CN107338265A - A kind of gene editing system and enter the method for edlin to Plant Genome using it - Google Patents
A kind of gene editing system and enter the method for edlin to Plant Genome using it Download PDFInfo
- Publication number
- CN107338265A CN107338265A CN201710599030.2A CN201710599030A CN107338265A CN 107338265 A CN107338265 A CN 107338265A CN 201710599030 A CN201710599030 A CN 201710599030A CN 107338265 A CN107338265 A CN 107338265A
- Authority
- CN
- China
- Prior art keywords
- cas9
- sgrna
- plant
- sequence
- transcriptional units
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8213—Targeted insertion of genes into the plant genome by homologous recombination
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Molecular Biology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
Enter the method for edlin to Plant Genome the invention discloses a kind of gene editing system and using it.The gene editing system includes sgRNA transcriptional units, and the DMC1p Cas9 transcriptional units formed from the DMC1 gene promoters and Cas9 genes of corn by being sequentially connected with, wherein, the target site of sgRNA identifications meets the sequence rules of 5 ' Nx NGG, 3 ' or 5 ' CCN Nx 3 ', N represents any one of A, T, C and G, 14≤X≤30, and X is integer, NXRepresent X continuous deoxyribonucleotides.The gene editing system of the present invention will be expected to improve the genome editorial efficiency in monocotyledon, promote crop genetic improvement and basic research.
Description
Technical field
The invention belongs to field of plant genetic, and in particular to a kind of gene editing system and application its to plant
The method that genome enters edlin.
Background technology
Traditional breeding technique depends on the hereditary variation of nature generation, is obtained by the means such as hybridizing and being returned
Possess the crop varieties of merit.But naturally variation is often random, limited.Nearest decades development turns base
Because technology provides good opportunity for crop breeding, genetic engineering breeding also achieves huge achievement.Transgenic technology is
Some merits are obtained by the way that the means of the target DNA fragment of external source manually to be conducted into the genome of plant
Such as pest-resistant, antiweed.The characteristics of transgenic technology, causes the inhereditary material that the genetically modified plants obtained contain external source, therefore
The safety of GM food is also worried by the public.Genome editor (Genome editing) technology to grow up in recent years
New opportunity is provided for crop functional genome research and breeding.
Genome editing technique main at present includes three kinds, i.e.,：Zinc finger nuclease (Zinc finger nuclease,
ZFN), class activating transcription factor effector nuclease (Transcription activator like effector
Nuclease, TALEN) and cluster regular intervals short palindrome repetitive sequence and its related system (Clustered
Regularly interspaced short palindromic repeats/CRISPR associated protein 9,
CRISPR/Cas9).These three technologies can realize that the special target site in genome produces DNA double chain fracture (Double
Strand break, DSB), by the DSB repair mechanisms of cellular endogenous, obtain insertion or the missing in genome specific site etc.
Variation, realize the editor of genome.It is extensive at present because the utilization of CRISPR/Cas9 systems is relatively easy and efficient
Basis and application study for plant.CRISPR/Cas9 systems are in main crops such as rice, wheat, corn and soybean
Deng having been obtained for successfully applying, powerful Breeding Application potentiality have been shown.
The editorial efficiency of CRISPR/Cas9 systems must take into consideration to it in crop basic research and application study
One factor.High editorial efficiency can not only save manpower and resources costs, also cause the extensive gene of full-length genome to compile
Collect and easily realize, thus promote the excavation of new gene or genetic locus.And the editorial efficiency of CRISPR/Cas9 systems mainly by
To the influence of following some factors：Such as it is used for the promoter for driving Cas9 to express, for the promoter for driving sgRNA to express, compiles
Target site collected etc..These factors are carried out with targetedly optimization (to open as found the more efficient expression in callus
Mover) potentiality of gene editing efficiency will be improved.CRISPR/Cas9 systems in the practical studies of crop to being used to drive at present
The main promoter for using two kinds of constitutive expressions of Cas9 gene expressions：The 35S promoter of tobacco mosaic virus (TMV) and corn
Ubiquitin gene promoters.In corn, the transformation system based on Agrobacterium, produce in existing report homozygous or double etc.
Position mutant plants average frequency be respectively<1% (35S), 13% (Ubi) and 30% (Ubi).The editor reported in wheat
Efficiency is based on via Particle Bombardment Transformation system 10% or so.Yet there are no in wheat based on Agrobacterium-mediated Transformation system
Report.It is used to drive the expression realization of Cas9 genes higher therefore, it is necessary to which new promoter is excavated and explored in crops
The genome editor of effect, so as to save time and human cost, the extensive editor of crop full-length genome is also caused to be easier in fact
It is existing, promote CRISPR/Cas9 systems to play more powerful effect in the basic and applied research of crop.
The content of the invention
In view of this, Plant Genome is entered it is an object of the invention to provide a kind of new gene editing system and using it
The method of edlin, to solve at least part technical problem present in above-mentioned prior art.
To achieve the above object, the present invention provides a kind of new gene editing system, and it includes sgRNA (Single
Guide RNA) transcriptional units, and the DMC1 gene promoters and Cas9 genes from corn by being sequentially connected with form
DMC1p-Cas9 transcriptional units, wherein, the target site of sgRNA identifications meets 5 '-Nx-NGG-3 ' or 5 '-CCN-Nx-3 ' sequences
Rule, it is integer that N, which represents any one of A, T, C and G, 14≤X≤30, and X, NXRepresent X continuous deoxyribose cores
Thuja acid.
Wherein, the DMC1 gene promoters are expressed as DMC1p, and its sequence is preferably as shown in SEQ ID NO.1.
The present invention also provides a kind of recombinant vector for including forementioned gene editing system.
The present invention also provides the construction method of foregoing recombinant vector, and it comprises the following steps：
(1) clone obtains DMC1 gene promoters and is sequentially connected with itself and Cas9 genes, composition DMC1p-Cas9 transcriptions
Unit；
(2) the sgRNA transcriptional units of structure identification target site, and DMC1p-Cas9 transcriptional units and sgRNA are transcribed into list
Member simultaneously import binary vector, obtain recombinant vector, wherein, sgRNA identification target site meet 5 '-Nx-NGG-3 ' or 5 '-
CCN-Nx-3 ' sequence rules, it is integer that N, which represents any one of A, T, C and G, 14≤X≤30, and X, NXRepresent X continuously
Deoxyribonucleotide.
In step (1), the sequence of the DMC1 gene promoters is preferably as shown in SEQ ID NO.1.
In step (1), clone obtains DMC1 gene promoters preferably with the following method：With corn B73 genomic DNA
It is that amplimer is reacted entering performing PCR using primer of the sequence as shown in SEQ ID NO.2 and SEQ ID NO.3 for template, amplification
Obtain fragment of the sequence as shown in SEQ ID NO.1, as DMC1 gene promoters.
It is described to be sequentially connected with itself and Cas9 genes preferably after clone obtains DMC1 gene promoters in step (1)
With the following method：Obtained DMC1 gene promoters are replaced into the 35S promoter in 35S-Cas9-SK carriers, obtained
PDMC1-Cas9-SK recombinant vectors, Xma I and EcoR two restriction enzyme sites of I are recycled to shift DMC1p-Cas9 transcriptional units
To binary vector pTF101.1, recombinant vector pDMC1-Cas9 is obtained.
Step (2) is preferably using following operation：5 '-Nx-NGG-3 ' or 5 '-CCN-Nx-3 ' sequence rules will be met
The DNA sequence dna of target site is connected into pU3-sgRNA carriers by primer annealing, then will by Hind III digestions site
SgRNA transcriptional units are subcloned into recombinant vector pDMC1-Cas9, obtain including sgRNA transcriptional units, and by sequentially
The recombinant vector for the DMC1p-Cas9 transcriptional units that the DMC1 gene promoters and Cas9 genes from corn of connection form.
The present invention also provides a kind of method that editor's activity to foregoing recombinant vector is identified, it includes：By described in
Recombinant vector transformed plant protoplast, by identifying the efficiency of the producer group editor in plasm so that it is determined that described heavy
Editor's activity of group carrier.
The present invention also provides a kind of genetic engineering bacterium for including foregoing recombinant vector.
The present invention also provides a kind of method for entering edlin to Plant Genome using forementioned gene editing system, and it is wrapped
Include：By forementioned gene engineering bacteria transformation receptor plant tissue, the transgenic plant material edited is obtained.
Wherein, the preferred monocotyledon of the plant, more preferably corn or wheat.
Wherein, the preferably immature rataria of the recipient plant tissue.
Compared with prior art, the present invention achieves following positive effect：
Gene editing system using the present invention enters edlin to Plant Genome, realizes in corn and is obtained in T0 generations
The plant of a high proportion of homozygous or double allelic variant bodies；The plant low containing mutant proportion obtained for T0 generations, the offspring of selfing
In T1 generations, can also produce new mutant plant；It can obtain for transformation system of the wheat based on Agrobacterium and be mutated containing certain proportion
Chimera plant, it is contemplated that can obtain the plant of homozygous or double allelic variant bodies in T1 generations.Plant is applied to the promoter
Genome editing system, does not have been reported that before making the present invention, and the invention belongs to pioneering.CRISPR/Cas9 after the improvement of the present invention
System will be expected to improve the genome editorial efficiency in monocotyledon, promote crop genetic improvement and basic research.
Brief description of the drawings
Fig. 1 is shown in embodiment 4 and obtained with corn gene 1 (international numbering is GRMZM2G027059) for target gene
4 transgenic events resistant calli (respectively taking 3 parts of samples) using PCR (Polymerase Chain Reaction)-
Digestion method be mutated the result of identification.
Fig. 2 shows the phenotypic results of the mutant of the homozygosis obtained in embodiment 4 or diallele.
Fig. 3 shows that T1 is mutated the PCR- digestion rear electrophoresis results identified for plant in embodiment 4.
Fig. 4 shows that T0 is for the result of deletion mutation in Transgenic plant of wheat in embodiment 4.
Embodiment
For the object, technical solutions and advantages of the present invention are more clearly understood, below in conjunction with specific embodiment, and reference
Accompanying drawing, the present invention is described in further detail.
The present inventor in the course of the study, practical proof by substantial amounts of screening experiment and repeatedly, in jade
The expression that a new promoter is used to drive Cas9 genes has been excavated in rice, while has constructed the recombinant vector for facilitating it to apply
And engineering strain.The transformation experiment of Agrobacterium shows, is compared with other promoters reported in the prior art, the present invention
The effect for carrying out gene editing using CRISPR/Cas9 systems in plant particularly monocotyledonous plant Zea mays can be significantly improved
Rate, can be regenerated in the kanamycin-resistant callus tissue in transgenosis T0 generations 100% and obtain homozygous or double allelic variant body plant, and homozygous or double equipotentials are dashed forward
Ratio of the variant plant in the plant that all regeneration obtain is more than 65%.The present invention is expected to more in wheat T1 generation acquisitions simultaneously
The homozygous or double allelic variant body plant of gene loci.
In following each embodiments, the source of used experiment material is as follows：
Plasmid 35S-Cas9-SK is in document " Feng Z.Y.et al., Efficient genome editing in
Mistake disclosed in plants using a CRISPR/Cas system.Cell Res 2013 "；
Plasmid pU3-sgRNA is in document " Feng C.et al., Efficient Targeted Genome
Disclosed in Modification in Maize Using CRISPR/Cas9 System.J Genet.Genomics 2016 "
Cross；
Plasmid pTF101.1 is in document " Paz M.M.et al., Assessment of conditions affecting
Agrobacterium mediated soybean transformation using the cotyledonary node
Mistake disclosed in explant.Euphytica 2004 "；
Agrobacterium strains EHA105 can be bought from commercial company, such as You Bao biotech firms, production code member：ST1140；
Corn variety HiII is in document " Armstrong C.L., Green C.E.＆Phillips R.L.Development
and availability of germplasm with high type II culture formation
Mistake disclosed in response.Maize Genet.Coop.News Lett.65,92-93 (1991) "；The public can be from maize genetic
Learn and genomic database (MaizeGDB) website obtains；
Corn variety B73 is in document " Russell W.A.Registration of B70 and B73 parental
Mistake disclosed in lines of maize.Crop Sci.12,721 (1972) "；The public can be from maize genetics and genomics number
Obtained according to storehouse (MaizeGDB) website；
Wheat breed Sumai 3 is recorded in " the optimal anti-source-Su Mai 3 of the wheat scabs such as Jiangsu TAI HU AREA institute of agricultural sciences
Number Jiangsu's agriculture science, 1988 " one texts, the public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research；
DNA extraction kit (article No.：DP305-03), plasmid extraction kit (DP103-03), EsayGeno are quickly weighed
Group Cloning Kit (article No.：) etc. VI201-02 it is purchased from TIANGEN Biotech (Beijing) Co., Ltd.；
Carrier T (the article No. of PCR primer clone：CT111-01) it is purchased from Beijing Quanshijin Biotechnology Co., Ltd；
Restriction enzyme is purchased from knob Great Britain biotechnology (Beijing) Co., Ltd；
Primer sequence is synthesized by matching your scientific and technological (China) Co., Ltd of silent winged generation；
Sequencing is completed by Beijing Bioisystech Co., Ltd of farsighted Boxing section；
If the other materials reagent or instrument and equipment used in experiment do not make specified otherwise, this area routine city is represented as
Selling to obtain.
The structure of the clone of DMC1 gene promoters and pDMC1-Cas9 recombinant vectors in the corn of embodiment 1
The sequence of corn DMC1 genes is obtained from maize genetics and genomic database (MaizeGDB) website
(gene I/D GRMZM2G109618).Primer dmc-F (sequences are designed according to 5 ' end noncoding regions of the sequence of total length in the gene
Row as shown in SEQ ID NO.2) and dmc-R (sequence is as shown in SEQ ID NO.3) be used as amplimer pair, with corn B73
(Zea mays L.) genomic DNA is template, and PCR amplifications obtain fragment (the sequence such as SEQ ID NO.1 of a 3614bp length
It is shown), the fragment is close to initiation codon.Utilize primer dmc-F2 (sequence is as shown in SEQ ID NO.4) and dmc-R2 (sequences
As shown in SEQ ID NO.5) amplimer pair is used as, the piece segment DNA of above-mentioned 3614bp length enters performing PCR amplification for template, then
It is using EsayGeno Quick Castings Cloning Kit (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.) that the fragment of amplification is whole
Close in 35S-Cas9-SK (digestions of Xho I) carrier and replace 35S promoter, the pDMC1-Cas9-SK carriers recombinated.Then
DMC1p-Cas9 transcriptional units are subcloned on binary vector pTF101.1 using I two restriction enzyme sites of Xma I and EcoR, obtained
To recombinant vector pDMC1-Cas9.
Meeting 5 '-Nx-NGG-3 ' or 5 '-CCN-Nx-3 ' in Maize genome, (N represents any one in A, T, C and G
Kind, 14≤X≤30, and X is integer, NXRepresenting X continuous deoxyribonucleotides) sites of sequence rules is selected as
The target site of CRISPR/Cas9 system editors.The DNA sequence dna of target site can be connected into pU3-sgRNA carriers by primer annealing
(digestions of Bbs I).Then sgRNA transcriptional units are subcloned into foregoing pDMC1-Cas9 by the restriction enzyme sites of Hind III to carry
Body.Then pDMC1-Cas9 carriers are transferred to agrobacterium strains EHA105, obtained recombination engineered strain is used for corn
Conversion.
The Corn Protoplast of embodiment 2 converts
The method of Corn Protoplast conversion is referring to existing report (Feng C.et al., Efficient Targeted
Genome Modification in Maize Using CRISPR/Cas9 System.J Genet.Genomics 2016)。
Corn material used is B73 in the present embodiment.Preparation of reagents and main step are as follows：
Main solution formula is as follows：
(1) enzymolysis liquid：1.5% cellulase, 0.4% macerozyme R10,0.4M mannitol, 20mM potassium chloride, 20mM fat
Sour methyl ester sulfonate, pH5.7.Add 10mM calcium chloride after 55 DEG C of water-bath 10min, 0.1% bovine serum albumin and 5mM's
β mercaptoethanols；
(2) W5 solution：154mM sodium chloride, 5mM potassium chloride, 125mM calcium chloride, 2mM MESs,
pH5.7；
(3) MMg solution：4mM MESs, 0.4M mannitol, 15mM magnesium chlorides, pH5.7；
(4) 40%PEG solution：40%PEG4000,100mM calcium chloride, 0.6M mannitol.
Experimental procedure is as follows：
Seedling is sprouted under (1) 25 DEG C of dark condition, treats that seedling is grown to 10cm or so the most tender blade of selection and is cut into 0.5mm
Wide fragment；
(2) blade cut is put into vacuum suction 0.5h in the enzymolysis liquid newly prepared, and then 40rpm is digested on shaking table
4h, finally handled 5 minutes under the conditions of 80rpm；
(3) with the protoplast after 41 μm of nylon wire membrane filtration enzymolysis into round bottom centrifuge tube, 100g centrifugation 3min,
Supernatant is drawn, is washed 2 times with W5 solution；
(4) protoplast after washing places 30min on ice, is then centrifuged for, sucks supernatant, it is molten to add appropriate MMg
It is 5 × 10 that liquid, which is resuspended to cell number,5g/ml；
(5) the recombinant vector pDMC1-Cas9 that embodiment 1 obtains is transferred to coli strain DH5 α, in case extracting matter
Grain.The plasmid extraction kit produced with TIANGEN Biotech (Beijing) Co., Ltd. extracts plasmid.Take 190 μ l protoplasts molten
Liquid, 10 μ l plasmids (being more than 5 μ g) are added, 200 μ l 40%PEG solution is added, is gently mixed with pipette tips, 25 DEG C of placements
18min；
(6) 1.4ml W5 solution is added, overturns and mixes, 100g centrifugation 3min, supernatant is discarded, adds 1.5ml W5 solution weights
It is outstanding, then protoplast suspension is transferred in 6 orifice plates, 1.5d is cultivated under 28 DEG C of dark.
The agriculture bacillus mediated maize genetic conversion of embodiment 3
Existing report (Frame the B.R.et al., Agrobacterium- of method Primary Reference of maize genetic conversion
mediated transformation of maize embryos using a standard binary vector
system.Plant Physiol.2002).The acceptor material of conversion is HiII.Preparation of reagents and main step are as follows：
Main solution and culture medium prescription are as follows：
(1) N6 vitamins stocks liquid (1000 ×)：2.0g/L glycine, 1.0g/L vitamin B1s, 0.5g/L vitamin B6s,
0.5g/L nicotinic acid, filtration sterilization；
(2) MS vitamins stocks liquid (1000 ×)：2.0g/L glycine, 0.5g/L vitamin B1s, 0.5g/L vitamin B6s,
0.05g/L nicotinic acid, filtration sterilization；
(3) culture medium is infected：4.0g/L N6 salt, 1ml/L N6 vitamins stock liquid, 1.5mg/L 2,4-D, 0.7g/L
L-PROLINE, 68.4g/L sucrose, 36.0g/L glucose, pH5.2, filtration sterilization, face with the acetyl cloves for adding 100.0 μM
Ketone；
(4) culture medium is co-cultured：4.0g/L N6 salt, 1.5mg/L 2,4-D, 0.7g/L L-PROLINEs, 30.0g/L sugarcanes
Sugar, 3.0g/L plant gels, pH5.8；1ml/L N6 vitamins stock liquid, 100.0 μM of acetyl fourths are added after autoclave sterilization
Ketone musk, 300.0mg/L Cys, 5.0 μM of silver nitrates；
(5) tranquillization culture medium：4.0g/L N6 salt, 1.5mg/L 2,4-D, 0.7g/L L-PROLINEs, 30.0g/L sucrose,
0.5g/L MESs, 8.0g/L agar, pH5.8；1ml/L N6 vitamins stocks are added after autoclave sterilization
Liquid, 100.0mg/L CTXs, 100.0mg/L vancomycins, 5.0 μM of silver nitrates；
(6) Selective agar medium I：4.0g/L N6 salt, 1.5mg/L 2,4-D, 0.7g/L L-PROLINEs, 30.0g/L sucrose,
0.5g/L MESs, 8.0g/L agar, pH5.8；1ml/L N6 vitamins stocks are added after autoclave sterilization
Liquid, 100.0mg/L CTXs, 100.0mg/L vancomycins, 5.0 μM of silver nitrates, 1.5mg/L herbicides；
(7) Selective agar medium II：4.0g/L N6 salt, 1.5mg/L 2,4-D, 0.7g/L L-PROLINEs, 30.0g/L sugarcanes
Sugar, 0.5g/L MESs, 8.0g/L agar, pH5.8；The storage of 1ml/L N6 vitamins is added after autoclave sterilization
Liquid storage, 100.0mg/L CTXs, 100.0mg/L vancomycins, 5.0 μM of silver nitrates, 3.0mg/L herbicides；
(8) regeneration culture medium I：4.3g/L MS salt, 1.0ml/L MS vitamins stock liquid, 100.0mg/L inositols,
60.0g/L sucrose, 3.0g/L plant gels, pH5.8；100.0mg/L CTXs, 3.0mg/L are added after autoclave sterilization
Herbicide；
(9) regeneration culture medium II：4.3g/L MS salt, 1.0ml/L MS vitamins stock liquid, 100.0mg/L inositols,
30.0g/L sucrose, 3.0g/L plant gels, pH5.8；Autoclave sterilization.
Major experimental step is as follows：
(1) Agrobacterium is infected
In the YEP of the restructuring EHA105 engineering strains that the previous day inoculation embodiment 1 for infecting experiment obtains to 10ml
In culture medium, 28 DEG C in shaking table, 200rpm shaken cultivations 16-20h.Agrobacterium is collected by centrifugation, is resuspended to and infects in culture medium
(OD550It is worth for 0.3-0.4).Immature rataria (1.5-2.0mm) is stripped out from immature ear, rataria is subsequently placed at containing agriculture
In the 2ml EP pipes for infecting culture medium of bacillus, 5min is overturned 5-10 times and then stood.
(2) co-culture
Rataria after infecting is subsequently placed on sterile filter paper and is and then transferred in the culture medium of co-cultivation, rataria
Scultellum is upward.Then it is put into incubator under 20 DEG C of dark and cultivates 3 days.
(3) tranquillization culture
After the co-cultivation of 3 days, all ratarias are shifted to tranquillization culture medium, are placed in incubator, under 28 DEG C of dark
Culture 7 days.
(4) the selection culture of kanamycin-resistant callus tissue
By the tranquillization culture of 7 days, all ratarias were shifted to Selective agar medium I, are placed in incubator, under 28 DEG C of dark
Culture 14 days.Then all callus are shifted to Selective agar medium II, is placed in incubator under 28 DEG C of dark and cultivates 14 days.
Callus repeats subculture 3-5 times in Selective agar medium II, until selecting all resistant callis successively.
(5) regeneration of resistant calli
Cultivated by selection, the resistant calli culture screened is placed in regeneration culture medium I is transferred to after sufficiently large
Cultivated 14 days under 25 DEG C of dark in incubator.Then choose the callus containing somatic embryo and be transferred to regeneration culture medium II,
It is placed in incubator and is cultivated under 25 DEG C of illumination, until regenerates all seedling.
The gene editing Efficiency testing of embodiment 4
(1) the transgenic corns callus converted in protoplast or embodiment 3 in collection embodiment 2, plant leaf
Sample and transgenic wheat sample.Extracted with DNA extraction agents box (being purchased from TIANGEN Biotech (Beijing) Co., Ltd.)
Genomic DNA；
(2) enter performing PCR with the primer in respective target site to expand, the PCR primer of acquisition takes a portion corresponding target position
The restriction enzyme (being purchased from NEB companies) of point does digestion experiment, the laggard row agarose gel electrophoresis of digestion (Fig. 1)；Fig. 1 is shown
With the kanamycin-resistant callus tissue that corn gene 1 (international numbering is GRMZM2G027059) is 4 transgenic events that target gene obtains
Organize (respectively taking 3 parts of samples) mutation qualification result；Expand to obtain the DNA fragmentation of the 651bp containing target site, such as infructescence by PCR
Row can then be digested into 501bp and 150bp two fragments without mutation, on the contrary then can not be cut open.Each swimming lane pair in Fig. 1
The sample answered is from left to right：4 transgenic event #1, #3, #4, #6 (each 3 parts of samples), control and Marker, can from Fig. 1
To find out, wherein 3 all contain homozygous or double allelic variants in 4 callus samples.
(3) for that can be directly sent to sequencing company containing homozygous or the mutation of diallele sample, PCR primer and enter
Row sequencing；For the other kinds of sample containing mutation, the mutant nucleotide sequence band that digestion is not cut is reclaimed, is cloned into commercialization
Carrier T, then picking monoclonal send company to be sequenced；
(4) result of digestion and sequencing is analyzed, counts mutation type and ratio.
A marker gene, the gene mutation are used as corn gene 1 (international numbering is GRMZM2G027059)
The phenotype of albefaction occurs in plant in theory.The transformation experiment of two batches obtains 10 transgenic events, 9 events therein altogether
In obtain the plant of albefaction phenotype, and be identified as homozygous or diallele mutant (Fig. 2)；In addition an event
In obtain the plant of yellowing leaf, identified and diallele mutant.Therefore, regeneration is obtained containing homozygous or double etc.
The transgenic event of position gene mutation plant is 100%；Homozygous or diallele mutant plant ratio in all regeneration plants
Example is more than 65%.The relatively low plant selfing of mutant proportion in T0 generations or hybridization are obtained into T1 generations, qualification result shows in T1 for portion
Mutant proportion in plant is divided to be significantly improved (Fig. 3) compared with T0 generations, what Fig. 3 was shown is a T0 for plant and the offspring of wild type crosses
The qualification result of target gene mutation；Left side is the result that T0 identifies for plant, and swimming lane counter sample is followed successively by transgenosis T0 plants
Strain, control and Marker；Right side is the result that T1 identifies for plant, and 8 transgenosis that swimming lane counter sample is followed successively by T1 generations are planted
Strain, control and Marker.
Wherein, mutant gene type statistics is shown in Table 1 in T0 generations：
Mutant gene type statistics in the embodiment 4 of table 1
For corn gene 2 (international numbering is GRMZM2G456570), it is known that the gene mutation can produce in theory
Lethal phenotype, this lethal phenotype is had proven in other species, converted substantial amounts of rataria but only obtained two and resist
The callus of sexual behavior part, transformation efficiency significantly reduce.This from showing in most positive transgenic event to a certain extent
Generate homozygous or diallele mutation.Find to exist in addition, carrying out the callus of two positive events mutation identification
The two samples are chimeric mutant, and mutant proportion is respectively also more than 50%.
For wheat, we have selected fhb1 genes in Sumai 3 material etc. and are used as target gene, to identified at present
T0 6 transfer-gen plants of generation in wherein 4 can detect deletion mutation (Fig. 4).
In summary, the invention provides a kind of new recombinant vector for plant gene editing.By in unifacial leaf
Experiment in plant particularly corn shows：Using genetic conversion system of the recombinant vector based on Agrobacterium, in T0 in corn
In generation, can obtain a high proportion of homozygous or double allelic variant body plant materials.And for T0 generation the plant containing low mutant proportion,
Mutant proportion can significantly improve in plant part in the T1 generations that selfing or hybridization obtain.
Particular embodiments described above, the purpose of the present invention, technical scheme and beneficial effect are carried out further in detail
Describe in detail bright, it should be understood that the foregoing is only the present invention specific embodiment, be not intended to limit the invention, it is all
Within the spirit and principles in the present invention, any modification, equivalent substitution and improvements done etc., the protection of the present invention should be included in
Within the scope of.
<110>Inst. of Genetics and Development Biology, CAS
<120>A kind of gene editing system and enter the method for edlin to Plant Genome using it
<130> IB177450
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 3614
<212> DNA
<213> Zea mays L.
<400> 1
ttttcaaagc gcatcctctc gcaaaggagc aggaagtttt tcagtataga acaaaatccc 60
ctggactaaa ccatccaggt atctcagcag ttttccactg gttttcattt tcaaaaacag 120
ctactggact tcccgtccat catagctcac gactaaacca ccggaccttt ttaaaaacca 180
cttttcaaaa gcaccttttt ttggaaaaca aaacactaat tgtcatacca cactagactt 240
gtccattcct gtggacatgg actattcgaa taggttttca aactctgcgt agaggtgtac 300
actttaccca ctagtccggc tctgcgatct catggccaat gagacccgaa accgaatctc 360
tttctttcct cgcacgtcct taccttaacg gttatatcgg aaggagtcag gccaccgcca 420
tgtccaaact agacaaaaca ttccccctcc ttatcctccc ggtgctcccc agccttcata 480
accctggggt ttggaccgca cgagttcaga ttgagtgtct acccatacag tctcgagtgg 540
ttgtacttat catgagtaca gttagtgaag gatgacaaac cagtccttat atgagaggac 600
aatccttctg ctcacaccta acctggctga gccaacacct gaggccctcc cctaaaccag 660
ggagtccctg atgatcccta ctcaaaggtg ataagggtga aaacccttca ttatacacat 720
tttgaaaagc attttctttt gatcccggct gggtggccat aataacttgt ctcaaaatca 780
tatcatgcat aaaataacag gctgagggtt gtggttgaaa aatcataggt aatttatgca 840
tcaaagggat ccagtgagtt tgtcgtgctt atccgacgaa gggggaagag gagctcgcgg 900
aactgtcttc tagctccact gcctggcgta gacttgcaga tctggcctcc acgagacgac 960
atgaacgctc cgataactat gcaacatgaa taagcaaaca tacaaaccaa caagtatacc 1020
aacaaatatt tagtatagtg gtcagaatag cgatatatgg atgggtagag tcgtgagtag 1080
aatctgtgtc atgtggtgtt gagatactac tagtggtgga gcggaggtgc ttaccaggag 1140
ggtggactgg aggcgaagcg acactatgcg tgtagccggc agagcggagt aactaagtga 1200
gtggggtgtt tgccttggct gagggtgttg agtgtgtgtg gagagggaga gggtagctgg 1260
gtgtatttat agctgggtgt atgtggtgta gcacaatgaa gttcattgtt ggtgagaata 1320
gtgacatatg aatcgtctga cttagtatga acaggagagt tatatgcaga atatgggaca 1380
agagtatttt gggagttttc tggaatatga accatactta agtgatgaca tggttggata 1440
gggaataatt tgataagaat ttagaaacaa gaattattgg aatcggagtt tggaagcctg 1500
gttcgaagga atctcaaagt taagcgtgct caacttagag aaacctggga tgggtgacca 1560
gatgggaagt tccctactgg aaggaaaatc acagtcaccg gagttcgtat gactgaaata 1620
tgggtctggc tggtcttagg tggactggat agcatgatgg atgagtagtt gaaatttagg 1680
gtgatcggat gatcgatgaa tagtgtcttg gaatgagaag gaagaaagca cgattgcaaa 1740
agctaagcga caagagcgac aaataacaca cagatcactc tctctctcaa gtcactaatc 1800
actaatgatc acttgtctta attgtggaac ttggagagat tggaagcttt gattgtgtct 1860
tggaatggat tgctagctct tgtattgaat gtgaaggatt ggaatgcttg ggtgtcatga 1920
atggaggtgg ttggggttgt atttatagcc ctcaaccact tcctagccgt tgctcctttt 1980
ctaccgaccg cggacggtcc gcgcccctgg tccggacagt ccgcccctgc acatcaacgg 2040
ctgaaatcgc aacgatcagc agtaacggct atatcaacgg ctatatagca tttaatgtgt 2100
cgtcagatgt cagataaaag cagtcgcaga cggtccggtc atgcaccccg gacggtccgc 2160
gaggatgcta taattcattt taccgaaccc gtcaccttcg ggtttttcgg ttcttcaccg 2220
acctgatggt ccgcgcctga ggccggatgg tccgcgcttg gtctcggacg gtgcttggct 2280
ttccatcgga cggtccgtag tgtagacttg tgtttttgta ttggttctgt cctaggctca 2340
ccctagtttc gcggacggtc cgccgcaagg gcccagacgg tccgcgctta tgtgattttc 2400
caaaaagctt ctcctgtcca gaataatcta cggtattccg gacagtcgac tttagaatag 2460
ttgtagatga acttatgcac ctgtggaatg atcaattaga caaaccggtt agtccacaag 2520
gtttgtgatg gtcgtcaaac accaaaaccg attataggga atattgaaac tatttccctt 2580
tcaattatat ttgtttagtt gaggttctaa attttttata gcaaggcccg tttggttaga 2640
gagactaatt ttagtccctg acttttagtc tcatttagtc tctattttgc caaacggaag 2700
gactaaagta gggactaatt ggttttaggg catgtttggt tcgttacctc aattgccaca 2760
ttttgcctaa cttttctgcc taaggttagt tattcaattc gaataactaa ccttaggcaa 2820
agtggggcac agttagccac aaaccaaaca agcccttagt ctttagtccc ttacatagat 2880
gctaaaaggg actaaagggg aatatttact ctaattaccc ttgcctagaa aactagtgtg 2940
aaacaaaaaa aagagtattt tgatctttat gtattacatt taatgtattt aaaatctgtt 3000
tagcccctac aactaaacaa tatagagact aaagtttagt ttagggacta aactttagtc 3060
ctaagactaa tggagccaaa cggggcccaa atctcgttta aaaatttgaa gcaaacacat 3120
ccttaacgga ccgtgggcaa tgagctgagt ctcccctgac tctgggccgc aaagtcctgg 3180
tacacttgat catgttggtc catcctatgg ctgaccgcga cttccctaga ctgaaatcaa 3240
cccattccta ggctcctagc ccagcccaac ggccgagcac acgatccgtt cgagagcgag 3300
aaggtcctcc ggcctcagca ccctcaagta cacagtacac cctcgcaccg gtaccgcggt 3360
ccgcggcaca cgacaccccc actcgtctgt cgactcacgt ctgatcgtct ctccccaaca 3420
atctctcgag tcttgccatt cgcctcagct tgcccctcct ccaagcgtcc aagccccacc 3480
cggccattgc ctcctcctcc tgccgcaggt aagctagctg ctccagcttc tcttgccatc 3540
gcgtgtcctg cactcaccgc cctcgcgcgt gtaactcctc ctccgtcccc cgatcgggct 3600
actagtgcag gcac 3614
<210> 2
<211> 20
<212> DNA
<213>Artificial sequence
<220>
<223> dmc-F
<400> 2
ttttcaaagc gcatcctctc 20
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence
<220>
<223> dmc-R
<400> 3
gtgcctgcac tagtagcccg atc 23
<210> 4
<211> 46
<212> DNA
<213>Artificial sequence
<220>
<223> dmc-F2
<400> 4
aattgggtac cgggcccccc cccgggtttt caaagcgcat cctctc 46
<210> 5
<211> 43
<212> DNA
<213>Artificial sequence
<220>
<223> dmc-R2
<400> 5
tcgtggtcct tatagtccat gtgcctgcac tagtagcccg atc 43
Claims (12)
1. a kind of gene editing system, it includes sgRNA transcriptional units, and the DMC1 bases from corn by being sequentially connected with
Because of the DMC1p-Cas9 transcriptional units that promoter and Cas9 genes form, wherein, the target site of sgRNA identifications meets 5 '-Nx-
NGG-3 ' or 5 '-CCN-Nx-3 ' sequence rules, it is integer that N, which represents any one of A, T, C and G, 14≤X≤30, and X,
NXRepresent X continuous deoxyribonucleotides.
2. gene editing system according to claim 1, wherein, the sequence such as SEQ ID of the DMC1 gene promoters
Shown in NO.1.
A kind of 3. recombinant vector for including gene editing system as claimed in claim 1 or 2.
4. the construction method of recombinant vector described in claim 3, it comprises the following steps：
(1) clone obtains DMC1 gene promoters and is sequentially connected with itself and Cas9 genes, forms DMC1p-Cas9 transcriptional units；
(2) the sgRNA transcriptional units of structure identification target site, and DMC1p-Cas9 transcriptional units and sgRNA transcriptional units are same
When import binary vector, obtain recombinant vector, wherein, sgRNA identification target site meet 5 '-Nx-NGG-3 ' or 5 '-CCN-
Nx-3 ' sequence rules, it is integer that N, which represents any one of A, T, C and G, 14≤X≤30, and X, NXRepresent that X continuously take off
Oxygen ribonucleotide.
5. construction method according to claim 4, in step (1), the sequence such as SEQ ID of the DMC1 gene promoters
Shown in NO.1.
6. construction method according to claim 5, in step (1), clone obtains DMC1 gene promoters and uses such as lower section
Method：Using corn B73 genomic DNA as template, using primer of the sequence as shown in SEQ ID NO.2 and SEQ ID NO.3 as expansion
Increase primer pair template and enter performing PCR reaction, amplification obtains fragment of the sequence as shown in SEQ ID NO.1, as DMC1 gene promoters
Son.
It is described after clone obtains DMC1 gene promoters in step (1) 7. construction method according to claim 4
Itself and Cas9 genes are sequentially connected with the following method：Obtained DMC1 gene promoters are replaced into 35S-Cas9-SK carriers
In 35S promoter, obtain pDMC1-Cas9-SK recombinant vectors, recycle two restriction enzyme sites of Xma I and EcoR I will
DMC1p-Cas9 transcriptional units are transferred to binary vector pTF101.1, obtain recombinant vector pDMC1-Cas9.
8. construction method according to claim 7, step (2) is using following operation：To meet 5 '-Nx-NGG-3 ' or
The DNA sequence dna of the target site of 5 '-CCN-Nx-3 ' sequence rules is connected into pU3-sgRNA carriers by primer annealing, is then passed through
SgRNA transcriptional units are subcloned into recombinant vector pDMC1-Cas9 by Hind III digestions site, and obtaining, which includes sgRNA, turns
Unit is recorded, and the DMC1p-Cas9 formed from the DMC1 gene promoters and Cas9 genes of corn by being sequentially connected with turns
Record the recombinant vector of unit.
A kind of 9. genetic engineering bacterium for including recombinant vector described in claim 3.
10. a kind of method that gene editing system using described in claim 1 enters edlin to Plant Genome, it includes：
By the genetic engineering bacterium transformation receptor plant tissue described in claim 9, the transgenic plant material edited is obtained.
11. according to the method for claim 10, wherein, the plant is monocotyledon, more preferably corn or wheat.
12. according to the method for claim 10, wherein, the recipient plant is organized as immature rataria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710599030.2A CN107338265B (en) | 2017-07-21 | 2017-07-21 | Gene editing system and method for editing plant genome by applying same |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710599030.2A CN107338265B (en) | 2017-07-21 | 2017-07-21 | Gene editing system and method for editing plant genome by applying same |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107338265A true CN107338265A (en) | 2017-11-10 |
| CN107338265B CN107338265B (en) | 2020-02-18 |
Family
ID=60217364
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710599030.2A Active CN107338265B (en) | 2017-07-21 | 2017-07-21 | Gene editing system and method for editing plant genome by applying same |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107338265B (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108841854A (en) * | 2018-06-11 | 2018-11-20 | 北京市农林科学院 | A method of obtaining carrot mutant |
| WO2019014917A1 (en) * | 2017-07-21 | 2019-01-24 | 中国科学院遗传与发育生物学研究所 | Gene editing system and method for editing plant genome by using same |
| CN109852635A (en) * | 2019-03-01 | 2019-06-07 | 安徽华明太合生物工程有限公司 | A kind of method of quick detection gene editing validity |
| CN113151346A (en) * | 2021-02-09 | 2021-07-23 | 中国农业科学院作物科学研究所 | CRISPR/Cas9 system-mediated wheat polygene knockout editing system |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195177A (en) * | 2014-08-05 | 2014-12-10 | 南京大学 | Method for remarkably improving fish genome editing efficiency |
| CN105177038A (en) * | 2015-09-29 | 2015-12-23 | 中国科学院遗传与发育生物学研究所 | CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode |
| CN106167810A (en) * | 2015-04-03 | 2016-11-30 | 内蒙古中科正标生物科技有限责任公司 | Monocot genes knockout carrier based on CRISPR/Cas9 technology and application thereof |
-
2017
- 2017-07-21 CN CN201710599030.2A patent/CN107338265B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104195177A (en) * | 2014-08-05 | 2014-12-10 | 南京大学 | Method for remarkably improving fish genome editing efficiency |
| CN106167810A (en) * | 2015-04-03 | 2016-11-30 | 内蒙古中科正标生物科技有限责任公司 | Monocot genes knockout carrier based on CRISPR/Cas9 technology and application thereof |
| CN105177038A (en) * | 2015-09-29 | 2015-12-23 | 中国科学院遗传与发育生物学研究所 | CRISPR/Cas9 system for efficiently editing plant gene groups in fixed-point mode |
Non-Patent Citations (7)
| Title |
|---|
| AYMAN EID等: "High efficiency of targeted mutagenesis in arabidopsis via meiotic", 《PLANT CELL REPORTS》 * |
| F.ETEDALI等: "Genome wide cloning of maize meiotic recombinase Dmc1 and its functional structure through molecular phylogeny", 《GENETICS AND MOLECULAR RESEARCH》 * |
| FENG C等: "Efficient Targeted Genome Modification in Maize Using CRISPR/Cas9 System", 《JOURNAL OF GENETICS AND GENOMICS》 * |
| JN033376.1: "Zea mays meiotic recombination protein DMC1 (DMC1) gene, promoter region, exons 1", 《GENBANK》 * |
| MASAFUMI MIKAMI等: "Comparison of CRISPR/Cas9 expression constructs for efficient", 《PLANT MOLECULAR BIOLOGY》 * |
| VICTOR I. KLIMYUK等: "AtDMCl, the Arabidopsis homologue of the yeast DMCl", 《THE PLANT JOURNAL BANNER》 * |
| 张慧展: "《基因工程》", 31 January 2017, 华东理工大学出版社有限公司 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2019014917A1 (en) * | 2017-07-21 | 2019-01-24 | 中国科学院遗传与发育生物学研究所 | Gene editing system and method for editing plant genome by using same |
| CN108841854A (en) * | 2018-06-11 | 2018-11-20 | 北京市农林科学院 | A method of obtaining carrot mutant |
| CN108841854B (en) * | 2018-06-11 | 2021-09-17 | 北京市农林科学院 | Method for obtaining carrot mutant |
| CN109852635A (en) * | 2019-03-01 | 2019-06-07 | 安徽华明太合生物工程有限公司 | A kind of method of quick detection gene editing validity |
| CN113151346A (en) * | 2021-02-09 | 2021-07-23 | 中国农业科学院作物科学研究所 | CRISPR/Cas9 system-mediated wheat polygene knockout editing system |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107338265B (en) | 2020-02-18 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| KR102085189B1 (en) | Sophisticated plant modification method through temporary gene expression | |
| CN104846010B (en) | A kind of method for deleting transgenic paddy rice riddled basins | |
| CN105063083B (en) | Prevent method for creating and its application of the Rice Engineering maintainer of genetic drift | |
| KR102107735B1 (en) | Method for producing whole plants from protoplasts | |
| CN105025701B (en) | The method for removing genetic linkage in plant | |
| CN106164272B (en) | Modified plants | |
| CN109943585B (en) | Method for utilizing plant heterosis | |
| CN104830847B (en) | For detecting the nucleic acid sequence and its detection method of corn plant DBN9936 | |
| EA038896B1 (en) | Method of conducting site-directed modification of plant genomes using non-inheritable materials | |
| CN110213961A (en) | Crop based on genome editor is engineered and produces plant of short stem | |
| CN104837334A (en) | Establishment of maintainer plant line and sterile line and use thereof | |
| CN107338265A (en) | A kind of gene editing system and enter the method for edlin to Plant Genome using it | |
| CN106470544A (en) | The melon plant that fruit yield improves | |
| CN100350042C (en) | Process of rapid variety-independent plant transformation | |
| CN104846009B (en) | A kind of construction method of Rice Engineering maintainer and its application | |
| CN107245495A (en) | The method for creating of the common line with genic sterile of paddy rice and application | |
| US11814632B2 (en) | Modified excisable MON87701 soybean transgenic insect resistance locus | |
| US20220364105A1 (en) | Inir12 transgenic maize | |
| CN113767847A (en) | Method for creating homozygous waxy maize germplasm by rapid site-specific mutagenesis and application thereof | |
| WO2019014917A1 (en) | Gene editing system and method for editing plant genome by using same | |
| CN106496313B (en) | Disease-resistance-related protein IbSWEET10 and its encoding gene and application | |
| CN106868036B (en) | A kind of method and its application of rite-directed mutagenesis initiative corn compact plant germplasm | |
| US20220033842A1 (en) | Inir12 transgenic maize | |
| Arockiasamy et al. | Breeding and biotechnological efforts in Jatropha curcas L. for sustainable yields | |
| CN106350536A (en) | Plant hybridization system and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |