CN1647624A - Method for in situ modification and breeding of fish genes - Google Patents

Abstract

The present invention aims at providing one homologous recombination method for in-situ fish gene modification breeding. The method includes the following steps: 1) separating specific fish gene flanking sequence; promoter regulating sequence DNA segments to constitute homologous recombination vector with actin gene promoter regulating sequence to replace growth hormone gene promoter regulating sequence; 2) microscopic injecting the homologous recombination vector to zebra fish embryo to screen positive homologous recombination replaced fish fry; and 3) analyzing relative gene expression level and genetic phynotype of the endogenous gene modified fish. The present invention is used in breeding fish variety.

Description

Method for in situ modification and breeding of fish genes

Technical field

The invention relates to a method for genetic engineering breeding, in particular to a method for in situ modification and breeding of fish genes.

Background technique

The process of genetic breeding is a process of genome manipulation. Genetic engineering breeding is based on the understanding of gene function and regulatory mechanism, and the genome is modified and transformed at the molecular level, so as to obtain directional genetically improved genetic varieties and get rid of the barriers of genetic fortresses between species. shackles, from passive use of genetic resources to active transformation and innovation. In 1982, Palmiter et al. transferred the rat growth hormone gene to the fertilized mouse eggs and bred fast-growing "super mice". This breakthrough achievement ushered in a new era of transgenic animal research. In 1985, the Institute of Hydrobiology of the Chinese Academy of Sciences took the lead in carrying out research on directed breeding of fish genetic engineering, established a complete theoretical model of transgenic fish and a complete experimental technology system, revealed the dynamic process of exogenous gene integration, proposed The model theory for the formation of transgenic fish and the countermeasures for cultivating homozygous transgenic fish strains have been established, which has laid a theoretical foundation for the breeding of transgenic fish. At present, dozens of laboratories around the world have carried out fish gene transfer research, established a variety of gene transfer methods such as microinjection, electrofusion, and sperm carrying methods, and obtained fast-growing transgenic carps, salmon, For rainbow trout, Atlantic salmon and tilapia, etc., large-scale breeding trials are in progress. Although breakthroughs have been made in genetic engineering directional breeding technology, the purpose of genetic engineering breeding has not yet been achieved. Issues such as random integration of exogenous genes, uncontrollable expression of exogenous genes, and biological safety of transgenic fish have seriously affected this gene. The effective application of engineering breeding technology in fish breeding has become a bottleneck restricting the further development and application of transgenic fish research. Therefore, the defect of the prior art is: it adopts the random integration of exogenous genes to complete the genetic engineering breeding.

Contents of invention

The technical problem to be solved by the invention is to overcome the deficiencies of the prior art and provide a method for in situ modification and breeding of fish genes by using homologous recombination.

The technical scheme adopted in the present invention is that the present invention comprises the following steps:

1) Isolate the β-actin gene start-up regulatory sequence, growth hormone gene start-up control sequence and flanking sequences of fish;

2) using the growth hormone gene promoter regulatory sequence in the fish gene as the target DNA fragment for homologous recombination;

3) replacing the growth hormone gene activation regulatory sequence with the β-actin gene activation regulatory sequence by homologous recombination;

4) Construction of homologous recombination vector:

A) The homologous recombination vector includes the 5' flanking sequence of the growth hormone initiation and regulation sequence, the 3' flanking sequence of the growth hormone initiation and regulation sequence, the β-actin gene initiation and regulation sequence, the green fluorescent protein forward marker gene and two homologous Directed Cre/loxP sequence, red fluorescent protein negative marker gene;

B) The 5' flanking sequence of the growth hormone activation regulatory sequence is used as a homologous long arm, and the 3' flanking sequence of the growth hormone activation regulatory sequence is used as a homologous short arm;

C) placing the two homologous Cre/loxP sequences on both sides of the green fluorescent protein forward marker gene, and then placing the green fluorescent protein forward marker gene on the homologous long arm and the inner side of the homologous short arm;

D) placing the red fluorescent protein negative marker gene outside the homologous long arm and the homologous short arm;

E) Digested with a restriction endonuclease to obtain a linear recombinant DNA fragment;

5) After artificial insemination of the fish, before the first cleavage, the homologous recombination vector is introduced into the fertilized eggs by microinjection, and then the fertilized eggs are hatched in a sterile environment at 26°C to 30°C in the laboratory ;

6) Under the fluorescent dissecting microscope, the embryos with only green fluorescence were screened to obtain homologous recombination chimeras;

7) Cells or embryos with homologous recombination confirmed by PCR and Southern hybridization.

The beneficial effects of the present invention are: because the present invention utilizes homologous recombination to carry out in-situ modification of fish genes for breeding, avoiding the transfer of exogenous genes, and will greatly eliminate people's doubts about the safety of transgenic organisms; and due to the present invention By constructing a homologous recombination vector and establishing an efficient gene homologous recombination technology, humans can make fixed-point and quantitative changes to the genetic structure of fish according to the design, thereby directional changing the genetic structure and characteristics of the cell or the whole itself. The present invention implements the in-situ modification of endogenous genes and the controllable expression of endogenous genes, and establishes a technical platform for fish genetic engineering breeding, which is useful for fish genetic engineering breeding, bioreactor development, gene function, expression regulation, and developmental organisms. Scientific research has great theoretical guiding significance and potential application value.

Description of drawings

Fig. 1 is the structure diagram of this fish genome;

Figure 2 is a schematic structural view of the homologous recombination vector of the present invention;

Fig. 3 is the structural representation of the recombinant body after homologous recombination screening of the present invention;

Fig. 4 is a schematic diagram of the construction process of the homologous recombination vector of the present invention.

Detailed ways

Taking zebrafish as an example, in Figure 1, Figure 2, and Figure 3, 1 represents the 5' flanking sequence (as a homologous long arm) of the zebrafish growth hormone gene (GH) regulatory sequence, and 2 represents the GH regulatory sequence ( GH-promoter) fragment, 3 represents the growth hormone gene (GH) promoter regulatory sequence 3' flanking sequence (as a homologous short arm) fragment, 4 represents the β-actin gene (β-actin) promoter regulatory sequence fragment, 5 represents Two Cre/loxP sequence fragments in the same direction, 6 represents the green fluorescent protein (GFP) positive marker gene fragment, 7 represents the red fluorescent protein (RFP) negative marker gene fragment, 8 represents the forward PCR detection primer, 9 represents the reverse For PCR detection primers, 10 represents the non-target DNA sequence fragment of the homologous short arm 3' flanking sequence.

Embodiments of the present invention are described in detail below in conjunction with the accompanying drawings:

1. Obtain the target gene

The promoter regulatory sequence of zebrafish β-actin gene (β-actin), the promoter regulatory sequence of growth hormone gene and their flanking sequences were amplified by PCR.

2. Homologous recombination vector construction

(A) Technical description of homologous recombination:

The invention adopts a double fluorescence screening method of gene homologous recombination, uses double fluorescence to screen cells with homologous recombination in embryos, and does not need the operation of embryonic stem cells and embryo chimeras. The present invention adopts the following technical measures: based on the principles of positive selection and negative selection, a double fluorescence screening homologous recombination vector is constructed, the steps of which are: (1) using the growth hormone promoter regulatory sequence (GH-promoter) in the zebrafish gene as the same (2) the green fluorescent protein (GFP) gene is a gene for positive selection, which is placed inside the homologous DNA fragment; (3) the red fluorescent protein (RFP) gene is a gene for negative selection, which is placed in the same outside of the source DNA fragment. (4) In the homologous recombination vector, the β-actin promoter (β-actin promoter) is connected to the green fluorescent protein (GFP), and then connected to the red fluorescent protein (RFP) through the homology arm. After digestion with restriction endonucleases, a linear recombination DNA fragment was obtained, and the homologous recombination vector was introduced into zebrafish fertilized eggs by microinjection. In cells with non-homologous recombination, green fluorescence and red fluorescence can be observed at the same time; in cells with homologous recombination, because the red fluorescent protein (RFP) gene placed outside the homologous DNA fragment Excised during recombination, only green fluorescence (GFP) can be observed. The embryos with only green fluorescence were screened under a fluorescent dissecting microscope to obtain homologous recombination chimeras.

(B) As shown in Figure 2, the construction process of the homologous recombination vector of the present invention is:

The positive and negative marker genes used in the homologous recombination vector are green fluorescent protein gene (GFP) and red fluorescent protein gene (RFP) respectively. In order to facilitate the connection of GFP and RFP to the plasmid pBluescript IISK(-)(pBSK) multiple cloning restriction site (MCS), first PCR out the strong promoters CMV, GFP/RFP and SV40 from pEGFP and pDSRFP respectively. Signal and its polyA, and then use fusion primers to PCR out the complete GFP/RFP gene fragment (PCMV-GFP/RFP-SV40-polyA) that deletes MCS. At the same time, by PCR, Apa I and Sph I restriction sites were introduced at the 5' end of RFP, and an ApaI restriction site was introduced at its 3' end. Apa I was single-digested and inserted into pBSK, and the clone (pBSK-RFP) in the correct direction (counterclockwise) was called out by sequencing. The introduction of Sph I was for the subcloning of the homologous short arm of growth hormone (GH) in the next step. In order to realize the "whole fish gene", the exogenous GFP gene sequence was removed from the positive fish integrated by site-directed homologous recombination, and Cre/loxp sequences in the same direction were introduced at both ends of the GFP fragment during PCR. A BamH I restriction site was added to the 5′ end of the GFP amplification primer, and an EcoR I restriction site was added to its 3′ end so that it could be cloned into pBSK-RFP by BamH I and EcoR I double restriction restriction (pBSK -GFP-RFP).

The promoter regulatory sequence (2251bp) of β-actin was obtained by PCR from the zebrafish genome, and cloned into pBSK-RFP-GFP (pBSK -GRP). The 3′ flanking sequence (1.7 kb, 2.8 kb) of the GH promoter regulatory sequence and the upstream sequence of the GH promoter regulatory sequence, namely the 5′ flanking sequence (2.5 kb), were PCRed out by genome walking method and directly connected to pGEM-T vector (pGEM -T-GH and pGEM-T-Pgh), the homologous short arm of GH passes through Sal I and Sph I, and its homologous long arm is the upstream sequence (5' flanking sequence) of the start regulatory sequence by using Sac II and BamH I respectively from pGEM -T-GH and pGEM-T-Pgh were excised and connected to pBSK-GRP by double enzyme digestion in sequence, and finally released by single digestion with BssHII using the BssHII enzyme digestion sites on both sides of the MCS on pBSK desired linear DNA fragment.

3. Microinjection of homologous recombination vector into zebrafish fertilized eggs

After artificial insemination of zebrafish, before the first cleavage, the homologous recombination vector was introduced into the fertilized eggs by microinjection, and then the fertilized eggs were hatched in a sterile environment at 26°C to 30°C in the laboratory. The best incubation temperature for fertilized eggs is 28°C

4. Fluorescence observation and homologous recombination positive embryo screening

In cells with non-homologous recombination, green fluorescence and red fluorescence can be observed at the same time; in cells with homologous recombination, because the red fluorescent protein (RFP) gene placed outside the homologous DNA fragment Excised during recombination, only green fluorescence (GFP) can be observed. The embryos with only green fluorescence were screened under a fluorescent dissecting microscope to obtain homologous recombination chimeras.

5. PCR detection of positive homologous recombination cells

The forward detection primer is set on the forward selection gene green fluorescent protein (GFP) gene of the carrier, and the reverse detection primer is set on the non-target DNA sequence of the homologous short arm of the genome. The cells or embryos with homologous recombination undergo PCR and Confirmation by Southern hybridization.

The homologous recombination efficiency of the present invention is 4/1600. The homologous recombination vector is microinjected into 5000 fertilized eggs, and 1600 fish are normally developed to the fry stage, and 4 of them are positive homologous recombination fish. The growth and development speed of the zebrafish with genetic in situ modification is twice as fast as that of the control group, that is, it takes about 3 months for the control group fish to grow to sexual maturity, while the genetically modified fish obtained by the present invention only needs about 1.5 months.

                    Zebrafish β-actin initiation regulatory sequence

1 tgtgcgcaca gattctgtgt gggcctagtc attacctata ctcaacgttt

51 actctgagaa acttccatat tttagaggta aaatgctcag aattgtaaaa

101 agtcttgtca aaatacttgg aatttgccaa aaacaaaata aaagtcacat

151 cacctcattg tccttggtat cagcaagcac ttttaacaca atcagctgtt

201 tttgtttata actcccactg aaggccatgt gcattactcc caagttacac

251 agtaaaaagt cacatgtagt ttatcatgat cactgctgct caagcacagc

301 tttatctgtt attcagtgtg gcaatatctg aaatgtgtgc aaaatattat

351 ttattgtagc tgatcaccaa cactttcagt gttttttttt tctactttta

401 atgagaatta tgttcttagg atttatcctt ataaacctct ttaaaaagaa

451 atgttatgtc tgcatagaca cgacatgaaa ttatgaacag aaaatttaac

501 tatttaagat caagacttga catataacat ttctaatgaa cattaaacct

551 gaggtaggcc cacttgcaaa aatgtaactg ctatcactca ctttatacat

601 cattgtgtaa cttgtacaca caaaaatagt gaagcaatgt ggcatggaat

651 gcaggtccca ttcattcaaa gttattaaag cactttctta aagtgcatta

701 ctactgagct aagaccagct agttgctcat acaacatact acaagtagtg

751 tcatcctaat ttggataaaa aaaatgtcca ttttatacat ggaaaagtac

801 tcattagatc ccccattgtt tgtattacgg tatttcgtga acacaagagg

851 taaatgacct acagagctgc tgttgtgtta gattgaaaac acaacacagg

901 atcatggagg catccatcat tatgacgcat tctgccactt gtaactctgc

951 acagactttt tgaaaagttt aattgagtct tggcgttgtc gcagaaaaat

1001 acgaaaaatt ctgtgttaaa atttgcaaat aaaaattcca gttttatagg

1051 aaatcgccat ggccttgttc tttaaccagt taagcttccc cttctttcac

1101 tctcaagttg caagaagcaa gtgtagcaat gtgcacgcga cagccgggtg

1151 tgtgacgctg gaccaatcag agcacagagc tccgaaagtt taccttttat

1201 ggctagagcc gggtatgtgc cgtcatataa aagagcgcgc ccagcttttc

1251 agcctcactt tgagctcctc cacacgcagc tagtgcggaa tatcatctgc

1301 ttgtaaccca ttctcttaag tcgacaaccc cccaaaccca aggtgagttg

1351 gttttacagt ttttaagcgt ttaattatag tttctatcta ttaatgttaa

1401 gtaaaatgtg taaatggata aatatgattt gatgaagtct ttagacagtt

1451 cataacggtc tctttgtagt tatctactag gtttgatta taagtggtgg

1501 ttcttttgat aatctatgtt acatttaata aatattggct gtatcttaac

1551 ttttttgtag cgttagtgga tgtggcaggt gagaatgtcg gtaatttaac

1601 gtgaccagtc gtaggcacga cattgaacat tgaataggcc ggtgtgaaat

1651 gggtgttaag tcttaatata aagttgtgtt ctggctaatc tctctttaac

1701 agccataaat gtgattttta actacagttt tgagggtgct ggtgagcatg

1751 ttgcacactt gatggatagc cggcatggga agttctttgt gcaggcagtg

1801 ctgcagcagg gtgtgaccta ctttagctag ccggctaacc agctctcatc

1851 tgatgtaacg taaaccccat caatctaaca tcggctcatt cgtgctttat

1901 aagatcgaag atcttgttat tagtaacggg tattggcatt tagttttaac

1951 ttggttaatt ttctttttgt tatgtgttct atttaaatgt agcctgcagc

2001 cccctctgtc tttaatgaag ctgaagatta acgttaacgt tagtgaagtg

2051 ttgtatccgc tatgaaagat ttgaatttta gaggtgtggt ccactttaat

2101 aacggctgcg gagttctctg gctgcacttc agccgctgtg actgtcgaag

2151 cagagcccat cagtttttca ggatctcggt gatttttgag gctagactat

2201 gaactgaacc gactgagagt gaccgtccag aaatgcttta gtgtgaagtc

2251 tccgctcacg aggagtccgt ggactgactg

  Zebrafish growth hormone gene sequence, initiation regulatory sequence and its 5′ flanking sequence

1 ccaggggggag ctctcgagac cttcctttac atagttatg accaggcggg agccctgggc

61 tctgttatct ccgagctcag ggttcttccc aggacagcat gtcaaacctg ctatagcgt

121 caagcatatt taagtgtcaa ctcttgaagc taaactgtaa aaacagtgta acatccataa

181 ttttacagtt tatttccggc agctagggtg ctgtaaattt accataaaat aacagacatt

241 aaattacaga aatttcctta aattgtaatt tcccccaaaa ttcaacctgt gttatttcac

301 agtaaatttc tgtattttgt ggccgttctt tcttttatgc cgtagctttc ttttacggca

361 tcccagctgg cagaaataag ctgtaaaatt actaattgtt ttttttacag tgtacggtta

421 gtgttgcaaa tcatggttga tttgtggttg ttcagttgtc tgtgaaaatg taaagctttt

481 cgtataggga cttcacagat gaagaccttt accctctaca gtggaatgag cgaacacaga

541 aacatatttc ttcaaagaaa actgtaagtt gtattttgtt acgttttcat ttatctctgat

601 ttctttataa gttgtgtgct tttcctgact gagtacttgc atcattccct caccttcgtt

661 tagacaaatg tgctgcaaat aaaaaaaaaa acactaacaa attaaacaca tcatcagtat

721 taaactctca caacacatgc aaacacagaa atgcattgca aatagcgcag aacccagaca

781 tccagaacat tcaggagaac ttgggaaatc cataatggct cctgtaaatt ggacaaaatg

841 tcccggaaat ggagtcgctt tcccaagcag gttgtgtttt tccatattta ttcttgcagt

901 gctcttgcgc tctctcagtc atcatataca agttattctt cttggaccac taaagagaat

961 tatagataat tccaaataat ggcagactct tttaacaatt acttaaaagt ataataggat

1021 tgttccatac atgtgcagaa tacttccact gtaattcatg tgttgtaaaa taaagttcag

1081 cgatgaacca accaagacat tatttattat tcaaaaaaaa atctgacatt gatggttcat

1141 gagactttta gcatggcatt atcagttttg agtgaataga ctctttaata cgtagactat

1201 tttaggctat atctaagtat atttttgtta aatatagctt ggctatttgt tttacacagt

1261 aaatatctgt attataattt ctaacctgca gtttgtcact ttctatttta tgtcctgtcc

1321 acatagaaaa cacacgccac cctgtggtca aaatagatta gattcatagc ggtgttctgg

1381 atctagactg tagacagcag tgttttactg tgatagtaca cacacagcag tacactgttg

1441 gggataaggg agaagtctta tagcttagta aaaaaaaata ttttaggatg aaacgtggct

1501 tctgtaagac attagtgact gtttctcacg ttctctttct ctccctcagg catacacaca

1561 caaggtcaca ttgccctcgc aggacgtagc ctagaagtgc tccggcatcc tctggatcac

1621 agccgccgga gttccatgca tacaaatgag gtcaggggtc agcagtgaag cgtgacctgc

1681 aggacagaca gacggtcagt gtgttcaccg cttctcctac accctgatga gtccaagccg

1741 actggtgtta atgtactcat tcattctcac tgactgaccc ggttagggcc gctataatcg

1801 aatattcagg caatcgtttt tacatgttgg gaacaaatta cagcaagtat aagtttgtta

1861 gaattagttt aacatttgct gaatacttta aaagcattta ggaatcttgc tttgtgttaa

1921 cgtaaatcct taccctgaaa tcagaggtcg tatatttgta atgttaatta atggacttaa

1981 atgtactatt tctttttctc agtagaaatg cttggaagtg cataccaaat gtacgtcaaa

2041 tgtgacctat ttaactgtct tattttgtta ttacattagc tattgacaaa atcgtaatgt

2101 ttttcttttt tctttttgga gattgttagt agaccagaat tagttggacc aattgaacaa

2161 gttcacatat acttgctaag ttaccaaatg catcctgtgg gaccatcaaa atgtttacag

2221 tttacagttt tagagtaaat gatattaaac tgtaaaataa tgctctaaag accaagttac

2281 ttggggtcaa attaaattat acctgcgttt attatttctg atttgggttt tatttttagg

2341 actcgtctat acttgatttt aaagaattgc ctgacacaca acatcaatat gtcaggaaca

2401 tttgtcccaa ccatcacaga tttggtgtac attaggtaac ttctctgaac aaacatgtag

2461 gaggtgtttt attaactgta atgaccagta ctgtacacta tgcttagcgc tgtcaggacg

2521 gcacacttga ccggcgcaac attttgtttg cttcctttgc tggctggaag ggaatgtgaa

2581 cggcctctcg ggggattcgg ccttcatgca agctaaggaa gtaaaggaga agctcgtccc

2641 atcaacgact ggagcctcca aaagcaaaga agccgcagct gcccagagag atgcaacatg

2701 agaccagtgg aggcctgtga tgagcagcct gtggaattgg agagcgggtg cgatggagtc

2761 agtgcgttcc agggaagatg tgtaacattg cctgctttga aactggagtt gacgagaagt

2821 gggaacaagg tggagtttgc tttaaaggaa gagggttact tgtgcagctc ataaagatcc

2881 cggaaccatt taagggcaac tttatggctg agagtgcagt caatggactg tttcctcttg

2941 gtgttattat ttgtatattt tcagacatca ttaagaccct cggttcatca cactactgta

3001 tgttacttta cgggagctct ctgagcatca cacaagttgt gctattcaaa gaacgaatcc

3061 atgtaaaaat ggatggttca cctcaaaatt gaaaatccat aataatttac tcacaattta

3121 cttgtttcaa acctttatct ttgagtcgtt ttcctctgtt aaatatgaaa taaggtatta

3181 tgaagaagtc tggaaacctg tatccactgc ttaatattat ggaagtcagt gtctacacat

3241 tttcagcttt caaaaaatta tgtgttcaac agaggaaaga aactactaaa ggtttaaaac

3301 catttgaggg tgggttgtga gtaaattgta atttttgggt gcatatttag atatttccct

3361 ttaatgctct ttactaatga gcaaaagaag agaatataga ttacagctat tatcatctgt

3421 ttaatgaagc aaagaatgaa taccttattt tggtgcgttt ttccacaaat gtcgcttaac

3481 aatcatgcgt ttggtcagaa tgtggagaat agttttacaa ctcactatct acacaacaac

3541 tttaccagtg aacattttac tccacatgtg tggaatttaa cacttttctg ttgtatattt

3601 gagtcccacc cgccaagtgc ccaggttaca ctttcataag tgttgaatat atacacctct

3661 gcagtgcaat atatatttca cttaagagtg taaaaatgca caccaaattt ttaacatctg

3721 ctgggcaaat tgtccagtgt atatacagtt gcatttagaa ttattagccc ccctttgatt

3781 cttttttctt tatttaatat tttccaaatt atgtttaaca gtgcaaggaa atttttacag

3841 tatgtctgat aatatatttt cttctggcta cagtcttatt tgttttattt cagttagaat

3901 aaaagcagtt tttaattttt taaacaccat ttaaggtca aatttattag ctcctttaag

3961 ctatattgtt ttttaatagt ctacagaaca catcgttgtt atacaataac attcctaatt

4021 accctaacct gcctagttaa cttaattaac ccagttaagc ctttaaatgt cactttaggt

4081 tatatagaag tgtctttaaa aatatctagt aaaatttgat ttactgtcat catggcaaag

4141 atcaaataaa tcagttcaaa gaaacgagtt gttaaaactg ttatgtttga aaatgtgttg

4201 aaaaaaaaat cttctctcca ttaaacagaa attgatgaaa aaaataaaca gggggctaat

4261 aattcaggag gtttaataat tctgattgca actatataac cccagagaag tcccaccagc

4321 ccagtgtgca agtgacactt tcgaaagtgt ttaatatata cacttaagca gagtaaataa

4381 ttttacacta agagtgcaaa aactgtatac tagattaaat gctttatcac cactgccagg

4441 tcatatttag cgttttcttt aaacagacat ggataaaagt taacattttc tccacataaa

4501 tttcacaatg gaaagtgtat ctggatattt taacactctg gaatgataca attaataaca

4561 ttataaataa attgacaata aaatgtcaac taactctttt ctgttacctg gctaaaatga

4621 gtgtttaata gtttaattta atccagatca tcgttaataa tagtatatac acttggaatt

4681 taacactata actattacaga ggtttacact tgaaattcaa cacctcaaaa tttgctgtgt

4741 acccttctat gtatttgaaa aaaaaaaaaa tgaaaaattg tggcccaaat tatgtttaaa

4801 tagtattcca ctaaagtttc ctggatggtt tgctatttcc ttcaaattca ctttttcttt

4861 ccccaaaaag ttcatacttt tacgactcag cacataagca tgtccacttg tgattgtatt

4921 gatgtcaaag gtcatcgttc actgtgtact taaacaggtg gaatcaggta ctgcctcttt

4981 aagaatgcta agcattctta atttattatt agagtttaag accctgactt actaacgaag

5041 ccaaaggtaa aaataaaacg agaaccagtg tgttaagaga gaggatgata ttcagtgaga

5101 tgcggaaggg gtaaacttgt ttaaatgttt ccggtatgta aagccgcctg catagatatg

5161 agatcagagc agctttgcac tgaaccacct taacatgaga tgacaacatt attagtagag

5221 ttaatggtgt gataaaggta gatgtaagca tatactgcag aactttaata cttttatta

5281 catttcaagt tgctaaaatg gtaaaatcca gaaattttca gtacgacaat tagctacttc

5341 ctgtaaatgg acgagacaat attttattag tcgcaaactg aatgtttttg agcatctaaa

5401 gtaaccaaag gcggcgttaa aatgcgcaac tctccccaag ttcatatgat gtagctattt

5461 cagagattta aggtaataga tgttgaattt attggtgccc tctggtgttt agaaatgtaa

5521 tgactagcaa agttattagc ttgttctcat aattcatgct cttactgttg gtttttgacg

5581 gaaccagctg acagggcttc ttcagggtgt attgactgac ggtacagtca tcagaaatgg

5641 caaatatgaa cagctgaagc atcttgaggg cttttctctt taacctgcat cattctgact

5701 gcaaccacat ggtggaaata taactcacaa tttctcaatg ctttcacctt ctcctgttcg

5761 ctctctctgt atgtgttttc ctggcaatta gatctgagac cagattctgc aaaaaaaata

5821 aataaataaa taaataaata aataaataaa taaataaata aacgtcagac tggtttttgg

5881 taagataaat gtggctaaaa gaatgatgca gtgtttgtga atctggacca caataggagt

5941 catagtgtca gtttacatga tgaaataatc tttcaactga tgtgtggttt gtatattatt

6001 caaagactac tgttttttga cgacactact gtttgatgaa ctagtatgtc aaaacaataa

6061 taaaatatat aaagtatata tatatatata tatataaaaa tatatatata tataaagaga

6121 gagagagaga gagagagaaa gaaagagagc catttattct ctatacagag aattaaatca

6181 tttacattta gagtagattt ggggtctctg tcaagttcat gtgtgaaaga gaagtgttac

6241 acacatgttg tctgctatat ggaatgccaa aactttgaag ctcattatcc caaaatcatt

6301 aagaacgcag atagaacctt ataattccaa ggggacataa tgctgtacaa gtattgttca

6361 ttctcatgtt agtaaataaa tcaactaata gacttattgt aaagtgtgac caattattca

6421 ttatctacat tataagcaaa atatatattg ttatgtagta aatatggtac cattcaccat

6481 gttctgtact ggtagtcttg ttgtaatgtt ttatctattt catttattaa cttcaactgc

6541 agaacacggt gctactctta taatgccata acctgaagtg acagcagctc taagattcag

6601 gtaaacagac tgttctctgt ttttccgtga aagggattaa actattcatg tcacactgac

6661 atttcacaac cccaagactg ctgaatatta aacagcactg aataggaaaa tcccctcaca

6721 tcccacagtg acaacaaaca cgtatgacct gatatcctct cataattatt gaaaacgttt

6781 cctttcggga gaagatgaca ccagttacat tacacacgtc ttctcattac atagacatta

6841 catgaaacat ttgtgccaac actaacttgt ttacagatta atttgattcg taatgtcaat

6901 gacaaataaa tccaacaaaa atatctgaat tgatggtaag aattttcag aatagaaaca

6961 tctcaacatt atttaacctt taatgttatt tattatagc aaaatgagca attackctac

7021 aataaataat taaatataat tagattagaa attagatttt tgctgctgct taaatctact

7081 taaatccaaa ttgttttact atttgtaatt attgtttttt aagtaaattt taccattttt

7141 ttctacacaa atattttttg ctacttttct aatataagca aaataaacaa ttcctttttg

7201 tttgtttgtt tgtttgtttg tttgtttgtt tggttgggtt tttttttttg gtggtgggggg

7261 ggggggtcaa ctctatcttc aatccacttg aaattgttaa aacaattaag ttaacttaat

7321 cgatttgtgt tgaaacaaca tgaaggaatc atgcggaacc gggcattatt tacagtgtac

7381 attatggata actgtgtttt aaaaaggcat aacagttgac aaaatactga tagataaagc

7441 aagtaacttg cttaaagtga tgtcagttcc tctggcggga aactctaaaa ggcactgagg

7501 catttcattt gttctaatat cattaatttg tgcaaaatgt ttagaataaa gcacatatag

7561 cacatttcat acacagtggc aatacaaagt gctttacata aacaagaaga aaaaaagaca

7621 agaaataaaa gataataaaa atgattaaaa acagattaaa atgtgttaaa acaggttgaa

7681 tgaaaaagaa aagaaagaca taatagtgag atctgtggga cgtagaacag tgctcattca

7741 ttaaaggcac agctaaacag atgtgttttc agtcttgatt tgaatgtgcc taatgttgga

7801 gcacatctga tcctttctgg aagctgattc cagcagtgag gggcatagta gctgaaagct

7861 gattcaccct gctttgactg aactcttgga atatatatta attatatgtc aactctgtta

7921 ttgtgaaatt tcacatgcag ttttagaaac aataatatga ggaaaatcat aaaaaatatg

7981 ctttagaccc gccatgccat atctggtttg agattatagtat cttgtgctaa agcataaaat

8041 ggcagacagt gtcaactttg taattaatta actttgctcg tggtttcaaa agttcaatga

8101 ttaattaagt aacaattagt aacacaatca attctctatt gcattttgca ttaatttaca

8161 ctaataatgt gtcaaattaa tttacactaa tacattctgc atttgttgta tatcattcca

8221 atttatggaa aattaataaa aaatgcctaa aacgcacaat tctagaatga gtcacatgtt

8281 acagataaaa acagtaataa tattttaagc agctcttaag tatgaactga aaacttacat

8341 ttgagcacgt taagtgaaca ttctggatgt caaaggagca aaacggtgtt aaaggtaaaa

8401 tcacaaggtc attcaaaggg acctgatcat gttccttcaa cactgtacaa aatgctgggt

8461 tccatacaat cgatttgtgt tgggtcaaca tgaaggaatt aagttaacct attcgttttt

8521 acaaatttaa gtggattaaa tataaaacaa ttaagttgtc ccagcgaaat ctcaagaatt

8581 gtgttgtttc cactcatttt aaataagtag tttgaacaag tgttttattt tcattcatgc

8641 attttctttt cggcttagtc cctttagaaa tctggggtcg ccacagcaga atgaaccacc

8701 aacttatcca gcatgttttc cagctgcaac ccatcactgg gaaatatcca tacacactca

8761 ttcacacaca tacactacgg caaatttagt ttaattcaaat cacatgcctt tggacttgtg

8821 ggggaaaccg gagcacccgt aggaaaccca catgcacaca gggagaacat gctaactcca

8881 cacagaaatg ccaaataacc cagccgaagc tcgaaccagc gactttcttg ctgtaaggcg

8941 attgtcctac ccactgcgcc accgtgatgc ctgtgtttca tattttaaaa attaatataa

9001 taattatttt agaaaaagaa tgtaatcttg cctgaattga aaaaaaaaga tacttttgct

9061 gcttgtttaa atagtaaata accacagaaa tgtattcttg ttttaatcag caagttgaag

9121 acaaatgaga tgctgtgatt tactgcctgc ttttccacct gggagaatca tctcgggagc

9181 tcattgaaga ctaccaggta cattaaaatg tctaaattac tttttacttt ggatatggtt

9241 atggttatgg tatggttctt ttattattaa agacaagtta gtatatggtg acgcagtggc

9301 acagtaggta gtgctgtcac ctcacagcaa gaaggtcgct gaatcgctgg tttgagtctc

9361 ggctgggtca gttgaagttt ctgtgtgcat attttccctg cgttcacgtg ggtttccttt

9421 gggttctccg gtttcctcca cagtccaaag acatgtggta caggtgaatt gggttggcta

9481 aattagccgt tgtgtatgag tgtgtggacg tttccccagag atgggttgcg gcaggaaggg

9541 catccgctgt gtaaaaacat gctggataag ttggcggttc attctgctgt ggtgaccccg

9601 gattaataaa gggactaagc cgaaaagaaa atgaatgaat gaatgaatga atgaatgaag

9661 ttagtatatt tatcccccaa tattgtaagt tttggctaac ttttaaaaac tttaatcact

9721 ttatgcaaca gatcttaaca tcaatacaat ctattatagc aaaaagcaag agataacgtt

9781 gatatatgac ccccatcatt ctgtctgagg ggacttttta acttgtttaa tgtaaagttt

9841 aattcaaatt aatagaaatg tctggcaagg caagttgcaa tgtatcacaa atttataaa

9901 ataacatttg tatgtaaagt ttactcagga taactaaata agataactag gactatttaa

9961 taaaaaaatt aatctataat atatttaaag gtacgactta aattttacaa acaaaaagtc

10021 aataacattt acttacaatt cccaagtaaa cttaacattt ctaaataaat gtactaataa

10081 ttatgtttag tcttttactt ggcaaaactt tgaagattta ctagcctttt ctgaatgaaa

10141 aaaagtaaac cctactcttc atttttttta caatctacct atataaaatt gagtttaaac

10201 aacacaattc ttcatttttt ttgggggggg gggggtggac aacttaattg ttttatgttt

10261 aatttactta aatttgtaaa accattaaat taaattagtt gttaatcgtt actaaatctt

10321 tttcaattga tacaaacatt ttcaatcata ttctgtcaat aaaacaatgc tcttttttct

10381 tttctgtgaa aaaaatacaa taaaagggta tgacaaaaaa agttaacaaa acaaagttaa

10441 tccgactaaa aatccatgct aaaaaaaagg cagcaagtgt agtggcaaca tcatcctgga

10501 gtcatggaaa tagccataca cataaatgac cggaatcaca acactatcct atggtgtcta

10561 aatacacaat gcatattaat gtaaagctca catggaaaaa tctcatgttt ctggataaaa

10621 tgaatctaaa acacgttcat gccacatata aatatcagta cacctgagcc agaaaaACAA

10681 ACATTTACCA ACCTTCAATC AAGAACGAGT TTGTCTATCT TGGACAAAAT GGCTAGAGgt

10741 acatttacca accttcaatc aagaacgagt ttgtctatct tggacaaaat ggctagaggt

10801 atgtctttgt gtagtgcact ttatttgtag cataccattg atgattatat ctcattatgc

10861 tttattttt gcttacatgc tttacttttt ttactgttca gatttattgg atcgtatttt

10921 aaaacaacag ttttgatgaa tgaatgtatt atggtttgat cttgattgcg ttggttaata

10981 tctgtgttgt cttctagCAT TGGTGCTGTT GCAGTTGGTG GTGGTTAGTT TGCTGGTGAA

11041 TCAGGGGAAA GCCTCCGAAA ACCAGCGGCT CTTCAACAAC GCAGTCATCC GTGTGCAACA

11101 CCTTCACCAG CTGGCTGCAA AAATGATTAA CGACTTTgta agtttcattc aaatcccttc

11161 agaactagtt gatgcaccct ctcatgcaac aatttgctat ttgttataca gGAGGAAGGT

11221 CTTATGCCTG AGGAACGCAG ACAGTTGAGT AAAATTCTTCC CTCTGTCGTT CTGCAACTCT

11281 GACTCCATCG AGACGCCGAC GGGAAAAGAT GAAACGCAAA AAAGCTCTgt gagttaacaa

11341 tatgcttcat taaacaaata tataattgtt gaacaaatgg tgcttcataa aaatgttaac

11401 actttggttt aagtattaat tctcactatt agtggcttat tacctgccta ttattaatat

11461 gttgctgttt attagtactt ttaagtacat attctgcact atctgatcct gcaaatccaa

11521 cccaatacct caacccaagt acaacattac taactgctca tgatcagcaa attatatact

11581 ttgttaaaat ttttgtaaat aatatttaac tgtaatataa actgtatttt gctgtatttt

11641 aaagtcgcat tgtgaaaatg actgtatatt tcacagtaaa gatatacaga caattctgta

11701 aaaacttaca gtataaaaca agataaattg tgttgcattt gtaagtacac tacttttgct

11761 gtaattgatt tacagaaagt aactggcaaa ctgctgccag caagttatactg aagttatcta

11821 aaggaaattg ttagttagtt agttagttag ttagttagtt agttagttag ttagttagtt

11881 agttagttag tttactagat ttatccctta tttaaaagtt taatttgcag catacaccaa

11941 cggggagatt tatataaata gatgcgtttt aaacccaagg tggtactaat ctaaataaat

12001 atccagtctg agcatcaact accacttaga gaggaggaat ttaaaatctt tattggttga

12061 gggatgaatt atataaatta atataaatta ttttgaccta ttccttacac atcacatcca

12121 gtgacacaat atcatcaacc tgatggtaac aaaattagga gtttgttaag ctaaaagtca

12181 cggtttgtta attgtacctg aaaataatgt gtgaaacaaa agaaaagatt cttgatatta

12241 atatagcaat gtgtccttta atttttttta tttcatttat atcccaagga gacaaagtta

12301 aaaaagacag aacatatatt tatgaacata tatatatata tatatatata tattcataaa

12361 tatatgtttt gtctttttta actttgtctc cttgggatat aaatgaaatg tataatttaa

12421 attataattt gtttattata tataaagaga tggtaacaaa catgtcaatg gaaaaaataa

12481 cttgtatatt aagaaataaa tgcataattt tgtaaagaga ttttggacct tttatgttgt

12541 ttttaaaagt tagtgaagtt ggaaatgttt gactagaaga ttagtaaaag aggtgatatt

12601 ctgatgtaaa atactgagaa gactttagtt ttgtttgttc atttgtttgc ttgttagttt

12661 atgttatatc tggaacatgg aaatgtgtat gtacgtgtta aaatataata gaaaggtatt

12721 ggaaaaagaa atgtttggcc gttaaaacca acatgcagtc aaagtgcaac attagattag

12781 agtacataca tgcaagcctt taatgttttt gctgttttca cacaattcaa ttttatttt

12841 tgctcttaaa ttgctcataa atcatataca tttttgcaat ttattttaca tgttgaagat

12901 gcaaaacatg ctaaataaaa aagtgtcaaa acaatcaatt cataattatc gatataaagg

12961 atttataaaa tattttaaat tgatttcctt tttaataata gagcatttgt aaatgtatta

13021 ttgtacacag tcaacacttt tgtgtttaaa gaccaattat tgccagcata aagagaagat

13081 tttttcaaca catttctaaa cataatagtt ttaaataata aataatattt gacttgatat

13141 ttttcaagac acttctatac agcttaaagt gacatttaaa ggcttaacta ggttaattag

13201 gctggttagg gtaattaggc aagttatgtgt ataatgatgg tttgtgctat ggactatcga

13261 aaaaaaaaag gggggggggg gtaataattt tgaccttaaa atgattttta aaaatttaaa

13321 actgctttta ttctagccaa aatgaaacaa ataagacttt ctccagaaga aaaatattat

13381 cagacatgct gtgaaaattt ccttgcttat cttttgggaa atataaaaaa aaagaatttt

13441 ttttaaaagg gggctaatat tgttgacttc aactgtgtgt gtgtctgtat atatatcaac

13501 agtggttaat cctaatcatg ttctcttttt tcagATGTTG AAGCTGCTTC GTATCTCTTT

13561 CCGCCTCATT GAATCCTGGG AGTTTCCCAG CCAGACCTTG AGCTCCACTA TCTCAAACAG

13621 CCTGACCATC GGAAACCCCA ACCAAATCAC TGAGAAACTG GCGGACCTGA AAATGGGCAT

13681 CAGCGTGCTC ATCAAGgtga agcgtactgt gcaagcttac ttgctcaaag ctgatttgtg

13741 ttcatccatt taaactgcca atgtcatgtc accacctggg ggtgatgatt tccaataact

13801 catgcaaagt tgccaatgat ttgaacattg caaaacaatt aatcattttg ggaatcagtt

13861 acttggaaaa actcaaaaca tttctctcca aataatcgca ttaaagttac tttcaaggag

13921 agtttggtca ctcacagcag ccatatctgc atcacctcag gacaactatt ttgggctcac

13981 aagagcaagt catttctgtt tcaatggaga atagcggaat ctaacaaact aagaactcac

14041 aattaaatca tatatggaat aatgttaatc tgacctaaat gtgtttagaa atctgtattt

14101 caggccagta cacaaaggta atcctaaatg ctgtttttaa tgggcgctga tttgacttta

14161 ccaattgctg attagctcct taaagtgtta aaacgtataa aacagttggt tgataaccta

14221 aacctcatct cgaaaggtag ttaagctact gttgagctac acttaaaaat aaatgaataa

14281 aaaaataaat aaacgtaggt tacaaggtac ttcctaaaag tagctagtta caccaaatct

14341 cctcatttat ttcacacaat tacaaaagca taatcgttct ttttaagtta aatatttggt

14401 acttaaaact gaaatacaat tataaggtca aagctcaatg ttctattaca cagctttcaa

14461 aattctcagg gtgtcaagag tcaaagtgca acaaatgtag cagtaacaaa ataaaatgta

14521 gatactgtag ctactggaat ggctacattg tatcataaat agctgagctg ataacatcta

14581 cattgtagct gagctactgt caagctaagg tttttctcca tccatattct ttttcttcaa

14641 agaggaaata gacttgtttg ttaaaccctt ctgtttgcat caattcaact tattataact

14701 gtgtttataa tggaatccat gagggaaaaa aactaattag ttataatgtt gtatagatag

14761 ctagttaaat agaacccaaa agctatgtgt agactcctct ctcaaaatac tgtaatattg

14821 ataacactta ttatgtgtaa ttaattatat atattatata gtatcatatg taatacataa

14881 tacaccttgt aagtggttgg tttggttcta aacttattaa gcttaaacac ccttgtgcaa

14941 aaaaaaatat gtatattatg tacgtacgta caaactccac tcagaattgc caattgaccc

15001 agtcggggct cgaaccagca accctcttgc tgtaagggca ttgtgctacc cactacgcca

15061 ccgtgctgtc ctaaatttta tttatttatt tttgtcacaa aggttttaat ttttacactt

15121 ctgagtattt ccatgtgcaa tatgattaag tctttgtata tttccctgac atcctttggc

15181 atttgatttt gcacagGGAT GTCTCGATGG ACAGCCAAAT ATGGATGACA ACGACTCCCT

15241 GCCGTTGCCT TTTGAGGATT TCTACCTGAC CGTAGGGGAG ACCAGTCTCA GAGAGAGCTT

15301 TCGCCTGCTG GCCTGCTTCA AGAAGGACAT GCACAAGGTG GAAACTTACC TGAGGGTTGC

15361 GAATTGCAGG AGATCTCTGG ATTCCAACTG TACCCTGTAG agggcgctaa tgtattgcta

15421 gtcaaagcct gctttatcct tttctgcaaa tctaagacca gtttgcatta tcaaaacata

15481 aactaattat tatctggtcc tatatatgca ggaaatatca agcaggcatg gctggatctg

15541 tactttatt cccttccata aaccttacac ctaccaccat tgtatttatt cttcttattg

15601 ggaagtatta tcatttcaag atgttcctta aaaacgtaaa tattgattct tatttaatat

15661 ccgaacctta ttcacagtgg tgcttagcaa tttctggcga tattttctta aatgtgccaa

15721 aattgactta aatcaaagtg ctaatattgt gctttggtgt atattatatc taaaacagtt

15781 aaagatcagt gttcaaaggg ttcactccca aatgtgtgaa tggaaacgtg tctgtctgat

15841 agattcttgc cttaatatta tcaactcatc ctgttctatt ctaactgtat caattaaagt

15901 tttaaaatgc attcagtgtg tcttctgttc tgtataacat gtttgtgtca gttacattta

15961 catttagtca tttagcagac gcttttatcc aaagcaactt acaaatgagg acaaggaagc

Claims (1)

1. A method for in situ modification and breeding of fish genes, characterized in that it comprises the following steps: 1) Isolate the β-actin gene start-up regulatory sequence, growth hormone gene start-up control sequence and flanking sequences of fish; 2) using the growth hormone gene promoter regulatory sequence in the fish gene as the target DNA fragment for homologous recombination; 3) replacing the growth hormone gene activation regulatory sequence with the β-actin gene activation regulatory sequence by homologous recombination; 4) Construction of homologous recombination vector: A) The homologous recombination vector includes the 5' flanking sequence of the growth hormone initiation and regulation sequence, the 3' flanking sequence of the growth hormone initiation and regulation sequence, the β-actin gene initiation and regulation sequence, the green fluorescent protein forward marker gene and two homologous Directed Cre/loxP sequence, red fluorescent protein negative marker gene; B) The 5' flanking sequence of the growth hormone activation regulatory sequence is used as a homologous long arm, and the 3' flanking sequence of the growth hormone activation regulatory sequence is used as a homologous short arm; C) placing the two homologous Cre/loxP sequences on both sides of the green fluorescent protein forward marker gene, and then placing the green fluorescent protein forward marker gene on the homologous long arm and the inner side of the homologous short arm; D) placing the red fluorescent protein negative marker gene outside the homologous long arm and the homologous short arm; E) Digested with a restriction endonuclease to obtain a linear recombinant DNA fragment; 5) After artificial insemination of the fish, before the first cleavage, the homologous recombination vector is introduced into the fertilized eggs by microinjection, and then the fertilized eggs are hatched in a sterile environment at 26°C to 30°C in the laboratory ; 6) Under the fluorescent dissecting microscope, the embryos with only green fluorescence were screened to obtain homologous recombination chimeras; 7) Cells or embryos with homologous recombination confirmed by PCR and Southern hybridization.

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