CN110408647A - A kind of construction method of biology mutant library - Google Patents

Abstract

The invention discloses a kind of construction methods of biological mutant library.The construction method of biology mutant library disclosed by the invention includes: that DNA fragmentation 1) is imported into target organisms, obtains genetically modified organism；The DNA fragmentation can express the required element for completing rite-directed mutagenesis target DNA in genetically modified organism；2) genetically modified organism is cultivated, biological mutant library is obtained；The biology is mictic organism, and genetically modified organism can the specifically rite-directed mutagenesis target DNA in reproduction cell.The mutant library that benefit is obtained by the present invention can screen the biology with following character: antibiont adverse circumstance, resisting abiotic adverse circumstance, high yield, good quality trait, high secondary metabolites yield etc..Therefore, method of the invention has huge application prospect.

Description

A kind of construction method of biology mutant library

Technical field

The present invention relates in technical field of bioengineering, a kind of construction method of biology mutant library.

Background technique

Gene editing technology, especially CRISPR/Cas9 technology, realize accurately gene editing in biological cell. Its technical principle is to combine to form RNA by one section of guidance RNA (sgRNA or gRNA) and DNA restriction endonuclease (such as Cas9, Cpf1) With the compound (abbreviation RNP) of albumen, which can search for the target sequence complementary on guidance RNA in the genome, So that accurately in the region, the DNA to combination is sheared DNA restriction endonuclease.According to different DNA restriction endonuclease characteristics, cut The result cut is varied, can be the double-strand DNA cleavage (DSB) of flat end or cohesive end, is also possible to single-strand DNA breaks (Nick).Organism will lead to insertion to the reparation of DSB or Nick or delete (Indel), to realize to target gene Precisely editor.

The plant of different editing type seeds can be generated by, which having at present, is directly obtained by transgenic protocol, i.e. agriculture Bacillus dips in T1 that colored method converts for Arabidopsis plant, or the T0 obtained by the methods of tissue cultures is for plant.Turn base Because of at high cost, the low efficiency of process, it is difficult largely to obtain this plant that can generate different editing type offsprings.

Summary of the invention

The technical problem to be solved by the present invention is to how prepare mutant library.Largely studies have shown that being directed to certain bits The reparation for the DSB that point generates is the result is that produce a large amount of different mutation types.In order to the mutated gene for keeping these new It is enough transmitted to the next generation, specifically starting is practiced shooting in reproduction cell, can theoretically be generated and largely be contained different mutated genes Offspring individuals.By taking plant as an example, if it is possible to which continual generation is largely with the seed of different mutation types, it will be able to Establish a mutant seeds library in the site.

In order to solve the above technical problems, being X1 present invention firstly provides a kind of construction method of biological mutant library) Or X2):

The method X1) include following X11) and X12):

X11 DNA fragmentation) is imported into target organisms, obtains genetically modified organism；The DNA fragmentation can be in the transgenosis The required element of rite-directed mutagenesis target DNA is completed in expression in biology；

X12 the genetically modified organism) is cultivated, biological mutant library is obtained；

The method X2) include following X21) and X22):

X21 DNA fragmentation) is imported into target organisms, obtains genetically modified organism；The DNA fragmentation can be in the transgenosis The required element of rite-directed mutagenesis target DNA is completed in expression in biology；

X22 the genetically modified organism) is cultivated, for selecting the target DNA to mutate from the genetically modified organism Body (being denoted as mutant) obtains biological mutant library, and selection contains the DNA fragmentation and the target from the genetically modified organism The individual (being denoted as mutant library deposit individual) that mark DNA does not mutate is bred, to expand the mutant library.

The mutant library deposit physical efficiency further generates the individual that the target DNA mutates and containing described The individual that DNA fragmentation and the target DNA do not mutate, the individual that the target DNA mutates can be used as mutant and return Enter biological mutant library, it is described containing the DNA fragmentation and individual that the target DNA does not mutate can be used as mutant library Deposit individual is further used for propagation of mutated body and mutant library deposit individual, and " will generate what the target DNA mutated Individual and containing the DNA fragmentation and individual that the target DNA does not mutate " this feature heredity go down.By numerous The mutant library deposit individual is grown, the quantity of mutant in the mutant library is expanded.The number of the breeding can be according to tool Body it needs to be determined that.

Specifically, the specific site generation mutation (i.e. rite-directed mutagenesis) of the target DNA can be by DNA restriction endonuclease to described DSB is repaired after target DNA cutting to generate, and is also possible to realize by base deaminase or the enzyme of other changeable bases.

In the above method, the biology can be mictic organism or apomictic organism, as plant, animal, fungi or Bacterium.

In the above method, the biology is mictic organism, and the genetically modified organism can be specifically fixed in reproduction cell Target DNA described in point mutation.

The plant can be dicotyledon or monocotyledon.The dicotyledon can be crucifer.

The reproduction cell includes but are not limited to egg cell, egg mother cell, pollen cell or pollen mother cell.

In the above method, at least one in the element is started by reproduction cell specific promoter to be expressed.

In the above method, concretely egg cell specific promoter, pollen cell are special for the reproduction cell specific promoter Different promoter or meiosis specific promoter.The egg cell specific promoter can be DD45 (SEQ ID NO:6), EC1.1 The promoter, fusion (SEQ ID NO:4) of (SEQ ID NO:3) or EC1.1-1.2.The pollen cell specific promoter is specific It can be rice Os 08g0560700 promoter (SEQ ID NO:7), tomato LAT52 promoter (SEQ ID NO:8) or arabidopsis AtSPL(SEQ ID NO:5).The meiosis specific promoter can for arabidopsis AT4G40020 (SEQ ID NO:9), The promoter of AT4G20900 (SEQ ID NO:10) or AT1G15320 (SEQ ID NO:11) gene.

In the above method, the element can be element needed for CRISPR/Cas method or CRISPR/Cpf1 method.

In the above method, element needed for the CRISPR/Cas method or CRISPR/Cpf1 method may each comprise a1) Or a2):

A1) DNA restriction endonuclease and sgRNA；

A2) the DNA restriction endonuclease, crRNA and tracrRNA.

The DNA restriction endonuclease can be Cas9, Cas9n, dCas9 or xCas9.Used in the CRISPR/Cpf1 method DNA restriction endonuclease can be Cpf1.

Specifically, at least a kind of in the DNA restriction endonuclease and the sgRNA start table by reproduction cell specific promoter Up to can be following a1), a2) or a3):

A1) the DNA restriction endonuclease is started by the reproduction cell specific promoter and is expressed, and the sgRNA is opened by composing type Mover starting expression；

A2) sgRNA is started by the reproduction cell specific promoter and is expressed, and the DNA restriction endonuclease is made of described The starting expression of type promoter；

A3) the DNA restriction endonuclease and the sgRNA are started by the reproduction cell specific promoter expresses.

In the above method, the sgRNA targets the target DNA.The target DNA can be the code area of gene, It can be the control region, such as promoter, enhancer, 5 ' UTR etc. of gene.The expression product of the code area can be albumen Matter, polypeptide or RNA.

In the above method, the biology is plant, and the reproduction cell specific promoter is egg cell specific promoter, X22) specific can include: ovum of the selection containing the DNA fragmentation and target DNA mutation from the genetically modified organism Cell and sperm (sperm can be free of the DNA fragmentation containing the DNA fragmentation) fertilization are formed by individual, as prominent Variant is selected in the mutant library；Selection is without containing the DNA fragmentation from the genetically modified organism and the target DNA is not sent out The egg cell of raw mutation and containing the DNA fragmentation and sperm fertilization that the target DNA does not mutate is formed by individual (being denoted as individual 1) lays in individual as mutant library.

Further, the method is in X22) after include the following steps X23a): select to contain from individual 1 offspring (sperm can contain the DNA fragmentation also not to the egg cell and sperm that the DNA fragmentation and the target DNA mutate Containing the DNA fragmentation) being fertilized is formed by individual, it is selected in the mutant library as mutant；From individual 1 offspring Selection is without containing the DNA fragmentation and the target DNA egg cell not mutated and contains the DNA fragmentation and the target The sperm fertilization that mark DNA does not mutate is formed by individual, is further used as mutant library deposit individual and is used to prepare mutation Body and with individual 1 feature can be used as mutant library deposit individual individual.

Repeat step X23a), can in mutant library described in further expansion mutant number.The duplicate number can For several times, number of repetition is determined according to specific needs.

In the above method, the biology is plant, and the reproduction cell specific promoter is spermatid specific promoter, X22) specific can include: essence of the selection containing the DNA fragmentation and target DNA mutation from the genetically modified organism Son is formed by individual with egg cell (egg cell can be free of the DNA fragmentation containing the DNA fragmentation) fertilization, as Mutant is selected in the mutant library；Selection does not contain the DNA fragmentation and the target DNA not from the genetically modified organism A sperm of mutation and containing the DNA fragmentation and fertilizing oocytes that the target DNA does not mutate are formed by Body (is denoted as individual 2), lays in individual as mutant library.

Further, the method is in X22) after include the following steps X23b): select to contain from individual 2 offspring (egg cell can contain the DNA fragmentation to the sperm that the DNA fragmentation and the target DNA mutate with egg cell Without the DNA fragmentation) being fertilized is formed by individual, it is selected in the mutant library as mutant；From individual 2 offspring Middle selection is without containing the DNA fragmentation and the target DNA sperm not mutated and contains the DNA fragmentation and the target The fertilizing oocytes that do not mutate of mark DNA are formed by individual, be further used as mutant library deposit individual be used to prepare it is prominent Variant and with individual 2 feature can be used as mutant library deposit individual individual.

Repeat step X23b), can in mutant library described in further expansion mutant number.The duplicate number can For several times, number of repetition is determined according to specific needs.

In the above method, the DNA fragmentation can contain b1) or b2):

B1 the expression cassette of the DNA restriction endonuclease) can be expressed and the expression cassette of the sgRNA can be expressed；

B2 the expression cassette of the DNA restriction endonuclease) can be expressed, the expression cassette of the crRNA can be expressed and described in capable of expressing The expression cassette of tracrRNA.

The DNA fragmentation can also express the selection markers that can be used for screening transgenic biology.

The expression cassette is the DNA for referring to express express target protein in host or purpose RNA, which not only may include The promoter (promoter is denoted as promoter S) for starting destination protein encoding gene or the transcription of purpose RNA encoding gene, may be used also Terminator including terminating destination protein encoding gene or the transcription of purpose RNA encoding gene.The promoter S can open for composing type Mover.

The expression cassette that the DNA restriction endonuclease will be expressed and the expression cassette that can express the sgRNA are denoted as expression cassette 1 respectively With expression cassette 2.When the biology is the plant or the animal, at least one in the expression cassette 1 and the expression cassette 2 Promoter in a expression cassette can be the tissue-specific promoter.

Specifically, the DNA fragmentation also contains and can express the expression cassettes of the selection markers and (expression cassette is denoted as expression Box 3).

In one embodiment of the invention, the selection markers are Hygromycin resistance marker.Further, the expression cassette 3 contain hygromycin gene.Further, the expression cassette 3 also opening containing the starting hygromycin gene expression Mover and the terminator for terminating the hygromycin gene expression.

When the DNA fragmentation has contained only the expression cassette 1 and the expression cassette 2, the expression cassette 1 and the expression cassette 2 upstream and downstream sequence is without particular/special requirement, as long as the DNA restriction endonuclease and the sgRNA can be expressed by being able to achieve.Described When DNA fragmentation contains the expression cassette 1, the expression cassette 2 and the expression cassette 3, the order of connection between each expression cassette is without spy It is different to require, as long as the DNA restriction endonuclease, the sgRNA and the resistance marker can be expressed by being able to achieve.The DNA piece Section is also containing the DNA sequence dna for connecting each expression cassette.

In the above method, the target DNA can for biotic stress, abiotic stress, yield traits, quality trait or secondary Raw metabolin correlation DNA.

The target DNA concretely with biotic stress, abiotic stress, yield traits, quality trait or secondary metabolism The relevant gene of object.

Further, the biological mutant library can be antibiont adverse circumstance mutant library, resisting abiotic adverse circumstance mutant library, production Measure character mutant library, quality trait mutant library or secondary metabolites mutant library.

The present invention also protects the method in screening antibiont adverse circumstance, resisting abiotic adverse circumstance, high yield, good quality or high order Application in raw metabolin mutant.

The biotic stress includes but are not limited to disease, worm, nematode, virus etc..The abiotic stress includes but not only It is limited to herbicide, antibiotic, insecticide, fungicide, drought, flood, cold, heat, salt, heavy metal etc..

The present invention provides a kind of construction methods of biological mutant library, and this method is by that will express completion rite-directed mutagenesis The DNA fragmentation of the required element of target DNA imports in target organisms, has obtained a kind of genetically modified organism.When target organisms are to have The sexual reproduction biochron, the promoter of at least one element is that reproduction cell is special in the required element of rite-directed mutagenesis target DNA Promoter, therefore, obtained genetically modified organism specifically can carry out rite-directed mutagenesis to target DNA in reproduction cell, according to The genotype and target practice situation of female and male gametophyte, and then the offspring with different genotype is obtained, occur including target gene The transgenic progeny that the non-transgenic offspring and target DNA that offspring, the target DNA of mutation do not mutate do not mutate. The offspring that target gene mutates can be used for screening the individual containing objective trait, and what target DNA did not mutated turns base Because offspring will continue rite-directed mutagenesis to target DNA in reproduction cell and reach its offspring, while it can also generate target The transgenic progeny that mark DNA does not mutate.This process can pass on from generation to generation, and during breeding, and every Dai Junke is produced The plant that raw a large amount of target DNA mutate, and it is mutated multiplicity, and then the prominent of a large amount of target DNA mutations can be formulated The generation process in variant library, plant mutant library is as shown in Figure 1.It, can be from obtained mutant library according to the difference of target DNA Middle screening has the biology of following character: antibiont adverse circumstance, resisting abiotic adverse circumstance, high yield, good quality trait, high secondary metabolism Produce amount etc..Therefore, method of the invention has huge application prospect.

Detailed description of the invention

Fig. 1 shows the generation process in plant mutant library；

Fig. 2, which is shown, extracts 13 plants of T1 for the DNA of plant, and the knot of PCR amplification and sequencing is carried out for the target area of ALS Fruit；

Fig. 3, which is shown, screens a large amount of antiweed mutant；

Fig. 4 shows the DNA of 17 plants of antiweed plant, carries out the result of PCR sequencing.

Specific embodiment

The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.Experimental method in following embodiments is unless otherwise specified Conventional method.Material as used in the following examples, reagent, instrument etc., are commercially available unless otherwise specified. Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

The building of embodiment 1. is directed to the knockout carrier of arabidopsis als gene, Cas9 gene specifically expressing in egg cell.

Wherein, target spot isttgccgatgatcccgagtgg, arabidopsis als gene DNA is shown in SEQ ID NO:1, Amino acid sequence is shown in SEQ ID NO:2.

By DNA fragmentation 5 'ttgccgatgatcccgagtgg3 ' be cloned into pHEE401E carrier refer to (1) Xing H L, Dong L,Wang Z P,et al.A CRISPR/Cas9toolkit for multiplex genome editing in plants[J].BMC Plant Biology,2014,14(1):327.(2)Wang Z P,Xing H L,Dong L,et al.Egg cell-specific promoter-controlled CRISPR/Cas9 efficiently generates homozygous mutants for multiple target genes in Arabidopsis in a single 2015,16 (1): generation [J] .Genome Biology in the sgRNA expression cassette in 144. }, is transferred to Agrobacterium In GV3101, with for use by dipping in colored method arabidopsis thaliana transformation.

Embodiment 2. is converted { the part method therefor and material, with reference to (3) Chen Y, Wang Z, Ni to arabidopsis H,Xu Y,Chen Q,Jiang L.2017.CRISPR/Cas9-mediated base-editing system efficiently generates gain-of-function mutations in Arabidopsis.Science China Life Sciences 60,520-523.}

After the carrier built is transformed into Agrobacterium GV3101 bacterial strain by freeze-thaw method, bacterium solution is passed through into method of scoring (containing kanamycin and gentamicin) is applied in YEP solid medium, after cultivating 36-48h under 28 DEG C of dark surrounds, picking Single colonie is inoculated in being added in the liquid YEB culture medium of kanamycins and gentamicin of 1ml, and overnight (28 DEG C of shaken cultivation 200rpm).Bacterium colony PCR identification is carried out at this time, and chooses wherein positive colony, is added in 50ml conical flask, and 25ml is added YEP fluid nutrient medium (containing kanamycin and gentamicin), shake culture under conditions of 28 DEG C of 200rpm, until OD600 Value is increased in 0.8-1.0.Bacterium solution centrifugation is taken to remove supernatant, and with 5% sucrose solution (100ml of same volume Aseptic deionized water adds 5g sucrose) thallus is resuspended, and SilwetL-77 (0.02%) is added in bacterium solution and mixes.It is quasi- by be suitable for After southern mustard inflorescence dips 0.5-1min in bacterium solution, black opaque-plastic bag is covered on arabidopsis seedling, creates dark item Part is placed in 22 DEG C of hot-house cultures and is transferred to illumination cultivation afterwards for 24 hours, harvests seed after seed is mature.The arabidopsis seed of harvest After disinfection, the screening transgenic plant on the MS culture medium containing hygromycin (25mg/L).Extract T1 for plant DNA, PCR amplification is carried out for the target area of ALS and is sequenced.

Embodiment 3. identifies T1 for the genotype of plant

It is sequenced by PCR product and hygromycin resistance screens the editing type determined to als gene, while separating tool There is T-DNA and als gene plant (magic seed plant) not to be edited.

As shown in Figure 1, type there are two types of the egg cells that transgenic plant generates, ratio 1:1, the first genotype is to contain There is T-DNA (DNA fragmentation for completing the required element of rite-directed mutagenesis target DNA can be expressed in the genetically modified organism), Due to the specifically expressed characteristic of egg cell, the target gene on genome will be edited and (be mutated), second of genotype It is not contain T-DNA, therefore the target gene on genome is not edited, and is still wild type.The genotype of pollen is also same There are two types of samples, ratio 1:1, the first genotype is containing T-DNA, due to the specifically expressed characteristic of egg cell, on genome Target gene will not be edited, be still wild type, second of genotype be, does not contain T-DNA, the target gene on genome For wild type.After the plant selfing, there are four types of different genotype, ratio 1:1:1:1 for the seed tool of harvest.The first gene Type is the T-DNA copied containing one and contains edited target gene；Second of genotype is containing there are two copies T-DNA and contain edited target gene；The third genotype is without containing T-DNA and not contain edited target spot Gene, i.e. WT lines；4th kind of genotype is the T-DNA copied containing one and does not contain edited target spot base Cause.The plant of 4th kind of genotype is identical with female genotype in generation, and the probability of appearance is 1/4, if sieved to T-DNA (pass through hygromycin selection) if choosing, then its probability occurred is 1/3.These plant can pass through the sequencing to target gene It picks out.The individual for having 1/2 is also contained into edited target gene in the offspring that these plant generate, 1/4 is wild Type, 1/4 is mother for genotype.The process can be gone down with Infinite Cyclic.This seed with the 4th kind of genotype can generate With the identical seed of oneself genotype, additionally it is possible to generate seed more with independent mutation events, therefore from one Seed starts that one can be established to may include unlimited individual mutant library, and the seed of this genotype is named as " magic kind Son ".In addition, not editing target gene containing T-DNA and containing an editor and one according to the technical principle of " magic seed " Plant, can continue to generate in offspring and a large amount of independent update event.

13 plants of T1 are extracted for the DNA of plant, PCR amplification is carried out for the target area of ALS and is sequenced.The result shows that about 70% T1 plant produces mutation in the desired location of ALS, as shown in Figure 2.

The characteristic specifically practiced shooting using egg cell it is found that these T1 plant when generating seed (T2 generation), can be to not The site ALS of editor re-starts editor, independent to generate new mutation type.Again technical principle (also known as " the magic kind practiced shooting Son " system) as shown in Figure 1.

Embodiment 4. screens antiweed mutant

The technology path of " magic seed " is utilized, T2 the mutant of antiweed occurs for seed.

By from above-mentioned T1 for the T2 harvested on plant for seed, be taped against after disinfection containing 0.24mg/L AC 263222 On MS culture medium, antiweed mutant is screened, as shown in figure 3, successfully screening a large amount of antiweed plant.

The random DNA for extracting wherein 17 plants of antiweed plant, PCR sequencing show that the als gene of these plant has occurred Gene mutation as shown in Figure 4.

The all publications and patents application referred in specification is both incorporated herein by reference, such as every publication or Patent application individually, is particularly incorporated herein by reference the same.

Although the mode of aforementioned invention by way of illustration and example carries out in more detail for the sake of being clearly understood that Description, but it will be apparent that can implement certain changes and modification within the scope of the appended claims, it is such to change Become and modification is all within the scope of the present invention.

Sequence table

<110>Qingdao Qing Yuan compound Co., Ltd

<120>a kind of construction method of biological mutant library

<130> 20190403

<150> 2018104006077

<151> 2018-04-28

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<212> DNA

<213>arabidopsis als gene DNA sequence dna (ALS)

<400> 1

atggcggcgg caacaacaac aacaacaaca tcttcttcga tctccttctc caccaaacca 60

tctccttcct cctccaaatc accattacca atctccagat tctccctccc attctcccta 120

aaccccaaca aatcatcctc ctcctcccgc cgccgcggta tcaaatccag ctctccctcc 180

tccatctccg ccgtgctcaa cacaaccacc aatgtcacaa ccactccctc tccaaccaaa 240

cctaccaaac ccgaaacatt catctcccga ttcgctccag atcaaccccg caaaggcgct 300

gatatcctcg tcgaagcttt agaacgtcaa ggcgtagaaa ccgtattcgc ttaccctgga 360

ggtgcatcaa tggagattca ccaagcctta acccgctctt cctcaatccg taacgtcctt 420

cctcgtcacg aacaaggagg tgtattcgca gcagaaggat acgctcgatc ctcaggtaaa 480

ccaggtatct gtatagccac ttcaggtccc ggagctacaa atctcgttag cggattagcc 540

gatgcgttgt tagatagtgt tcctcttgta gcaatcacag gacaagtccc tcgtcgtatg 600

attggtacag atgcgtttca agagactccg attgttgagg taacgcgttc gattacgaag 660

cataactatc ttgtgatgga tgttgaagat atccctagga ttattgagga agctttcttt 720

ttagctactt ctggtagacc tggacctgtt ttggttgatg ttcctaaaga tattcaacaa 780

cagcttgcga ttcctaattg ggaacaggct atgagattac ctggttatat gtctaggatg 840

cctaaacctc cggaagattc tcatttggag cagattgtta ggttgatttc tgagtctaag 900

aagcctgtgt tgtatgttgg tggtggttgt ttgaattcta gcgatgaatt gggtaggttt 960

gttgagctta cggggatccc tgttgcgagt acgttgatgg ggctgggatc ttatccttgt 1020

gatgatgagt tgtcgttaca tatgcttgga atgcatggga ctgtgtatgc aaattacgct 1080

gtggagcata gtgatttgtt gttggcgttt ggggtaaggt ttgatgatcg tgtcacgggt 1140

aagcttgagg cttttgctag tagggctaag attgttcata ttgatattga ctcggctgag 1200

attgggaaga ataagactcc tcatgtgtct gtgtgtggtg atgttaagct ggctttgcaa 1260

gggatgaata aggttcttga gaaccgagcg gaggagctta agcttgattt tggagtttgg 1320

aggaatgagt tgaacgtaca gaaacagaag tttccgttga gctttaagac gtttggggaa 1380

gctattcctc cacagtatgc gattaaggtc cttgatgagt tgactgatgg aaaagccata 1440

ataagtactg gtgtcgggca acatcaaatg tgggcggcgc agttctacaa ttacaagaaa 1500

ccaaggcagt ggctatcatc aggaggcctt ggagctatgg gatttggact tcctgctgcg 1560

attggagcgt ctgttgctaa ccctgatgcg atagttgtgg atattgacgg agatggaagc 1620

tttataatga atgtgcaaga gctagccact attcgtgtag agaatcttcc agtgaaggta 1680

cttttattaa acaaccagca tcttggcatg gttatgcaat gggaagatcg gttctacaaa 1740

gctaaccgag ctcacacatt tctcggggat ccggctcagg aggacgagat attcccgaac 1800

atgttgctgt ttgcagcagc ttgcgggatt ccagcggcga gggtgacaaa gaaagcagat 1860

ctccgagaag ctattcagac aatgctggat acaccaggac cttacctgtt ggatgtgatt 1920

tgtccgcacc aagaacatgt gttgccgatg atcccgagtg gtggcacttt caacgatgtc 1980

ataacggaag gagatggccg gattaaatac tga 2013

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Tyr Val Gly Gly Gly Cys Leu Asn Ser Ser Asp Glu Leu Gly Arg Phe

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Val Glu Leu Thr Gly Ile Pro Val Ala Ser Thr Leu Met Gly Leu Gly

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Ser Tyr Pro Cys Asp Asp Glu Leu Ser Leu His Met Leu Gly Met His

340 345 350

Gly Thr Val Tyr Ala Asn Tyr Ala Val Glu His Ser Asp Leu Leu Leu

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Ala Phe Gly Val Arg Phe Asp Asp Arg Val Thr Gly Lys Leu Glu Ala

370 375 380

Phe Ala Ser Arg Ala Lys Ile Val His Ile Asp Ile Asp Ser Ala Glu

385 390 395 400

Ile Gly Lys Asn Lys Thr Pro His Val Ser Val Cys Gly Asp Val Lys

405 410 415

Leu Ala Leu Gln Gly Met Asn Lys Val Leu Glu Asn Arg Ala Glu Glu

420 425 430

Leu Lys Leu Asp Phe Gly Val Trp Arg Asn Glu Leu Asn Val Gln Lys

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Gln Lys Phe Pro Leu Ser Phe Lys Thr Phe Gly Glu Ala Ile Pro Pro

450 455 460

Gln Tyr Ala Ile Lys Val Leu Asp Glu Leu Thr Asp Gly Lys Ala Ile

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Ile Ser Thr Gly Val Gly Gln His Gln Met Trp Ala Ala Gln Phe Tyr

485 490 495

Asn Tyr Lys Lys Pro Arg Gln Trp Leu Ser Ser Gly Gly Leu Gly Ala

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Met Gly Phe Gly Leu Pro Ala Ala Ile Gly Ala Ser Val Ala Asn Pro

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Leu Leu Leu Asn Asn Gln His Leu Gly Met Val Met Gln Trp Glu Asp

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Arg Phe Tyr Lys Ala Asn Arg Ala His Thr Phe Leu Gly Asp Pro Ala

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Gly Ile Pro Ala Ala Arg Val Thr Lys Lys Ala Asp Leu Arg Glu Ala

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Ile Gln Thr Met Leu Asp Thr Pro Gly Pro Tyr Leu Leu Asp Val Ile

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Cys Pro His Gln Glu His Val Leu Pro Met Ile Pro Ser Gly Gly Thr

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cgatttcttc ttcctctgtt cttcggcgtt caatttctgg ggttttctct tcgttttctg 60

taactgaaac ctaaaatttg acctaaaaaa aatctcaaat aatatgattc agtggttttg 120

tacttttcag ttagttgagt tttgcagttc cgatgagata aaccaatacc atggactttg 180

ataaatgttc ctcgctgacg taagaagaca ttagtaatgg ttataatata tagctttcta 240

tgaatgtatg gtgagaaaat gtctgttcac tgattttgag tttggaataa aagcatttgc 300

gtttggttta tcattgcgtt tatacaagga cagagatcca ctgagctgga atagcttaaa 360

accattatca gaacaaaata aaccattttt tgttaagaat cagagcatag taaacaacag 420

aaacaaccta agagaggtaa cttgtccaag aagatagcta attatatcta ttttataaaa 480

gttatcatag tttgtaagtc acaaaagatg caaataacag agaaactagg agacttgaga 540

atatacattc ttgtatattt gtattcgaga ttgtgaaaat ttgaccataa gtttaaattc 600

ttaaaaagat atatctgatc tagatgatgg ttatagactg taattttacc acatgtttaa 660

tgatggatag tgacacacat gacacatcga caacactata gcatcttatt tagattacaa 720

catgaaattt ttctgtaata catgtctttg tacataattt aaaagtaatt cctaagaaat 780

atatttatac aaggagttta aagaaaacat agcataaagt tcaatgagta gtaaaaacca 840

tatacagtat atagcataaa gttcaatgag tttattacaa aagcattggt tcactttctg 900

taacacgacg ttaaaccttc gtctccaata ggagcgctac tgattcaaca tgccaatata 960

tactaaatac gtttctacag tcaaatgctt taacgtttca tgattaagtg actatttacc 1020

gtcaatcctt tcccattcct cccactaatc caacttttta attactctta aatcaccact 1080

aagcttcgaa tccatccaaa accacaatat aaaaacagaa ctctcgtaac tcaatcatcg 1140

caaaacaaaa caaaacaaaa caaaaacccc aaaaagaaag aataagctag atggattaca 1200

aggaccacga cggggattac aaggaccacg acattgatta caaggatgat gatgacaaga 1260

tg 1262

<210> 4

<211> 1614

<212> DNA

<213>EC1.1-1.2 promoter, fusion (EC1.1-1.2)

<400> 4

cgatttcttc ttcctctgtt cttcggcgtt caatttctgg ggttttctct tcgttttctg 60

taactgaaac ctaaaatttg acctaaaaaa aatctcaaat aatatgattc agtggttttg 120

tacttttcag ttagttgagt tttgcagttc cgatgagata aaccaatacc atggttatac 180

tagtgaataa aagcatttgc gtttggttta tcattgcgtt tatacaagga cagagatcca 240

ctgagctgga atagcttaaa accattatca gaacaaaata aaccattttt tgttaagaat 300

cagagcatag taaacaacag aaacaaccta agagaggtaa cttgtccaag aagatagcta 360

attatatcta ttttataaaa gttatcatag tttgtaagtc acaaaagatg caaataacag 420

agaaactagg agacttgaga atatacattc ttgtatattt gtattcgaga ttgtgaaaat 480

ttgaccataa gtttaaattc ttaaaaagat atatctgatc taggtgatgg ttatagactg 540

taattttacc acatgtttaa tgatggatag tgacacacat gacacatcga caacactata 600

gcatcttatt tagattacaa catgaaattt ttctgtaata catgtctttg tacataattt 660

aaaagtaatt cctaagaaat atatttatac aaggagttta aagaaaacat agcataaagt 720

tcaatgagta gtaaaaacca tatacagtat atagcataaa gttcaatgag tttattacaa 780

aagcattggt tcactttctg taacacgacg ttaaaccttc gtctccaata ggagcgctac 840

tgattcaaca tgccaatata tactaaatac gtttctacag tcaaatgctt taacgtttca 900

tgattaagtg actatttacc gtcaatcctt tcccattcct cccactaatc caacttttta 960

attactctta aatcaccact aagctagtaa cgcctatcat gaattagctc tactaaatct 1020

agcaaccttt caaatttgca gtattgcagg tgtctctgtg tctttaaaat agttgcctta 1080

tgatttcttc ggtttcaaga tgatcaaata gttatagatt tcatgctcac acatgctcat 1140

tagatgtgta catactttac ttacccaaat ctattttctc gcaaagattt tgatggtaaa 1200

gctgatttgg ttctattgaa ctaaatcaaa cgagtttcag actgagtgat tctaatccgg 1260

cccattagcc cctaaacaga cccactaatt acgcagcttt taatagagta attacaccta 1320

gtttacccac taaaccacta agcactaatt atctcacaat ctaatgagct tccctcgtaa 1380

ttacttgggc tttcactcta ccatttattt gtaacagtca agtctctact gtctctatat 1440

aaactctcta aagttaacac acaattctca tcacaaacaa atcaaccaaa gcaacttcta 1500

ctctttcttc tttcgacctt atcaatctgt tgagaaatct agatggatta caaggaccac 1560

gacggggatt acaaggacca cgacattgat tacaaggatg atgatgacaa gatg 1614

<210> 5

<211> 2016

<212> DNA

<213>AtSPL promoter (AtSPL)

<400> 5

ggatccgttt gtttgttttt taatcgtttt catcaacatg attgatatat atatagtttt 60

tgcacttgaa aaagttttga tttttattta tgtaaaaaac tgcagaagaa acgtttggat 120

ggtgatcaga ataatgtagt tcgatccaac ggtggtggat tttcgaaata cacaatgatt 180

cctcctccga tgaacggcta cgatcagtat cttcttcaat cagatcatca tcagaggagc 240

caaggtttcc tttatgatca tagaatcgct agagcagctt cagtttctgc ttctagtact 300

actattaatc cttatttcaa cgaggcaaca aatcatacgg tactaagtat agtccattta 360

ttaatactca tatataggta tatatgtata taactgttga tcttatttga tttaactggt 420

gggtttaggg accaatggag gaatttggga gctacatgga aggaaaccct agaaatggat 480

caggaggtgt gaaggagtac gagttttttc cggggaaata tggtgaaaga gtttcagtgg 540

tggctaaaac gtcgtcactc gtaggtgatt gcagtcctaa taccattgat ttgtccttga 600

agctttaaat gttttatctt tctatattga tttaaacaaa atcgtctctt taaagaaaaa 660

acattttaag tagatgaaag taagaaacag aagaaaaaaa agagagagcc ttttttggtg 720

tatgcatctg agagctgagt cgaaagaaag attcagcttt tggattaccc ttttggttgt 780

ttattatgag attctaacct aaacactcag acatatatgt tctgttctct tccttaattg 840

ttgtcatgaa acttctctct ccctctctct ctctaaagtt gtgtcagatc ttggggcttg 900

tatccatttt gtgtttcttt agttaggcaa tgatcctgtc atgattcatg ttatcaccta 960

attaatcctc ggttttcgaa acggtgtcat gcatatctga ttaatcttgt tatctggtgt 1020

gagataaatg tatcaaattg ctcttgttta tagcttttcc aattaatgtc attgcatagc 1080

atatgggatg tacagcttcg ttttcgattt ataaaattta tacatgtgtt tggtacgtac 1140

tgttttgttt tggatcagaa ttataaattt agtacagtat aaactacttt aaaaattggc 1200

caatgagaat tttgagttta tcttcttttt ataagttgat tgattttgct ttgtttattt 1260

atagaaaata tgaaatttta tcaattattt ttgggatgct tttttattga ttttagttcg 1320

gctaaaatta gttagatgtt taaatttatt aaaacagata tgcaaaaatt aactcgatgg 1380

tttttttttt tttttttagg gggataaaaa tcgaactata tagttaatta aaacttaaaa 1440

tgtttttctg aacggctatt tcactgattt tttagtagtt taaatgtttt tcaccgaaaa 1500

taaaaacaga ataggaaaca atactataga agtctaacca aaaaaagttc aatatccagt 1560

agttcaatat ttctaaaaag aaaatttcta cacaaagttt atggcaaaaa cctaagttat 1620

tatggagaaa accaagttat tgaaagttat agcgtactgt atgtacaatc tttcatcgtg 1680

atttcatgta ttatagataa aattattcat tgtggatgta atgcaaatag aaccatccgt 1740

aaatccatgt attacggaaa aattattcat cacaactctt atgtttgaaa atagttagat 1800

tcggtcttag atcattattt taaaaataca aataaacaaa tacgatcgac aacaattgct 1860

cagcaaattt gacatactga taaagtcatt gtttgacaaa acacatatgg gccttaatat 1920

caaaacacac ggcccattat aaaagtaaac tgataagata aaacagttcc ctatgattcc 1980

tcaataagca gctgctcagc acgttctcgt ggtacc 2016

<210> 6

<211> 1020

<212> DNA

<213>DD45 promoter (DD45)

<400> 6

aaatgttcct cgctgacgta agaagacatt agtaatggtt ataatatata gctttctatg 60

aatgtatggt gagaaaatgt ctgttcactg attttgagtt tggaataaaa gcatttgcgt 120

ttggtttatc attgcgttta tacaaggaca gagatccact gagctggaat agcttaaaac 180

cattatcaga acaaaataaa ccattttttg ttaagaatca gagcatagta aacaacagaa 240

acaacctaag agaggtaact tgtccaagaa gatagctaat tatatctatt ttataaaagt 300

tatcatagtt tgtaagtcac aaaagatgca aataacagag aaactaggag acttgagaat 360

atacattctt gtatatttgt attcgagatt gtgaaaattt gaccataagt ttaaattctt 420

aaaaagatat atctgatcta gatgatggtt atagactgta attttaccac atgtttaatg 480

atggatagtg acacacatga cacatcgaca acactatagc atcttattta gattacaaca 540

tgaaattttt ctgtaataca tgtctttgta cataatttaa aagtaattcc taagaaatat 600

atttatacaa ggagtttaaa gaaaacatag cataaagttc aatgagtagt aaaaaccata 660

tacagtatat agcataaagt tcaatgagtt tattacaaaa gcattggttc actttctgta 720

acacgacgtt aaaccttcgt ctccaatagg agcgctactg attcaacatg ccaatatata 780

ctaaatacgt ttctacagtc aaatgcttta acgtttcatg attaagtgac tatttaccgt 840

caatcctttc ccattcctcc cactaatcca actttttaat tactcttaaa tcaccactaa 900

gcttcgaatc catccaaaac cacaatataa aaacagaact ctcgtaactc aatcatcgca 960

aaacaaaaca aaacaaaaca aaaaccccaa aaagaaagaa taatggcttc taacacaagt 1020

<210> 7

<211> 263

<212> DNA

<213>rice Os 08g0560700 promoter (Os08g0560700)

<400> 7

aaaaagggga tcgagaggaa gaggaagctt tgtgagtttg ggcctttatt tggtggaggc 60

ccattgattc ctacgtgggc tgggccgaac acggtccagg tagttggtcc acattccagg 120

ttgaacgatt ccagcggcgg cggcgagtgg tggcgcgcga tcatctccca ctcccaccgt 180

tgttcctcct caacacgcgc cactcctcac attcatttcc acctgctcat ctatatctat 240

ctatcatcaa gctgatcatc cat 263

<210> 8

<211> 603

<212> DNA

<213>tomato LAT52 promoter (LAT52)

<400> 8

tgtcgacata ctcgactcag aaggtattga ggaatgatcg attctgggtc atttgtgtgg 60

ttaatcaccc tccaaatcaa ctaagtcatc ctgaaggaca atatcctatt ttttctctcg 120

taggtttatc atttaaatta ctatcgcgtg ataattttgt aacgtagaaa aataatacca 180

ttaatccaaa cgttatattc attaaaataa ttatgataca tttaaaaata tttcgtgacc 240

tctcaattat tgcaaattct aagccatccc aagttttgag gctaattttt tttactatac 300

tatttttaca accacaaaaa cataaaaaat aaaaaataaa aaaaataaac cgagtcaatt 360

gctacaatca cttcattatt aattttaatt aatattatgt ggttatatat gaaactgtta 420

gagaaataat agctccacca tatttttttc tcaatttatt ttcactataa aaaggctatt 480

tcattataat caaaacaaga cacacacaaa gagaaggagc aataaaataa aagtaaacaa 540

caatttgtgt gtttaaaaaa aaaaaaaaag tacacacacc aaaaaaaaaa attccaattt 600

aaa 603

<210> 9

<211> 1553

<212> DNA

<213>AT4G40020 promoter (AT4G40020)

<400> 9

ggggtttagg tctttccatt actttttaat gttttttctg ttactgtctc cgcgatctga 60

ttttacgaca atagagtttc gggttttgtc ccattccagt ttgaaaataa aggtccgtct 120

tttaagtttg ctggatcgat aaacctgtga agattgagtc tagtcgattt attggatgat 180

ccattcttca tcgttttttt cttgcttcga agttctgtat aaccagattt gtctgtgtgc 240

gattgtcatt acctagccgt gtatcgagaa ctagggtttt cgagtcaatt ttgccccttt 300

tggttatatc tggttcgata acgattcatc tggattaggg ttttaagtgg tgacgtttag 360

tattccaatt tcttcaaaat ttagttatgg ataatgaaaa tccccaattg actgttcaat 420

ttcttgttaa atgcgcagat ggctcgtacc aagcaaaccg ctcgtaagtc caccggaggt 480

aaagctccaa ggaagcaact tgctactaag gttttgttcc ttcttgtctc ttttttcaaa 540

taatacttgt gttgtgaagt tgaatgttaa tctccttctt tattaacctc aggctgctcg 600

taaatctgca ccaactactg gtggagtcaa gaaaccacat cgttaccgtc ctggaactgt 660

tgctctccgg tttgtccctt cttcgatttg tatgtgattc tttgagatta tgtaacattg 720

tgtgttaaca ttcctcttat cttttggtgt tcagtgaaat ccgtaagtac cagaagagta 780

ctgagttgct tatcaggaaa ctgccatttc agaggctagt ccgtgagatt gctcaagatt 840

tcaagactga tttgcgtttc cagagccatg ctgtcttagc tctccaggaa gctgcagaag 900

catatcttgt tggtctcttt gaagacacta acctttgtgc cattcatgcc aagcgtgtga 960

ccataatgcc caaagacatt cagctcgctc gtcgtatcag aggtgaacgc gcttaagcca 1020

accaagaatc cgagatttgg ttcaagtagt ttttgttttt tatgaaagca agatcttaat 1080

tgctgtgttt tttaaatctc tgggtatgta gtagtaagag tagtaagaag tctgcaatct 1140

aagaatgttg tgttctttaa gcttattatt atgtgtgttg cgaactcttt ttaactcttt 1200

tgttaaattc ttatgttttc ttaactcgtt tgttcgtcac cattgctttc tcttcaaata 1260

atggctgatt tttttctata attttggatt tgttgaatgc tttcttttaa aagaatcaga 1320

ttttggactt ttgaccaaag aaaataataa tatcagacga taaaatagac ggctctcgat 1380

aaaactaacc ctaaaaataa ggaaataagt tcctctttga accaaatttt ctttctttga 1440

ccaatagatc tttttgtcaa cctcttaaat atattcttag tcaatcttct aataaaccca 1500

ttggccatta ccaaaaattc ctcggaaacg ctgaataaaa aacattctat cat 1553

<210> 10

<211> 621

<212> DNA

<213>AT4G20900 promoter (AT4G20900)

<400> 10

ctcggcaaac gccataacta cagcgtcgag tttagcggtg ttggtgggtc tggtgaagta 60

gaacccggat ttgcttctca gatagctatt tttagacggg aagcttcatc tgttgagaat 120

gttgcagaaa gctcaatgca gccttataaa gtcatctggg agtggaagaa agaagatgta 180

gaaaagaaaa agactgatct ttgactagag tagtatctac cagagaatat atatattgca 240

agcaatgtga tgacgtgttc atctataaaa ctcaaagctc gtttagttct tttgccatga 300

gcttttgcag attaaccata aaatcgaacc tttatgttta caagatcctt ttgtctccaa 360

gtctactggt ccgttgaagt ttaaagatta gatcagtaga ttaccctttt tgcccctacg 420

ttaaagtttg tttatttaaa tacaagtcct tgtgattttt atcactttct cacttcttca 480

acgacactga ttcgttttca aagcatttgc gttcttgatc tttttcacga gggcgatttc 540

tgaagaacac gagaatttga attctgggaa aagctttctc gaactttatc tgagtaaatt 600

gacagagaga atcgaaaaag a 621

<210> 11

<211> 1320

<212> DNA

<213>AT1G15320 promoter (AT1G15320)

<400> 11

caactcacca cctccctctc cttccccgaa gttttctcgg tgaactccag agctgtgagt 60

ggcagccacg tttgcaactc cggtaggcga catcctttca aatcagttgc tgaaaacgta 120

cctatcactt ttctatgtct cccctgcaat ctcaaaaaac acaaaatgag taaacattat 180

atattaacca aagtatatat gtaaaatgaa gcctacattg actagctgca gatgatcctc 240

ttgtgctata tcaggtgcat gaacgatggg gacagcgttg agcagagcac ccttcataac 300

gttgatggca ttggaaacag tggttctctc tgtgatggcg taaacggaat cattaacagc 360

tccgagatca gagatggagc gtgagagaac agttttgagg tcatcaaagt gatggtcttt 420

aaggaaacgc aagagatcca tctgagtaag catcttgtaa gcagaggcag actcaatcaa 480

ctcaactcct gcaatagtgt tattgctctc tatgctgctc tccactggca ctaaagctcg 540

atggatccct ttgctaaaca cttccatgca ctctaacacg ctggtattag gattcagtgt 600

ccagagactg agtccttcga ggcaatgacc gatgatggat gacacctgtg aactcatctt 660

gcgatcaaga tcagagagat tggaatcttc accggcgata tgagcaagta tgtcaagcat 720

cgtgagaatc ccgatatagt gttttcttac aactccagtt tgtttgtctg attccatgat 780

catggaacca ccggctccga tccagtggcc tggaggtgca gccactggga gggctgagat 840

ggagttagcc accaatgtgt tcattgcgtg ggaaagtgta gccgtgtatg gtacctcgac 900

gagccgtcgg tttctgaccg ttagatcttt ggccgtgaca ttgatcagac ggctgtgatc 960

ttctttgctc ttctcttctt gcatagtgtt ctactactgt tgtcgtagct tgaacttgtt 1020

gctttgagaa cctttttaag tgtcgatctt ggctcgaaat ttgatgtgaa tggatcagga 1080

cgccgtgtga tgatgatcta gaagcacgtg gcaatcgata cttgctaatg acatgcaaca 1140

cgtggcattt ttcattgggc cttagctaac ggcccagtag tatgtttaat gacccaaata 1200

acccttcgtc tctgctttgt ttttatttct taaaatcagt atctccctcg cgtccgcaaa 1260

tgaaacgacc tgagaatcca agaacgtagg aaagaaatca gtgctgaaga agaaacacga 1320

Claims (10)

1. a kind of construction method of biology mutant library, it is characterised in that: the method X1) or X2):

The method X1) include following X11) and X12):

X11 DNA fragmentation) is imported into target organisms, obtains genetically modified organism；The DNA fragmentation can be in the genetically modified organism The required element of rite-directed mutagenesis target DNA is completed in middle expression；

X12 the genetically modified organism) is cultivated, biological mutant library is obtained；

The method X2) include following X21) and X22):

X21 DNA fragmentation) is imported into target organisms, obtains genetically modified organism；The DNA fragmentation can be in the genetically modified organism The required element of rite-directed mutagenesis target DNA is completed in middle expression；

X22 the genetically modified organism) is cultivated, the individual of target DNA mutation is selected to obtain from the genetically modified organism To biological mutant library, selection is containing the DNA fragmentation from the genetically modified organism and the target DNA do not mutate Individual is bred, to expand the mutant library.

2. according to the method described in claim 1, it is characterized by: the biology is that mictic organism or asexual reproduction are raw Object.

3. according to the method described in claim 1, the transgenosis is raw it is characterized by: the biology is mictic organism Object can the specifically target DNA described in rite-directed mutagenesis in reproduction cell.

4. method according to claim 1 to 3, it is characterised in that: at least one in the element is thin by reproduction The starting expression of born of the same parents' specific promoter.

5. according to the method described in claim 4, it is characterized by: the reproduction cell specific promoter is that egg cell specifically opens Mover, pollen cell specific promoter or meiosis specific promoter.

6. any method in -5 according to claim 1, it is characterised in that: the element be CRISPR/Cas method or Element needed for CRISPR/Cpf1 method.

7. according to the method described in claim 6, it is characterized by: the CRISPR/Cas method or CRISPR/Cpf1 method Needed for element include a1) or a2):

A1) DNA restriction endonuclease and sgRNA；

A2) the DNA restriction endonuclease, crRNA and tracrRNA.

8. any method in -7 according to claim 1, it is characterised in that: the DNA fragmentation contains b1) or b2):

B1 the expression cassette of the DNA restriction endonuclease) can be expressed and the expression cassette of the sgRNA can be expressed；

B2 the expression cassette of the DNA restriction endonuclease) can be expressed, the expression cassette of the crRNA can be expressed and described in capable of expressing The expression cassette of tracrRNA；

Further, the DNA fragmentation can also express the selection markers that can be used for screening transgenic biology.

9. -8 any method according to claim 1, it is characterised in that: the target DNA is and biotic stress, abiotic Adverse circumstance, yield traits, quality trait or secondary metabolites correlation DNA.

10. in claim 1-9 any the method screening antibiont adverse circumstance, resisting abiotic adverse circumstance, high yield, good quality or Application in high secondary metabolites mutant.