CN104846010B - A kind of method for deleting transgenic paddy rice riddled basins - Google Patents
A kind of method for deleting transgenic paddy rice riddled basins Download PDFInfo
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Abstract
The present invention provides a kind of methods for deleting transgenic paddy rice marker gene, belong to plant genetic engineering field.The present invention establishes a method that high efficiency deletes transgenic paddy rice marker gene in plant level using the genome fixed point editing technique of CRISPR/Cas9 System-mediateds.Whole segment mark gene can be effectively deleted, and pointedly can only delete the expression cassette of marker gene using the method for the present invention, the expression without changing other compositions in transgenic paddy rice.It therefore can be with the transgenic paddy rice of high-efficiency breeding screening marker-free gene, so as to completely eliminate safety doubt of the people to riddled basins using the method for the present invention.
Description
Technical field
The present invention relates to biotechnologys and field of plant genetic.It specifically, can be with the present invention relates to one kind
The method for deleting Elimination of Selective Marker Genes from Transgenic Plants.
Background technology
In transgenic plants, the foreign gene in plant is imported other than target gene, also selection markers base
Cause and other related genes.Target gene is the gene for optimizing or assigning plant specific trait；Riddled basins then can
It assigns transfer-gen plant and resists the characteristics such as certain antibiotic or herbicide, the use in transgenic protocol, which can greatly improve, turns base
Because of the efficiency of foliage filter screening.In addition to target gene people are concentrated mainly on riddled basins the worry of genetically modified plants,
Although exact evidence confirms that riddled basins are implicitly present in security risk not yet, the safety of riddled basins is asked
Topic is paid attention to by countries in the world government and tissue, and has become the master for limitation genetically modified plants industrialization and commercialized development
Want bottleneck.Therefore, how to delete riddled basins to improve the safety of genetically modified plants is always genetic engineering research
One of hot spot.
For a long time, whether riddled basins and its product can have an impact environment or human health, cause people
Extensive concern.For solve may the safety problem caused by selected marker, some scientists employ safer gene
It alternatively marks, such as 6- Phophomannose isomerase genes, xylose isomerase gene.But this replacement policy has still failed
It totally disappeared except safety doubt of the people to riddled basins, fundamentally, the screening mark in complete deletion genetically modified plants
Remember gene, cultivate the genetically modified plants of marker-free, be only the most basic strategy for solving selected marker safety concerns.Cause
This, the research and cultivation of transgenic plant without selective marker have become a trend of plant genetic engineering field in the world.Mesh
Before, applied to the molecule deletion system efficiency in rice system than relatively low, and need to carry out induction processing in the tissue cultures stage,
It is unfavorable for scale operation.
Invention content
In consideration of it, the present invention utilizes the genome of CRISPR/Cas9 System-mediateds dexterously, in a way characteristic to the invention
Fixed point editing technique establishes a method that high efficiency deletes transgenic paddy rice riddled basins in plant level.
Principles and methods
CRISPR/Cas is a kind of acquired immune system being widely present in bacterium or Archimycetes.CRISPR sites are
A series of one section of specific DNA sequence on bacterial chromosome or on plasmid, by short highly conserved direct repetitive sequences
The similar intervening sequence of length is alternatively arranged composition therewith.Cas9 genes be connected with CRISPR repetitive sequences it is conservative
CRISPR related protein genes.Specific recognition cutting characteristics based on CRISPR/Cas9 systems, at present application are more successfully
By the CRISPR/Cas9 systems for carrying out self-produced Streptococcus pyrogenes of transformation.Jinek etc. is realized for the first time based on CRISPR/Cas9 systems
The DNA double chain fracture that system induces, the further application for CRISPR/Cas9 provide the foundation.Cong etc. utilizes CRISPR/ for the first time
The EMX1 of Cas9 system on human 293T cells and the Th of mouse cell realize rite-directed mutagenesis, at present CRISPR/Cas9 technologies
It has been successfully applied in Escherichia coli, Diplococcus pneumopniae, zebra fish, drosophila and mouse.Recently, CRISPR/Cas9 is in plant
It is to apply also to achieve important breakthrough, Li et al. is also successfully realized genome using CRISPR/Cas9 in arabidopsis and tobacco
Rite-directed mutagenesis, and find that mutation efficiency is related with the expression quantity of gRNA；Prove that CRISPR/Cas9 systems can be simultaneously to more simultaneously
Gene or monogenic multiple sites carry out fixed point editor.Nekrasov etc. is successfully realized cigarette using CRISPR/Cas9 systems
The rite-directed mutagenesis of careless gene PDS, mutation rate 1.8%-2.4%.
CRISPR has been applied to the bases such as rice, wheat, arabidopsis at present as a kind of new target gene modification technique
In the pointed decoration research of cause, but there has been no the researchs that the important crop economical character of progress is improved.
In view of CRISPR/Cas9 technical characteristics, the present invention is dexterously applied to transgenic paddy rice selection markers base
The deletion of cause, the high efficiency for realizing transgenic paddy rice riddled basins are deleted.
Specifically, high efficiency deletes transgenic paddy rice riddled basins in plant level the present invention provides one kind
Method, using CRISPR/Cas9 System-mediateds genome pinpoint editing technique, establish screening marker-free gene rice turn
Gene system, the transgenic paddy rice new varieties to cultivate tool bio-safety provide a new way, turn for safe and efficient rice
Gene breeding provides basis.The present invention can be used for deleting the riddled basins in transgenic paddy rice gene or its segment, under
Such gene or segment can also be known as original vector in text.
Further, it the described method comprises the following steps：
1) first is chosen in the original vector marker gene encoder block upstream sequence area that rice riddled basins are transferred to
Target fragments, and choose the second target fragments, first and second target fragments in marker genes encoding frame downstream sequence
A chain be respectively provided with 5 '-(N)X- NGG-3 ' structures, wherein (N)XRepresent the base sequence { N that number is X1, N2……
NX, N1, N2……NXIn each represent any one in base A, G, C, T, N is appointing in base A, G, C, T in NGG
Meaning one；
2) it puts in order according to the nucleic acid of first and second target fragments, structure is practiced shooting for paddy gene
CRISPR/Cas9 recombinant vectors, the recombinant vector include having the guide rna expression frame of the target fragments and Cas9 cores
Sour expression of enzymes frame；
3) callus of the transgenic paddy rice containing the riddled basins is prepared, it is spare；
4) the recombinant vector steps for importing 3 for being obtained step 2)) in obtain callus in rice cell, induction
The guide rna expression frame and Cas9 enzyme nucleic acid expression frames of the target fragments co-express in rice cell, to be deleted
The region in marker genes encoding frame promoter upstream and terminator downstream is realized and is sheared simultaneously, causes double-strand break.It triggers simultaneously
The DNA repair functions of rice cell itself cause missing base in target site, realize intracellular screening marker gene, upstream
The fragment deletion of promoter and downstream terminator；
5) regeneration plant is obtained by protoplast instantaneous conversion or agriculture bacillus mediated stable conversion, passes through genome
DNA section containing target fragments in PCR method clonal turnover plant, and amplified production is sequenced.
6) transfer-gen plant that selection riddled basins encoder block lacks completely.
In a kind of preferred implementation, 5 ' in first target fragments-(N)XThe numerical value of the X of-NGG-3 ' structures with
5 ' in second target fragments-(N)XThe numerical value of the X of-NGG-3 ' structures is different from each other, in another implementation, the two
It is mutually the same.
In a kind of preferred implementation, X is 19 or 20.
In a kind of preferred implementation, for the CRISPR/Cas9 recombinant vectors that paddy gene is practiced shooting, for double target spot weights
Group carrier.
In a kind of preferred implementation, first target fragments are located at the promoter of driving riddled basins expression
Upstream, for 5 '-GTGGACGAGATTAGATAGCC-3 ', original vector can be used for assembling other efficient locus specificities sequence
Identify sequence.
In a kind of preferred implementation, second target fragments are located at the terminator for terminating riddled basins expression
Downstream, sequence 5 '-TAGCCCTGCAGGAAATTTAC-3 ', original vector can be used for assembling other efficient locus specificities
Identify sequence.
In a kind of preferred implementation, Reporter gene GUS is contained in original vector, original vector, which can be used for assembling, has spy
Determine the gene of function.
In a kind of preferred implementation, it is different that the riddled basins refer to hygromycin gene, anti-herbicide gene, xylose
Structure enzyme gene, antibiotics resistance gene etc..
In a kind of preferred implementation, the gene with specific function refers to anti insect gene, disease-resistant gene, anti-weeding
Agent gene, adversity gene, anti-aging gene, quality function improvement gene and the fusion being made of said gene or polyad
Cause.
In a kind of preferred implementation, the rice refers to long-grained nonglutinous rice and japonica rice.
During the specific implementation of the above method, the present invention is obtained using carrier pCAMBIA1300 with selection markers
The Nipponbare transfer-gen plant of gene.
On the other hand, the present invention also provides a kind of CRISPR/Cas9 recombinant vectors practiced shooting for paddy gene, the loads
Body contains skeleton carrier, target fragments 1 and target fragments 2.
Preferably, the nucleotides sequence of the first target fragments is classified as 5 '-GTGGACGAGATTAGATAGCC-3 ', the second target
The nucleotides sequence of segment is classified as 5 '-GTAAATTTCCTGCAGGGCTA-3 '.First target fragments are located at riddled basins
The upstream region of promoter, the second target fragments are located at riddled basins terminator downstream area；Preferably, target fragments lean on
Nearly promoter or terminator region, and be non-Rice Genome Sequence or homologous sequence.
The present invention imports above-mentioned recombinant vector in the rice cell containing riddled basins, several by cytothesis
Rice plant.By rice protoplast instantaneous conversion of the method for recombinant vector Introduced into Rice cell for PEG mediations or Agrobacterium
The Rice Callus stable conversion of mediation.Also, the present invention passes through piece containing target in Genomic PCR method clonal turnover plant
The DNA section of section, and amplified production is sequenced.Effect is deleted by the way that evaluation and screening marker gene is sequenced.In addition, to reach complete
Delete riddled basins effect, the plant that riddled basins encoder block and promoter and terminator lack completely.
Technique effect
The method that the present invention establishes a deletion transgenic paddy rice riddled basins.The method of the present invention is to target site
Complementary pairing of the identification dependent on base between nucleic acid, can to the target sequence of any 20bp for following NGG closely into edlin, and
Distribution frequency of its target spot in genome is very high；The method of the another present invention can be simultaneously to the different loci of same gene or multiple
The site of gene is oriented editor, makes it with more flexible；In addition method of the invention is simple and quick, rule preferably
Modelling, high-throughput operation.In short, the method for the present invention orients editing technique as a kind of new gene, wide hair is presented
Open up potentiality and application prospect.Relative to other riddled basins delet methods, method of the invention has the advantage that：
1. deleting, effect is thorough, and the present invention uses the recombinant vector of special designing, with reference to CRISPR/Cas9 Knockout technologies,
The expression cassette that efficiently orientation deletes riddled basins is realized, including riddled basins sequence, starts selection markers base
Because of the promoter of expression, the terminator of riddled basins expression is terminated, so as to thoroughly knock out the transcription of riddled basins
And expression.Present inventor has also carried out similar experiment using other recombinant vectors, and effect can not show a candle to the present invention and be adopted
Carrier.
2. with strong points, the present invention only deletes the expression cassette of riddled basins, without changing its in transgenic paddy rice
His ingredient does not influence the economical character of transgenic paddy rice.
3. the period is short, i.e., it can be removed riddled basins in the transgenosis present age.
4. riddled basins safe to use, that CRISPR/Cas9 target practices recombinant vector introduces can be divided by passage
From being deleted without carrying out other deleting techniques.
5. applicability is wide, different rice varieties, such as Nipponbare, 9311 can be suitble to.In addition, it is applicable not only to list plant
Object, such as rice, wheat, barley, corn, are also adapted to dicotyledon, such as tobacco, potato, cotton, tomato etc..
Existing CRISPR/Cas9 technologies are merely capable of knocking out certain several gene with realizing fixed point, can not
Realize the whole removal of riddled basins.And method using the present invention can completely cut off entire riddled basins piece
Section, the missing without causing other useful genes.Also, applicants have discovered that primer using the present invention can make excision
It is more efficient, upstream and downstream segment retain it is more complete, and use other primers then efficiency be substantially reduced.
The present invention can eliminate may bring harm due to the use of riddled basins to ecology, environment, food security etc.
Risk.Also, a new way is provided to cultivate the transgenic paddy rice new varieties of tool bio-safety and edible safety using the technology
Diameter also provides basis for safe and efficient Transgenic Rice breeding.
Description of the drawings
Carry out the specific embodiment that the present invention will be described in detail below in conjunction with the accompanying drawings, illustrate.In attached drawing：
Fig. 1 is the CRISPR/Cas9 recombinant vector schematic diagrames of the present invention.1 He of target fragments is driven with OsU3 promoters
The expression of sgRNA, the expression of OsU6 promoters driving target fragments 2 and sgRNA.
Fig. 2 is the result schematic diagram to the CRISPR/Cas9 recombinant vector digestion verifications of the present invention.Wherein, recombination is carried
Body carries out BsaI single endonuclease digestions, and what large fragment represented is the CRISPR/Cas9 targeting vectors of linearisation, and small fragment includes target sequence
1 and sequence 2 including segment.
Fig. 3 is by deleting riddled basins in agriculture bacillus mediated transgenic paddy rice strain as a result, PCR detection parts
Result figure.Wherein, 500bp amplified or so bands represent that PMI encoder blocks are removed.And 3730bp amplified or so items
Band represents that PMI encoder blocks are not removed.There is 2 500bp or so band in figure, illustrate successfully to delete transgenic rice plant
In riddled basins PMI.
Fig. 4 is by deleting riddled basins sequencing result, wherein WT in agriculture bacillus mediated transgenic paddy rice strain
It is expressed as PMI encoder blocks in transgenic paddy rice strain not to be deleted, the sequence for deleting mutation has occurred in "-" expression.Sequencing result
Illustrate, which has not only successfully deleted the marker gene PMI in transgenic paddy rice, but also target sequence also occurs in various degree
Base deletion.
Fig. 5 is rice paddy seed GUS colored graphs before and after deletion marker gene.A is the transgenic paddy rice deleted before marker gene
The GUS colored graphs of seed；B be delete marker gene after transgenic paddy rice seed GUS colored graphs.As a result illustrate, mark base
Because before and after deletion, the embryo and endosperm of transgenic paddy rice seed suffer from color, and coloration result does not have apparent difference.Illustrate this
Invention does not change the expression of other compositions such as reporter gene in transgenic paddy rice after successfully marker gene is deleted.
Specific embodiment
Illustrate the present invention referring to specific embodiment.It will be appreciated by those skilled in the art that these embodiments are only
For illustrating the present invention, do not limit the scope of the invention in any way.
Experimental method in following embodiments is conventional method unless otherwise specified.Medicine used in following embodiments
Material raw material, reagent material etc. unless otherwise specified, are commercially available products.
The structure of carrier
In the present embodiment, the Nipponbare transfer-gen plant with selection markers is obtained using carrier pCAMBIA1300.And
And it illustrates by taking riddled basins PMI, the Reporter gene GUS as an example.Turned according to acquired containing PMI marker gene
The characteristic of PMI encoder blocks in trans-genetic hybrid rice, chooses suitable target practice site, and the present invention has selected target fragments 1：5’-
GTGGACGAGATTAGATAGCC-3 ' and 5 '-TAGCCCTGCAGGAAATTTAC-3 ' is respectively synthesized OsU3 promoters, target sequence
(synthesis of above-mentioned each segment is using normal for row 1, the encoder block of SgRNA, OsU6 promoters, target sequence 2, the encoder block of SgRNA
Rule method carries out, and is not discussed in detail here) and it is loaded with BsaI restriction enzyme sites (Fig. 1) in the synthesis segment.Simultaneously with limit
Property restriction endonuclease BsaI processed carries out digestion to CRISPR/Cas9 targeting vectors plasmid, will synthesize the big of segment and targeting vector plasmid
Segment is attached.Screen by resistance culture base and (build successful carrier and only grown on Kana, do not grown on Spec),
Bacterium colony PCR screens recon and digestion verification (the results are shown in Figure 2) correctly, and Beijing six directions Hua Da Gene science share is sent to have
Limit company is sequenced and will sequencing.The CRISPR/Cas9 recombinant vectors as constructed by the present invention are correctly cloned in verification.It should
Recombinant vector contains the guide RNA encoder blocks of target sequence 1, the guide RNA encoder blocks of target sequence 2 and Cas9 nucleases
Encoder block.The transfer vector plasmid is extracted for converting Agrobacterium EHA105.Picking positive colony is stored in -80 DEG C with glycerine,
And for rice transformation.
In above-mentioned each segment
The sequence of OsU3 promoters is:
AAGGGATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATG
CATGGATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCA
AATGCTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAG
CATTTCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAA
CAAAGACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGT
GGCA
The sequence of OsU6 promoters is:
GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGAT
TCTCTTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGG
CGAGTGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAA
ACCCGCGCGCTGTCGCTTGTGTG
The sequence of SgRNA is:
gttttagagctatgctgaaaagcatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgag
tcggtgcttttttttagtagtagcatctgacggtgaagggggcggccgcgg
Agriculture bacillus mediated rice transformation：
(1) acquisition of PMI kanamycin-resistant callus tissues：To have obtained the Nipponbare transgenic seed with PMI selection markers as material.
It chooses and goes glume without scab, the good transgenic paddy rice seed containing PMI marker gene of embryonic development, the seed after disinfection is used
Sterile water soaked overnight under 30 DEG C of dark conditions, embryo is peeled be placed in the inducing culture containing PMI selective agents with scalpel
On.12 embryos are uniformly placed per ware, 2~3 weeks evoked callus are placed under 30 DEG C of dark conditions, until growing yellowish coloured particles
Shape callus.
(2) acquisition of transfer-gen plant：Using agrobcterium-mediated transformation, CRISPR/Cas9 will be built
The recombinant vector of target practice is converted into the transgenic paddy rice callus containing PMI marker gene, and specific conversion process is with reference to document
Duan et al. is in " An Efficient and High-throughput Protocol for Agrobacterium-
mediated Transformation based on Phosphomannose Isomerase Positive Selection
Method disclosed in Japonica Rice (Oryza sativa L.) .Plant Cell Reports (2012) ".Wherein,
Using to selective agent for hygromycin, the coding production of the hygromycin gene included in the recombinant vector practiced shooting for CRISPR/Cas9
Object.
In conversion, as a result of the recombinant vector constructed by the present invention and specific target fragments, can induce
The guide rna expression frame and Cas9 enzyme nucleic acid expression frames of target fragments 1,2 co-express in rice cell, to mark to be deleted
The region in note gene code frame promoter upstream and terminator downstream is realized and is sheared simultaneously, causes double-strand break.Trigger water simultaneously
The DNA repair functions of rice cell itself cause missing base in target site, realize that intracellular screening marker gene, upstream open
The fragment deletion of mover and downstream terminator.
Marker gene deletes effect identification：
(1) PCR amplification detects：Using Plant Genome Mini Kit (production of Tiangeng biochemical corp), institute is extracted
Obtain the genomic DNA of 42 regeneration plants.Using the DNA as template, with Phusion high-fidelity DNA polymerases, (NEB companies give birth to
Production), PCR amplification includes the sequence of targeting regions, and partial results are as shown in Figure 3.Primer wherein used in PCR amplification is：
gc CHECK FP:5’-GAGGTAGATACGGTAACAAGG-3’
gc CHECK RP:5’-CGCCTTGAGCGACACGAATT-3’
(2) sequencing detection：Pcr amplified fragment direct Sequencing is obtained using gc CHECK FP by primer pair, analyzes targeting regions
Sequence deletion situation.Sequencing result shows that in 42 plant are surveyed 18 plant carry the missing of different size segment.
It is 2 plants that the regeneration strain lacked, which all occur, in two of which equipotential PMI expression cassettes, while deletion equipotential marker gene efficiency occurs and be
4.9%.The PMI encoder blocks contained in transgenic paddy rice and target sequence position and 2 plants of equipotential PMI codings are shown in Fig. 4
The schematic diagram that frame is deleted.
(3) GUS dyeing detection：The transgenic paddy rice of marker gene to be deleted has converted pCAMBIA1300 carriers, contains
There is gus gene, so having inserted gus gene in transgenic paddy rice genome.After GUS chemistry tissue stainings, seed
Endosperm and embryo tissue are displayed in blue.The present invention successfully delete transgenic paddy rice marker gene after, equally to gained plant into
GUS dyeing is gone.The results show that before the endosperm and embryo tissue of the seed of gained plant are displayed in blue and delete marker gene
Tissue GUS dye levels do not have apparent difference.Therefore illustrate, the present invention does not interfere with it while marker gene is deleted
The expression of his ingredient.Gene elmination is marked using pCAMBIA1300 carriers in the present invention, and efficiency is very high.
Specific description of embodiments of the present invention above is not intended to limit the present invention, and those skilled in the art can be according to this
Invention is variously modified or deforms, and without departing from the spirit of the present invention, should all belong to the model of appended claims of the present invention
It encloses.
Claims (8)
- A kind of 1. method of Transgenic Rice system for establishing screening marker-free gene, it is characterised in that：The method utilizes The genome fixed point editing technique of CRISPR/Cas9 System-mediateds establishes the Transgenic Rice system of screening marker-free gene, institute The method of stating includes the following steps：1) the first target is chosen in the original vector marker gene encoder block upstream sequence area that rice riddled basins are transferred to Segment, and marker genes encoding frame downstream sequence choose the second target fragments, the one of first and second target fragments Chain is respectively provided with 5 '-(N)X- NGG-3 ' structures, wherein (N)XRepresent the base sequence { N that number is X1, N2……NX, N1, N2……NXIn each represent any one in base A, G, C, T, N is any one in base A, G, C, T in NGG It is a,；2) be respectively synthesized OsU3 promoters, the first target fragments, the encoder block of SgRNA, OsU6 promoters, the second target fragments, The encoder block of SgRNA is simultaneously loaded with BsaI restriction enzyme sites in the synthesis segment, so as to build targeting vector plasmid, with restricted interior Enzyme cutting BsaI carries out digestion to targeting vector plasmid, and the large fragment for synthesizing segment and targeting vector plasmid is attached, and profit With the constructed carrier of resistance culture base screening；3) callus of the transgenic paddy rice containing the riddled basins is prepared, it is spare；4) the recombinant vector steps for importing 3 for being obtained step 2)) in rice cell in the callus that obtains, described in induction The guide rna expression frame and Cas9 enzyme nucleic acid expression frames of first and second target fragments co-express in rice cell, treat The riddled basins encoder block promoter upstream of deletion and the region in terminator downstream are realized shears simultaneously, and double-strand is caused to break It splits, while triggers the DNA repair functions of rice cell itself, cause missing base in target site, realize intracellular screening mark Remember the fragment deletion of gene, upstream promoter and downstream terminator；5) regeneration plant is obtained by protoplast instantaneous conversion or agriculture bacillus mediated stable conversion, by Genomic PCR side DNA section containing target fragments in method clonal turnover plant, and amplified production is sequenced；6) transfer-gen plant that selection riddled basins encoder block lacks completely,Wherein,The sequence of OsU3 promoters is:AAGGGATCTTTAAACATACGAACAGATCACTTAAAGTTCTTCTGAAGCAACTTAAAGTTATCAGGCATGCATG GATCTTGGAGGAATCAGATGTGCAGTCAGGGACCATAGCACAAGACAGGCGTCTTCTACTGGTGCTACCAGCAAATG CTGGAAGCCGGGAACACTGGGTACGTTGGAAACCACGTGATGTGAAGAAGTAAGATAAACTGTAGGAGAAAAGCATT TCGTAGTGGGCCATGAAGCCTTTCAGGACATGTATTGCAGTATGGGCCGGCCCATTACGCAATTGGACGACAACAAA GACTAGTATTAGTACCACCTCGGCTATCCACATAGATCAAAGCTGATTTAAAAGAGTTGTGCAGATGATCCGTGGCAThe sequence of OsU6 promoters is:GGATCATGAACCAACGGCCTGGCTGTATTTGGTGGTTGTGTAGGGAGATGGGGAGAAGAAAAGCCCGATTCTC TTCGCTGTGATGGGCTGGATGCATGCGGGGGAGCGGGAGGCCCAAGTACGTGCACGGTGAGCGGCCCACAGGGCGAG TGTGAGCGCGAGAGGCGGGAGGAACAGTTTAGTACCACATTGCCCAGCTAACTCGAACGCGACCAACTTATAAACCC GCGCGCTGTCGCTTGTGTGThe sequence of SgRNA is:gttttagagctatgctgaaaagcatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcac cgagtcggtgcttttttttagtagtagcatctgacggtgaagggggcggccgcgg。
- 2. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： 5 ' in first target fragments-(N)XThe numerical value of the X of-NGG-3 ' structures and 5 '-(N) in second target fragmentsX-NGG- The numerical value of the X of 3 ' structures is different from each other.
- 3. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： It is characterized in that, X is 19 or 20.
- 4. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： For the CRISPR/Cas9 recombinant vectors that paddy gene is practiced shooting, for double target spot recombinant vectors.
- 5. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： First target fragments are located at the upstream of the promoter of driving marker gene expression, sequence for 5 '- GTGGACGAGATTAGATAGCC-3‘。
- 6. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： Second target fragments are located at the downstream of the terminator of end mark gene expression, and sequence 5 '- TAGCCCTGCAGGAAATTTAC-3‘。
- 7. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： Contain Reporter gene GUS in original vector, original vector can be used for gene of the assembling with specific function；The riddled basins Refer to hygromycin gene, anti-herbicide gene, xylose isomerase gene, antibiotics resistance gene；The gene with specific function refers to anti insect gene, disease-resistant gene, anti-herbicide gene, adversity gene, anti-aging base The fusion formed because of, quality function improvement gene and by said gene or multivalent genetic.
- 8. the method for Transgenic Rice system according to claim 1 for establishing screening marker-free gene, it is characterised in that： The rice refers to long-grained nonglutinous rice and japonica rice.
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| US9163284B2 (en) | 2013-08-09 | 2015-10-20 | President And Fellows Of Harvard College | Methods for identifying a target site of a Cas9 nuclease |
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| US9340800B2 (en) | 2013-09-06 | 2016-05-17 | President And Fellows Of Harvard College | Extended DNA-sensing GRNAS |
| US9388430B2 (en) | 2013-09-06 | 2016-07-12 | President And Fellows Of Harvard College | Cas9-recombinase fusion proteins and uses thereof |
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