein the first expression frame is a hygromycin resistance gene expression frame and consists of a constitutive promoter 35S promoter P35S from tobacco mosaic virus, the nucleotide sequence of the hygromycin resistance gene expression frame is shown as SEQ ID NO. 2 in a sequence table, the nucleotide sequence of an artificially synthesized hygromycin resistance gene Hyp is shown as SEQ ID NO. 3 in the sequence table, the nucleotide sequence of a terminator T35S from tobacco mosaic virus is shown as SEQ ID NO. 4 in the sequence table, and the first expression frame 35S is composed of Hyp:: T35S; the second expression frame is a Talen-L expression frame which is composed of a constitutive promoter ubiquitin promoter Pubi from a maize ubiquitin gene, the nucleotide sequence of the expression frame is shown as SEQ ID NO. 5 in the sequence table, an artificially synthesized Talen-L gene and a Fok1 fusion protein TALEN-L are shown as SEQ ID NO. 6 in the sequence table, and the nucleotide sequence of a rbcs terminator Trbcs from arabidopsis thaliana is shown as SEQ ID NO. 7 in the sequence table, so that the second expression frame Ubi is composed of TALEN-L, TALEN-L and Trbcs; the third expression frame is a Talen-R expression frame which is composed of a constitutive promoter 35S promoter P35S from tobacco mosaic virus, the nucleotide sequence of the third expression frame is shown as SEQ ID NO. 2 in the sequence table, the nucleotide sequence of the third expression frame is shown as SEQ ID NO. 8 in the sequence table of an artificially synthesized Talen-R gene and a Fok1 fusion protein TALEN-R, and the nucleotide sequence of the third expression frame is shown as SEQ ID NO. 9 in the sequence table of a bacteria-derived Nos terminator Tnos, so that the second expression frame 35S is formed, wherein TALEN-R is TALEN-R and Tnos.

After the element sequencing verification of each expression frame, the fragments are sequentially connected into a pCAMBIA1301 vector to finally obtain a plant expression vector pOsBel, which is shown in figure 2.

Example 3 Bel Gene analysis of pOsBel vector-transferred Rice plants

Carrying out heat shock transformation on the agrobacterium strain EHA105 by using the recombinant vector pOsBel obtained in the example 2 to obtain recombinant agrobacterium containing the recombinant vector pOsBel, and naming the recombinant agrobacterium as EHA 105/pOsBel; 100 independent single-copy transgenic plants are obtained by an agrobacterium infection callus method, the genome DNA of the transgenic plants is used as a template, and a primer Ben-F: 5'-ACAGAAACACATCACACATTCG-3' (SEQ ID NO:11) and Ben-R: 5'-TAGTAGTGGAGCAAGAAGAGGATAG-3' (SEQ ID NO:12), performing PCR amplification, and HRM analysis on the amplification product to find that Bel gene melting curves of plants No. 4, 10, 18, 19, 24, 36, 41, 44, 56, 66, 67, 76, 78 and 81 are changed, wherein the specific result is shown in figure 3, WT is a melting peak diagram of the Bel gene of wild rice, and the material of the Bel gene is wild plants and plants which are transgenic but have NO mutation in the Bel gene are marked by WT; 24 and Mutants are indicated by melting peaks after mutation of the Bel gene.

HRM (high Resolution melt), the translation of Chinese to "high Resolution melting" is the latest SNP and mutation research tool that has emerged internationally in recent years. The detection method is not limited by mutation base sites and types, does not need sequence specific probes, and can finish the analysis of the genotype of the sample by directly running high-resolution melting after the PCR is finished. According to the invention, a plurality of pairs of detection primers are designed according to target genes, and through experimental comparison, the specificity of the detection primers Ben-F and Ben-R is the best, and the nucleotide sequences are respectively shown as SEQ ID NO. 11 and SEQ ID NO. 12. The HRM analysis was performed as follows:

1. and preparing a PCR buffer solution. Typically a 10. mu.l system comprising: 10. mu.l of Buffer, 0.1. mu.l of dNTP, 0.2. mu.l of Primer, 0.1. mu.l of Taq enzyme, 0.1. mu.l of LCGreen, 1. mu.l of high and low temperature internal standard DNA, 0.2. mu.l of genomic DNA and 7. mu.l of water.

2. PCR reaction was performed.

3. The PCR product was detected with a LightScanner instrument and the results were analyzed.

Example 4 sensitivity analysis of POsBel vector-transferred Rice plants on Bendalon

When the transgenic plants grow to 3-4 leaves, bentazon with the concentration of 1200mg/L is sprayed, after one week, the plant phenotype is observed and photographed, the result is shown in figure 4, the left wild type plants grow basically normally, and the right wild type plants are plants with mutant Bel genes obtained by HRM screening and are shown to be sensitive to bentazon (the leaves are dry and the growth is slow). Wherein, the progeny partial plants of the partially mutated plants such as the plants of No. 4, 18, 20 and 24 are withered, which shows that the sensitivity of the plants to the bentazon is enhanced, and the wild plants grow normally.

Example 5 application of Bendalong sensitive Rice plants in hybrid seed production

The method for breeding hybrid rice by transferring the bentazon sensitive rice into the restoring line can be innovated: the restoring line with bentazon sensitive gene is used as male parent, and after free pollination, the restoring line of male parent can be killed by spraying bentazon, so that the mixed seeding and mixing of male and female parents in the hybrid rice seed production can be realized, the outcrossing rate can be improved, the labor intensity can be reduced, and the mechanized operation can be realized. If the two-line hybrid rice is transferred into a two-line sterile line, the major problems of insufficient purity and production loss caused by temperature change in the seed production of the two-line hybrid rice can be solved.

The method takes a bentazon sensitive plant (Wuyujing No. 7) as a male parent and a Huanghuazhan mutant male sterile line as a female parent, adjusts the sowing period to ensure that the flowering periods of the bentazon sensitive plant meet, two rows of female parents, one row of male parents and 20 m row length, plants 30 rows in total, sprays 1200mg/L bentazon on 28 days in 8 months, completely withers and dies the father after one week, and mechanically harvests in 29 days in 9 months. 1kg of dried seeds are taken for seedling culture, bentazon is sprayed in the 3-leaf period, and no wilting dead plants are found after one week. Indicating that the harvested seeds have no seeds produced by the selfing of the hybrid male parent.

SEQUENCE LISTING

<110> Ming Xingwang System crop design front laboratory (Beijing) Co., Ltd

Shenzhen market crop molecular design breeding research institute

Shenzhen xingwang biological species limited company

Xingwang investment Co Ltd

<120> fixed-point knockout system of rice Bel gene and application thereof

<130>

<160> 16

<170> PatentIn version 3.3

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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> Artificial Synthesis

<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> Arabidopsis 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> Artificial Synthesis

<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> Artificial Synthesis

<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> Rice (Oryza Sativa)

<400> 10

cagcttagcc atggataacg cctacattat tgccattctc tctgtagct 49

<210> 11

<211> 22

<212> DNA

<213> Artificial Synthesis

<400> 11

acagaaacac atcacacatt cg 22

<210> 12

<211> 25

<212> DNA

<213> Artificial Synthesis

<400> 12

tagtagtgga gcaagaagag gatag 25

<210> 13

<211> 102

<212> DNA

<213> Artificial Synthesis

<400> 13

cttactcctg agcaagttgt agcgattgct agtaatattg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

<210> 14

<211> 102

<212> DNA

<213> Artificial Synthesis

<400> 14

cttactcctg agcaagttgt agcgattgct agtcatgacg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

<210> 15

<211> 102

<212> DNA

<213> Artificial Synthesis

<400> 15

cttactcctg agcaagttgt agcgattgct agtaatgggg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

<210> 16

<211> 102

<212> DNA

<213> Artificial Synthesis

<400> 16

cttactcctg agcaagttgt agcgattgct agtaacaatg gtggcaaaca ggctcttgaa 60

acagtccaac gactacttcc agttctctgt caagcccacg gc 102

Claims (4)

1. A site-directed knockout system of a paddy rice bentazon gene comprises a DNA binding domain of TALE and a DNA cutting domain of Fok I, namely a fusion protein TALEN-L of TALEN-L and Fok I, wherein the gene coding sequence of the fusion protein TALEN-L is shown as 6, and a fusion protein TALEN-R of TALEN-R and Fok I, wherein the gene coding sequence of the fusion protein TALEN-R is shown as 8.

2. An expression vector comprises gene coding sequences of a DNA binding domain of TALE and a DNA cutting domain of Fok I, coding genes of fusion proteins TALEN-L of TALEN-L and Fok I respectively, and nucleotide sequences of the coding genes are shown as 6, and coding genes of fusion proteins TALEN-R of TALEN-R and Fok I respectively, and nucleotide sequences of the coding genes are shown as 8.

3. The expression vector according to claim 2, wherein a Ubiquitin promoter is linked in front of the coding gene of TALEN-L, the nucleotide sequence of which is shown in SEQ ID NO. 5, and a 35S promoter is linked in front of the coding gene of TALEN-R, the nucleotide sequence of which is shown in SEQ ID NO. 2.

4. Use of the site-directed knockout system of claim 1 or the expression vector of any one of claims 2 to 3 for obtaining a rice bentazon-sensitive plant.