CN106636182A - Construction of CRISPR-Cas9 system of tomato PSY 1 gene and application thereof - Google Patents
Construction of CRISPR-Cas9 system of tomato PSY 1 gene and application thereof Download PDFInfo
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Abstract
The invention belongs to the field of gene engineering and genetic modification and relates to construction of a CRISPR-Cas9 system of a tomato PSY 1 gene and an application thereof. The construction includes the following steps: (1) according to a cDNA conserved sequence of the tomato PSY 1 gene, designing a DNA sequence of sgRNA, which is represented as the SEQ ID No.1; (2) designing a primer combination used for amplifying a tomato PSY 1 gene target zone in the sgRNA; and (3) performing sgRNA amplification with the primer and purifying an amplified product to obtain a CRISPR-Cas9 Level-1 carrier and a CRISPR-Cas9 Level-2 carrier, which contain the sgRNA of the specific targeted tomato PSY-1 gene. The construction is beneficial to researching on effect that the gene participates in fruit maturity of tomatoes, so that the molecular mechanism of the fruit maturity of tomatoes can be known furthermore. The system can be used for producing PSY 1 gene deleted transgenic tomatoes, and has important effect of culturing a new tomato variety which is tolerant to storage and transportation in future.
Description
Technical field
The invention belongs to genetic engineering and gene genetic modification field, specifically, are related to a kind of tomatoPSY 1Gene
CRISPR-Cas9 system constructions and its application.
Background technology
Tomato（Solanum lycopersicumMill）, also known as tomato, tomato, kind persimmon, June persimmon, Mao Laguo
Deng, belong to Solanaceae tomato genus, originate in South America.At present tomato is to cultivate the most maximum vegetable crop of wide, consumption figure in the world,
Being that China is main plants one of vegetable species.Lycopene is a kind of natural carotenoid of induction biosynthesis, and it is widely present in kind
In the vegetables such as eggplant, capsicum, carrot, pumpkin.Its content highest in tomato, is 0.2-20mg per 100g fresh weights content.Tomato
Red pigment is the isomers of carrotene, is synthesized by isoprene metabolic pathway, and δ-Hu Luo can be transformed in the presence of cyclase
The carotenoid such as Bu Su, gamma carotene, alpha-carotene, beta carotene, lutein, luteole, violaxanthin, neoxanthin.
Phytoene synthetase（Phytoene synthase, PSY）It is promotion kind in Carotenoid in Plants biosynthesis pathway
The upstream key enzyme of Lycopene synthesis, is catalyzed the Mang ox base Mang ox base pyrophosphoric acid of two molecules（geranylgerany
Pyrophosphate, GGPP）Condensation forms phytoene,PSYGene separates identification most in tomato, at present from
Isolated 2 encoding genes in tomato,PSY 1WithPSY 2（Kato, 2004）.
CRISPR（Clustered regularly interspaced short palindromic repeats）It is wide
The general special repetitive dna sequence being present in bacterium and archeobacteria genome, its sequence is by a leader（Leader）, it is multiple
Short and highly conserved repetitive sequence area（Repeat）With multiple spacer regions（Spacer）Composition.Leader is normally at
CRISPR upstreams, are the regions rich in AT, length for 300-500bp, it is considered to be the promoter sequence of CRISPR.Repeat
Length is 21-48bp, due to containing palindromic sequence, can form hairpin structure.By length for 26-72bp's between repetitive sequence
Spacer separates.Spacer regions are made up of the foreign DNA captured, and are similar to immunological memory, when the foreign DNA containing same sequence
During invasion, can be recognized by bacterium body, and carry out shearing and be allowed to expression silencing, reach protection inherently safe purpose.Cas
（CRISPR associated）Albumen is present in CRISPR location proximates, is a kind of double-stranded DNA nuclease, can be in guide RNA
（Small guide RNA, sgRNA）Target site is cut under guiding.Special Cas albumen can recognize ectogenic heredity
There is prototype intervening sequence adjacent area in material（Protospacer adjacent motifs, PAM）DNA fragmentation, pass through
The DNA that Cas albumen holds PAM 5 '（That is protospacer）Short DNA fragmentation is processed into, the weight of its CRISPR sequence is inserted into
Between complex sequences, finally cause bacterium by spacer recognition sequences and subsequently target these external original papers to be cut off.
CRISPR-Cas systems give prokaryotic and are directed to foreign DNA specific immunity, and this species specificity is determined by intervening sequence.
The CRISPR-Cas systems of 3 types are had now been found that, according to the tissues-derived difference of Cas locus genes, this 3 type is again
10 kinds of hypotypes can be further separated into, for different interference different albumen compositions can be expressed.Wherein, II types system is constituted most
For simple, it is only necessary to which 1 Cas9 albumen can cutting DNA double-strand.Cas9 restriction endonucleases are under the guiding of sgRNA molecules to specific
The DNA in site is cut, and forms double-stranded DNA breach, and then cell can be by homologous recombination machinery（homologous
Recombination, HR）Or non-homologous end joining mechanism（Non-homologous end joining, NHEJ）To disconnected
The DNA for splitting is repaired.If cell is repaired by HR mechanism, the DNA that can fill up fracture with other section of DNA fragment lacks
Mouthful, thus one section of new hereditary information can be introduced.CRISPR technologies are not only the sharp weapon of gene functional research, and can be used for base
Because editing to improve crops, set up genopathy animal model and carry out gene therapy.This technology is chosen as 2013 by Science
One of year ten big sciences progress, and its function is summarized as " Genetic Microsurgery for the Masses ".
At present, to be successfully applied to Escherichia coli, Diplococcus pneumopniae, saccharomyces cerevisiae, zebra fish, mouse thin for the technology
In born of the same parents, mouse, mankind's various kinds of cell system, fruit bat, nematode, rat, paddy rice, wheat, arabidopsis and this life cigarette, but
CRISPR-Cas9 systems are still blank out on tomato is studied.
The content of the invention
It is an object of the invention to provide tomatoPSY 1The CRISPR-Cas9 system constructions of gene and its application.
The present invention adopts the following technical scheme that realization：A kind of tomatoPSY 1The CRISPR-Cas9 system structures of gene
Build, the method is comprised the following steps：
（1）According to tomatoPSY 1The cDNA conserved sequences of gene design the DNA sequence dna of sgRNA, and the sgRNA is located at tomatoPSY 1
On first extron of gene, centre has a restriction enzyme site Sty I, as shown in SEQ ID NO. 1；
（2）It is designed for expanding the tomato of sgRNAPSY 1The primer combination in gene target area, thereon, downstream primer nucleotides sequence
Row are as shown in SEQ ID NO. 2 and SEQ ID NO. 3；
（3）SgRNA amplifications are carried out using primer, and purifies amplified production；
（4）By sgRNA amplified productions after purification and pICSL01009::AtU6p and pICH47751 connects, and obtains containing special
Opposite sex targeting tomatoPSY 1The carriers of CRISPR-Cas9 Level 1 of gene sgRNA；
（5）By step（4）Product again with pICH47732:NPT II、pICH47742:Cas 9, pICH41766 and
PAGM4723 connects, and obtains containing selectively targeted tomatoPSY 1The carriers of CRISPR-Cas9 Level 2 of gene sgRNA.
Described tomatoPSY 1The CRISPR-Cas9 systems of gene are preparing tomatoPSY 1The transgenosis kind of gene mutation
Application in eggplant.
Described tomatoPSY 1The CRISPR-Cas9 systems of gene answering in the transgene tomato for preparing yellow fruit
With.
Described tomatoPSY 1The CRISPR-Cas9 systems of gene are preparing tomatoPSY 1The transgenosis kind of gene mutation
Application in eggplant, CRISPR-Cas9 systems are converted after Agrobacterium, for detecting CRISPR-Cas9-PSY 1Gene mutation
Primer is combined, and thereon, downstream primer nucleotide sequence is as shown in SEQ ID NO. 5 and SEQ ID NO. 6.
The present invention is first according to tomatoPSY 1The cDNA conserved sequences of gene design the DNA sequence dna of sgRNA, the sgRNA positions
InPSY 1On first extron, centre has a restriction enzyme site, is Sty I, according toPSY 1First extron of gene
DNA sequence dna devise sgRNA primers, then construct the CRISPR-Cas9 carriers containing the sgRNA, can be rightPSY 1Enter
Row gene editing, causes the gene that frameshift mutation occurs, and realizesPSY 1The silence of gene, causes funtion part or all disappearance.
In order to further verify effect of the present invention, by the conversion Agrobacterium of the CRISPR-Cas9 carriers containing the sgRNA, then use
Agrobacterium-mediated Transformation tomato cotyledon containing CRISPR-Cas9 carriers, using PCR and sequencing technologies, successfully be detected generation base
The transfer-gen plant of disappearance, itsPSY 1Gene lacks functionality.
The present invention contributes to studying the effect during gene participation Fruit Ripening of Tomato, further appreciates that tamato fruit
Ripe molecule mechanism, can be used to preparePSY 1The transgene tomato of gene delection, to following storage tolerance New Tomato Variety is cultivated
Play an important role.
Description of the drawings
Fig. 1 is the agarose gel electrophoresis figure of sgRNA amplified productions；
Fig. 2 is the agarose gel electrophoresis figure of the amplified production of the carrier bacterium solutions of CRISPR-Cas9 Level 1 of sgRNA；
Fig. 3 is the agarose gel electrophoresis figure of the amplified production of the carrier bacterium solutions of CRISPR-Cas9 Level 2 of sgRNA；
Fig. 4 is the agarose gel electrophoresis figure of the special primer amplified production of Agrobacterium bacterium solution；
Fig. 5 is the agarose gel electrophoresis figure of the special primer amplified production of transfer-gen plant；
Fig. 6 is the sequence alignment figure of mutant strain and wild type DNA target area；
Fig. 7 is the sequence map of mutant strain；
Fig. 8 is mutant strain tamato fruit photo；
In above-mentioned Fig. 1 to Fig. 5, M：Marker；1、2、3、4：The different of same sample are repeated.
Specific embodiment
Following examples are used to illustrate the present invention, but are not limited to the scope of the present invention.Without departing substantially from spirit of the invention
In the case of essence, the modification made to the inventive method, step or condition or replacement belong to the scope of the present invention.
If not specializing, material used, biological chemical reagent are conventional commercial reagent in enforcement, institute in embodiment
Technological means is that those skilled in the art know conventional meanses.
Embodiment 1：TomatoPSY 1The sgRNA designs of gene C RISPR-Cas9
1st, PAM is found（proto adjacent motif）Motif
PSY 1Find PAM motifs, i.e. NGG in the exon 1 of gene order the 1st.
2nd, sgRNA sequences are determined
One section of sequence of 20 bp is held to be sgRNA sequences in PAM positions 5 ', U6 starts used in general tomato CRISPR-Cas9
Son is transcribing sgRNA, therefore the base at 5 ' 20, the ends of preferential NGG is G, i.e. G (N)19NGG.TomatoPSY 1Gene the 1st is outer aobvious
The sgRNA sequences of sub-district are as shown in SEQ ID NO. 1.
3rd, sgRNA primers synthesis
Sequence is respectively as shown in SEQ ID NO. 2 and SEQ ID NO. 3.
4th, sgRNA amplification reaction systems
By the primer of synthesis, deionized water is added respectively so as to which concentration is 10 μM, then prepares sgRNA amplification reaction systems such as
Under：
5th, sgRNA amplification reaction conditions
6th, product detection
SgRNA pcr amplification products are detected with 2% Ago-Gel, size is 174 bp, as shown in Figure 1.
Embodiment 2：The gene sgRNA of tomato PSY 1 are connected on the carriers of CRISPR-Cas9 Level 1
1st, sgRNA amplified productions purifying
Using GenElutTMPCR Clean-Up Kit（SIGMA）Carry out PCR primer purifying.
2nd, the coupled reaction systems of CRISPR-Cas9 Level 1
3rd, the coupled reaction conditions of CRISPR-Cas9 Level 1
4th, connection product is transformed in Escherichia coli
Take 40 μ L bacillus coli DH 5 alphas（DH8142）Competent cell and the connection products of 5 μ L Level 1 are mixed, ice bath 15
Min, afterwards 42 DEG C of s of water-bath 45, then ice bath 5 min.Then plus 600 μ L LB culture mediums, 37 DEG C of h of shaken cultivation 1.5.Draw
100 μ L nutrient solutions are uniformly coated onto LB flat boards（Containing 100 mg L-1 Xgal and 100 mg L-1 Carbenicilin）On, 37 DEG C of trainings
Support overnight.
5th, select monoclonal and shake bacterium
White monoclonal is selected on LB flat boards in 10 mL LB culture mediums（Containing 100 mg L-1 Carbenicilin）, 37 DEG C
Overnight incubation.The identification of bacterium solution PCR, upstream and downstream primer sequence such as SEQ ID NO. 2 and SEQ ID are carried out with specific primer
NO. shown in 3.PCR programs：98 DEG C, 30 s；94 DEG C, 10 s；60 DEG C, 30 s；72 DEG C, 30 s；35 circulations；72 DEG C, 2
min；4 DEG C, hold.
PCR reaction systems：
Then with 2% Ago-Gel come testing goal PCR primer, correct purpose fragment is 174 bp, as shown in Fig. 2 extracting
The DNA of positive bacterium solution, sequencing.
6th, plasmid connection confirms
Analyses and comparison sequencing result, detects whether that successfully double-strand sgRNA is connected on U6-sgRNA carriers, as a result such as SEQ ID
NO. shown in 4.As a result illustratePSY 1- sgRNA is connected on U6-sgRNA carriers, is obtained containing selectively targeted tomatoPSY 1
The carriers of CRISPR-Cas9 Level 1 of gene sgRNA.
The gene sgRNA of 3 tomato PSY of embodiment 1 are connected on the carriers of CRISPR-Cas9 Level 2
1st, the coupled reaction systems of CRISPR-Cas9 Level 2
2nd, the coupled reaction conditions of CRISPR-Cas9 Level 2
3rd, connection product is transformed in Escherichia coli
Take 40 μ L bacillus coli DH 5 alphas（DH8142）Competent cell and the connection products of 5 μ L Level 2 are mixed, ice bath 15
Min, afterwards 42 DEG C of s of water-bath 45, then ice bath 5 min.Then plus 600 μ L LB culture mediums, 37 DEG C of h of shaken cultivation 2.Draw
100 μ L nutrient solutions are uniformly coated onto LB flat boards（Containing 100 mg L-1 Kanamycin）On, 37 DEG C of overnight incubations.
4th, select monoclonal and shake bacterium
Picking white monoclonal is in 10 mL LB culture mediums on LB flat boards（Containing 100 mg L-1 Kanamycin）, 37 DEG C of trainings
Support overnight.Enter performing PCR identification, upstream and downstream primer sequence such as SEQ ID NO. 2 and SEQ ID with specific bacterium solution PCR primer
NO. shown in 3.PCR programs：98 DEG C, 30 s；94 DEG C, 10 s；60 DEG C, 30 s；72 DEG C, 30 s；35 circulations；72 DEG C, 2
min；4 DEG C, Hold.
PCR system：
Then with 2% Ago-Gel come testing goal PCR primer, correct purpose fragment is 174 bp or so, as shown in Figure 3.
Select 3 positive bacterium solutions, sequencing.
5th, plasmid connection confirms
Analyses and comparison sequencing result, detects whether that successfully double-strand sgRNA is connected on Cas9 carriers.As a result CRISPR- is illustrated
The carriers of Cas9 Level 2 include NPT II, Cas 9, sgRNA and L3E, and do not occur base mutation part, to be contained
Selectively targeted tomatoPSY 1The carriers of CRISPR-Cas9 Level 2 of gene sgRNA.
Embodiment 4：The carriers of 1 Level of tomato CRISPR-Cas9-PSY 2 convert Agrobacterium
1st, high concentration CRISPR-Cas9-PSY 1 The extraction of the plasmids of Level 2
Shake the mL of bacterium 10（LB+Kanamycin）So as to OD600 is about 1.5 or so, then according to PuriLink Quick
Plasmid DNA Miniprep Kits（Invitrogen）Extract and be connected to obtained in embodiment 3PSY 1- sgRNA's
CRISPR-Cas9 plasmids, make final plasmid concentration be more than 100 ng μ L-1。
2nd, the plasmids of Level 2 are transformed in Agrobacterium
Take 40 μ L Agrobacterium EHA105 competent cells and the DNAs of 2 μ L Level 2 are mixed, and be transferred to the electricity of precooling and turn
In cup, converted on electroporation（The μ FD of 2.5V, 400 Ω, 25）.Add the LB culture mediums of 1 mL precoolings immediately afterwards, shift
To in 1 mL centrifuge tubes, 28 DEG C of h of shaken cultivation 3.Draw 100 μ L nutrient solutions and apply LB flat boards（Containing 100 mg L-1
Kanamycin）, 28 DEG C of overnight incubations.
3rd, select monoclonal and shake bacterium
Picking white monoclonal is in 10 mL LB culture mediums on LB flat boards（Containing 100 mg L-1 Kanamycin）, 28 DEG C of trainings
Support overnight.
4th, conversion confirms
The identification of bacterium solution PCR, upstream and downstream primer sequence such as SEQ ID NO. 5 and SEQ ID NO. 6 are carried out with specific primer
It is shown.PCR programs：98 DEG C, 30 s；94 DEG C, 10 s；60 DEG C, 30 s；72 DEG C, 30 s；35 circulations；72 DEG C, 2 min；4 DEG C,
Hold。
PCR system：
Then with 2% Ago-Gel come testing goal PCR primer, correct purpose fragment is 1800 bp or so, such as Fig. 4 institutes
Show.
5th, Agrobacterium preserves
Glycerine of the bacterium solution with 50% mixes, -80 DEG C of preservations.
Embodiment 5：The Agrobacterium-mediated Transformation tomato cotyledons of CRISPR-Cas9-PSY 1
1st, explant prepares
The surface of the seed sterilization is carried out by material of tomato wild type AC++ seed.
（1）（50% ethanol：50% water）The middle immersion mins of seed 10；
（2）The mins of seed 20 is soaked in saturation trisodium phosphate solution, with the distilled water flushing seed of sterilizing on superclean bench
5 times；
（3）（50% sodium hypochlorite：50% water）The middle immersion mins of seed 10.The distilled water flushing seed sterilized with 1 L.
Sow on MSR3 culture mediums, density is 3 cm-2.Germination culture environment condition：26 DEG C, the h of illumination length 16
d-1.After planting 10-12 d（5-6 d after germination）, taking cotyledon before the 1st true leaf grows carries out preculture.In order to reduce damage,
First cotyledon is soaked in water, then with the sharp tip for suitably cutting cotyledon and base portion.The cotyledon for cutting is placed on into 3C5ZR
Upper 26 DEG C of light cultures 24 h of culture.
MSR3 culture mediums：
R3 vitamins：
3C5ZR culture mediums：
2nd, Agrobacterium activation
The μ L of Agrobacterium 10 of -80 DEG C of preservations are taken, adds the fresh LB culture mediums of 10 mL, 28 DEG C of d of shaken cultivation 2.Detection agriculture bar
Bacteria concentration, and with MS culture mediums Agrobacterium concentration OD value is adjusted to 0.2.
3rd, tomato cotyledon is converted
Pre-incubated cotyledon is put in agrobacterium liquid and is soaked after 15 mins, with filter paper unnecessary bacterium solution is sucked, then be put back into original
26 DEG C of d of light culture 2 on the 3C5ZR culture mediums for coming.
4th, selective culture
Cotyledon is transferred to and is added with 80 mg L-1 Kanamycin and 500 mg L-1 The 3C5ZR culture mediums of Carbenicilin
Middle culture.Environmental condition：26 DEG C, the h d of illumination length 18-1.Guarantee that cotyledon cut ends are contacted with culture medium.Per 3-4 w transfers
Cotyledon is into fresh culture medium.
5th, culture of rootage
When there is plantlet to grow from callus, it is transferred to and is added with 0.87 mg L-1 IAA、80 mg·L-1
Kanamycin and 500 mg L-1 Culture of rootage in the MSR3 culture mediums of Carbenicilin.
6th, rooting culture
Good bottle seedling of taking root carefully is taken out from culture medium, is transplanted in matrix, and it is wet to put clean polybag holding
Degree.Gradually leak informaton in polybag perforate after 3 d, make plant adapt to external environment.
Embodiment 6：Abrupt climatic change
1st, the extraction of genomic DNA
Using DNeasy Plant Mini Kit（QIAGEN）The genomic DNA of wild type and transfer-gen plant is extracted, and is detected
Concentration（50 ng μ L need to be typically more than-1）, for subsequent experimental detection.
2nd, PCR specific amplifications purpose fragment
With extract genomic DNA as substrate, with specificity detection primer（Such as SEQ ID NO. 7 and the institutes of SEQ ID NO. 8
Show）Enter performing PCR amplification.
Pcr amplification reaction system
Pcr amplification reaction condition
Then detected with 2% Ago-Gel, purpose fragment is about 830 bp or so, as shown in Figure 5.
3rd, PCR primer purifying
Using GenElutTMPCR Clean-Up Kit（SIGMA）Carry out PCR primer purifying.
4th, it is sequenced
The PCR primer for drawing 15 μ L purifying send company（Eurofins MWG Operon）Sequencing.Sequencing primer such as SEQ ID
NO. shown in 7.
5th, sequence alignment
The result of sequencing and wild type are compared using DNAMAN softwares, analysis determines the interval catastrophe of purpose, such as
Fig. 6, shown in 7.The plasmid vector can in tomato cotyledon function cuttingPSY 1, so as to mutagenesis.Wherein existPSY 1The target sequence in exon 1 area occurs 1 base deletion causes the gene function to be lost.
6th, phenotypic character identification
Normal management genetic transformation plant, when its fruit maturation, observes fruit colour, as shown in figure 8,PSY 1Gene the 1st
There is the transformed plant of 1 base deletion in the target sequence of exon 1, fruit colour shows as yellow, and lower section fruit is in figure
Yellow.
Although above having used general explanation, specific embodiment and test, the present invention is made to retouch in detail
State, but on the basis of the present invention, it can be made some modifications or improvements, this is to those skilled in the art apparent
's.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to claimed
Scope.
Sequence table
The ＞ Vegetables Inst., Shanxi Academy of Agricultural Science of ＜ 110
A kind of tomatoes of the ＞ of ＜ 120PSY 1The CRISPR-Cas9 system constructions of gene and its application
〈160〉8
〈210〉1
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
〈223〉sgRNA
〈400〉1
GGCAGGCAGC CTTGGTGAAG 20
〈210〉 2
〈211〉 56
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to expand the upstream primer of sgRNA
〈400〉2
TGTGGTCTCA ATTGGCAGGC AGCCTTGGTG AAGGTTTTAG AGCTAGAAAT AGCAAG 56
〈210〉3
〈211〉31
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to expand the downstream primer of sgRNA
〈400〉3
TGTGGTCTCA AGCGTAATGC CAACTTTGTA C 31
〈210〉4
〈211〉256
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 contain selectively targeted tomatoPSY 1The carriers of CRISPR-Cas9 Level 1 of gene sgRNA
〈400〉4
GAAGACAAAC TAGAATTCGA GCTCGGAGTG ATCAAAAGTC CCACATCGAT CAGGTGATAT
ATAGCAGCTT AGTTTATATA ATGATAGAGT CGACATAGCG ATTGGCAGGC AGCCTTGGTG
AAGGTTTTAG AGCTAGAAAT AGCAAGTTAA AATAAGGCTA GTCCGTTATC AACTTGAAAA
AGTGGCACCG AGTCGGTGCT TTTTTTCTAG ACCCAGCTTT CTTGTACAAA GTTGGCATTA
CGCTTTACTT GTCTTC
256
〈210〉5
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to expand CRISPR-Cas9-PSY 1Upstream primer
〈400〉5
GAAATTTAGA TTGAAGCTGG 20
〈210〉6
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to expand CRISPR-Cas9-PSY 1The downstream primer of gene mutation
〈400〉6
GTGTTTTCAA CTGGGTGTTC 20
〈210〉7
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to detectPSY 1The upstream primer of gene mutation
〈400〉7
TTGGTTTGCC TGTCTGTGGT 20
〈210〉8
〈211〉20
〈212〉DNA
The ＞ artificial sequences of ＜ 213
〈220〉
The ＞ of ＜ 223 are used to detectPSY 1The downstream primer of gene mutation
〈400〉8
TGTGTTTGTG CGCGTAACTG 20
Claims (4)
1. a kind of tomatoPSY 1The CRISPR-Cas9 system constructions of gene, is characterized in that the method is comprised the following steps：
（1）According to tomatoPSY 1The cDNA conserved sequences of gene design the DNA sequence dna of sgRNA, and the sgRNA is located at tomatoPSY 1
On first extron of gene, centre has a restriction enzyme site Sty I, as shown in SEQ ID NO. 1；
（2）It is designed for expanding the tomato of sgRNAPSY 1The primer combination in gene target area, thereon, downstream primer nucleotides sequence
Row are as shown in SEQ ID NO. 2 and SEQ ID NO. 3；
（3）SgRNA amplifications are carried out using primer, and purifies amplified production；
（4）By sgRNA amplified productions after purification and pICSL01009::AtU6p and pICH47751 connects, and obtains containing special
Opposite sex targeting tomatoPSY 1The carriers of CRISPR-Cas9 Level 1 of gene sgRNA；
（5）By step（4）Product again with pICH47732:NPT II、pICH47742:Cas 9, pICH41766 and
PAGM4723 connects, and obtains containing selectively targeted tomatoPSY 1The carriers of CRISPR-Cas9 Level 2 of gene sgRNA.
2. a kind of tomatoPSY 1The CRISPR-Cas9 systems of gene are preparing tomatoPSY 1In the transgene tomato of gene mutation
Application.
3. a kind of tomato according to claim 1PSY 1The CRISPR-Cas9 systems of gene are preparing turning for yellow fruit
Application in transgenic tomato.
4. a kind of tomato according to Claims 2 or 3PSY 1The CRISPR-Cas9 systems of gene are preparing tomatoPSY 1
Application in the transgene tomato of gene mutation, is characterized in that, CRISPR-Cas9 systems are converted after Agrobacterium, for detecting
CRISPR-Cas9-PSY 1The primer combination of gene mutation, thereon, the downstream primer nucleotide sequence such as and of SEQ ID NO. 7
Shown in SEQ ID NO. 8.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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| CN108728486A (en) * | 2018-06-20 | 2018-11-02 | 江苏省农业科学院 | A kind of construction method of eggplant CRISPR/Cas9 gene knockout carriers and application |
| CN109207506A (en) * | 2017-06-29 | 2019-01-15 | 中国科学院遗传与发育生物学研究所 | A method of tomato red fruit material is changed by steamed dumpling with pork, mushrooms and bamboo shoots material by gene editing |
| CN109423498A (en) * | 2017-08-30 | 2019-03-05 | 中国科学院遗传与发育生物学研究所 | A method of the purple black fruit tomato material of high anthocyanidin is formulated by gene editing |
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Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450774A (en) * | 2014-12-04 | 2015-03-25 | 中国农业科学院作物科学研究所 | Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification |
-
2016
- 2016-10-21 CN CN201610918480.9A patent/CN106636182B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104450774A (en) * | 2014-12-04 | 2015-03-25 | 中国农业科学院作物科学研究所 | Construction of soybean CRISPR/Cas9 system and application of soybean CRISPR/Cas9 system in soybean gene modification |
Non-Patent Citations (1)
| Title |
|---|
| JINJIE ZHU ET AL.: "Efficiency and Inheritance of Targeted Mutagenesis in Maize Using CRISPR-Cas9", 《JOURNAL OF GENETICS AND GENOMICS》 * |
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