Method for constructing recombinant expression vector simultaneously expressing a plurality of genes
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A carrier and gene technology, applied in the field of constructing recombinant expression vectors that express multiple genes at the same time, can solve the problems of laborious, time-consuming and expensive multi-gene assembly
Inactive Publication Date: 2011-02-23
CHINA AGRI UNIV
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However, multigene assembly using the methods described above...
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[0046] 1. Construction of CmR gene expression cassette
[0047] 1. Using CmF01 and CmR01 as primers and using pGWC as a template to perform PCR amplification to obtain PCR amplification products;
[0048] 2. Use CmF02 and CmR01 as primers, and use the PCR amplification product in step 1 as a template to perform PCR amplification to obtain a PCR amplification product;
[0049] 3. Digest pL34R12-CmR-ccdB with restriction endonucleases BamHI and SalI; recover the vector backbone, digest it with T4 DNA polymerase to produce blunt ends; connect the blunt-ended vector backbone to the PCR amplification product in step 2 , to obtain the recombinant plasmid pL34R12-Cm+.
[0050] 2. Remove the oriR6KγHindIII site
[0051] 1. Construction of recombinant plasmid pL4R2mVT
[0052] ① Using CmF03 and CmR02 as primers and using pL34R12-Cm+ as a template for PCR amplification, the obtained PCR amplification product was digested with rest...
Embodiment 2
[0188] Embodiment 2, MISSA experiment
[0189] 1. Construction of Donor Vector
[0190] 1. Construction of donor vector pL-Hyg
[0191] ① Use MarkerF and MarkerR as primers and pMDC99 as a template to carry out PCR amplification, and the obtained PCR amplification product is digested with restriction endonucleases HindIII and EcoRI to obtain the digested product;
[0192] ② Digest pL-ccdB with restriction endonucleases HindIII and EcoRI to recover the vector backbone;
[0193] ③ Ligate the digested product of step ① with the vector backbone of step ② to obtain the recombinant plasmid pL-Hyg.
[0194] 2. Construction of donor vector pR-TM2
[0195] ① Digest pL12R34sB-MAR with restriction enzymes HindIII and EcoRI, and recover a fragment of about 983bp;
[0196] ② Digest pR-ccdB with restriction endonucleases HindIII and EcoRI to recover the vector backbone;
[0197] ③ Ligate the small fragment in step ① with the vector backbone in step ② to obtain the recombinant plasmid pR-...
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Abstract
The invention discloses a method for constructing a recombinant expression vector simultaneously expressing a plurality of genes. An MISSA system consists of a strain and a vector, wherein the strain comprises a donor strain and an acceptor strain; the vector comprises a donor vector and an acceptor vector; replication initiation protein carried by a chromosome of the donor strain is responsible for replicating and amplifying a suicidal donor vector, and the carried conjugal transfer protein Tra is responsible for conjugal transfer of the donor vector; the acceptor strain can induce and express two sets of locus specific recombinant proteins, namely Cre recombinant enzyme and lambda phage locus specific recombinant proteins; when the donor strain is mixed with the acceptor strain, the donor strain and the acceptor strain are conjugated, and the donor strain is transferred into the acceptor vector because the donor vector in the donor strain carries an oric element; and the donor vector and the acceptor vector undergo recombination reaction in the acceptor strain under the action of the two sets of locus specific recombinant proteins, so that a target gene carried by the donor vector is assembled onto the acceptor vector. The process is repeated, so a plurality of target genes can be assembled onto the acceptor vector according to the preset direction and order.
Description
technical field [0001] The invention relates to a method for constructing a recombinant expression vector for simultaneously expressing multiple genes. Background technique [0002] Many agronomic traits, including crop yield traits, metabolic pathways, protein complexes and signal transduction pathways, are controlled by polygenes. Genetic manipulation of multiple genes has important application prospects for both basic research and bioengineering. Golden rice (Ye X, Al Babili S, Kloti A, Zhang J, Lucca P, Beyer P, Potrykus I (2000) Engineering the provitamin A (beta-carotene) biosynthetic pathway into (carotenoid-free) riceendosperm. Science 287: 303- 305) and purple tomato (Butelli E, Titta L, Giorgio M, Mock HP, Matros A, Peterek S, Schijlen EG, Hall RD, Bovy AG, Luo J, Martin C (2008) Enrichment of tomato fruit with health-promoting anthocyanins by expression of select transcription factors.Nat Biotechnol 26:1301-1308) are two successful cases of multi-gene transgenic...
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