Anthracene nucleus antibiotic producing strain dnrX gene fragment, gene breaking engineering bacterium prepared from the same and application thereof
A technology of gene fragments and antibiotics, applied in the field of genetic engineering, can solve problems such as low conversion rate, environmental pollution, complex process, etc., and achieve the effect of increasing production
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Embodiment 1
[0032] Embodiment 1 PCR amplification dnrX' gene
[0033] Since only the dnrX gene sequence (accession number AF048833) derived from the daunorubicin-producing strain Streptomyces persei ATCC29050 has been published in GenBank, and there is no relevant report on the gene sequence outside the final codon. Given that the purpose of amplifying the gene is to block mutations, an incomplete gene can be amplified. Based on this, primers dnrX1 and dnrX2 were designed.
[0034] dnrX1 sequence: 5'-GGCCGCCAGCCGCTGTCCGA-3' (SEQ ID No.5)
[0035] dnrX2 sequence: 5'-GTGGTTCCAGGCGAAGAGCAGCGCATAGTC-3' (SEQ ID No.6)
[0036] PCR amplification was performed using the SIPI-1482 genome of S. coeruleorubidus as a template, and the reaction system was shown in Table 1:
[0037] Table 1 Composition of PCR reaction system
[0038]
[0039] The reaction conditions were: pre-denaturation at 97°C for 5 min, denaturation at 95°C for 0.5 min, annealing at 60°C for 1 min, extension at 72°C for 1 min,...
Embodiment 2
[0042] Preparation and sequencing of the gene fragment dnrX" of embodiment 2dnrX'
[0043] The plasmid pYG957 was digested with Nco I, and the large fragments were recovered for self-ligation, transformed into DH5α competent cells, and the intermediate plasmid pYG962 (such as Figure 4 shown). The intermediate plasmid pYG962 was verified by double digestion with Kpn I and HindIII, and the result of double digestion with Kpn I and HindIII of pYG957 was used as a control. Such as Figure 5 As shown, two bands of 1203bp and 2651bp were obtained by double enzyme digestion of pYG962, and two bands of 1080bp and 2651bp were obtained by double enzyme digestion of pYG957. correct.
[0044] Then, dnrX" was obtained by digestion with KpnI+HindIII from plasmid pYG962. The measured sequence is shown in SEQ ID No.3 in the sequence listing, and the encoded amino acid sequence is shown in SEQ ID No.4.
Embodiment 3
[0045] Example 3 Construction and Verification of Destroying dnrX Using Single Exchange to Block Mutation Plasmid pYG963
[0046] The dnrX" gene fragment obtained from plasmid pYG962 digested with KpnI+HindIII and the apramycin resistance gene apr (GenBank accession number: AJ566337, commercially synthesized by Yingjun Company) were connected to the vector pBluescript2KSM and constructed into recombinant plasmid pYG963 (Such as Figure 6 shown), transform DH5α competent cells, and select transformants by apramycin and ampicillin resistance. Extract the plasmid for PCR verification and enzyme digestion verification, such as Figure 7 shown. Using dnrX1 and dnrX2 as primers for PCR, using the plasmid pYG957 as a template can amplify the partial gene dnrX' (1080bp) of the dnrX gene, but not using pYG963, such as Figure 7 (A), consistent with the expected results. Use the common enzyme cutting site Sal I on the original vector and the insert to carry out pYG963 enzyme digesti...
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