CN103952405A - Goat MSTN (myostatin) gene fixed-point modification system and application thereof - Google Patents
Goat MSTN (myostatin) gene fixed-point modification system and application thereof Download PDFInfo
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Abstract
The invention discloses a goat MSTN (myostain) gene fixed-point modification system and an application method thereof. The goat MSTN gene fixed-point modification system can effectively identify and modify transcription activator like effector nucleases (TALENs) of a goat MSTN gene target sequence, the TALENs provided by the invention is composed of nucleases TALEN-R and TALEN-L which can respectively identify identification modules TALMEX-1F and TALMEX-1R; the nuclease TALEN-L is a protein coded by a nucleotide sequence shown in SEQ ID NO.2 or SEQ ID NO.3, and the nuclease TALEN-R is a protein coded by a nucleotide sequence shown in SEQ ID NO.4 or SEQ ID NO.5.The invention also discloses a method for obtaining a muted cell line by carrying out targeted modification on a goat cell MSTN gene by adopting the knock-out system. According to the goat MSTN gene fixed-point modification system, the goat MSTN gene is knocked out or modified for obtaining a new variety with a high high-quality meat factor, so that the goat MSTN gene fixed-point modification system has an important application value on mechanism study of an MSTN gene regulation and control mechanism.
Description
Technical field
The present invention relates to gene engineering technology field, relate in particular to a pair of transcriptional activation increment effector nuclease and encoding gene and application, utilize specifically a pair of transcriptional activation effector nuclease pointed decoration goat MSTN gene.
Background technology
Flesh generates statin (Myostatin, MSTN) and is extensively present in animal skeleton intramuscular, is the negative regulatory factor of Skeletal Muscle Growth, and MSTN gene has expression in the skeletal muscle of Embryonic Development in Animal phase and Adulthood.The cDNA of the MSTN gene of mouse, rat, people, ox, pig, sheep, chicken, turkey, zebra fish is cloned, checks order.Result of study shows, MSTN gene contains 3 exons, and between different plant species MSTN gene order high conservative.Knock out MSTN gene and can cause animal myofiber popularity hyperplasia and hypertrophy, skeletal muscle amount significantly increases.The sudden change of MSTN gene coded sequence origination point or the phase shift mutation of the species such as mouse, rat, people, pig, chicken, ox, sheep, baboon and zebra fish, all present " two flesh symptom " (double muscling), show the proterties that muscle mass significantly increases.In the species such as people, ox, sheep, dog, found at present the sudden change of abiogenous MSTN gene, ox as blue in Belgium (Belgian blue), Piemonte ox (Piedmontese) etc. are undergone mutation in exon3 sequence, and body shows double-muscling shape.
According to mankind's wish, genome is carried out to the dream that directed modification is many scientists always.By genetic modifications such as MSTN gene are suddenlyd change, lacks, knocked out, obtain gene mutant animals, can construct on the one hand there is " two flesh symptom " animal model for biological study and pathogenic mechanism research, can reach on the other hand the object of improving muscle quality and weight, produce the improved seeds domestic animal with commercialization character.
People are finding simple method efficiently always genome are being carried out to targeting modification.Traditional gene targeting depends in cell the exchange of recombinating at random of abiogenous homologous chromosomes, and the ratio that hits is extremely low, conventionally only has 10
-6-10
-8, be not widely used.The genome targeting modification technical development that nuclease instructs is in recent years rapid.This class nuclease consists of a DNA recognition structure territory and a non-specific endonuclease structural domain conventionally, by the special identification target site in DNA recognition structure territory, nuclease is navigated to the genome area that need to edit, then by non-specific endonuclease, cut off DNA double chain, cause DNA self-regeneration mechanism, thereby cause the sudden change of gene order and the generation of promotion homologous recombination.
Artificial Zinc finger nuclease (Zinc Finger Nucleases, ZFN) technology is the genome pointed decoration technology that development in recent years is got up.ZFN is that a kind of structural domain and Fok I restriction endonuclease structural domain by identifying special DNA sequence dna merges the albumen forming, by identifying specific DNA sequence dna, make Fok I endonuclease form dimer generation endonuclease activity at target site, cause target site DNA double chain break, thereby cause non-homologous end joining and homologous recombination repair mechanism.The phase shift mutation producing in repair process can be used to goal gene to carry out gene knockout, and the gene that homologous recombination follows the insertion of foreign gene can be used to carry out specific site is knocked in.It is significant to gene function or generation transgenic animal that ZFN technology was once considered to, but be full of, wish with disappointed, because ZFN is subject between Context-dependent effects and target spot DNA sequence dna in conjunction with unstable, can not target all sequences, before use, need a large amount of screening and identification work, and the transgenation possibility that missing the target property of generation cutting introducing cannot be expected is high.In addition, it is expensive that business is bought efficient special ZFN, and the very difficult designed, designed of investigator goes out special and efficient ZFN.
Research is in recent years found, transcriptional activation sample effector (transcription activator-like effector, TALE) there is DNA binding specificity, and its recognition code has modularization and simplification feature, TALE-DNA binding domains is comprised of the repeating unit of connecting, and most of unit is containing 34 amino acid, the 12nd and 13 amino acids alterable heights of unit, become repetition variable region (repeat variable diresidues, RVDs).4 bases of the RVDs identification DNA sequence dna of TALE have height specificity, and the 13rd amino acids directly and the base specific combination of DNA.Investigator can build special TALE-DNA identification in conjunction with territory according to DNA sequence dna in any site, can be widely used in gene order sudden change modification and gene targeting etc.
Set DNA target sequence, TALE-DNA is in conjunction with territory in assembling, merges the non-specific DNA cutting territory of Fok I restriction endonuclease, is assembled into TALE nuclease (tanscription activator-like effector nucleases, TALENs).TALENs target is in conjunction with DNA, and Fok I Non-specific cleavage DNA sequence dna, produces DNA double splitting of chain (DNA double-srand breaks, DSBs).In eukaryotic cell, DSBs activates two conservative DNA repair pathways: non-homologous end joining reparation (non-homologous end joining, NHEJ) karyomit(e) of fracture can be reconnected, in connection procedure Fracture site, likely introduce loss or the insertion of small segment base, thereby affect gene function or produce gene knockout effect.If now introduce homologous recombination vector; Homology instructs reparation (homology-directed repair, HDR) can utilize similar DNA profiling to instruct and repairs, and substitutes breaking point DNA sequence dna around, realizes specific sudden change or fixes a point to import foreign DNA.
Summary of the invention
The object of the invention is to provide the gene site-directed modification system of a kind of goat MSTN and the application method in goat MSTN genetic modification thereof.
In the present invention, term goat MSTN gene refers to goat myogenesis arrestin (Myostatin, MSTN) gene.
In order to address the above problem, the present invention has taked following technical scheme:
The gene site-directed modification system of goat MSTN, described pointed decoration system is the transcriptional activation sample effector nuclease TALENs of a pair of goat MSTN gene target sequence, can effectively shear goat MSTN gene target sequence;
Described goat MSTN gene target sequence nucleotide sequence as shown in SEQ ID NO.1,23-40 position Nucleotide is identification module TALMEX-1F, and the reverse complementary sequence of 57-74 position Nucleotide is identification module TALMEX-1R, and 41-56 position Nucleotide is intervening sequence;
Described transcriptional activation sample effector nuclease TALENs is comprised of with the nuclease TALEN-R of the described identification module TALMEX-1R of identification the nuclease TALEN-L that identifies described identification module TALMEX-1F;
Described nuclease TALEN-L, is characterized in that, by TALMEX-1F recognition structure territory TALE-L, is merged and forms with the engineered DNA scinderin Fok-L of process; Described nuclease TALEN-R, is characterized in that, by TALMEX-1R recognition structure territory TALE-R, is merged and forms with the engineered DNA scinderin Fok-R of process;
Described recognition structure territory TALE-L is the protein of 229-2022 position nucleotide coding shown in SEQ ID NO.2 or SEQ ID NO.3; Described DNA scinderin Fok-L is the protein of 2023-2814 position nucleotide coding shown in SEQ ID NO.2 or SEQ ID NO.3; Described recognition structure territory TALE-R is the protein of 229-2022 position nucleotide coding shown in SEQ ID NO.4 or SEQ ID NO.5; Described DNA scinderin Fok-R is the protein of 2023-2808 position nucleotide coding shown in SEQ ID NO.4 or SEQ ID NO.5.
The present invention also provides the above-mentioned goat MSTN preparation method of gene site-directed modification system, comprises the following steps:
(1) according to nucleotides sequence shown in goat MSTN gene Selection SEQ ID NO.1, classify target sequence as, shown in SEQ ID NO.1, in nucleotide sequence, 23-40 position Nucleotide is identification module TALMEX-1F, with the sequence of 57-74 position Nucleotide reverse complemental be identification module TALMEX-1R, 41-56 position Nucleotide is intervening sequence;
(2) according to step (1) gained identification module TALMEX-1F and the synthetic transcriptional activation sample effector nuclease TALENs that shears described target sequence of identification module TALMEX-1R; Described transcriptional activation sample effector nuclease TALENs is comprised of with the nuclease TALEN-R of the described identification module TALMEX-1R of identification the nuclease TALEN-L that identifies described identification module TALMEX-1F;
The nucleotide coding sequence of described nuclease TALEN-L is as shown in SEQ ID NO.2 or SEQ ID NO.3, and the nucleotide coding sequence of described nuclease TALEN-R is as shown in SEQ ID NO.4 or SEQ ID NO.5;
The invention also discloses and contain gene site-directed recombinant expression vector, recombinant bacterium or the recombinant cell lines that knocks out system coding gene of above-mentioned goat MSTN;
Disclose that described goat MSTN is gene site-directed knocks out the application that system is modified goal gene nucleotide sequence in target cell.Described MSTN is gene site-directed knocks out the application of system in other mammalian cells MSTN genetic modification.
Fixed point knocks out a method for goat MSTN gene, is included in the step that the above-mentioned fixed point of goat cells knocks out system.
The mRNA that can express described TALENs is further provided, has it is characterized in that, described mRNA sequence is classified as and is transcribed template with the nucleotides sequence shown in TALEN-L and TALEN-R.And described mRNA deletes the application of MSTN gene in goat cell or zygote.
Compared with prior art, beneficial effect of the present invention is:
1, have been found that the natural sudden change of MSTN gene appears in the species such as ox, sheep and people, and can cause double-muscling shape, but be limited to technical factor, application homology target practice technology or ZFN method are difficult to realize the efficient sudden change of goat MSTN gene.Therefore, the present invention carries out TALENs by TALEN technology to goat MSTN gene and edits, and obtains the clone that MSTN gene target sequence is undergone mutation, for further verifying that the regulatory mechanism of MSTN gene pairs goat muscle growth provides feasible materials and methods.
2, the clone that the MSTN gene target sequence that the present invention obtains is undergone mutation, can prepare as body-cell neucleus transplanting the nuclear donor cell of MSTN transgenation goat, for improving goat meat and improving goat dressing percentage, provide new technological approaches, for MSTN gene being knocked out or transform to obtain the new variety of premium muscle rate and the Regulation Mechanism of research MSTN gene pairs goat muscle growth on goat, there is major application and be worth.
3, TALENs structure of the present invention is the recombinant nucleic acid enzyme for goat MSTN gene particular target sequence, in special site, cuts off gene DNA, causes non-homologous end joining reparation, and then produces MSTN gene Knock-out sudden change.He has overcome conventional ZFN method can not identify arbitrary target gene order, and miss rate is high, and recognition sequence is often subject to the problems such as upstream and downstream sequence affects; Also overcome homologous recombination workload large, mutation rate is low, need to introduce the problems such as foreign gene or marker gene; And there is ZFN, equate or higher activity, do not introducing in foreign gene or marker gene situation, make MSTN site-directed point mutation become more simple, convenient, and use this type of mutant cell in subsequent applications, can obtain the gene mutant animals strain that biological safety is higher.
Accompanying drawing explanation
Fig. 1 is the action principle schematic diagram of TALENs pointed decoration goat MSTN gene;
Fig. 2 is pTALEN-L and pTALEN-R plasmid schematic diagram;
Fig. 3 is cell sample amplified production electrophorogram;
Fig. 4 is cell sample amplified production AluI restriction enzyme digestion and electrophoresis figure;
Fig. 5 is the active order-checking peak figure that detects of TALENs;
Fig. 6 is mutant clone L4 order-checking peak figure
Fig. 7 is L4, L6, L7, L10, L11, L13 sample TA cloning and sequencing result; Note: WT represents wild type gene sequence; △ represents to occur deletion mutant; Fragmesshift represents to occur Substitution;
Fig. 8 is L9, L16 sample order-checking peak value figure; Note: peak value is single without cover peak, shows to occur the sudden change situation that diallele occurs in full accord;
Embodiment
Below in conjunction with accompanying drawing and concrete case study on implementation, the present invention is described in further detail, but not as a limitation of the invention, specific operation process is carried out according to step below:
The first step, the gene order knocking out according to need are designed has specific sequence TALEN target spot;
The TALEN target spot that second step, basis are designed constructs TALEN plasmid;
The 3rd step, by TALEN plasmid transfection recipient cell;
The 4th step, to turning cell then, screen, cultivation, picking mono-clonal, mono-clonal enlarged culturing, identifies monoclonal cell genome mutation situation, picks out and meets the positive cell clone that sudden change requires, and in order to subsequent experimental, uses.
The experimental technique using in following example is ordinary method if no special instructions.
In following example, material used, reagent etc., if no special instructions, all can buy acquisition from commercial channels, and available class reagent or test kit substitute.
Example Goat Fetus inoblast MSTN genetic modification
MSTN gene is relevant with muscle growth regulation and control.The present invention's application TALENs technology, carries out codon mutation operation for the AGCT site of goat MSTN gene exon1, thereby realizes the modification of MSTN gene.
1, this example material requested and reagent
FastTALETM TALEN Assembly kit test kit is by Sidansai Biotechnology CO., and LTD (http://www.sidansai.com/) provides.This test kit is by 8 skeleton carriers, the compositions such as 172 RVD monomer identification modules and 5 kinds of module attended operation liquid.
Competent cell is purchased from life emerging biotechnology (Nanjing) company limited, Cla I, AluI restriction endonuclease, DNA Marker etc. are purchased from the raw biotechnology (Dalian) of treasured company limited, order-checking and primer are synthetic to be completed by Hua Da gene (Beijing) Science and Technology Ltd., other biochemical reagents are purchased from life work biotechnology (Shanghai) limited-liability company, Multiporator, Hypotonic ElectroporationBuffer and electroporation cuvettes are purchased from Eppendorf company, and Goat Fetus inoblast is taken from conceived 35～40 days Sa energy milk goats.
PL15, pR11 skeleton carrier all derive from FastTALETM TALEN Assembly kit.
2, the selection of goat MSTN gene specific TALENs target spot
This example for goat MSTN gene exon1 sequence, adopt TAL effector Nucleotide Targer2.0 (https: //tale-nt.cac.cornell.edu/node/add/talen) design identification module, 5' end retains T base.Choosing TALMEX-1F and TALMEX-1R is that identification module is modified the AGCT site of goat MSTN gene exon1 sequence.
TALMEX-1F?CCTCAGTAAACTTCGCCT
TALMEX-1R?TATAGCATCTTTGCTGAT
The RVD sequence that identification module TALMEX-1F is corresponding:
HD-HD-NG-HD-NI-NN-NG-NI-NI-NI-HD-NG-NG-HD-NN-HD-HD-NG or
HD-HD-NG-HD-NI-NH-NG-NI-NI-NI-HD-NG-NG-HD-NH-HD-HD-NG
The RVD sequence that identification module TALMEX-1F is corresponding:
NG-NI-NG-NI-NN-HD-NI-NG-HD-NG-NG-NG-NN-HD-NG-NN-NI-NG or
NG-NI-NG-NI-NH-HD-NI-NG-HD-NG-NG-NG-NH-HD-NG-NH-NI-NG
Wherein HD identifies C, and NI identifies A, and NG identifies T, NN or NH identification G.
The nucleotide sequence of this example code TALEN-L cog region protein is as shown in SEQ ID NO.2 or SEQ ID NO.3 sequence 229-2022 position Nucleotide, the nucleotide sequence of coding TALEN-R cog region protein is as shown in SEQ ID NO.4 or SEQ IDNO.4 sequence 229-2022 position Nucleotide, and (TALEN-L identifies CCTCAGTAAACTTCGCCT to 18 Nucleotide in identification MSTN gene that respectively can be special; TALEN-R identifies TATAGCATCTTTGCTGAT), by TALEN-L and TALEN-R, jointly form TALENs abruptly-changing system, its action principle is as shown in Figure 1.
3, the structure of TALENs expression vector
With reference to FastTALETM TALEN Assembly kit operation instruction, according to TALMEX-1F and TALMEX-1R identification module, select corresponding link block, TALMEX-1F selects 9 identification form modules (CC1-T2-CA3-CT4-AA5-AC6-TT7-CG8-CC9) to be connected with pL15 skeleton carrier, builds pTALEN-L plasmid; TALMEX-1R selects 9 identification form modules (TA1-T2-AG3-CA4-TC5-TT6-TG7-CT8-GA9) to be connected with pR11 skeleton carrier, builds pTALEN-R plasmid.
The transformed competence colibacillus cell row filter of going forward side by side, ClaI enzyme is cut and is identified and order-checking obtains and connects correct pTALEN-L and pTALEN-R plasmid, and pTALEN-L and pTALEN-R schematic diagram are as shown in Figure 2.
4, the acquisition of the active detection of TALENs and mono-clonal mutant clone
PTALEN-L and pTALEN-R plasmid amplification, extraction and purification carry out with reference to the method for < < molecular cloning experiment guide > > second edition.
Operation method is got 35 age in days Sa energy milk goat fetuses, in Bechtop, with D-Hank ' s liquid, wash three times, with eye scissors, cut off fetal head, four limbs and internal organ, residue tissue is cut into the little block organization of 1mm3 with scissors, move in 10ml centrifuge tube and add 0.25%Trypsin Digestive system to hold piping and druming digestion 15min, after standing 2min, draw the larger tissue block in bottom to new centrifuge tube, repeat digestion operation, supernatant 1500r/min, 5min is centrifugal, supernatant discarded, adds fresh DMEM nutrient solution (containing 10%FBS+1%PS) re-suspended cell, is diluted to 3 * 10
5individual/ml, bed board is cultivated (37 ℃, 5%CO
2, saturated humidity CO
2).Growth of Cells during to degree of converging 80%, is processed with 0.25% tryptic digestion, and it is standby that washing obtains Goat Fetus inoblast after collecting.
In the cell of collecting, add 1ml Electroporation Buffer suspension cell, the centrifugal 5min of 1500r/min, abandon supernatant liquor, cell is resuspended in the transfection liquid (Hypotonic Electroporation Buffer contains respectively pTALEN-L and the pTALEN-R plasmid of 20 μ g/ml) of 400 μ L, making cell density is 1～3 * 10
6individual/ml, the cell suspension of 400 μ l is transferred in electroporation cuvettes (the wide 2mm in footpath), puts into Multiporator electrotransfection groove and carry out electrotransfection (transfection pattern (Mode) is: Eukaryotes " ⊙ ", Voltage (V) 400V, Time constant (τ) 300us, No.of pulse (n) 1 time).After electricimpulse, make cell suspension standing 5min under room temperature condition in cuvette (transfection cup); Cell suspension is added to normal cell nutrient solution, be laid on 6 orifice plates and be placed on 37 ℃, 5%CO
2, saturated humidity cultivates.
After bed board 24h, change screening and culturing liquid (DMEM+10%FBS+1%PS+3 μ g/ml Puro), after screening 72h, being replaced by normal cell nutrient solution continues to cultivate, digestion is collected 3 porocytes and is carried out activity detection, be numbered L1～L3, all the other cells continue enlarged culturing 5～10 days, when 96 of picking cell clones to 48 orifice plates continue to be cultured to degree of converging 70%, collect the detection that suddenlys change of part cell, be numbered L4～L45, collect part normal cell as a control group simultaneously, be numbered W1～W3.
By unicellular PCR method, carrying out activity detects and suddenlys change and detect.The cell sample of collecting is numbered: L1～L3 is for screening the cell of collecting after 72h; L4～L45 is monoclonal cell strain; W1～W3 is non-transfected cells.Cell sample is placed in 200 μ l centrifuge tubes, and centrifugal removal nutrient solution, adds 10 μ L cell pyrolysis liquids (in Table 1), hatches 15min for 45 ℃, hatches the DNA profiling directly detecting as regular-PCR after 20min for 96 ℃, and-20 ℃ save backup.
Table 1 cell pyrolysis liquid
| Moiety | Volume (μ l) |
| 10×PCR?buffer | 1 |
| 1％tween20 | 1 |
| 0.1％triton | 1 |
| Protin?K(25mg/ml) | 0.2 |
| Distilled water | 6.8 |
Get 2 μ l split products and carry out PCR reaction as template, PCR primer designs in goat MSTN genome, and it is good that goat cellular genome is extracted quality, all can amplify object band, and primer sequence is in Table 2, and reaction system is in Table 3.
Table 2PCR primer
Table 3PCR reaction system
| Moiety | Volume (μ l) | Final concentration |
| 10×PCR?buffer | 4.8 | 1× |
| dNTP(10Mm) | 2 | 200μM |
| TAL-2se(10μM) | 0.3 | 0.1μM |
| TAL-2an(10μM) | 0.3 | 0.1μM |
| Taq enzyme (5U/ μ l) | 0.3 | 2.5U/reaction |
| Template | 2 | ? |
| ddH 2O | 40.3 | ? |
PCR reaction conditions is: 95 ℃ of 5min; 95 ℃ of 50sec, 52 ℃ of 30sec, 72 ℃ of 45sec, totally 35 circulations; 72 ℃ of 10min; 4 ℃ of preservations.Get 5 μ l PCR products and carry out agarose gel electrophoresis detection, as shown in Figure 3, L1, L4～L17, L19, W1 cell sample all amplify 439bp specificity object band to partial detection.Get 10 μ l L1, L4～L17, W1 sample P CR product carries out enzyme with AluI restriction enzyme and cuts evaluation, electrophoresis detection enzyme is cut situation, detected result as shown in Figure 4, result shows: L1 sample P CR product only can be partly cut across, illustrate that PCR product is mixed with multiple fragment, there is the sudden change of AGCT restriction enzyme site in Partial Fragment, can not be cut open; L4, L5, L6, L8, L11, L12, L14 sample P CR product only can be partly cut across, and illustrate that PCR product is for mixing fragment, obtain cell clone be monoallelic sudden change; L7, L9, L10, L13, L16 sample P CR product can not be cut open, and illustrate that the diallele that sports existing in obtained cell suddenlys change; L15, L17, W1 sample P CR product can be cut completely, illustrate that L15, L17, W1 cell sample do not undergo mutation.
By L1, L4, L6, L11, L7, L9, L10, L13, L16 cellular genome amplified production, and after amplified production connection T carrier, send the order-checking of Hua Da gene (Beijing) Science and Technology Ltd., sequencing primer is CX-se:TTGGCTTGGCGTTACTCA.
Sequencing result shows:
(1) L1 cellular genome amplified production sequencing result as shown in Figure 5, in goat MSTN gene exon1 sequence A GCT site, there is obviously cover peak, explanation has multiple sequence signal to occur in this site, exist special sequence to change, prove that the TALENs of this example design has MSTN transgenation activity;
(2) L4, L6, L7, L9, L10, L11, L13, L16 sample sequencing result show: L4, L6, L7, L9, L10, L11, L13 amplified production direct Sequencing occur base deletion and replacement near being presented at MSTN gene exon1 sequence A GCT site, occur overlapping peaks (peak figure is shown in Fig. 6 as the order-checking of L4 sample) simultaneously; Amplified production connects the demonstration of TA cloning and sequencing result L4, L6, L11 sample monosome gene is undergone mutation, and L7, L10, L13 diplochromosome allelotrope are all undergone mutation, and the sudden change situation that every sample contains as shown in Figure 7; The sudden change situation of L9, L16 sample allelotrope target site is in full accord, is homozygous mutation, and sudden change situation as shown in Figure 8.
(3) when detected result explanation is used TALENs of the present invention to process Goat Fetus inoblast, can efficiently fix a point to goat myostatin (Myostatin, MSTN) gene is modified, thereby realizes the transformation of MSTN gene coded sequence or knock out.
Above example is only exemplary embodiment of the present invention, is not used in restriction the present invention, and protection scope of the present invention is defined by the claims.Those skilled in the art can make various modifications or be equal to replacement the present invention in essence of the present invention and protection domain, this modification or be equal to replacement and also should be considered as dropping in protection scope of the present invention.
Claims (7)
1. the gene site-directed modification system of goat MSTN, is characterized in that efficient gene pointed decoration system is for shearing the transcriptional activation sample effector nuclease TALENs of goat MSTN gene target sequence;
Described goat MSTN gene target sequence nucleotide sequence as shown in SEQ ID NO.1,23-40 position Nucleotide is identification module TALMEX-1F, with the complementary sequence of 57-74 position Nucleotide be identification module TALMEX-1R, 41-56 position Nucleotide is intervening sequence;
Described transcriptional activation sample effector nuclease TALENs is comprised of with the nuclease TALEN-R of the described identification module TALMEX-1R of identification the nuclease TALEN-L that identifies described identification module TALMEX-1F;
Described nuclease TALEN-L, is merged and forms with the engineered DNA scinderin Fok-L of process by TALMEX-1F recognition structure territory TALE-L; Described nuclease TALEN-R, is merged and forms with the engineered DNA scinderin Fok-R of process by TALMEX-1R recognition structure territory TALE-R;
Described recognition structure territory TALE-L is the protein of 229-2022 position nucleotide coding shown in SEQ ID NO.2 or SEQ ID NO.3; Described DNA scinderin Fok-L is the protein of 2023-2814 position nucleotide coding shown in SEQ ID NO.2 or SEQ ID NO.3; Described recognition structure territory TALE-R is the protein of 229-2022 position nucleotide coding shown in SEQ ID NO.4 or SEQ ID NO.5; Described DNA scinderin Fok-R is the protein of 2023-2808 position nucleotide coding shown in SEQ ID NO.4 or SEQ ID NO.5.
2. the preparation method of the gene site-directed modification system of goat MSTN described in claim 1, is characterized in that, comprises the following steps:
(1) according to nucleotides sequence shown in goat MSTN gene Selection SEQ ID NO.1, classify target sequence as, the Nucleotide of nucleotide sequence 23-40 position shown in SEQ ID NO.1 is identification module TALMEX-1F, is that identification module TALMEX-1R, 41-56 position Nucleotide are intervening sequence, 47-50 position Nucleotide is for shearing target spot and can be used as AluI digestion with restriction enzyme sudden change detection site with the sequence of 57-74 position Nucleotide complementation;
(2) according to step (1) gained identification module TALMEX-1F and the synthetic transcriptional activation sample effector nuclease TALENs that shears described target sequence of identification module TALMEX-1R; Described transcriptional activation sample effector nuclease TALENs is comprised of with the nuclease TALEN-R of the described identification module TALMEX-1R of identification the nuclease TALEN-L that identifies described identification module TALMEX-1F;
Described nuclease TALEN-L is the protein of nucleotide coding shown in SEQ ID NO.2 or SEQ ID NO.3, described nuclease TALEN-R be shown in SEQ ID NO.4 or SEQ ID NO.5 shown in the protein of nucleotide coding.
3. the recombinant expression vector, recombinant bacterium or the recombinant cell lines that contain the gene site-directed modification system of goat MSTN described in claim 1.
4. the application of the gene site-directed modification system of goat MSTN in the identification of target cell goal gene, modification described in claim 1.
5. described in 1 or 3, carrier or nucleotide sequence are synthetic or transcribe the mRNA that can express TALENs described in claim 1 as requested, it is characterized in that, described mRNA sequence is classified as and transcribed template with the nucleotides sequence shown in TALEN-L or TALEN-R.
6. the application of mRNA in goat cell MSTN genetic modification according to claim 5.
7. the application of the gene site-directed modification system of MSTN in other mammalian cells MSTN genetic modification according to claim 1.
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