CN107312793A - The tomato dna editor carrier of Cas9 mediations and its application - Google Patents
The tomato dna editor carrier of Cas9 mediations and its application Download PDFInfo
- Publication number
- CN107312793A CN107312793A CN201710540810.XA CN201710540810A CN107312793A CN 107312793 A CN107312793 A CN 107312793A CN 201710540810 A CN201710540810 A CN 201710540810A CN 107312793 A CN107312793 A CN 107312793A
- Authority
- CN
- China
- Prior art keywords
- tomato
- cas9
- dfd
- dna
- seq
- 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.)
- Pending
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
- C12N15/79—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)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- 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
Abstract
The invention belongs to gene engineering technology field, tomato dna editor carrier and its application of a kind of Cas9 mediations are specifically disclosed.Invention, by Cas9 Gene into Tomato M82, gene editing is carried out to tomato itself DFD genes by building CRISPR/Cas9 tomato plants expression vectors.A kind of method for creating storage endurance transgene tomato is provided, the New Tomato Variety of storage endurance is obtained with this, tamato fruit resistant storage properties are improved, while not influenceing growth and other qualities of plant, the weak point of forefathers' research approach can be made up.
Description
Technical field
The invention belongs to gene engineering technology field, specifically disclose a kind of Cas9 mediations tomato dna editor carrier and
It is applied.
Background technology
The limitation of accumulating time and shelf life is the subject matter that gardening product is faced, when extension vegetables and fruit accumulating
Between huge interests will be brought to retailer and ultimate consumer from peasant to whole seller to links in supply chain, particularly
Production and the not high Chinese fresh present situation on product of circulation efficiency, research show that raw vegetable is entirely supplying chain link just
The proportion of goods damageds be up to 20%-30%, can be improved by the technological means of transgenosis and with huge market potential.Tomato be
Extremely wide vegetables are cultivated in the world, are also one of main culturing vegetable of China.
Ripe phenotype is the summation that fruit development changes to latter stage biochemistry and physiology, and this phenotype becomes organ
Edible, and Dependent Animals propagate seed, while making it that there is certain value as agricultural products.These changes, although
It is different between different species, but generally comprise the change of Ultrastructure of cell wall and quality, Starch Conversion be sugar, increase fruit
Reality is to the sensitiveness of postharvest pathogen, the biosynthesis and accumulation for changing pigment, the content of raising aromatic substance and discharges perfume (or spice)
Taste.In these Mature properties, there are some to be intended to reduce the shelf life of fruit, increase harvesting, shipping and the cost of storage, especially
It is the change and ripening fruits comprehensively resistance of the reduction to microbial infection of the hardness of fruit.It is estimated that from leave farmland to
Retail, fruit and victual can lose 12 one 20%.The technology of the undesirable mature characteristic of existing frequently-used reduction is included before maturation
Harvesting, air conditioned storage, insecticide are applied and in good time ripe come regulating fruit using chemical substance induction.Unfortunately, these
Means often add the expense of production, shipping and processing, while also reducing the quality of fruit, are to existing agricultural production water
Flat competitiveness and the challenge of sustainable development.
With the increase of the multiple target breeding difficulty such as tomato high yield, high-quality, degeneration-resistant, tomato is realized using hybrid complementary effect
The synchronous breakthrough of the characters such as yield, quality, resistance a, it has also become important development direction of tomato breeding research.Therefore, in order to
The genetic resources of abundant tomato in China kind, improves the resistant storage properties of tomato, and seed selection storage endurance is strong, the kind sampled is always
The important goal of tomato breeding.Be present the kind of storage endurance tomato in the market, be substantially by traditional crossbreeding
Mode cultivate, but the resistant storage properties of these kinds can only be substantially qualified, the time of transgenic product storage endurance
Will be relatively long, and product can effectively improve this mainly asking in storage transport of the bacterium infection after picking fruit
Topic.Although the arguement on transgenic technology just never stopped from the date of birth, the speed of development in science and technology is
Make rapid progress, along with developing rapidly for its biological economy, the sustainable growth year by year of global genetically modified crops shows transgenosis invariably
The main flow characteristic in breeding of technology.
The content of the invention
The mesh of the present invention is：With newest gene editing technology, there is provided a kind of establishment storage endurance transgene tomato
Method.This method uses Agrobacterium transgenic technology, by building CRISPR/Cas9 plant expression vectors and conversion tomato, with
This obtains the New Tomato Variety of storage endurance.
Technical scheme：One kind is by building CRISPR/Cas9 tomato plants expression vectors, the tomato plants
Expression vector nucleotide sequence is as shown in SEQ ID NO.1.
Tomato plants carrier is, by the regulation and control of CaMV 35S promoters, to be expressed by AtU6 promoters, described 35S promoter
Primer sequence is as shown in SEQ ID NO.2；AtU6 promoter primers sequence is as shown in SEQ ID NO.3.
A kind of method of orthomutation tomato dna, using above-mentioned tomato plants expression vector, comprises the steps：
（1）CRISPR/Cas9 carriers are connected with DFD-gRNA fragments, tomato plants expression vector is built with 35S promoter；
（2）The Agrobacterium LBA4404 that the CRISPR/Cas9 tomato plants expression vector of structure is converted infects tomato hypocotyl；
（3）Metainfective tomato hypocotyl, which is placed on MS culture mediums, carries out callus, budding, culture of rootage；
（4）The transformation seedlings of taking root are transplanted into cultivation of burying, PCR identifications；
（5）After field planting, F1 generation selfing, stable transgenosis self-mating system is formed, the good transgene tomato of resistant storage properties is obtained
New varieties.
Sense primer DFD-JCF in the above method in PCR identifications：Acaaacataaagtagtggaccca such as SEQ ID
Shown in NO.4；Anti-sense primer DFD-JCR：Acctctttcggctatttcgtata is as shown in SEQ ID NO.5.
Identified in the above method using PCR, in its 20 μ L reaction system,
The μ L of genomic DNA 1.0 of the transfer-gen plant of the gene containing DFD
PCR Master Mix 10μL
The μ L of 10 pM sense primers 1.0
The μ L of 10 pM anti-sense primers 1.0
ddH2O is mended to 20 μ L.
PCR working procedures are in the above method：94 DEG C of min of pre-degeneration 3；94 DEG C of denaturation 35 s, 55 DEG C of anneal 35 s, 72
DEG C extension 1 min, 35 circulation；72 DEG C extend 10 min, 4 DEG C of preservations eventually.
Beneficial effect：The present invention is by building CRISPR/Cas9 plant expression vectors, by Cas9 Gene into Tomato M82
In, gene editing is carried out to tomato itself DFD genes.Improve tamato fruit resistant storage properties, at the same do not influence the growth of plant with
Other qualities, can make up the weak point of forefathers' research approach, start the beginning that gene editing uses genetically modified plants, thus
The technology path has the relatively strong, novelty in forward position.
Brief description of the drawings
Accompanying drawing 1 participates in the schematic diagram explanation of expression vector to CRISPR/Cas9：Spcas9 by CaMV 35S promoters control
System, participation is expressed by atu6 promoters；Double-crossed marks the target sequence of DFD target sites, the PAM of single marking, and lower stain is
DFD/DFD gene mutation sites；Accompanying drawing 2, DFD/DFD gene mutation sites：A replaces with T and edited in gene；Accompanying drawing 3, Agrobacterium
Convert tomato Hypocotyl Tissues culture regeneration；The DFD gene PCRs detection of accompanying drawing 4, transformation tissue culture tomato plants；Accompanying drawing 5 is DFD
Genetic fragment sequencing result；Accompanying drawing 6, the result of transgene tomato pair and storage.
Embodiment
Tomato has turned into staple vegetable crop in the world, because the seasonality of tomato production and the anniversary of demand are harmonious
Contradiction, causes the tomato busy season to list, postharvest decay loss up to 50%.The present invention is mutually tied using gene editing technology with transgenosis
Close the method for improving the out-of-date storage endurance of tomato.By agriculture bacillus mediated and tomato hypocotyl tissue culture technology, CRISPR/Cas9 is planted
Thing expression vector is transferred to tomato, using Cas9 albumen editor's DFD genes, improve tamato fruit resistant storage properties obtain it is available
In the metamaterials of storage endurance New Tomato Variety seed selection.
Embodiment, 1）, material
The direct sources of tomato M82 seeds are cultivated by Horticulture Gop Inst., Xinjiang Academy of Agriculture and preserved, and primary source is
In September, 2008 is introduced by Ke Wang Bai from the hereditary center of U.S. tomato.
Exo+ polymerase, DNA QIAquick Gel Extraction Kits and cloning vector used in PCR is limited by the golden biotechnology of the full formula in Beijing
Company buys.Restriction enzyme and ligase are purchased from TaKaRa companies, and PCR primer synthesis is given birth to the equal student on commission's work of sequencing fragment
Thing engineering (Shanghai) limited company completes.
2）, expression vector establishment
SpCas9 carriers are obtained at the high rosy clouds researcher of the Chinese Academy of Sciences, and this carrier is open carrier, and general Study personnel can
Introduced by online plasmid storehouse（http://www.addgene.org/）, by the direct compounding design of Beijing Kang Wei biotech firms
Target gene.Main carriers for combination are that Horticulture Gop Inst., Xinjiang Academy of Agriculture's Biotechnology Experiment room is preserved
PCAM1301 carriers, this carrier is open carrier, and common laboratory can all be introduced.The sequence of DFD genes is open sequence, typically
Researcher can be obtained by online Biotechnology Information center（https://www.ncbi.nlm.nih.gov/, is logged in
Number：AY573803）.The donor sequences of DFD genes are building up on pCAM1301 carriers using polymerase ring-type extension clone.Carrier
Upper spcas9 genes are driven by 35S promoter, and gRNA is driven by arabidopsis U6 promoters.The carrier built is turned by heat shock method
Change into escherichia coli DH5a bacterial strain amplification.Sequence verification carrier sequence, then Agrobacterium EHA105 bacterial strains are converted by freeze-thaw method,
Amplification is in case infect utilization.
3）, tomato genetic transformation and transformant regeneration
A, sowing
The tomato seeds distilled water flushing of picking health for several times after, first with 70% alcohol 30s, then with 20% NaClO surface sterilizations
20min, finally with sterile water wash 3-4 times, is seeded in 1/2MS culture mediums.Environmental requirement 16h/8h, light intensity 1800Lx, 26
±1℃。
B, preculture
14-16d seedling is taken, sterile hypocotyl is taken in superclean bench, blade and petiole is cut, explant about 0.5cm length,
The preculture 2d on the MS differential mediums containing 3.0 mg/L 6-BA and 0.2 mg/L NAA.
C, co-cultivation
The hypocotyl of preculture is punctured and is placed in OD6008-12min in=1 agrobacterium suspension, unnecessary bacterium solution is blotted with filter paper,
It is placed on co-cultivation base, 28 DEG C of light culture 2d.
D, differentiation culture are with taking root
Hypocotyl after co-cultivation is placed in differential medium（Contain 2.0 mg/L 6-BA+1.0 mg/L IAA+3 mg/L tides
Mycin+100mg/L carbenicillins）.It is to be screened go out resistance regeneration bud grow to 2cm it is high when, be transferred to MS+0.1 mg/L
In the root media of the mg/L carbenicillins of 2 mg/L hygromycin of IAA++80, transplanted after under growth root.
4）, transfer-gen plant Molecular Detection
PCR is detected：It is C0-1, C0-2 etc. by contemporary transformed plant numbering.Each strain self progeny be C1-1, C1-2, C2-1,
C2-2 etc. is by that analogy.Transformed plant leaflet tablet 0.2g is gone to extract genomic DNA with Tiangeng DNA extraction kit.Utilize DFD-
In DFD genes in JCF/DFD-JCR primers amplification transfer-gen plant, 20 μ L systems, including MIX 10 μ L, ddH2The μ L of O 7, primer
Each 1 μ L.PCR reaction conditions：94 DEG C of min of pre-degeneration 3；94 DEG C of denaturation 35 s, 55 DEG C of annealing 35 s, 72 DEG C of 1 min of extension,
35 circulations；72 DEG C extend 10min, 4 DEG C of preservations eventually.
After PCR terminates, point sample progress electrophoresis observation is distinguished in 1% Ago-Gel, then PCR primer is reclaimed and converted,
Bacterium solution send Sangon Biotech (Shanghai) Co., Ltd. to be sequenced.Base A is changed into T in acquired results DFD gene orders, thus
Prove the success of tomato transgenic plant gene editing.
5）, transfer-gen plant character observation and fruit storage endurance test
By the way that to transfer-gen plant, compared with the character observation of WT lines M82 fields, both are thick, solid in tomato plant height, stem
Shape thing, pulp thickness, all do not have significant difference on compression resistance.It is also very nearly the same from the point of view of the fruit development cycle.By two groups of fruits
After real harvesting, placed under same environment after 40d, wilting, which substantially occur, in wild type M82 fruits to soften, and transgene tomato is obvious
Than compareing storage endurance, as shown in Figure 6.
SEQUENCE LISTING
<110>Horticulture Gop Inst., Xinjiang Academy of Agriculture
<120>The tomato dna editor carrier of Cas9 mediations and its application
<160> 5
<170> PatentIn version 3.5
<210> 1
<211> 4144
<212> DNA
<213>Artificial sequence
<400> 1
atggctccta agaagaagcg gaaggttggt attcacgggg tgcctgcggc tatggataag 60
aagtacagca ttggtctgga catcgggacg aattccgttg gctgggccgt gatcaccatg 120
agtacaaggt cccttccaag aagtttaagg ttctggggaa caccgatcgg cacagcatca 180
agaagaatct cattggagcc ctcctgttcg actcaggcga gaccgccgaa gcaacaaggc 240
taaagaaccg caaggagacg gtatacaaga aggaagaata ggatctgcta cctgcaggag 300
attttcagca acgaaatggc gaaggtggac gattcgttct ttcatagatt ggaagaaagt 360
ttcctcgtgg gaagataaga agcacgagag gcatcctatc tttggcaaca ttgtcgacga 420
ggttgcctat cacgaaaagt accccacaat ctatcatctg cggaagaagc ttgtggactc 480
gactgataag gcggattaga ttgatctacc tcgctctggc acacatgatt aagttcaggg 540
gccattttct gatcgagggg gatcttaacc cggacaatag cgatgtggac aagttgttca 600
tccagctcgt ccaaacctac aaagctcttt gaggaaaacc caattaatgc ttcaggcgtc 660
gacgccaagg cgatcctgtc tgcacgcctt tcaaagtctc gccggcttga gaacttgatc 720
gctcaactcc cgggcgaaaa gaagaacgct gttcgggaat ctcattgcac tttcgttggg 780
gctcacacca aacttcaaga gtaattttga tctcgctgag gacgcaaagc tgcagctttc 840
caaggacact tatgacgatg acctggataa cctttgccca aatcggcgat cagtacgcgg 900
acttgttcct cgccgcgaag aatttgtcgg acgcgatcct cctgagtgat attctccgcg 960
tgaacaccga gattacaaag gccccgctct cggcgagtat atcagcgcta tgacgagcac 1020
catcaggatc tgaccctttt gaaggctttg gtccggcagc aactcccaga gaagtacaag 1080
gaaatcttct ttgatcaatc caagaacggc tacgctggtt atattgcggc gggcatcgca 1140
ggaggaattc tacaagttta tcaagccaat tctggagaag atggatggca cagaggaact 1200
cctggtgaag ctcaataggg aggacctttt gcggaagcaa agaactttcg atacggcgca 1260
tccctcacca gattcatctc ggggagctgc acgccatcct gagaaggcag gaagacttct 1320
acccctttct taaggataac cgggagaaga tcgaaaagat tctgacgttc agaattccta 1380
ctattcggac cactcgcccg gggtaattcc agatttgcgt ggatgaccag aaagagcgag 1440
gaaaccatca caccttggaa cttcgaggaa gtggtcgata agggcgcttc cgcacagagc 1500
ttcatgagcg ctgacaaatt ttgacaagaa cctgcctaat gagaaggtcc ttcccaagca 1560
ttccctcctg tacgagtatt tcactgttta taacgaactc acgaaggtga agtatgtgac 1620
cgagggaatg gcaagccccc ttcctgagcg gcgagcaaaa gaaggcgatc gtggaccttt 1680
tgtttaagac caatcggaag gtcacagtta agcagctcaa ggaggactac ttcaagaaga 1740
ttgaatgctt cgattcgttg agatcgcggc gtggaagaca ggtttaacgc ctcactgggg 1800
acttaccacg atctcctgaa gatcattaag gataaggact tcttggacaa cgaggaaaat 1860
gaggatatcc tcgaagacat tgcctgactc ttcgttgttt gaggataggg aaatgatcga 1920
ggaacgcttg aagacgtatg cccatctctt cgatgacaag gttatgaagc agctcaagag 1980
aagaagatac accggatggg gaaggctgcc cgcaagcttt caatggcatt agagacaagc 2040
aatcagggaa gacaatcctt gactttttga agtctgatgg cttcgcgaac aggaatttta 2100
tgcagctgat tcacgatgac tcacttactt tcaagaggat atccagaggc tcaagtgtcg 2160
ggacaaggtg acagtctgca cgagcatatc gccaaccttg cgggatctcc tgcaatcaag 2220
aagggtattc tgcagacagt caaggttgtg gatgagcttg gaaggtcatg ggaggcataa 2280
gcccgagaac atcgttattg agatggccag agaaaatcag accacacaaa agggtcagaa 2340
gaactcgagg gagcgcatga agcgcatcga ggaaggcatt aaggagtggg gagtcagatc 2400
ttaaggagca cccggtggaa aacacgcagt tgcaaaatga gaagctctat ctgtactatc 2460
tgcaaaatgg cagggatatg tatgtggacc aggagttgga tattaaccgc cttcggatta 2520
cgacgtcgat catatcgttc ctcagtcctt ccttaaggat gacagcattg acaataaggt 2580
tctcaccagg tccgacaaga accgcgggaa gtccgataat gtgcccagcg aggaagtcgt 2640
aagaagatga agaactactg gaggcaactt ttgaatgcca agttgatcac acagaggaag 2700
tttgataacc tcactaaggc cgagcgcggg gtctcagcga actggacaag gcgggcttca 2760
ttaagggcaa ctggttgaga ctagacagat cacgaagcac gtggcgcaga ttctcgattc 2820
acgcatgaac acgaagtacg atgagaatga caagctatcc gggaagtgaa ggtcatcacc 2880
ttgaagtcaa actcgtttct gacttcagga aggatttcca attttataag gtgcgcgaga 2940
tcaacaatta tcaccatgct catgacgcat acctcaacgc tgtgtcggaa cagcattgat 3000
taagaagtac ccgaagccga gtccgaattc gtgtacggtg actataaggt ttacgatgtg 3060
cgcaagatga tcgccaagtc agagcaggaa attggcaagg ccactgcgaa tatttctttt 3120
actctaacat tatgaatttc tttagactga gatcacgctg gctaatggcg aaatccggaa 3180
gagaccactt attgagacca acggcgagac aggggaaatc gtgtgggaca aggggaggat 3240
ttcgccacag tccgcaaggt tctctctatc ctcaagtgaa tattgtcaag aagactgaag 3300
tccagacggg cgggttctca aaggaatcta ttctgcccaa gcggaactcg gataagctta 3360
tcgcagaaag aaggactggg atccgaagaa gtatgaggtt tcgactcacc aacggtggct 3420
tactctgtcc tggttgtggc aaaggtggag aagggaaagt caaagaagct caagtctgtc 3480
aaggagctcc tggtatcacc attatggaga ggtccagctt caaaagaatc cgatcgattt 3540
tctcgaggcg aagggatata aggaagtgaa gaaggacctg atcattaagc ttccaaagta 3600
cagtcttttc gagttggaaa cggcaggaag cgcatgttgg cttccgcgga gagctccaga 3660
agggtaacga gcttgctttg ccgtccaagt atgtgaactt cctctatctg gcatcccact 3720
acgagaagct caagggcagc ccagagataa cgaacagaag caactgtttg tgggcaacac 3780
aagcattatc ttgacgagat cattgaacag atttcggagt tcagtaagcg cgtcatcctc 3840
gccgacgcga atttggataa ggttctctca gctacaacaa gcaccgggac aagcctatcg 3900
agagcaggcg gaaaatatca ttcatctctt caccctgaca aaccttgggg ctcccgctgc 3960
attcaagtat tttgacacta cgattgatcg gaagagataa cttctacgaa ggaggtgctg 4020
gatgcaccct tatccaccaa tcgattactg gcctctacga gacgcggatc gacttgagtc 4080
agctcggtgg cgataagaga cccgcagcaa ccaagaaggc agggcagcaa agaagaagaa 4140
gtga 4144
<210> 2
<211> 485
<212> DNA
<213>Artificial sequence
<400> 2
ggatcctcta gagtcccccg tgttctctcc aaatgaaatg aacttcctta tatagaggaa 60
gggtcttgcg aaggatagtg ggattgtgcg tcatccctta cgtcagtgga gattccagat 120
aggcctaacg cttgtccaag atctattcag gatatcacat caatccactt gctttgaaga 180
cgtggttgga acgtcttctt tttccacgat gctcctcgtg ggtgggggtc catctttggg 240
accactgtcg gcagaggcat cttcaacgat ggcctttcct ttatcgcaat gatggcattt 300
gtaggagcca ccttcctttt ccactatctt cacaataaag tgacagatag ctgggcaatg 360
gaatccgagg aggtttccgg atattaccct ttgttgaaaa gtctcaattg ccctttggtc 420
ttctgagact gtatctttga tatttttgga gtagacaagt gtgtcgtgct ccaccatgtt 480
gacga 485
<210> 3
<211> 313
<212> DNA
<213>Artificial sequence
<400> 3
ttccgtggga gaaatctcaa aattccggca gaacaatttt gaatctcgat ccgtagaaac 60
gagacggtca ttgttttagt tccaccacga ttatatttga aatttacgtg agtgtgagtg 120
agacttgcat aagaaaataa aatctttagt tgggaaaaaa ttcaataata taaatgggct 180
tgagaaggaa gcgagggata ggcctttttc taaaataggc ccatttaagc tattaacaat 240
cttcaaaagt accacagcgc ttaggtaaag aaagcagctg agtttatata tggttagaga 300
cgaagtagtg att 313
<210> 4
<211> 23
<212> DNA
<213>Artificial sequence
<400> 4
acaaacataa agtagtggac cca 23
<210> 5
<211> 23
<212> DNA
<213>Artificial sequence
<400> 5
acctctttcg gctatttcgt ata 23
Claims (6)
1. one kind is by building CRISPR/Cas9 tomato plants expression vectors, it is characterised in that：The tomato plants expression vector
Nucleotide sequence is as shown in SEQ ID NO.1.
2. the tomato plants carrier as shown in claim 1 is, by the regulation and control of CaMV 35S promoters, to be expressed by AtU6 promoters, institute
The 35S promoter primer sequence stated is as shown in SEQ ID NO.2；AtU6 promoter primers sequence is as shown in SEQ ID NO.3.
3. a kind of method of orthomutation tomato dna, using the tomato plants expression vector described in claim 1, its feature exists
In comprising the steps：
（1）CRISPR/Cas9 carriers are connected with DFD-gRNA fragments, tomato plants expression vector is built with 35S promoter；
（2）The Agrobacterium LBA4404 that the CRISPR/Cas9 tomato plants expression vector of structure is converted infects tomato hypocotyl；
（3）Metainfective tomato hypocotyl, which is placed on MS culture mediums, carries out callus, budding, culture of rootage；
（4）The transformation seedlings of taking root are transplanted into cultivation of burying, PCR identifications；
（5）After field planting, F1 generation selfing, stable transgenosis self-mating system is formed, the good transgene tomato of resistant storage properties is obtained
New varieties.
4. a kind of method of orthomutation tomato dna as claimed in claim 3, it is characterised in that in the PCR identifications
Sense primer DFD-JCF：Acaaacataaagtagtggaccca is as shown in SEQ ID NO.4；Anti-sense primer DFD-JCR：
Acctctttcggctatttcgtata is as shown in SEQ ID NO.5.
5. a kind of method of orthomutation tomato dna as claimed in claim 3, it is characterised in that using PCR identifications, it 20
In μ L reaction systems,
The μ L of genomic DNA 1.0 of the transfer-gen plant of the gene containing DFD
PCR Master Mix 10μL
The μ L of 10 pM sense primers 1.0
The μ L of 10 pM anti-sense primers 1.0
ddH2O is mended to 20 μ L.
6. a kind of method of orthomutation tomato dna as claimed in claim 5, it is characterised in that PCR working procedures are：94
DEG C min of pre-degeneration 3；94 DEG C of denaturation 35 s, 55 DEG C of annealing 35 s, 72 DEG C of 1 min of extension, 35 circulations；72 DEG C of extensions 10 eventually
Min, 4 DEG C of preservations.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710540810.XA CN107312793A (en) | 2017-07-05 | 2017-07-05 | The tomato dna editor carrier of Cas9 mediations and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710540810.XA CN107312793A (en) | 2017-07-05 | 2017-07-05 | The tomato dna editor carrier of Cas9 mediations and its application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107312793A true CN107312793A (en) | 2017-11-03 |
Family
ID=60181059
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710540810.XA Pending CN107312793A (en) | 2017-07-05 | 2017-07-05 | The tomato dna editor carrier of Cas9 mediations and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107312793A (en) |
Cited By (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| CN109097387A (en) * | 2018-08-08 | 2018-12-28 | 华南农业大学 | A kind of methods and applications with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants |
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| WO2020038384A1 (en) * | 2018-08-22 | 2020-02-27 | 中国科学院遗传与发育生物学研究所 | Plant having improved sugar content |
| CN110878324A (en) * | 2019-12-09 | 2020-03-13 | 新疆农业科学院园艺作物研究所 | Preparation method of tomato material with high fruit solid content |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| CN110923231A (en) * | 2019-12-09 | 2020-03-27 | 新疆农业科学院园艺作物研究所 | Method for preparing tomato material with high fruit solid content |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| CN113025646A (en) * | 2021-03-12 | 2021-06-25 | 浙江大学 | LOB1 gene editing method and application thereof in preparation of tomato with storage, transportation and easy after-ripening |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009143155A2 (en) * | 2008-05-19 | 2009-11-26 | Cornell University | Delayed fruit deterioration allele in plants and methods of detection |
| CN102533853A (en) * | 2012-03-02 | 2012-07-04 | 江苏省农业科学院 | Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology |
| CN104988160A (en) * | 2015-07-31 | 2015-10-21 | 中国农业科学院蔬菜花卉研究所 | Preparation method of pink tomato material |
| CN106086062A (en) * | 2016-04-19 | 2016-11-09 | 上海市农业科学院 | A kind of tomato dna group that obtains pinpoints the method knocking out mutant |
| CN106636182A (en) * | 2016-10-21 | 2017-05-10 | 山西省农业科学院蔬菜研究所 | Construction of CRISPR-Cas9 system of tomato PSY 1 gene and application thereof |
-
2017
- 2017-07-05 CN CN201710540810.XA patent/CN107312793A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009143155A2 (en) * | 2008-05-19 | 2009-11-26 | Cornell University | Delayed fruit deterioration allele in plants and methods of detection |
| CN102533853A (en) * | 2012-03-02 | 2012-07-04 | 江苏省农业科学院 | Method for cultivating anti-TYLCV (Tomato Yellow Leaf Curl Virus) tomato plant by using RNAi technology |
| CN104988160A (en) * | 2015-07-31 | 2015-10-21 | 中国农业科学院蔬菜花卉研究所 | Preparation method of pink tomato material |
| CN106086062A (en) * | 2016-04-19 | 2016-11-09 | 上海市农业科学院 | A kind of tomato dna group that obtains pinpoints the method knocking out mutant |
| CN106636182A (en) * | 2016-10-21 | 2017-05-10 | 山西省农业科学院蔬菜研究所 | Construction of CRISPR-Cas9 system of tomato PSY 1 gene and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| CHANGTIAN PAN等: ""CRISPR/Cas9-mediated efficient and heritable targeted mutagenesis in tomato plants in the first and later generations"", 《SSCIENTIFIC REPORTS》 * |
| MONTSERRAT SALADIE等: ""A Reevaluation of the Key Factors That Influence Tomato Fruit Softening and Integrity"", 《PLANT PHYSIOLOGY》 * |
Cited By (44)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10323236B2 (en) | 2011-07-22 | 2019-06-18 | President And Fellows Of Harvard College | Evaluation and improvement of nuclease cleavage specificity |
| US11920181B2 (en) | 2013-08-09 | 2024-03-05 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10954548B2 (en) | 2013-08-09 | 2021-03-23 | President And Fellows Of Harvard College | Nuclease profiling system |
| US10508298B2 (en) | 2013-08-09 | 2019-12-17 | President And Fellows Of Harvard College | Methods for identifying a target site of a CAS9 nuclease |
| US11046948B2 (en) | 2013-08-22 | 2021-06-29 | President And Fellows Of Harvard College | Engineered transcription activator-like effector (TALE) domains and uses thereof |
| US10858639B2 (en) | 2013-09-06 | 2020-12-08 | President And Fellows Of Harvard College | CAS9 variants and uses thereof |
| US11299755B2 (en) | 2013-09-06 | 2022-04-12 | President And Fellows Of Harvard College | Switchable CAS9 nucleases and uses thereof |
| US9999671B2 (en) | 2013-09-06 | 2018-06-19 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10597679B2 (en) | 2013-09-06 | 2020-03-24 | President And Fellows Of Harvard College | Switchable Cas9 nucleases and uses thereof |
| US10912833B2 (en) | 2013-09-06 | 2021-02-09 | President And Fellows Of Harvard College | Delivery of negatively charged proteins using cationic lipids |
| US10682410B2 (en) | 2013-09-06 | 2020-06-16 | President And Fellows Of Harvard College | Delivery system for functional nucleases |
| US10465176B2 (en) | 2013-12-12 | 2019-11-05 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US11053481B2 (en) | 2013-12-12 | 2021-07-06 | President And Fellows Of Harvard College | Fusions of Cas9 domains and nucleic acid-editing domains |
| US11124782B2 (en) | 2013-12-12 | 2021-09-21 | President And Fellows Of Harvard College | Cas variants for gene editing |
| US10704062B2 (en) | 2014-07-30 | 2020-07-07 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11578343B2 (en) | 2014-07-30 | 2023-02-14 | President And Fellows Of Harvard College | CAS9 proteins including ligand-dependent inteins |
| US11214780B2 (en) | 2015-10-23 | 2022-01-04 | President And Fellows Of Harvard College | Nucleobase editors and uses thereof |
| US10113163B2 (en) | 2016-08-03 | 2018-10-30 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US10947530B2 (en) | 2016-08-03 | 2021-03-16 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11702651B2 (en) | 2016-08-03 | 2023-07-18 | President And Fellows Of Harvard College | Adenosine nucleobase editors and uses thereof |
| US11661590B2 (en) | 2016-08-09 | 2023-05-30 | President And Fellows Of Harvard College | Programmable CAS9-recombinase fusion proteins and uses thereof |
| US11542509B2 (en) | 2016-08-24 | 2023-01-03 | President And Fellows Of Harvard College | Incorporation of unnatural amino acids into proteins using base editing |
| US11306324B2 (en) | 2016-10-14 | 2022-04-19 | President And Fellows Of Harvard College | AAV delivery of nucleobase editors |
| US10745677B2 (en) | 2016-12-23 | 2020-08-18 | President And Fellows Of Harvard College | Editing of CCR5 receptor gene to protect against HIV infection |
| US11820969B2 (en) | 2016-12-23 | 2023-11-21 | President And Fellows Of Harvard College | Editing of CCR2 receptor gene to protect against HIV infection |
| US11898179B2 (en) | 2017-03-09 | 2024-02-13 | President And Fellows Of Harvard College | Suppression of pain by gene editing |
| US11542496B2 (en) | 2017-03-10 | 2023-01-03 | President And Fellows Of Harvard College | Cytosine to guanine base editor |
| US11268082B2 (en) | 2017-03-23 | 2022-03-08 | President And Fellows Of Harvard College | Nucleobase editors comprising nucleic acid programmable DNA binding proteins |
| US11560566B2 (en) | 2017-05-12 | 2023-01-24 | President And Fellows Of Harvard College | Aptazyme-embedded guide RNAs for use with CRISPR-Cas9 in genome editing and transcriptional activation |
| US11732274B2 (en) | 2017-07-28 | 2023-08-22 | President And Fellows Of Harvard College | Methods and compositions for evolving base editors using phage-assisted continuous evolution (PACE) |
| US11319532B2 (en) | 2017-08-30 | 2022-05-03 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11932884B2 (en) | 2017-08-30 | 2024-03-19 | President And Fellows Of Harvard College | High efficiency base editors comprising Gam |
| US11795443B2 (en) | 2017-10-16 | 2023-10-24 | The Broad Institute, Inc. | Uses of adenosine base editors |
| CN109097387A (en) * | 2018-08-08 | 2018-12-28 | 华南农业大学 | A kind of methods and applications with CRISPR/Cas9 gene editing system initiative purple fruit Tomato mutants |
| CN112969791B (en) * | 2018-08-22 | 2023-11-17 | 苏州齐禾生科生物科技有限公司 | Plants with increased sugar content |
| WO2020038384A1 (en) * | 2018-08-22 | 2020-02-27 | 中国科学院遗传与发育生物学研究所 | Plant having improved sugar content |
| CN112969791A (en) * | 2018-08-22 | 2021-06-15 | 中国科学院遗传与发育生物学研究所 | Plants with increased sugar content |
| US11643652B2 (en) | 2019-03-19 | 2023-05-09 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11795452B2 (en) | 2019-03-19 | 2023-10-24 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| US11447770B1 (en) | 2019-03-19 | 2022-09-20 | The Broad Institute, Inc. | Methods and compositions for prime editing nucleotide sequences |
| CN110878324A (en) * | 2019-12-09 | 2020-03-13 | 新疆农业科学院园艺作物研究所 | Preparation method of tomato material with high fruit solid content |
| CN110923231A (en) * | 2019-12-09 | 2020-03-27 | 新疆农业科学院园艺作物研究所 | Method for preparing tomato material with high fruit solid content |
| US11912985B2 (en) | 2020-05-08 | 2024-02-27 | The Broad Institute, Inc. | Methods and compositions for simultaneous editing of both strands of a target double-stranded nucleotide sequence |
| CN113025646A (en) * | 2021-03-12 | 2021-06-25 | 浙江大学 | LOB1 gene editing method and application thereof in preparation of tomato with storage, transportation and easy after-ripening |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107312793A (en) | The tomato dna editor carrier of Cas9 mediations and its application | |
| CN105755021A (en) | Rice cadmium-tolerant gene OsGSTU37 and application thereof | |
| CN110573623A (en) | Expression of phosphate transporters for increasing plant yield | |
| CN109609527A (en) | CDPK18L gene is improving the application in tomato bacterial leaf spot resistance and high temperature resistance as negative regulatory factor | |
| CN102725414A (en) | Method for providing mature transgenic recalcitrant plants via induction of bbm during regeneration | |
| CN110358772A (en) | The OsEBP89 gene and preparation method of raising rice abiotic stress resistance and application | |
| CN112996804A (en) | Resistance modifier gene of Beet Necrotic Yellow Vein Virus (BNYVV) | |
| CN103739686A (en) | Protein related to plant yield improvement and quality improvement, and encoding gene and application thereof | |
| CN107304422A (en) | Control the application of the OsSBR1 genes and its RNA interference fragments of resisting rice sheath blight | |
| CN117082972A (en) | Plant resistance genes and means for their identification | |
| Kang et al. | A robust genome-editing method for wild plant species Nicotiana attenuata | |
| CN107406844A (en) | The transgenic rice plant of improvement | |
| CN111996192A (en) | Method for creating green-free green-shoulder tomato material and application thereof | |
| CN102732553B (en) | Improve the gene engineering method and material of plant products | |
| CN1307312C (en) | Tetraploid transgenic locust and quick cultivation | |
| CN102559676B (en) | Rice root specific promoter and application thereof | |
| CN108486112A (en) | A kind of promoter with anther tissue specificity | |
| Averkina et al. | Pinpointing regulatory protein phosphatase 2A subunits involved in beneficial symbiosis between plants and microbes | |
| CN104861051B (en) | Plant development associated protein AtUBP15 and its encoding gene and application | |
| CN106892973A (en) | Plant adversity resistance related protein GhMYB4 and encoding gene and application | |
| Nora et al. | Melon regeneration and transformation using an apple ACC oxidase antisense gene | |
| JP5164093B2 (en) | Method for increasing resistance of rice to pathogens and pathogen-resistant rice transformants | |
| Suri et al. | High frequency regeneration and Agrobacterium tumefaciens-mediated transformation of broccoli (Brassica oleracea var. italica) | |
| CN104450732B (en) | Gene DnFCA-belta of dendrobium nobile and application thereof to regulation of plant growth and development | |
| CN104558132B (en) | Peanut DELLA gene families and its encoding gene and application |
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 | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20171103 |
|
| RJ01 | Rejection of invention patent application after publication |