Carrier for coexpression of multiple genes of chlamydomonas reinhardtii and construction method thereof
A multi-gene, co-expression technology, applied in the field of genetic engineering, can solve the problems of backward expression ability of exogenous genes, inability to cope with simultaneous expression, and insufficient expression efficiency of exogenous genes, so as to achieve high gene expression efficiency and improve the effect of expression
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example 1
[0026] Example 1 Construction of carrier pJD124 of the present invention
[0027] The vector pMD18-T simple was purchased from Takara Company, endonuclease and T4 DNA ligase were purchased from Thermo Science Company, and primers and artificial genes were synthesized at Shanghai Sangon Bioengineering Company.
[0028] BleF (SEQ ID NO: 1) and BleR (SEQ ID NO: 2) are PCR amplification primer sequences for amplifying the bleomycin resistance gene.
[0029] RBCSTF (SEQ ID NO: 3) and RBCSTR (SEQ ID NO: 4) are PCR amplification primer sequences for amplifying terminators.
[0030] ProF (SEQ ID NO: 5) and ProR (SEQ ID NO: 6) are PCR amplification primer sequences for amplifying the promoter P h5-rb .
[0031] SEQ ID NO: 7 is the DNA sequence of the artificially synthesized bleomycin resistance gene.
[0032] SEQ ID NO: 8 is the DNA sequence of the artificially synthesized terminator Ter.
[0033] SEQ ID NO: 9 is the artificially synthesized promoter P h5-rb DNA sequence.
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example 2
[0041] The construction of the carrier pJD124-GFP that example two expresses GFP gene alone
[0042] Vector pEGFP-N1 was purchased from Clontech Company.
[0043] 1. Use the vector pEGFP-N1 as a template, F1 and R1 as primers (F1: 5' tct aagctt atggtgagcaagggcgagg 3', R1: 5' tat gaattc ttacttgtacagctcgtc 3', underlined to amplify the GFP gene, and the amplified product was purified with a PCR product purification kit.
[0044] 2. Digest the purified GFP amplification product with HindIII and EcoRI, and recover the 730bp fragment; digest the vector pJD124 with HindIII and EcoRI, and recover the large fragment; connect and transform the recovered large vector fragment with the 730bp fragment to obtain the recombinant vector , denoted as pJD124-GFP, see image 3 , the vector can be used to express the GFP gene alone.
example 3
[0045] The construction of the vector pJD124-RED of example three expressing RED gene alone
[0046] Vector pDsRed2-N1 was purchased from Clontech Company.
[0047] 1. Use the vector pDsRed2-N1 as a template, F2 and R2 as primers (F2: 5' tct aagctt ATGGTGCGCTCCTCCAAG 3', R2: 5' tat gaattc CTACAGGAACAGGTGGTGG 3', the underlined part is the endonuclease recognition site) to amplify the Red gene, and the amplified product was purified with a PCR product purification kit.
[0048] 2. Digest the purified Red amplification product with HindIII and EcoRI, and recover the 680bp fragment; digest the vector pJD124 with HindIII and EcoRI, and recover the large fragment; connect and transform the recovered large fragment with the 680bp fragment to obtain the recombinant vector, Denoted as pJD124-Red, see Figure 4 . The vector can be used to express the Red gene alone.
[0049] Image 6 The enzyme digestion verification results of the GFP gene vector and the RED gene vector con...
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